Antioxidant Activity Evaluation and

Bioavailability Enhancement of Bioactive

Components from Selected Medicinal Plants

BY

Umer Younas

DEPARTMENT OF CHEMISTRY
UNIVERSITY OF SARGODHA
SARGODHA, PAKISTAN
2019
i
 Antioxidant Activity Evaluation and
Bioavailability Enhancement of Bioactive
Components from Selected Medicinal Plants

A thesis submitted in partial fulfillment of the requirements for the degree of

DOCTOR OF PHILOSOPHY

IN

CHEMISTRY

BY

Umer Younas

Department of Chemistry
University of Sargodha
Sargodha, Pakistan
ii
 O Lord!
Advance me in Pure knowledge & Lead me to the Straight Path
(Ameen)

iii
 Dedicated to

my parents (especially Mother and NaNi Maa)

and teachers, all those, whose affection,

encouragement and prayers made me able to

accomplish this task

iv
 DECLARATION

I hereby declare that the work described in this thesis was carried out by me under the

supervision of Dr. Shahid Iqbal and co-supervision of Dr. Jamshed Akbar,

Department of Chemistry, University of Sargodha, Sargodha, for the award of degree

of Doctor of Philosophy in Chemistry.

I also hereby declare that the work mentioned in this thesis titled “Antioxidant

activity evaluation and bioavailability enhancement of bioactive components from

selected medicinal plants” is original and nothing has been stolen /copied/ plagiarized

from any other source.

___________

Umer Younas
Ph.D. Scholar
Department of Chemistry,
University of Sargodha, Sargodha

v
 CERTIFICATE OF ORIGNIALITY OF RESEARCH WORK

It is certified that the research work mentioned in this thesis titled “Antioxidant

activity evaluation and bioavailability enhancement of bioactive components from

selected medicinal plants” by Mr. Umer Younas is original and nothing has been

stolen/copied/plagiarized from any source.

___________

Supervisor
Dr. Shahid Iqbal
Assistant Professor,
Department of Chemistry,
University of Sargodha, Sargodha

___________

Co-Supervisor
Dr. Jamshed Akbar
Associate Professor,
Department of Chemistry,
University of Sargodha, Sargodha

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vii
 ACKNOWLEDGEMENT
All the praises to Almighty Allah, the Creator and Sustainer of universe, who is the Origin of all
knowledge and wisdom. All regards to Holy Prophet Muhammad (S. A. W.) who paved us to right
path with essence of faith in Allah.
I would like to express my gratitude and appreciation to my supervisor, Dr. Shahid Iqbal Rana,
and co-supervisor Dr. Jamshed Akbar for their sympathetic attitude, sincere cooperation,
meticulous criticism, indefatigable zeal, patronizing concerns and providing the available facilities
throughout my Ph.D.
My sincerest thanks are to Prof. Dr. Farooq Anwar, Chairman, Department of Chemistry,
University of Sargodha, Sargodha, for providing facilities during the conduct of this research
work.
It is immense pleasure to express my profound indebtness and gratitude to Madam Fozia batool,
for guiding me in computational and statistical analysis. I am thankful to Dr. Muhammad Sher
for providing me available instrumental facilities at Hi-Tech laboratory UOS. I am also tankful to
Dr. Raja Adil Sarfaraz, for helping me in HPLC analysis. I would also like to express my
gratitude to Prof. Dr. M.H. Qazi (Late) VC, The University of) Lahore, for providing me Lab
access.
I pay my special thanks to my research group fellows Mr. Hafiz Abdul Jabbar, Zohaib Saeed, for
their support during my research work. I am thankful to Mr. Asif Saleem, Mr. Rizwan kausar,
Mr. Fakhar udin Akabar, Mr. M. Jameel, Dr. M. Sarfaraz, and Dr. Adnan Ashraf for being
supportive to me. I pay my special thanks to Muhammad Arshed for helping me in computational
optimization of phenolic acid molecules.
I would like to express my gratitude to my colleagues at Department of Chemistry, UOL, Prof.
Dr. Zaid Mahmood, Prof. Dr. M. Zuber, Dr. Faiza Hassan, Ms. Maimoona Jillani, Mr. Qaisar
Manzoor, Mr. Zahid Ali, for their support.
I acknowledge my heartily thanks to my Family for their loving and convivial behaviors which
always sooth & calm me. I would like to thank my all friends who were always available for
sharing joys and sorrows during this duration. I am highly indebted for their support, cooperation
and nice attitude.
A cordial thank to all who directly or indirectly helped me during my lab work. May Allah bless all
above mentioned personalities long, happy and peaceful life. (Ameen)

Umer Younas

viii
 Table of Contents
List of Figures XV
List of Tables XVI
Abstract XVII
Chapter 1 1
Introduction
1.1. Significance of Medicinal Plants 2
1.2. Bioactive Compounds 3
1.2.1. Antioxidant bioactives 3
1.2.2. Mechanism of Action of Antioxidants 5
1.3. Extraction of Antioxidant Components from Botanical Material 7
1.3.1. Hot continuous extraction (soxhlet extraction) 9
1.3.2. Orbital shaker-assisted extraction 9
1.3.3. Microwave-assisted extraction 10
1.4. Optimization of Extraction Processes using Response Surface 11
Methodology
1.5. Fractionation of Crude Extract 13
1.6. Bioavailability Enhancement of Bioactive Compounds 14
1.7. Stabilization Efficiency of Plant Extracts in Different Model 17
Systems
1.7.1. Mechanism of lipid peroxidation 18
1.7.2. Prevention from lipid peroxidation 19
1.8. Medicinal Flora of Pakistan 20
1.9. Medicinal Plants Selected for Current Study 21
1.9.1. Mulberry 21
1.9.2. Artemisia Annua 22
1.9.3. Periploca Aphylla 23
1.9.4. Kenaf 23
1.10. Quantitative Structure Activity Relationship QSAR 25
1.11. Objectives of Study 26
1.12. Significance of Current Study 27
Chapter 2 28
Review of Literature
Part I 29
2.0. Determination of Proximate Composition and Antioxidant
Potential of Selected Medicinal Plants
Part II 37
2.2. Optimization of Extraction Process for Bioactive Compounds
from Leaves of Morus nigra and Artemisia annua Using RSM
Part III 40
2.3. Bioactive-Enriched Fractionation of Periploca Aphylla Crude
Extract Followed by Identification and Quantification of Novel
Antioxidant Compounds
Part IV 42
2.4. Nano-encapsulation of Defatted Kenaf Seed Meal (DKSM)
Extract to Enhance its Bioavailability and Antioxidant Activity
Part V 45

ix
2.5. Stabilization Efficiency of Selected Extracts on Shelf-life of
different Food Items (Oil and Meatballs)
Part VI 48
2.6. Structure-activity Relationship of Selected Phenolic Acids in
Order to Propose Mechanism of Their Antioxidative Action
Chapter 3 51
Materials and Methods
3.1. Chemicals and Reagents 52
3.2. Sample collection 52
Part I 53
3.3. Determination of Proximate Composition and Antioxidant
Potential of Selected Medicinal Plants
3.3.1. Determination of proximate composition of selected medicinal 53
plants
3.3.1.a. Ash content 53
3.3.1.b. Moisture 53
3.3.1.c. Lipid content 53
3.3.1.d. Alkaloid content 53
3.3.1.e. Protein content 54
3.3.1.f. Total carbohydrate content 54
3.3.1.g. Phytate content 55
3.3.2. Determination of Antioxidant Potential 56
3.3.2.a. Extraction of Samples 56
3.3.2.b. Determination of total phenolic content (TPC) 56
3.3.2.c. Determination of total flavonoid content (TFC) 56
3.3.2.d. Determination of ascorbic acid content (AAC) 57
3.3.2.e. Determination of total tannins content (TTC) 58
3.3.3. Determination of Antioxidant Activities 58
3.3.3.a. DPPH free radical scavenging activity assay 58
3.3.3.b. ABTS radical cation scavenging activity assay 59
3.3.3.c. Ferric Reducing Antioxidant Power (FRAP) Assay 60
3.3.3.f. Lipid peroxidation 60
Part II 61
3.4. Optimization of Extraction Process for Bioactive Compounds
from Leaves of Morus nigra and Artemisia annua Using RSM
3.4.1. Response Surface Methodology (RSM) 62
3.4.2. Orbital shaker-assisted extraction 63
3.4.3. Microwave-assisted extraction 63
3.4.4. Determination of antioxidant potential 63
Part III 64
3.5. Bioactive-Enriched Fractionation of Periploca Aphylla Crude
Extract Followed by Identification and Quantification of Novel
Bioactive Compounds
3.5.1. Fractionation of Periploca aphylla (PA) crude extract 64
3.5.2. Determination of antioxidant potential 64
3.5.3. Quantification of phenolic acids in Periploca aphylla fractions 65
by HPLC
3.5.3.a. Sample preparation 65

x
 3.5.3.b. HPLC analysis 65
3.5.4. Sub-fractionation of PA ButOH fraction 65
3.5.5. Determination of bioactive compounds in PA sub-fraction by 66
GC-MS
Part IV 67
3.6. Stability of Antioxidant Components from Defatted Kenaf
(Hibiscus cannabinus L.) Seed Meal (DKSM) Under Simulated
Gastrointestinal pH Conditions
3.6.1. Preparation of defatted kenaf seed meal (DKSM) 67
3.6.2. Preparation of defatted kenaf seed meal (DKSM) extracts 67
3.6.3. Determination of antioxidant potential of treated and untreated 68
DKSM
3.6.4. HPLC-DAD analysis of major phenolic compounds in treated 68
and untreated DKSM extracts
3.7. Nano-encapsulation of DKSM Extract to Enhance its 69
Bioavailability and Antioxidant Activity
3.7.1. Preparation of DKSM extract loaded nanoparticles 69
3.7.2. Characterization of nanoparticles 70
3.7.3. Determination of extract loading efficiency 70
3.7.4. In-vitro release of phenolic compounds from DKSM extract 70
loaded nanoparticles
Part V 71
3.8. Stabilization Efficiency of Encapsulated and Non-encapsulated
A. annua Leave Extract on Shelf-life of Sunflower Oil
3.8.2. Oil Stabilization Analysis 72
3.8.2.a. Weight gain analysis 72
3.8.2.b. Determination of peroxide value (PV) 72
3.8.2.c. Determination of acid content 73
3.8.2.d. Determination of p-anisidine value 73
3.8.2.e. Conjugated dienes and conjugated trienes 74
3.8.2.f. Thiobarbituric acid reactive substances (TBARS) 74
assay
3.9. Cinnamon Bark Deodorised Aqueous Extract as Potential 75
Natural Antioxidant in Meat Emulsion System
3.9.1. Preparation of cinnamon bark deodorised aqueous extracts 75
(CinDAE)
3.9.2. Chicken meatball processing and storage 75
3.9.3. Oxidative stability assessment 76
Part VI 76
3.10. Structure-activity Relationship of Selected Phenolic Acids in
Order to Propose Mechanism of Their Antioxidative Action
3.10.1. Optimization of 3-D molecules of phenolic acids 77
3.10.2. Artificial neural network (ANN) 77
Chapter 4 78
Results and Discussion
Part I 79
4.1. Proximate Composition and Antioxidant Potential of Selected
Medicinal Plants

xi
4.1.1. Proximate Composition and Antioxidant Potential of Leaves 79
from Three Mulberry Varieties
4.1.1.a. Proximate composition 79
4.1.1.b. Total phenolic content (TPC) 80
4.1.1.c. Total flavonoid content (TFC) 81
4.1.1.d. Ascorbic Acid Content (AAC) 81
4.1.1.e. DPPH radical scavenging activity 81
4.1.1.f. ABTS radical cation scavenging activity 82
4.1.1.g. Ferric ion reducing antioxidant power (FRAP) 82
4.1.1.h. Correlation among antioxidant components and 83
assays
4.1.2. Artemisia annua L. Leaves: Chemical Composition and 85
Antioxidant Potential of Different Solvent Extracts
4.1.2.a. Chemical composition 85
4.1.2.b. Total phenolic Content (TPC) 87
4.1.2.b. Total flavonoid content (TFC) 87
4.1.2.c. Ferric reducing antioxidant power (FRAP) assay 88
4.1.2.d. Trolox equivalent antioxidant capacity (TEAC) 89
assay
4.1.2.e. DPPH radical-scavenging potential 90
4.1.2.f. Lipid peroxidation 91
4.1.2.g. Statistical analysis 92
Part II 93
4.2. Optimization of Extraction Process for Bioactive Compounds
from Leaves of Morus nigra and Artemisia annua Using RSM
4.2.1. Experimental design for orbital shaker assisted optimized 94
extraction of antioxidant compounds from A. annua using
response surface methodology
4.2.2. Optimization of orbital shaker assisted extraction of 94
antioxidants from A. annua leaves using response surface
methodology
4.2.2.a. Total phenolic content 94
4.2.2.b. Total flavonoid content (TFC) 95
4.2.2.c. Ascorbic acid content (AAC) 95
4.2.2.d. DPPH radical scavenging activity 98
4.2.2.e. ABTS radical cation scavenging activity 99
4.2.3. Experimental design for microwave-assisted optimized 101
extraction of antioxidant compounds from Morus nigra using
response surface methodology
4.2.4. Optimization of microwave assisted extraction of antioxidants 101
from M. nigra leaves using response surface methodology
4.2.4.a. Total phenolic content (TPC) 101
4.2.4.b. Total flavonoid content (TFC) 102
4.2.4.c. Ascorbic acid content (AAC) 102
4.2.4.d. DPPH radical scavenging activity 103
4.2.4.e. ABTS radical cation scavenging activity 103
Part III 107
4.3. Bioactive-Enriched Fractionation of Periploca Aphylla Extracts

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 Followed by Identification and Quantification of Novel
Antioxidant Compounds
4.3.1. Antioxidant potential of different solvent fractions from 107
Periploca aphylla (PA) crude extract
4.3.1.a. Total phenolic content (TPC) 107
4.3.1.b. DPPH radical scavenging activity 108
4.3.1.c. ABTS radical cation scavenging activity 109
4.3.1.d. HPLC quantification of phenolic acids from 109
Periploca aphylla solvent fractions
4.3.2. Antioxidant activity of sub-fractions from Periploca aphylla 112
butanol fractions
4.3.2.a. DPPH radial scavenging potential of sub-fractions of Periploca 112
aphylla butanol fraction
4.3.2.b. ABTS radical cation scavenging activity of sub-fractions of 113
Periploca aphylla butanol fraction
4.3.2.c. Determination of bioactive compounds butanol sub-fraction 12 114
by GC-MS after derivatization
Part IV 120
4.4. Stability of Antioxidant Components from Defatted Kenaf
(Hibiscus cannabinus L.) Seed Meal (DKSM) Under Simulated
Gastrointestinal pH Conditions
4.4.1. Effect of simulated GI pH conditions on extract yield and TPC 120
of DKSM
4.4.2. Effect of simulated gastrointestinal conditions on antioxidant 121
potential of DKSM
4.4.2.a. DPPH radical scavenging potential 121
4.4.2.b. ABTS radical cation scavenging assay 122
4.4.2.c. Ferric Ion Reducing Antioxidant Power 123
4.4.2.d. Iron Chelating Potential 124
4.4.2.e. Hydroxyl radical scavenging potential 125
4.4.2. Effect of simulated GI pH conditions on Phenolic composition 127
of DKSM
4.5. Nano-encapsulation of DKSM Extract to Enhance its 130
Bioavailability and Antioxidant Activity
4.5.1. Characterization of DKSM extract loaded nanoparticles 130
4.5.1.a. SEM analysis of nanoparticles 130
4.5.1.b. Encapsulation efficiency and loading efficiency 130
4.5.1.c In-vitro release of phenolic compounds from 131
DKSM extract loaded nanoparticles
Part V 134
4.6.1. Stabilization Efficiency of Encapsulated and Non-encapsulated
A. annua Leave Extract on Shelf-life of Sunflower Oil
4.6.1.a. Weight gain analysis (WGA) 134
4.6.1.b. Peroxide value (PV) 135
4.6.1.c. Free fatty acids (FFA) content 136
4.6.1.d. Conjugated dienes and trienes (CD & CT) 137
4.6.1.e. Thiobarbituric acid reactive substances (TBARS) 139
4.6.2. Cinnamon bark deodorised aqueous extract as potential natural 140

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 antioxidant in meat emulsion system
Part VI 145
4.7. Structure-activity Relationship of Selected Phenolic Acids in
Order to Propose Mechanism of Their Antioxidative Action
4.7.1. Descriptors calculation and selection 145
4.7.2. QSAR model generation 146
4.7.3. Interpretation of model 147
4.7.3.a ABTS radical cation scavenging activity 147
4.7.3.a DPPH radical scavenging activity 148
CONCLUSION 156
REFERENCES 159

xiv
 List of Figures

Figure 4.1.1. Yield and contents of phenolics, flavonoids and ascorbic acid 82
in extracts from leaves of three Morus varieties (P < 0.05) (85)
Figure 4.1.2. Antioxidant activity (mMTE) of leaves from three species of 83
Morus (Mulberry) against DPPH, ABTS and FRAP assay
Figure 4.1.3. Variation in contents of total phenolics and total flavonoids in 88
various solvent extracts of Artimisia annua leaves, with
increasing polarity of solvent
Figure 4.1.4. Ferric reducing antioxidant power and trolox equvilant 90
equvilant antioxidant capacity of various solvent extracts of
Artimisia annua leaves
Figure 4.1.5. DPPH radical scavenging potential of various solvent extracts 91
of Artimisia annua leaves
Figure 4.1.6. Lipid peroxidation in extracts of Artimisia annua leaves 91
prepared in different solvents
Figure 4.2.1. Response Surface plots showing the effect of orbital shaker- 96
assisted extraction parameters on TPC, TFC, AAC, %
inhibition (DPPH & ABTS) of 70 % MeOH extracts of A.
annua leaves
Figure 4.2.2. Response Surface plots showing the effect of microwave- 104
assisted extraction parameters on TPC, TFC, AA and %age
inhibition (DPPH• and ABTS•+) of 70 % MeOH extracts of M.
nigra leaves
Figure 4.3.1. Total phenolic content in solvent fractions of Periploca 108
aphylla (PA)
Figure 4.3.2. DPPH radical %age inhibition of different solvent fractions 108
from Periploca aphylla (PA) crude extract
Figure 4.3.3. ABTS radical cation %age inhibition of different solvent 109
fraction from Periploca aphylla (PA) crude extract
Figure 4.3.4. DPPH radical scavenging activity of sub-fractions from 113
Periploca aphylla butanol fraction
Figure 4.3.5. ABTS radical cationscavenging activity of sub-fractions from 113
Periploca aphylla butanol fraction
Figure 4.3.6. Mass spectrum of butanol fraction 114
Figure 4.3.7. Structures of compounds identified and quantified in sub- 117
fraction of PA from butanol fraction
Figure 4.4.1. Antioxidant activities of defatted kenaf seed meal as affected 124
by simulated gastrointestinal pH condition
Figure 4.4.2. ESR spectra of hydroxyl radical scavenging activity of treated 126
and untreated defatted kenaf seed meal
Figure 4.5.1. SEM images of nanoparticles loaded with DKSM extract 130
Figure 4.5.2. Percentage release of total phenolic content from encapsulates 133
at different pH
Figure 4.5.3. Percentage release of total phenolic content from encapsulates 133
at different Temperature
Figure 4.6.1. Variation in weight gain of control and stabilized sunflower 135
oil samples
Figure 4.6.2. Variation in peroxide value (PV) of control and stabilized oil 136

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 samples
Figure 4.6.3. Variation in free fatty acid content in control and stabilized oil 137
samples
Figure 4.6.4.a. Variation in conjugated diene content of control and treated 138
oil samples
Figure 4.6.4.b. Variation in conjugated triene content of control and treated 139
oil samples
Figure 4.6.5. Variation in TBARS in control and stabilized oil samples 140
Figure 4.6.6.a. Change in peroxide value of chicken meatballs during storage 144
Figure 4.6.6.b. Change in TBRAS value of chicken meatballs during storage 144
Figure 4.7.1. Plot of experimental results vs ANN predicted response 154
(ABTS•+)
Figure 4.7.2. Plot of experimental results vs ANN predicted response 155
(DPPH•)

List of Tables
Table 4.1.1 Proximate composition of leaves from three varieties of morus 80
(Mulberry)
Table 4.1.2 Pearson correlation among chemical/antioxidant constituents 85
and scavenging activities for leaves from three varieties of
mulberry
Table 4.1.3. Chemical composition of Artemisia annua leaves 86
Table 4.1.4. Correlation coefficient between TPC, TFC, Lipid peroxidation 92
FRAP, TEAC and DPPH assays
Table 4.2.1 The coded values and corresponding actual values of the 94
optimization parameters used in Response Surface Analysis
Table 4.2.2. Measured and predicted TPC, TFC, AAC and % inhibition 97
(DPPH & ABTS) values for A. Annua leaves using orbital
shaker-assisted extraction technique
Table 4.2.3. Polynomial equations and statistical parameters for A. annua 98
leaves using orbital shaker-assisted extraction technique
Table 4.2.4. Optimized conditions to achieve maximum antioxidant potential 100
A. annua leaves
Table 4.2.5. The coded values and corresponding actual values of the 101
optimization parameters used in Response Surface Analysis
Table 4.2.6. Measured and predicted TPC, TFC, AAC, %age inhibition 105
(DPPH• and ABTS•+) values for M. nigra leaves extract prepared
using microwave-assisted extraction technique
Table 4.2.7. Polynomial equations and statistical parameters for M. nigra 106
leaves using microwave-assisted extraction technique, leaves
dried under hot air oven
Table 4.2.8. Optimized conditions to achieve maximum antioxidant potential 107
A. annua leaves
Table 4.3.1. Phenolic composition of Periploca aphylla different solvents 111
fractions by HPLC
Table 4.3.2. GC-MS analysis of Periploca aphylla butanol fraction 118
Table 4.4.1. Extract yield and total phenolic content of defatted kenaf seed 121
meal as subjected to simulated gastrointestinal pH condition

xvi
Table 4.4.2. Phenolic composition of defatted kenaf seed meal as affected by 129
simulated gastrointestinal pH condition
Table 4.7.1. Descriptors Selected for Modeling Purpose (ABTS•+ %age 146
inhibition)
Table 4.7.2. Descriptors Selected for Modeling Purpose (ABTS•+ %age 147
inhibition)
Table 4.7.3. Architecture and Specifications used for ANN (ABTS•+ %age 149
inhibition
Table 4.7.4. Architecture and Specifications used for ANN (DPPH• %age 149
inhibition)
Table 4.7.5. Experimental and predicted % ABTS•+ inhibition for selected 150
phenolic acids
Table 4.7.6. Experimental and predicted % DPPH• inhibition for selected 151
phenolic acids
Table 4.7.7. Parameters of ANN (ABTS•+ %age inhibition) 152
Table 4.7.8. Parameters of ANN (DPPH• %age inhibition) 153

xvii
Abstract
Current research is carried out to explore indigenous, abundantly available resources

of bioactive compounds from different botanical materials. For the purpose, plant

samples collected from different areas of Pakistan were analyzed for their

physicochemical, nutritive, functional and antioxidant characteristics. A range of

assays was employed for quantification of bioactive contents as well as assays for

evaluation of antioxidant potential. Potential was tested against different substrate

systems.

As representative of soft fruit, three species of Mulberry namely, Morus alba L.,

Morus nigra L. and Morus rubra L. were investigated for their nutritive and

antioxidant attributes with mutually significant differences but M. nigra L. was found

to be having highest antioxidant potential among the varieties.

To explore the effect of extraction solvents on antioxidant potential, A. annua leaves

were studied. A clear variation was observed regarding efficiency of different solvents

for extraction of antioxidants from leaf samples and the highest potential was

recorded for MeOH extract. However, structural diversity of bioactive compounds

suggested optimization of extraction media and extraction techniques for securing the

maximum yield. To accomplish this objective, extraction of bioactives from leaves of

Artemisia annua and Morus nigra was optimized using RSM. Results revealed that

extraction conditions strongly influence the recovery of antioxidant components from

plants matrix and their antioxidant potential as well.

To prepare bioactive-enriched fractionation, whole plant, Periploca aphylla (PA)

crude extracts was focused. Different solvent fractions of PA crude extract were

prepared and analyzed using different assays and HPLC. Butanol fraction exhibited

the highest content of antioxidants and this fraction was further fractionated using

xviii
column chromatography. Obtained sub-fractions were analyzed for their antiradical

(DPPH• and ABTS•+) activities Results revealed the presence of different bioactive

compounds in sub-fraction.

To study the impact of simulated gastrointestinal pH conditions on antioxidant

potential, defatted kenaf seed meal (DKSM) was studied. Antioxidant potential was

measured using multiple assays besides determination of hydroxyl radcial scavenging

potential using ESR technique. Results revealed that simulated gastrointestinal pH

conditions caused significant decrease in antioxidant potential of DKSM samples.

In order to enhance bioavailability of bioactive components, encapsulation of DKSM

extract was done. Chitosan was used as encapsulating material. Results of scanning

electron microscope confirmed the formation of rod shape nanoparticles.

Encapsulation efficiency and loading capacity was recorded up to 73% and 48%

respectively. Study of release profile under different pH and temperature conditions

confirmed controlled or sustained release of phenolic content from encapsulated

extract. On the basis of results, it is concluded that bioavailability of bioactive

components can be enhanced using encapsulation technique.

Stabilization efficiency of A. annua leaves extract (ALE) was assessed using

sunflower oil model system. Sunflower oil was administered with ALE, synthetic

antioxidants (BHA & BHT) and encapsulated ALE. Stabilization efficiency was

monitored at regular intervals, by measuring peroxide value, free fatty acids,

conjugated diene & trienes and TBRS. Efficiency of ALE at concentration of 1000

ppm was found comparable with synthetic antioxidants.

For QSAR studies, large pool of descriptors was reduced rationally by heuristic

method. The QSAR models were established by applying SMLR on the reduced

datasets. For QSAR of ABTS•+, 6 descriptors while 5 descriptors for DPPH• were

xix
used for modeling purposes. The descriptors obtained in both the cases are

representative of basic molecular properties which are crucial for antiradical potential

of selected phenolic acids. The descriptors were used to develop Artificial Neural

Network (ANN) models. QSAR Models generated in this study showed high

statistical significance, robustness and good predictive abilities.

xx
 CHAPTER-1

INTRODUCTION

1
1.1. Significance of Medicinal Plants

Since prehistoric era, plants have been being used for the treatment of different

ailments in addition to their multipurpose uses including food, clothes, building

materials. Humans are reported to have been on earth for some two or three million

years and they have struggled throughout the ages to establish knowledge about the

effectiveness of the plants. During the greater portion of this era, humans have

developed non-scientific differentiation between useful and harmful plants.

Information about these medicinal plants has been in the knowledge of elders and

wise men of those times. With the evolution in culture and custom, forms of

medicines have changed; but plants remaining the base materials [1]. Medicinal plants

are considered “backbone” of traditional system of medication, and more than 3.3

billion consumers have been reported using plant-based medicines. Recently,

scientists and researchers have focused on medicinal flora for the recovery and

utilization of natural therapeutic agents. [2]. Medicinal plants are considered rich in

bioactive ingredients which can be used in development and synthesis of modern

medicines. For example, salicylic acid, precursor of aspirin was first obtained from

white willow bark. Due to diversified topography, plants with unique composition of

bioactive compounds have been reported in sub-continental areas including India and

Pakistan [3]. However, the exploitation of higher plants for medicinal purposes is still

poorly explored. Small portion of these plants have been studied for their

pharmacological properties. Only 5000 species have been studied properly for their

content of bioactive ingredients [4]. The main attraction of scientists to screen the

medicinal flora in terms of recovery and isolation of active ingredients followed by

evaluation of their biological activities for pharmaceutical applications. Consistent

2
findings need to be executed to discover a probable abundance of medicinal plant

extracts containing novel bioactive compounds.

1.2. Bioactive Compounds

Bioactive compounds are particular chemical compounds that can cause a reaction,

trigger any biological function in living organisms. Commonly known bioactive

compounds are polyphenols, flavonoids, tannins, lycopene, lignin, polysaccharides,

caffeine, lichens and indoles.

These compounds are unique in their action, induce prolonged effects, act as defender

against different disease and significantly contribute in maintenance of health [5]. The

majority the world’s population on a regular basis consumes different parts of a plant

(fruits, vegetables, nuts, stems, barks, teas, seeds, flowers) containing variety of

bioactive compounds [6]. Despite of development of modern sciences, these naturally

occurring bioactive compounds are traditionally being used for the preparation of

herbal formulations. During last two decades, the medicinal plants have gained

significant importance in scientific community, as source of novel and unexplored

bioactive compounds [2, 7].

1.2.1. Antioxidant bioactives

Bioactive compounds have been proven to exhibit many biological activities such as

antiinflammatory, antimicrobial, antifungal, anticancer and antioxidant activities.

Researchers have proved role of bioactive compounds in treating asthma, allergies,

urticarial and many diseases. Among these bioactive compounds, there is a class of

compounds named antioxidants that help human body to overcome the state of

oxidative stress induced by free radicals and reactive oxygen species. Disturbance in

the pro-oxidant and antioxidant balance leads to viable damage in human cells. Pro-

oxidants perform the main function in damaging the human cell. These pro-oxidants

3
enhance the formation of free radicals including NO•, O2•-, H2O2 and –OCl [8].

Generation of these Reactive Oxygen Species (ROS) is owed to normal metabolic

process of body. On the basis of epidemiological studies, now days, there is ample

evidence that these reactive species are responsible for number of diseases, and

disorders associated with oxidative stress. A balance between production and

scavenging of these species is required, which is sometimes accomplished by

supplementing with antioxidants [9]. For the purpose, number of chemical compounds

have been being used, but safety concerns associated with their utilization prompted

the researchers to explore some alternate sources from natural materials [10]. These

concerns prompted food scientists to search alternative sources of antioxidants based

on natural origin, which may be safer, effective and economical, preferably from plant

materials based on indigenous resources. Consequently, number of plant materials and

their constituents were explored as promising sources of antioxidants. Plant extracts

are rich in bioactive compounds, possessing anti-oxidative properties, which are being

investigated with more sophisticated techniques [11].

Generally, consumption of botanical edibles is useful for health promotion due to

presence of protective antioxidants [12]. These concerns prompted food scientists to

search for alternative sources of antioxidants based on botanical materials, which are

supposed to be inherently safe. Several sources of antioxidants had been reported but

still exploration is going on and different botanical materials exhibited varying degree

of antioxidant activities [13]. In many studies, these extracts from natural sources

have been reported to be more effective than some synthetic antioxidants. The use of

natural antioxidants might be a good alternative source of synthetic antioxidants in

respect of low cost, highly compatible for dietary intake and no harmful effects in the

human body. Many attempts had been made to evaluate antioxidant potential of a

4
wide variety of vegetables (potato, spinach, tomatoes, and legumes and so forth) fruits

(berries, cherries, citrus, prunes, and olives) and teas [14-16].

Though some preliminary studies have been conducted on phytochemical attributes of

polyphenols, [17] but further studies are needed to develop more effective extraction

procedures to obtain a more detailed profile of the phenolic composition of extracts.

Although many synthesized drugs are in the market for curing different diseases but

their side-effects are also coming into evidence with the passage of time. Like many

other developing countries, a significant part of Pakistani population have been using

medicinal plants to improve their state of health and curing diseases since ancient

times. Traditional medicine use has further increased, with the increase in prices of

commercial medicine. Traditional medicine still remains the main alternative for a

large majority of people for treating health problems. Many plant materials had

already been reported to be containing versatile compounds with different structural

diversity, e.g. coumarins, curcuminoids, xanthones, and terpenoids [18].

1.2.2. Mechanism of Action of Antioxidants

There are two major classes of antioxidants. Both primary and secondary antioxidant

properties are also found in some antioxidants. Initiation, propagation and the β-

scission reaction are inhibited by primary antioxidants because they have ability to

scavenge the free radicals. The following mechanism shows the interaction of free

radical scavengers with peroxy radicals as described by Liebler [19].

ROO˙ + FRS ROOH + FRS˙

The following mechanism represents the interaction of alkoxy radicals and FRSs

RO˙ + FRS ROH + FRS˙

The hydrogen atom of FRSs have low energy, therefore it have great tendency to

donate hydrogen to the free radicals. It is an essential ability of FRSs for its

5
antioxidants activity. Any species will be able to donate its hydrogen if its reduction

potential is lower than that of free radicals. To avoid the FRS˙ radical ability to react

with oxygen and to oxidize other unsaturated fatty acids, this radical should be of low

energy. The resonance delocalization results in formation of low energy FRS˙ [20]. A

termination product produced due to interaction of FRS˙ with another FRS˙ or with

additional lipid radical and peroxyl/alkoxyl radicals also inactivated by interference

with FRS. These are properties of an ideal antioxidant to inactivate at least two

radials.

Ascorbic acid, carotenoids, ubiquinone (Q10), flavonoids, lignans, propyl gallate

(PG), butylated hydroxytoluene (BHT), butylated hydroxyanisole (BHA) and

tocopherols are some known antioxidants having FRS properties [20]. The oxidation

intermediates or prooxidants react with secondary metabolites and delay the oxidative

process. Copper and iron are examples of prooxidants

Fe3+ + ROOH Fe2+ + ROO˙ + H+

The metal redox cycling can be prevented by metal chelator or by providing steric

hindrance between metal and oxidation intermediate or fatty acid and by forming

insoluble metal complex. Citric acid and EDTA are common metal chelators. As Fe2+

oxidize fatty acids easily as compare to Fe3+ therefore when iron is present and EDTA

in low concentration then it will act as prooxidants and favor the chelation of Fe3+ .

While the oxidative ability of Fe2+ decreases and favor the chelation of Fe2+, when

EDTA is present in high concentration [21].

When we use combined antioxidants, it is termed as synergism. The primary FRS

regenerates the antioxidant activity therefore the level of fatty acid radicals is reduced

[22]. The concept of replacement of synthetic antioxidants with natural antioxidants

has been introduced despite of consistent quality, efficiency and low cost of synthetic

6
antioxidants. Carotenoid astaxanthin, rosemary extracts and α-tocopherol are

examples of natural alternatives. Astaxanthin behave both as a primary and as a

secondary antioxidant while rosemary and α-tocopherol extracts are both primary

antioxidants.

1.3. Extraction of Antioxidant Components from Botanical Material

Antioxidant compounds, present in plants, are of diverse structure and their

extractability is strongly influenced by their chemical structure. So, different

extraction media i.e. solvent systems, may provide varying yields of extracts with

selective recovery of antioxidants; depending upon the structure of antioxidant

compounds present in plant material under investigation [23]. For the purpose,

different solvent systems are used as extraction media for the maximum recover of

component of interest from plant matrix. Generally, it is known that a single plant

contains numerous secondary metabolites i.e. bioactive compounds. It is therefore

necessary to develop a best and rapid extraction method for the recovery of these

bioactive compounds [24]. Besides development of new methods, scientists and

researchers have also tried to develop extraction methods for different plants and

reported that efficiency of extraction method, solvent and conditions vary from plant

to plant or even from specie to specie of the same plant. Extraction of antioxidants is

being carried out using traditional solid-liquid techniques including maceration,

soxhlet extraction, and orbital shaker-assisted extraction. But these techniques have

required longer time, excessive solvent consumption and greater risk of thermal

degradation of labile bioactives. Soxhlet extraction has been considered to be the best

technique among all the conventional techniques but it needs extensive energy

consumption and long extraction times (up to 6 hours or more) which significantly

decreases sample amount and is a big problem in terms of marketable applicability.

7
Other extraction techniques have been focused with objectives to lower the solvent to

solid ratio, extraction time and maximum recovery of targeted compounds. These

extraction techniques include microwave-assisted extraction (MAE), supercritical

fluid extraction (SFE), accelerated solvent extraction (ASE), ultrasound-assisted

extraction (UAE) and solid-phase micro extraction (SPME).

Solid-liquid extraction (SLE) is used for the recovery of antioxidant compounds from

botanical materials. Solid-liquid extraction (SLE) involves the extraction of a

preferred constituent (solute) by leaching procedure by using a solvent that is capable

of dissolving the solute. Leaching is the oldest extraction technique and it has been

used since ancient times for separation of minerals from their ores and for the

extraction of sugar beet. Extraction takes place in different steps including solvent to

solid-matrix penetration, solute dissolution. The interaction time of solvent with solid

matrix is necessary for the recovery of solute which depends on the solubility of

solute, extraction temperature, flow and viscosity of solvent as well as the surface

area of the material being extracted [25]. SLE techniques have been used by the

researchers for the extraction of phenolic antioxidants from different botanical

materials since many years. It was found that polar compounds like phenolic acid,

flavonoids etc. can be extracted with polar solvents such as water, methanol, ethanol,

and mixtures of these solvents. Many reviews had been published about the efficiency

of different solvents but the solvents efficiency depends on different factors including

the solubilizing ability of solute with the solvent, particle size of the solute, extraction

time and temperature. Therefore, efficient, cost-effective methods for the extraction of

these natural antioxidant/bioactive compounds are essential to establish in order to

enhance commercialization of natural antioxidant. A brief introduction of all the

extraction techniques is given below.

8
1.3.1. Hot continuous extraction (soxhlet extraction)

A thimble made of paper is filled with finely ground crude medicinal plant/any part

followed by placing that thimble soxhlet apparatus. The extracting solvent is added in

round bottom flask, its vapors arises due to heating at boiling point. Vapors get

condensed after striking with the walls of the condenser. The condensed solvent come

in contact with crude plant material and process of extraction continues. After

reaching up to maximum level of solvent in siphon tube, the liquid contents of

chamber siphon into the flask and in the same way extraction continues. The

advantage of this technique is that large amounts of the sample can be extracted with

a smaller amount of solvent.

1.3.2. Orbital shaker-assisted extraction

This method is simple and have been widely preferred by many researchers or even it

is also used for commercial purposes. Its instrument is low cost, need less power

consumption but having efficiency comparable to many other methodologies. In this

method, the dried and sieved plant powder of known mesh size is generally extracted

with solvent through holding this flask in a shaker with/without hot air bath. Solvents

of varying polarities are used and sometimes solvent compositions of different

solvents are prepared. Solvent is added to the conical flask having dried powder.

Orbital shaker is started after adding glass balls into the flask. In this technique,

extraction can be carried out at different variables i.e. shaking speed can be adjusted,

extraction time can be varied, in some cases extraction at different temperature can be

conducted. Mechanism for this process is very simple i.e. during shaking molecules of

solvents penetrate in to the body of powdered sample and solubilize the bioactive

compounds and as a result extraction takes place. For solvent, maximum surface is

9
available for the extraction of bioactive compounds. The solvent extracts are usually

filtered with Whatman filter paper to obtain a dark green color solution [26].

1.3.3. Microwave-assisted extraction

Among modern techniques of extraction, microwave assisted extraction (MAE) is the

most promising technique. It was observed that MAE exhibit highest extraction

efficiency among all the tested extraction methodologies. [27]. Microwaves are

electromagnetic radiations, which serves as a potential source of energy. Microwave

radiations diffused through the botanical material and converted to heat. Among

advanced techniques, microwave-assisted extraction is rapid, more uniform and

energy efficient [28]. Two mechanisms have been proposed for microwave heating

such as dipole rotation and ionic polarization. Partial positive and negative ends of a

molecule are effected by microwave energy i.e. rotation of these charged ends is

observed. The typical example is that water consists of two hydrogen atoms carrying

partial positive charges and oxygen carrying a partial negative charge. Dipoles orient

themselves in line with the polarity of the field. Frictional heat produced due to the

rotation of molecules. In the case of ionic compounds, salts flow at an accelerated

speed in reaction to the discontinuous electric field and as a result, thermal energy

produced. It is clear from the mechanism that molecules and solvents having

permanent dipole moment heat efficiently in the microwave. The amount of applied

magnetic field to which a given material interacts is usually dependent on the

substances dielectric properties and those include dielectric constant and the dielectric

loss factor. The dielectric constant describes the capability of a material to keep

electrical energy while the dielectric loss factor defines the capacity of the material to

waste electrical energy. Attention towards MAE has increased in present years

10
because of a decrease in extraction time and solvent quantity over conventional

extraction techniques [29].

Conventional solvent extraction techniques depend on conduction and convection

method to heat the sample. These techniques require longer extraction times and

therefore increase the risk of thermolabile compounds degradation. While in MAE,

microwave energy directly heats the samples in short time. MAE is reported to be the

most efficient technique for the extraction of bioactive compounds from botanical

material. Temperature of the solvent increases rapidly with the application of

microwave radiation that decreases surface tension and viscosity of the solvent and it

conversely increases solvating power and improves its matrix penetration [30].

Different ratios of organic solvents and water are used for the maximum recovery of

bioactive component using microwave radiations [31].

MAE is considered as better technique for the recovery of bioactive compounds from

plant matrix when compared with conventional techniques. The reasons for better

efficiency of this technique may be less extraction time (rapid heating), less solvent

consumption, 70% power saving as compared to traditional extraction, leaving less

environmental pollution because of easy power transfer to matrix that results in

improved extraction yield and satisfactory product. There have been a lot of reported

studies regarding improvement of this technique for number of plant samples.

Therefore, for the current research project MAE technique is taken into consideration

a promising technique [32].

1.4. Optimization of Extraction Processes using Response Surface

Methodology

Response surface methodology (RSM) is a technique that involves a complex process

for the optimization. In this technique, a suitable experimental design is developed

11
that help to study the effect of independent variables. Data from the experimental

procedures is added to set of equations that provide theoretical value of an output. By

means of regression analysis, independent variables decide about the outputs, and

response of dependent variable can be estimated on the basis of new values of

selected independent variables [33, 34]. RSM was first reported by Box and Wilson in

1951 [35] that helped in reducing experimental runs as compared to the number of

runs conducted using empirical approach . As a result, this technique has widely been

used many researchers it helped in decreasing experimental runs, and the obtained

results are claimed to be statistically authentic [36]. Application of RSM method in

the optimization process helped to test all of the variables only in a short period of

time. In addition, those variables can be easily identified that has strong effect on the

system under observation, which is helpful for the researcher to select those variables

that can play a role in achieving maximum output.

Generally, one variable can be under the influence of many other variables involved

in process/ experimental design. To find the output-input connection, it is necessary to

find out the parameters that actually have significant relationship with one another.

Experiments based on single parameters are not so sufficient to determine the such

interactions [37]. RSM is a route to study such relationship in detail as well as

interactions of parameter affecting a process. RSM can be applied in three ways (1)

selection of experimental design (2) regression analysis to find out model equations

and (3) optimization of selected parameters using model equation [38].

Considering one factor/parameter as variable at a time is simple, expensive, and time

consuming approach, as it does not interpret the relationship among selected

variables. In order to study such relationships, Response Surface Methodology (RSM)

is a versatile tool to assess the effects prompted by various factors and their linkage

12
on achieving an optimum response of a parameter of interest. [39]. RSM is faster and

more informative than the classical one-variable-at-a-time approach or the use of full

factorial designs [40].

For the recovery of bioactives from botanical material in the form of extract, both

quantity and quality of extract is strongly affected by extraction protocol used. Factors

including particle size of the solute, nature of solvent, liquid-to-solid ratio,

temperature, pH and number of extraction steps must be taken in to count prior

deciding about the efficacy of the extraction procedure adopted. The selected

extraction parameters must ensure comprehensive recovery of the compounds of

interest avoiding any chemical modification [41]. Extraction techniques, procedure,

solvent, extraction conditions are usually selected the structure and distribution of

bioactives present in plant matrix. However, extraction of maximum bioactive

compounds may be not possible, if we rely on a single technique, solvent and set of

extraction conditions. Extraction is of bioactives is achieved using different solvents

(non-polar to polar), combination of solvents, multiple techniques and different set of

conditions. Scientists made all these efforts to find out appropriate parameters for the

extraction of bioactives especially antioxidants [42].

1.5. Fractionation of Crude Extract

Bioactivity-guided fractionation is important when trying to isolate bioactive of

interest, but it may eliminate many other compounds having promising biological

activities. Single compound may not exhibit biological activity with that strength, as

exhibited by a group of compounds due to synergism or pharmacokinetic influences.

For example, saponin fractions obtained from ginseng, were reported more potent

than the isolated saponins [4]. In addition, bioactivity guided isolation of one

compound of interest, usually ignore many other compounds having multiple

13
activities. For example, catharanthus roseus was initially reported for its anti-diabetic

activity but latter on it was found containing powerful anti-tumor agents and now a

days it is clinical use.

Previously, isolation of bioactive compounds from crude extracts have been the focus

of researchers for long time. But since last decade, number of limitations associated

with the isolation process, including consumption of high volumes of solvents,

requirement of the state-of-art instrumentation, besides low yield/ loss of target

compounds, especially those present in minor quantities, has resulted in decreased

interest of researchers in isolation. Furthermore, a number of studies have revealed

that, in comparison to isolated compounds, group of compounds may exhibit higher

activity due to synergistic effects. Based on above-mentioned factors, nowadays,

preparation of bioactive-rich fractions from crude extracts is generally preferred over

isolation and improved activities have been reported for enriched fractions as

compared to crude extracts or isolated compounds in number of cases [4].

1.6. Bioavailability Enhancement of Bioactive Compounds

There exists a difference between concentrations of antioxidants needed for in-vitro

efficiency are generally greater than those required for in-vivo efficiency. Efficiency

of these compounds depend on way of administration, integrity and bioavailability.

Orally administered bioactives are absorbed to limited extent, insufficient gastric

residence time, low permeability and/or low solubility. While, studies of

gastrointestinal tract demonstrate pH to be the most critical factor, which affects the

activities of a biomolecule in the human body. Therefore, antioxidant potential of

studied extracts or compounds may vary under gastrointestinal conditions [43].

Hence, reported data about antioxidant attributes of chemical extracts may not be

helpful for understanding their potential after ingestion. Keeping in view these

14
deficiencies, in vitro digestion and in vitro GI models have been developed to

understand the bioavailability and antioxidant properties during gastric and intestinal

stages [44, 45]. The results obtained, using these models, can be well correlated with

results of in vivo studies [44]. Employing these models, many authors have reported

variation in content of phenolics as well as antioxidant potential of extracts or

compounds under gastrointestinal conditions; as major beneficial effects of phenolics

are strongly dependent on their bioavailability and metabolic fate.

On the other hand, instability of active ingredients during different procedures

involved in its extraction, distribution or storage, limits benefits associated with of

antioxidant molecules. These molecules are at risk in living organisms due to multiple

factors including gastrointestinal pH, presence of other nutrients. Natural antioxidants

are delicate in their nature and are sensitive to environment having biological,

chemical species and other conditions. Oxidative deterioration of these molecules is

rapid, that results in appearance of color and/or odors. All these problems motivated

researcher to explore different masking methods before their exploitation. Hence,

prior administration, protecting layer is developed that may be helpful to maintain the

structural identity of the antioxidant compounds. Protective layers are designed to

secure their taste, enhance solubility and bioavailability [46].

For the purpose, encapsulation has gained an important place, as encapsulated

products instead of free compounds are considered to be more efficient. Nowadays,

various micro/nano-encapsulation techniques are in use for the preparation of

encapsulated products. These products are of multiple nature and applications, like

agricultural products, biotechnology, biomedical, industrial chemicals, sensor

industries and veterinary medicine [47]. Encapsulated products may carry solid, liquid

15
or gaseous active ingredients having size from 1 millimeter to 1 micron or from 1

nanometer to 1 micrometer [48].

Nano/micro encapsulation of bioactive ingredients increases their effectiveness,

specificity and targeting ability. Nanocarriers (NCs) protect biomolecules from

premature degradation in the biological environment, contribute to prolong existence

in blood, enhance bioavailability and cellular uptake. Many methods have been

reported for the synthesis of nanoencapsulates. Choice of synthesis method depend

upon the chemical structure of therapeutic agent, type of application, and time of

retention inside the body [49]. Different sized NCs can be synthesized by using

different encapsulation materials. Size and surface chemistry of NCs is responsible for

their in vivo performance. Size and size distribution of NCs is one of the important

parameters of that determine their cellular uptake and crossing across biological

barriers. Drug release mechanisms are equally important from the drug-NC-

formulation due to proposed application in drug delivery. Release mechanism can also

be modulated depending upon the nature of therapeutic agent and type of NCs [50,

51].

Encapsulation protect sensitive compounds from environmental factors, consumer

from harmful effects of drugs, secure aromas, helpful in keeping two irreconcilable

compounds within same medium. Furthermore, encapsulation was carried out to

achieve controlled release of the molecules. Targeted drug delivery can be achieved

that allow the release of bioactive after reaching at diseased site. Food applications

can be the objective, encapsulation technique enhances applications of bioactives as

food additive and preservative.

The scientists and researchers are using large number of encapsulation methods.

Those methods are; Physical methods (centrifugal extrusion, extrusion-

16
spheronization, fluid bed coating, spray-drying), Physicochemical methods: hot melt

coating, ionic gelation, spray-cooling, and solvent evaporation extraction and

Chemical methods: interfacial polycondensation, in situ polymerization, interfacial

polymerization, interfacial cross-link.

1.7. Stabilization Efficiency of Plant Extracts in Different Model Systems

Lipids are the important part of our diet, in the form of oils as well as fats. In human

body, lipids regulate many physiological functions; In addition, lipids provide variety

of fatty acids that are involved in many hormones such as prostaglandins.

Polyunsaturated fatty acids (PUFAs) have been found associated with heart diseases

because they shield against enhanced plasma level. Metabolism of pre-β–lipoproteins

and triglycerides monitor carbohydrate metabolism as well as diabetes mellitus [52,

53].

Disease preventive and health promoting role of dietary fatty acids is important. In

order to maintain the level of these fatty acids in human body, food industries are

encouraged to produce food products containing, α-linolenic acid, eicosapentanoic

acid (EPA) and docosahexanoic acid (DHA). But phenomenon of oxidative

deterioration of fatty acids is the main hindrance to produce large quantity of fatty

acid products with better quality. Oxidation of lipid containing products disturb

nutritional and sensory qualities of food. Therefore, there is an intense need to work

on the stabilization of these food items. Innovative methods and technologies have

been introduced to save PUFA by means of refining, packaging, adequate addition of

antioxidants, as lipids oxidize, so their use is limited [54].

Process of oxidation increases with an increase in concentrations of polyunsaturated

fatty acids in lipids. Lipids undergo autooxidation, when they come in contact with

oxygen, photooxidation may occur when exposed to light and enzymatic oxidation

17
may take place when exposed to lipooxygenases [21]. As far as, photooxidation and

enzymatic oxidation is concerned, these two are not the major problems because light

only disturb the lipids when directly exposed to sunlight, whereas enzymes become

deactivated during refining process. Hence, the primary reason of oxidative

deterioration is autooxidation in lipids. The rate and extent of lipid peroxidation is

controlled by many factors including peroxidases, concentration of oxygen, surface

area, water activity, temperature and composition of fatty acids [22]. Peroxides are

formed as a result of lipid peroxidation, which convert themselves into different

organic compounds including aldehydes, alcohols, acids, hydrocarbons or ketones.

These oxidation products reduce the availability of many nutrients and also cause

multiple diseseas (heart diseases, DNA damage, cancer, tumor promotion and aging

etc) [54].

1.7.1. Mechanism of lipid peroxidation

Lipid peroxidation follows a free radical mechanism which occurs in three steps i.e.

initiation, propagation and termination. Initiation step involves production of alkyl

radical (R˙) after removal of hydrogen atom from fatty acids or acyl glyserol. In case

of PUFA, rapid increase in degree of unsaturation is observed due to the removal of

hydrogen in the initiation step [55].

The initiation step is

RH R˙ + H˙

In propagation step, reaction between alkyl radical and molecular oxygen takes place.

Singlet and the triplet states, both, of molecular oxygen participate in the oxidation

process. Due to presence of empty outer antibonding orbital, singlet oxygen (1O2) is

very reactive toward PUFAs [56]. However, triplet oxygen does not react fatty acids

directly but it forms high-energy peroxyl radicals (ROO˙) after reacting with alkyl

18
radicals, which then propagate the removal of hydrogen from other fatty acid

molecules.

The propagation steps is given as

R˙ + 3O2 ROO˙

ROO˙ + RH ROOH + R˙

Formation of non-reactive compounds (fatty acid dimers) by the combination of

different radicals, it is called as termination step.

The process of termination can be written as

ROO˙ + R˙ ROOR

R˙ + R˙ RR

1.7.2. Prevention from lipid peroxidation

For the prevention of oxidative deterioration of the lipid containing food systems,

antioxidants are the best choice as food additives/ preservatives. Initially synthetic

antioxidants were used to inhibit oxidative deterioration. BHA and BHT are suitable

for thermally processed foods due to their stability at high temperature. But reports

revealed that use of synthetic antioxidants can be toxic and carcinogenic. Therefore

researchers aimed to search alternative, economical but safer and natural sources of

antioxidant compounds [57, 58].

Antioxidant molecules may prevent or delay the oxidation of biomolecules and

different cell components by hydrogen atom or by donating an electron pair to free

radicals and reactive oxygen species. Therefore, antioxidants are used to inhibit

oxidative deterioration of lipids (fats and oils), thus enhancing the shelf life of food

items. Antioxidants play an important role in food industry not only as food

preservatives but also as dietary supplements for its health promoting effects. These

days scientist are focusing on extraction of antioxidants from natural sources. These

19
natural antioxidants have comparable efficiency with the synthetic one. Natural

antioxidants are obtained from both animals and plants. The natural antioxidants are

extracted and purified before they are used as food additives [59].

1.8. Medicinal Flora of Pakistan

The floristic evaluation revealed that out of identified 500,000 plant species, 120,000

plant species are used for the treatment of various ailments worldwide. These flora are

used because of the presence of compounds that are biologically active [60]. Pakistan

is very rich in medicinal flora because of the various agro-climatic conditions wherein

more than 6000 plant species have been identified.

Pakistan covers an area of 803,944 km2 and lies among 24°-37° north range and 61°-

78° east longitude. Pakistan is physiographically divided into four regions wherein

Indus Plain that is rich in agricultural entities. It covers an area of 520,000 km2 in the

east and extends to at least 1,100 km from northern Pakistan southward to the Arabian

Sea. Pakistan covers different eco regions starting from mangrove forests extending

from the Arabian Sea to high mountains of Western Himalayas, Hindu Kush, and the

Karakorum. Himalayan region is the most important mountain system in the world

with endless and unique world resources of various plant species.

The plants of Himalayas vary with climate, rainfall, altitude, and soils. The

geographical area of Pakistan has produced a completely unique flora of 4950 plant

species, out of which 300 species are medicinal and are spread all over the country.

The land has been occupied for over thousands of years by humans who established

medicinal uses for many plants. Out of three hundred traditional medicinal plant life,

only 25% had been studied for their chemical components [61].

Natural drugs are effective, safer, and less expensive and are gaining attractiveness

among the rural and urban areas at the same time as awareness of harmful effects of

20
artificial drugs is increasing. According to an estimate, almost 2,500 medicinal plants

are of importance in international trade. Pakistan is one of the minor raw material

producing countries of South Asia. In Pakistan, 300 plant species out of 4950 native

species are used as a drug; which is roughly 6.1% of the world’s consumption of

medicinal plant species. In Pakistan, more than 2,000 plant species are used medicinal

purposes with 400–600 documented scientifically [61].

1.9. Medicinal Plants Selected for Current Study

For the current study, different medicinal plants from different regions, having

different topographical and agro-climatic conditions, are selected and investigated for

their bioactive compounds in addition to evaluation of their antioxidant, analgesic and

antimicrobial activities using preliminary assays based on different mechanistic

principles. On the basis of these investigations, three most potent medicinal plants

were selected for further investigations.

1.9.1. Mulberry

Mulberry, belongs to family Moraceae and genus Morus [62]. It has fast growth and

short proliferation period. In temperate region of Asia, 10–16 species are found [63].

Some species have high foliage yield, while few have ornamental importance while

some species are grown for their fruits. Commonly, three species of Morus, Morus

alba, Morus nigra and Morus rubra, are found in different regions of Pakistan e.g.,

Azad Kashmir to Quetta [64].

In Chinese traditional medicine system, different parts of Morus alba L. are used for

the treatment of different ailments [65]. Reports revealed the presence of precious

phytochemicals including coumarins, flavonoids, phenols have in leaves of Morus

alba L. Leaves have been reported to reduce cholesterol level that is helpful in

maintaining blood pressure.

21
Morus nigra L. is a wildly growing rustic plant, also grown in gardens and is used as

sericulture [63]. It is used for the treatment of diabetes and rheumatis in Chinese folk

medicine. Antioxidant activity have been reported for different parts of this specie and

in addition, separation and isolation of two new flavonoids have been achieved from

the leaves of this specie [66].

Morus rubra L. is a tree of 15–20 m height. Its leaves are used for the preparation of

many folk medicines. Many phytochemical constituents as well as biological

activities have been reported for different parts of this specie [67].

1.9.2. Artemisia Annua

A. annua; a single stem herb having vibrant growing ability and height up to 2-3

meters. Many bioactive compounds including artemisinin, artemisinic acid,

dihydroaritemisinic acid and arteannuin B have been found accumulated in leaves

[68]. Artemisinin is a well-known antimalarial drug and is available in market as well.

Synthetically prepared derivatives of artemisinin exhibited excellent pharmacological

activities against pathogens that instigate AIDS [68]. This plant is also known to have

anti-inflammatory, antitumor and allelopathic properties [69].

Many species of Artemisia L. [70] are found in central Asia while Pakistan hosts 25

species which are found in northern regions i.e. Abbottabad, Chitral, Gilgit,

Himalaya, Karakorum mountains Kashmir, Rawalpindi, Skardu and Swat [71].

Locally name of this plant is Afsantin or Afsantin jari and traditionally it is used

against malaria; especially leaves are used for cold, cough, cold, diarrhea and malaria

[70].

Antioxidant properties of Artemisia annua leaves extracts has been presented in many

articles. But no detailed investigation on its antioxidant activity as function of

different extraction solvents for maximum recovery of antioxidant components is

22
presented so far. Therefore, a part of this dissertation was planned to evaluate the

impact of solvent polarity on extraction of antioxidant compounds from A. annua

leaves along with determination of antioxidant activity through multiple assays.

1.9.3. Periploca Aphylla

Periploca aphylla, is member of genus Periploca (Asclepiadaceae). Antitumor

compounds have been reported from root bark of Periploca sepium. These

compounds are also effective for congestive heart failure [72]. Some biological

activities including, antimicrobial, insecticidal and antioxidant properties have been

reported for its essential oil [73]. Periploca laevigata, a specie of this family, is

consumed as tea and is used in herbal formulation for the treatment of diabetes and

headache and may also exhibit antioxidant activities.

Common name of Periploca aphylla is “Bata” or “Barara”. Milky juice is found in

this plant which is used for the treatment of swellings and tumors. In addition, swollen

joints, cough, flu and diseases including ulcer, skin infections and constipation can

also be treated by using this plant. Two new lupane derivatives, have been isolated

from the stems of Periploca aphylla. Different compounds extracted from P.

aphylla have shown inhibitory effect of α-glucosidase type VI and antibacterial

properties [74].

1.9.4. Kenaf

Kenaf (Hibiscus cannabinus L.) is a plant of family Malvaceae. It is a rapidly growing

annual herbaceous plant of tropical and temperate regions having excellent growth up

to 5-6 m, within 6-8 months [75]. It is found in USA, China, India, Thailand and

Malaysia and is of great industrial importance. Many bioactive compounds including

tannins, steroids, alkaloids, saponins and polyphenols have been determined from

different parts of this plant [76]. Leaves of this plant contain precious volatile

23
constituents and bark extracts have shown strong anticancer activity against tumoural

cell llines. Kenaf seeds are usually considered as agro-wastes or used as animal feed

due to high protein content [77]. Kenaf seed oil and flour are used for making many

functional confectionary products of human consumption. Kenaf seed oil contains

alpha linolenic, omega-3 fatty acid, phospholipids and phytosterols, having anti-

inflammatory, antithrombotic, antioxidant and anticancer activities and is, therefore,

known as chemopreventive agent [76]. The presence of these bioactive compounds

have been reported to possess significantly higher antioxidant potential compared to

many other commercially available cooking oils [78]. Kenaf seed oil and extracts

have been well-studied by number of authors and is reported to be a promising source

of antioxidants.

Kenaf is a biennial crop and its reported production is 1.2 tons per annum per hector.

According to an estimate, worldwide production of kenaf was up to 0.33 million ton

in 2005-06. In most of the developed and developing countries, kenaf is preferred for

its stem having high proportion of fiber, which is of great industrial significance.

Whereas, kenaf crop also provides seeds, which contain high content of oil (16-22%)

as compared to many other seeds. Now there is growing interest in manufacturing of

edible oil from kenaf seeds, which has resulted in extraction of oil from kenaf seeds at

semi-industrial scales [77]. But, after the extraction of oil, meal is just discarded or is

used in poultry feeds. Upto our best, no potential use of kenaf seed meal could be

searched out so far.

1.10. Quantitative Structure Activity Relationship QSAR

The Quantitative structure–activity relationship (QSAR) methodology has been

extensively used to find out function between any property of a molecular system and

its structural characteristics. This methodology is based on the assumption that

24
different properties, biological activities and in-vivo activity of any molecule depend

on its structure [79].

QSAR models have been developed using the knowledge of chemistry, statistics and

property of the molecular systems. Requirements for the creation of the QSAR model

are a data set, providing experimental measurements for a molecular system. These

datasets normally derived using hundred or fewer molecules having specific

properties such as inhibition efficiency, intestinal absorption or biological activity.

First report on structure-activity relationships was published by corwin Hansch in

1962 and 1963 [80].

In order to develop structure activity relationship (SAR), molecules from same class

of compound having same mechanism of any biological activity. For all the

compounds, initially biological activity is evaluated followed by calculation of

molecule descriptors. The obtained data obtained is then processed using different

SAR.

The concept of QSAR, for the development of new drug molecules, has gained a lot

of attention, for correlating molecular information with biological activities. QSPR is

a methodology in which structure and any activity of a class of compounds is

correlated. QSAR associates biological activities with group or molecular descriptors

in the series of compounds as it includes structure representation, descriptive analysis

and modeling. Molecular descriptors, being important results of a logical and

mathematical processing which transforms structural information to a readable data,

having a code for the symbolic representation of a molecule. These chemical

structural features are called molecular descriptor, and they are claimed to be closely

related to any activity of the molecules that they exhibit. Molecular descriptors may

include absorption, conformational, distribution, electronic, receptor, fragment

25
constants, graph-theoretic, information-content, molecular shape analysis, pKa,

quantum mechanical, spatial, structural, topological, thermodynamic, and receptor

surface analysis descriptors.

Two-dimensional descriptors are usually considered to understand the atomic

arrangement within a molecule, which encode in numerical form the information

about branching, molecular size, presence of heteroatoms, shape and multiple bonds.

Such information are considered useful during the designing of a new drug. However,

descriptors related to geometry of the molecule, or 3-D descriptors in general provide

significant information and judgment power than topological descriptors for similar

molecular structures [81].

1.11. Objectives of Study

Keeping in view the objectives, current study is divided into six parts as follows.

Part I: Determination of proximate composition and antioxidant potential of selected

medicinal plants.

Part II: Optimization of extraction process for bioactive compounds from leaves of

Morus nigra and Artemisia annua using RSM

Part III: Bioactive-enriched fractionation of Periploca aphylla extracts followed by

identification and quantification of novel bioactive/ antioxidant compounds.

Part IV: Nano-encapsulation of DKSM extract to enhance its bioavailability and

antioxidant activity.

Part V: Stabilization efficiency of encapsulated and non-encapsulated A. annua leave

extract on shelf-life of selected food items (Oil and Meatballs).

26
Part VI: Structure-activity relationship of selected phenolic acids in order to propose

mechanism of their antioxidative action

1.12. Significance of Current Study

This research project will make an important contribution by identifying the

antioxidant potential of indigenous medicinal plants. Information about presence of

certain bioactive compounds may be used for drug development. By encapsulation of

extracts, targeted delivery and bioavailability of extracts is expected to be enhanced,

which will be a value-added aspect of extracts for possible use as drugs. Oxidative

stabilization efficiency of plant extracts in selected in vitro model systems (meatballs,

vegetable oils) will be evaluated to assess their industrial significance as food

additives and preservatives. Medicinal plants with promising bioactive contents and

antioxidant attributes may possibly be exploited as potential sources of functional

foods and nutraceuticals.

27
 Chapter 2

REVIEW

OF

LITERATURE

28
Part I

2.1. Determination of Proximate Composition and Antioxidant Potential of

Selected Medicinal Plants

Botanical Materials as Source of Antioxidant Compounds

A literature review provides a roadmap of past research work conducted in a specific

research field. It offers us information about what type of work and what sort of work

has already been done so far in the selected research. Literature review highlights the

gaps that need to be addressed while working in that specific area of research.

Keeping in view the goals of the current study, review of literature was conducted and

compiled in this section. Many plants have been explored as source of bioactive

compounds for their antioxidant potential using a variety of methods. Some of these

are discussed below.

Herbs and Spices as Natural Sources of Bioactive Compounds

Herbs and spices, important sources of condiments since ancient times, are still used

as flavour enhancers. These herbs and spices contain different bioactive compounds.

Different essential biological active compounds had been found from herbs and spices

e.g. rosmanol, carnosic acid, carnosol, and epirosmanol. These are identified bioactive

compounds exhibited good antioxidant potential and are effective in reducing lipid

oxidation [82]. These bioactive compounds had been evaluated and approved by

incorporating them in various food products by comparing their efficiency with the

synthetic antioxidants. It was found that increasing the concentration of rosemary

extract resulted in an 8-fold decrease in development of thiobarbituric acid reactive

substances (TBARS) compared to either trolox or ascorbic acid. Additionally It has

been observed that oxidative stability of foods is improved by the addition of extract

of rosemary due to the presence of different bioactive compounds [83].

29
Salihoglu et al., (2013) determined the biological activity of various herbal plants

using Folin-Ciocaltue and DPPH antioxidant activity assays. Therefore, a conclusion

was made that herbs and spices can be a potent source of biologically active

compounds having like anti-bacterial, anti-inflammatory and anti-fungal activities. A

positive correlation was also reported between antioxidant activity and total phenolic

contents [84].

Matkowsk et al., (2008) determined the bioactivity of five herb extracts belongs to

Lamiaceae, sub family Lamioideae, while extracts were prepared using methanol as a

solvent. The crude extracts were further fractioned using various solvents using

liquid-liquid methodology. The plants under study exhibited the effective antioxidant

protection by free radical scavenging and metal ion reduction mechanism. L. cardiaca

and B. nigra polar fractions were found to be the most effective fractions with respect

to other. Therefore, all tested herbs are found to be a good sources of biological active

compounds that can be further investigated for pharmacological properties [85].

Wojdyło et al., (2007) evaluated antioxidant activity of 32 selected herbs from 21

botanical families, using multiple assays. HPLC method was used for the analysis of

a number of phenolic acids and flavonoids. It was found that different spices have

significant amount of total phenolics and antioxidant activity. Authors reported a

strong correlation between TEAC (ABTS and FRAP) values [86].

Cereals and Legumes as Natural Sources of Bioactive Compounds

Oxidation of biomolecules may cause different degenerative diseases like aging,

damages membrane, cancer and heart diseases in living organisms [87]. Antioxidant

compounds are major constituents that reduces the oxidative damages [88]. It has

been known for most of the 20th century that cereals and legumes have significant

30
quantities of bioactive compounds. Different types of natural bioactive compounds

exist in cereals and legumes such as phenols, phenolic acids and their derivatives [89].

Carlsen et al., (2010) screened different herbs and spices along with different

botanical materials, to identify their antioxidant compounds. The results indicate that

there is a significant difference in the antioxidant potential of selected botanical

materials. Based on results, researcher developed a database and concluded that foods

that have herbs and spices extracts can be a potential source of antioxidants in human

diet [90].

Hossain et al., (2008) determined the antioxidant activity of 30 spices extract and

phenolics and compared with five synthetic antioxidants. Clove extracts exhibited

highest antioxidant activity while garlic powder extract exhibited lowest values out of

all the examined spices. It was found that rosmarinic acid and eugenol exhibited

higher activity than synthetic antioxidants while activities of kaempferol, curcumin,

capsaicin, thymol gingerol were found 1comparable with synthetic antioxidants.

Therefore these spice phenolics are good source of antioxidant ingredients that can be

incorporated in foods to prevent the oxidative deterioration [91].

Shan et al., (2005) determined the antioxidant potential twenty six common spice

extracts, extracted from 12 botanical families. RP-HPLC was used for the

quantification of major phenolics in selected spice. Many species exhibited high

antioxidant activity and this activity can be attributed to presence off different

phenolic constituent’s e.g. different flavonoids, phenolic acids, diterpens and volatile

oils. One of the main phenolics acid (Rosmarinic acid) was found in all six species of

this family. Antioxidant activity and composition of phenolics confirms species as

potential sources of antioxidant molecules [92].

Fruits and Vegetables as Promising sources of Bioactive Compounds

31
A number of fruits (apple, peach, strawberry etc) and vegetables (spinach, burdock,

radish, cabbage, carrot etc) have investigated for their potential of antioxidant activity

and determination of biological active compounds [93]. Different health and food

related agencies advised that regular use of plant based foods (fruits & vegetables)

decreases the risk of various diseases. Osawa et al., (1992) studied the biological

activities of vegetables (purple radish, long white radish, Brussels sprout, onion,

welsh onion, carrot, etc.) extracts using Thiobarbuturic acid (TBA) method. The

sweet potato was found to be a rich source of β-carotene and its antioxidant potential

is thrice times stronger than carrot and pumpkin extracts [94]. Similarly Rababah et

al. (2005) also investigated different kind of fruits, including strawberry, peach and

apple and measured the values of total phenolics, anthocyanins and oxygen radical

absorbance capacities (ORAC). The effects of dehydration, ascorbic acid treatments

and the fruits color effect on the levels of these compounds. Fresh strawberry

exhibited the highest level of total phenolics and fresh apple and peach exhibited

lower levels [93].

Hamzah et al., (2013) investigated the phytochemical compounds and in vitro

biological properties of the methanolic extracts of Blighia sapida, Vitellaria paradoxa

and Vitex doniana fruits. Results showed that presence of bioactives like alkaloids,

flavoniods phenols, saponins and tannins in all the samples. Biological activities was

studied in a dose dependent way using DPHH free radical scavenging, and lipid

peroxidation assay. Vitellaria paradoxia exhibited the highest capacity to inhibit

lipids peroxidation than all thhe other extracts. Therefore MeOH extracts of fruit of

these species can be used as quality source of antioxidant bioactives [95].

Araújo et al. (2013) determined the total phenolic content and mineral elements in the

fruit peels of Myrciaria cauliflora. Extracts were prepared using different solvents

32
and combination of solvents. DPPH, FRAP, ABTS and β-carotene in-vitro assays

were used for the analysis. Fruit peels extract was found to be a potential source of

bioactive compounds. Fruit peels of M. cauliflora was found to be a good source of

bioactives and essential elements for the human daily use and for incorporation into

the diet for food improvement [96].

Leccese et al., (2011) research the effect of two solvents on extraction efficiency and

biological activity of the apricot fruit. Total phenolic contents were calculated by

Folin-Ciocalteu assay and biological potential with the Trolox equivalent antioxidant

capacity (TEAC) assay. A linear correlation was present between biological activity

and total phenolic contents. It was found that extraction of total polyphenols were

independent of the solvent extraction methods and depends on the hydrophilic or

lipophilic composition food [97].

Song et al., (2010) reported antioxidant potential of different fruits and vegetables and

dietary supplements in terms of total phenolic content, oxygen radical absorbance

capacity. Beets, broccoli, and red pepper exhibited the maximum biological activity

values while cucumber showed the moderate values. An important correlation was

observed b/w cellular antioxidant activity and total phenolic contents. Potatoes have

the highest contents of phenolics & biological activity in vegetables. Therefore high

consumption of fruits and vegetables is an effective way to increase the bioactive

intake [98].

Jakobe et al., (2007) evaluated the biological potential of red fruits (blueberry,

blackberry, chokeberry, sweet cherry, strawberry, raspberry, black currant, sour

cherry, elderberry and red currant) using DPPH and ABTS radicals assays.

Distribution of all these bioactive compounds in red fruits was identified by using

HPLC equipped with photodiode array detector. A significant correlation was

33
established between antioxidant activity and total poly-phenols while there was not a

direct correlation was originated between total anthocyanin, total hydroxycinnamic

acids. Therefore red fruits can be a good quality source of polyphenols in humans diet

and can be regarded as a good candidate for nutritional supplement formulation[99].

Yan et al., (2006) reported antioxidant properties (total phenolic contents, ascorbic

acid contents, antiradical activities) of two varieties of guava fruit grown locally, and

compared the results with some other local fruits such as banana, dragon fruit, sugar

apple, star fruit, apple and orange. Guava exhibited high amount of primary bioactives

when compared with other fruits. Banana has lower amount of primary bioactives and

having powerful secondary bioactives compounds. Hence, consumption of fruits,

vegetables and spices on regular basis can be helpful to maintain health [100].

Medicinal Plants as Potential Source of Bioactive Compounds

The ingestion of bioactive compounds can be increased by increasing consumption of

medicinal plants, their leaves and medicinal herbs. Medicinal plant i.e. Murraya

koenigii (curry) leaves are commonly used in daily life due to its taste but it also have

medicinal values. Leaves of curry plant exhibited the highest phenolic content, free

radical scavenging potential, ferric reducing antioxidant power (FRAP) and cupric

reducing antioxidant capacity. [101]. Similarly, Moringa oleifera have been

investigated by many researchers and traditionally it is also used for the treatment of

several diseases. Mechanism of different biological action of the Moringa oleifera

leaves extracts were determined and it was found that leaves extract is capable to

prevent oxidative damage to biomolecules by scavenging free radicals [102].

Nashiela et al., (2015) studied the biological potential of selected herbal tea sample

prepared from Cosmos caudatus at different maturity stages namely i) young ii)

mature and iii) old leaves. TPC, TFC, FRAP and DPPH radical scavenging assays

34
were applied for the analysis of herbal tea extracts. It was observed that herbal tea

prepared from young leaves showed significantly strong bioactivity potential for all

assays. A positive correlation was observed for TPC and TFC with reducing power.

Herbal tea preparation from young leaves was recommended more favorable and

good due to strong biological potential because maturation could reduce biological

activity in C. caudatus leaves [103].

Kaur et al., (2014) evaluated TPC & TFC, antiradical activity and antimicrobial

properties of seven selected medicinal Plants (Asparagus racemosus, Cassia fistula,

Ocimum sanctum, Piper betel, Catharanthus roseus, Citrus aurantifolia and

Polyalthia longifolia) using the alcoholic extracts. The highest bioactivity was found

in Citrus auantifolia. Antimicrobial potential of all the plant extracts against

Escherichia coli and Staphylococcus aureus strains was found significant. Therefore

these results concluded that the tested medicinal plants may be considered as valuable

sources of antioxidant and antimicrobial compounds [104].

HO et al., (2012) investigated the in vitro antioxidant and chemical properties of

thirty-one wetland medicinal plants. Methanol and water were used as solvents for

extract preparation. Different assays were employed for antioxidant evaluation. Out of

thirty-one selected medicinal plants samples, only seven samples exhibited promising

antioxidant potential. Therefore some of the selected medicinal plants can be a good

quality source of natural antioxidants in pharmaceutical and medical industry point of

view [105].

Moussa et al., (2011) investigated leaf extracts of 124 Egyptian medicinal plant

species belonging to fifty-six families and compared their bioactivities using different

antioxidant assays. Among 124 plant species tested, leaves extracts of the promising

plant species (eighteen plants) were subjected for the determination of contents of

35
phenolics and flavonoids. Punica granatum extract exhibited the highest bioactivity

and this extract was edible and safe, when tested on albino mice [106].

Olutayo et al., (2011) studied the phytochemicals and biological activities of five

Nigerian medicinal plants. Tested plants extracts have moderate to strong biological

potential and/or free radical scavenging activity. Therefore the selected medicinal

plants are good quality sources of bioactive compounds [107].

Kratchanova et al., (2010) investigated antioxidant activity of different solvents

extracts of twenty-five Bulgarian medicinal plants. It was found for all the antioxidant

assays conducted, high efficiency was exhibited by acetone extracts followed by

water extracts. Research work concluded that the solvent significantly affects the

recovery of polyphenols. All the tested medicinal plants have were reported as rich

sources of polyphenol compounds that can be exploited as a good source of

antioxidant compounds at commercial purpose in food [108].

Duda et al., (2009) observed the antioxidant potential and total poly-phenols of fifteen

medicinal plants using water and methanol as an extracting solvent. These herbs were

selected due to their good therapeutic potential. Methanol was more efficient solvent

for extraction of bioactive compounds from selected medicinal plants. Authors

recommended the use of these medicinal plants for further isolation of active

ingredients as well as commercial exploitation [109].

Katalinic et al., (2006) tested seventy medicinal plant extracts and evaluated their

antioxidant activity potential This work concluded that total phenolic content and total

antioxidant activity differs significantly among seventy selected medicinal plant

extracts/infusions. Melissae folium extracts/infusions exhibited highest phenolic

contents highest level of FRAP and total phenolic contents. Melissae folium

extracts/infusions also showed significantly reducing power and free radical

36
scavenging activity in vitro assays when compared with red wine or beverages like

tea. Therefore, Melissae folium infusion/extract was found to be good dietary source

of bioactives for human consumption [110].

Shyu et al., (2005) investigated the biological activities of twenty-six medicinal plants

used in Taiwan. Out of 26 tested medicinal plants, only 6 showed good biological

activities against DPPH and ABTS free radical scavenging potential. DNA damage by

hydroxyl radicals has also been tested and it was observed that specific plant extracts

showed effective prevention against damages. Results indicates that plant extracts of

Ludwigia octovalvis and Vitis thunbergii are potential source of biological activities

[111].

Part II

2.2. Optimization of Extraction Process for Bioactive Compounds from

Leaves of Morus nigra and Artemisia annua Using RSM

In part II of the dissertation, extraction techniques, for bioactive compounds from M.

nigra and A. annua leaves, was optimized. Orbital shaker assisted extraction

technique was used for A. annua leaves while microwave assisted extraction

technique for M. nigra leaves. Effect of different drying methodologies, extraction

solvents and extraction conditions on recovery of antioxidant components from

selected botanical materials has been studied. The same has been reported by many

authors, as summarized below.

Kaneria et al., (2012) studied the effect of solvents (ether, toluene, ethyl acetate,

acetone, water) and extraction techniques (Successive extractions, Individual cold

percolation, decoction methods) on antioxidant activity of leaf and stem of

Pomegranate (Punica granatum L.). Successive extraction resulted as a best option

37
for the extraction of antioxidants from leaf and stem of pomegranate and it was

evaluated using DPPH, FRAP and superoxide anion radical scavenging assay.

Antioxidant content including TFC and TPC was also measured. Decoction method

was found to be convenient and time saving for the aqueous extraction for both parts

of pomegranate. Solvents used for extraction showed marked differences as far as

antioxidants extraction is concerned. Comparably, leaf showed higher levels of

antioxidant activities as compared to stem showing that the leaf may serve as a

tremendous source of natural antioxidants [112].

Hussain et al., (2012) studied the effect of various extraction techniques on

antioxidant potential of peanut (Arachis hypogaea L.) hulls. Different extraction

techniques were compared for their extraction potential. Different solvent systems

like methanol/water and ethanol/water were also used. Determination of antioxidant

potential was achieved by applying multiple assays. Significant variations were

recorded in the antioxidant activity due to variation in extraction methods and solvent

systems. Sonicated-assisted stirring produced extract with better yield and antioxidant

potential as compared to other methods of extractions. Resultantly, it was found that

extracts of peanut hulls exhibited different antioxidant potential which is based on the

type of extraction technique used [113].

Bimakr et al., (2011) compared the effect of different extraction techniques including

conventional soxhlet extraction and supercritical carbon dioxide extraction on

bioactive flavonoids from spearmint (Mentha spicata L.) leaves. Different extraction

parameters were monitored for their effect on the selected extraction of flavonoids

from spearmint was investigated full factorial. Extracts obtained by the both

extraction techniques were analyzed by high performance liquid chromatography

(HPLC) for identification and quantification of major bioactive flavonoid compounds

38
profile. After using optimized parameters for both the techniques, leave extracts were

evaluated using HPLC and it was found that supercritical cabondioxide extraction is

better than conventional soxhlet extraction as former extraction technique showed 7

flavoniods with high concentration, while later showed 5 flavonoids [114].

Musa et al., (2011) studied influence of extraction techniques and solvents on the

antioxidant activity of Pink-Flesh Guava (Psidium guajava L.). Various extraction

techniques like homogenization, sonication, maceration, shaking and magnetic

stirring for 1 to 3 days. Different solvent systems were used for extraction followed

by determination of antioxidant potential using spectrophotometric assays. It was

found that homogenization and ultrasonic techniques were best for extraction of

antioxidant from guava fruit. Results showed that solvent systems in combination

played significant role for antioxidant extraction rather in the pure form. Acetone

(50%) was found as a best solvent for extraction. Moreover, pink-flesh guava showed

promising antioxidant activities, demonstrating it as an exceptional dietary source of

natural antioxidants [115].

Alothman et al., (2009) reported the effect of different solvent systems on the

extraction of phenolic compounds from three tropical fruit pulps (Honey pineapple,

Banana, Thai seedless gauve) from Malaysia. Solvent systems used for extraction

included methanol, ethanol and acetone with varying concentrations and distilled

water. Significant effects of different solvent systems on the extraction efficacy were

noticed. Enhanced phenolic contents significantly correlated with high antioxidant

capacity [116].

Pompeu et al (2009) optimized extraction of antioxidative compounds from Euterpe

oleracea L. using response surface methodology. Fruits of Euterpe oleracea L.

showed good antioxidant status due to the presence of phenolic compounds. For

39
optimization purpose, a rotatable central composite design having three variables was

used [117].

Wang et al., (2013) carried out optimized recovery of phenolic compounds from

pomegranate (Punica granatum L.) leaves. Initially, four solvents were used for the

extraction purpose and observed the effect of solvent polarity on the extraction yield.

Optimization extraction of phenolic compounds with three independent variables

(solvent concentration, temperature, extraction time) was done using response surface

methodology. Optimization of conditions were done based on absorption and

desorption ability of phenolics. Resultantly, a highly achievable and effective

extraction and enrichment methods were found from the leaves of pomegranate.

Macroporus resin chromatography was first time used for enrichment of phenolics in

pomegranate leaves. Furthermore, DPPH and ABTS abilities of resulting extracts

varied significantly [118].

Part III

2.3. Bioactive-Enriched Fractionation of Periploca Aphylla Crude Extract

Followed by Identification and Quantification of Novel Antioxidant

Compounds

In third part of this research work, bioactive enriched fractionation of periploca

aphylla crude extract has been presented. Antioxidant potential determination

followed by identification of novel antioxidant compounds has been reported.

Preparation of bioactive enriched fractions is now a days focused by many scientists

and some of the work is given as.

Erkan (2012) studied antioxidant activities and phenolic content in Portulaca

oleracea crude extract fractions by reversed-phase separation. Five fractions were

40
obtained and classified based on optical absorption between wavelengths of 200–400

nm. It was found that fraction 3 displayed highest absorption. Different phenolic acids

were detected in Fraction 3, In addition, highest lipid peroxidation, values for TBARS

assay were found associated with fraction 3 [119].

Cho et al., (2011) reported the effect of fractionation on antioxidant potential of

Enteromorpha prolifera L. Using the method of partitioning (n-hexane (HX),

chloroform (CF) and ethylacetate (EA)) different solvent fractions were prepared.

Fractions of CF showed highest DPPH and hydroxyl radical scavenging activities and

these were comparable to the potential of positive controls [120].

Yan et al., (2011) showed the antioxidant activities of EtOH extract fractionated in

water extract (WE), ethanol extract (EE), residue water extract (RWE) and petroleum

ether (PF), ethyl acetate (EF), n-ButOH (BF) and water (WF) in Eupatorium

lindleyanum DC for the first time. Various antioxidant parameters were evaluated for

all the extracts and fractions. All extracts and fractions showed different antioxidant

potential. High antioxidant abilities were also showed by BF for all the antioxidant

assays. Authr reported BF rich in phenolics, while WE and RWE were having highest

amount of tannins. [121].

Wei et al., (2011) measured the antioxidant activities of four different solvent sub-

fractions prepared from EtOH crude extract from Tuber indicum. All the fractions

were analyzed for their antioxidant potential. Significant difference was reported by

the authors among different fractions. Butyl alcohol fraction showed the most potent

on all antioxidant activities. [122].

Li et al., (2009) reported the effect on antioxidant activities of MeOH extracts and

fractions from Lysimachia foenum-graecum. Significant variations was recorded

among the fractions for the antioxidant efficiency. The decreasing order of antioxidant

41
activities among the fractions was reported as ethyl acetate fraction > n-ButOH

fraction > methanol extract > water extract. [123].

Mariod et al., (2009) studied antioxidant activity and phenolic content of fractions

obtained from black cumin (Nigella sativa) seedcake. Crude methanolic extract

(CME) was fractionated and investigated for antioxidant activity The CME and EAF

showed highest antioxidant activities. In addition, The effect of extract/fractions on

the oxidative stability of corn was assessed against standard butylated hydroxyanisole

(BHA) and promising results were reported [124].

Part IV

2.4. Nano-encapsulation of Defatted Kenaf Seed Meal (DKSM) Extract to

Enhance its Bioavailability and Antioxidant Activity

Bioactive compounds, when administered to human body, experience gastrointestinal

conditions before reaching active site. Survival of bioactive compounds through

gastrointestinal conditions and their bioactivity at target site is a matter of serious

concern. Therefore, part IV of this thesis, fate of bioactives from DKSM under

simulated gastrointestinal conditions has been determined followed by preparation of

nano-encapsulates for bioavailability enhancement. Many authors have reported

encapsulation as a valuable method for the enhancement of bioavailability of

bioactive compounds, summarized as.

Ballesteros et al., (2017) carried out encapsulation of antioxidant compounds obtained

from coffee grounds using different coating materials. For encapsulation two different

methods were used. Antioxidant potential of the encapsulated samples were analyzed

in order to verify the efficiency of each studied condition. Results of this study

revealed that both the technique and coating materials contributed towards

42
encapsulation of antioxidant compounds and best results were recorded by freeze-

drying method using maltodextrin [125].

Nanoencapsulation of green tea catechins was achieved by Bhushani et al., (2017) and

controlled release including in-vitro permeability was studied. Green tea catechins

encapsulation was performed by utilizing electrospraying technique, which is a

biocompatible, biodegradable macromolecule. The nanoencapsulated catechins

exhibited enhanced bioactivity under in-vitro gastrointestinal conditions. In addition,

the study provided a one-step approach for synthesis of green tea catechin

nanoencapsulates with promising sustained release and increased permeability

properties [126].

Tulini et al., (2016) prepared solid lipid microparticles loaded with a cinnamon

extract (Cinnamomum zeylanicum). In this study, vegetable fat were used as carrier to

prepare solid lipid microparticles (SLMs) by spray chilling technique carrying

proanthocyanidin-rich cinnamon extract. SLMs exhibited ability to mask the bitter

taste of bioactives present in cinnamon extract. Conclusions of these results showed

the potential of SLM loaded with cinnamon proanthocyanidins can be used for

improving shelf life of food items [127].

Gibis et al., (2016), reported in-vitro release of polyphenols, encapsulated in chitosan-

coated particles. In this work, more stable liposomes was obtained by applying

chitosan coating (1% w/w). Grape-seed extract (GSE), a having rich source for

polyphenols, was loaded into liposomes using high-pressure homogenization.

Encapsulation efficiency for uncoated liposomes was lower than coated liposomes.

Coated liposomes released the GSE in controlled manner that indicate the

bioavailability enhancement due to encapsulation. [128].

43
Pulicharla et al., (2016) performed encapsulation of polyphenols and in biodegradable

chitosan nanoformulation. Interaction of strawberry extract polyphenols (PPs) with

positively protonated amino groups of chitosan help in achieving maximum

encapsulation. The particles size was found between 300 and 600 nm and continuous

release of PPs was recorded at pH 7.4 of in vitro release studies. Authors reported that

encapsulation technique contribute towards controlled release and enhancement of

bioavailable fraction of polyphenols [129].

Díaz-Bandera et al., (2015) studied release patron of spray-dried Roselle extract

encapsulated with different carrier agents. In this study, investigation were carried out

to check the effect of different carriers on the nature of encapsulates. Among the

tested encapsulation material, researcher reported that pectin as most appropriate

transporter for Roselle's phenolic compounds compared to the other carriers used

[130].

Gómez-Estaca et al., (2015) reported encapsulation of curcumin in gelatin

microspheres to enhance bioaccessibility. Curcumin is used as a food colorant due to

its low solubility in water, which permits its dispersion in food matrices. It is also

used as functional ingredient but its bioaccessibility needed to be enhanced. Increase

in both water solubility and bioaccessibility of curcumin were recorded after

encapsulation. In test of coloring ability of microencapsulated curcumin exhibited

better dispersion in microencapsulated form [131].

Bryła et al., (2015) encapsulation elderberry extract into phospholipid nanoparticles.

For the purpose of food application, suitability of different lecithins for

nanoencapsulation of elderberry extract in liposomes was checked. Encapsulation

efficiency, size distribution and zeta potential were calculated for the liposome

characterization. It was concluded that among three tested lecithins, soybean lecithin

44
was of best stability. The efficiency of encapsulation in this case was of 25% and

liposomes were uniform both in terms of size and structure. Significantly higher

efficiency was reported for encapsulation, if sunflower and egg yolk lecithins is used

for the purpose [132].

Serrano-Cruz et al., (2013) encapsulated the extract of Roselle (Hibiscus sabdariffa

L.) extract and checked its controlled release and antioxidant activity. Release of TPC

after encapsulation in binary and ternary mixtures of different encapsulation material

was determined. The results of this study confirmed that the release of polyphenols

from encapsulated extract can be controlled by changing the composition of the

mixtures [133].

Belščak-Cvitanović et al., (2011) encapsulated different extracts in alginate–chitosan

system. The characterization of the encapsulated and nonencapsulated plant extracts

were performed by means of antioxidant potential evaluation. All extract

encapsulating microbeads have high encapsulation efficiency and obtained

microbeads exhibited significant antioxidant potential that may enhance the daily

intake of antioxidants and its use as food additive [134].

Part V

2.5. Stabilization Efficiency of Selected Extracts on Shelf-life of different Food

Items (Oil and Meatballs)

In food industry, rancidity of food items is a serious issue and for the stability of food

items antioxidants are used as food additives. Carcinogenic nature of synthetic

antioxidant has diverted the interest of food scientist towards the use of natural

antioxidants for shelf life enhancement of food items. In this part two different

extracts have been used to enhance shelf life of two different food items i.e. cooking

45
oil and meatballs. Many authors have reported enhancement in shelf life of different

food items that is summarized below.

Baştürk et al., (2018) studied the oxidative stability of corn oil having some herbal

extracts in it. Oxidative stability was monitored under accelerated oxidation

conditions and synthetic antioxidants were used for the sake of comparison. Total 10

corn oil samples were prepared, 2 having synthetic antioxidants while 6 samples were

administered with herbal extracts and a refined corn oil was taken as control. Samples

were stored at 60 °C and analysis were carried out for 6 weeks. Results of peroxide

value, thiobarbituri acid reactive substances (TBARS), conjugated dienes (CD) and

trienes (CT) revealed that extracts of sumac, thyme and mint efficiently retarded the

process of oxidation and authors recommended the use of these extracts in food

industry for the stabilization of oil samples [135].

Yang et al., (2016) used rosemary extract to improve shelf life of vegetable oil against

oxidative deterioration. The ability of rosemary extract to inhibit oxidation of

vegetable oils was found comparable with synthetic antioxidants. Results revealed

that rosemary extract effectively inhibited oxidative deterioration of oil by enhancing

antioxidant capacity. Authors suggested the use of rosemary extract as a potent source

of natural antioxidants [136].

Guo et al., (2016) evaluated antioxidant efficacy of rosemary extract in palm oil under

accelerated storage and frying conditions. Stability of palm oil having different

concentrations of rosemary ethanol extract were examined at 65 ◦C and 180 ◦C in

comparison with palm oil having synthetic antioxidants. A significant antioxidant

effect of extract in palm oil was recorded. During frying process rosemary ethanol

extract effectively reduced the peroxide value, P-anisidine value, free fatty acid value.

Authors also reported that production of saturated fatty acids was inhibited till 25 h of

46
frying and 24 d of accelerated storage. Rosemary ethanol extract prevented the

process of oxidation in palm oil sample, same as the synthetic antioxidants do.

Therefore, authors recommended the use of rosemary ethanol extract instead of

synthetic antioxidants [122].

Esposto et al., (2015) reported the effect of olive extract on the oxidative stability of

vegetable oils. The potential of different concentrations of phenolic extract to reduce

the negative effects of frying on refined olive oil was studied. Polyphenolic extracts

reduced the process of oxidation and the emission of low-molecular-weight aldehydes

better than BHT [137].

Teruel et al., (2015) studied the effect of different solvent extracts obtained from

rosemary on the quality of frozen chicken. Significant difference in concentration of

bioactives (total phenolic content, carnosol and carnosic acid content) and antioxidant

activity (FRAP and ABTS assays) was observed for three kind of extracts, prepared

using powder-acetone, liquid-methanol, liquid-acetone. Furthermore, effect of the

three extracts on quality parameters (physical–chemical and sensory) of frozen

chicken nuggets was assessed. Authors reported that rosemary extracts were unable to

modify the quality (pH, colour, sensory quality) of chicken nuggets [138].

Spigno et al., (2013) structured nano-encapsulates of grape extract to improve its

antioxidant efficiency in hazelnut paste. Three different nanoemulsion (oil/water

nanoemulsion; maltodextrin-assisted; ethanol/solid–lipid nanoemulsion, were used for

the encapsulation purpose. After adding the encapsulated and non-encapsulated

extracts to the hazelnut paste samples, half-life test at 60 ºC was conducted till 98

days with peroxides value testing. Paste oxidation was significantly inhibited by

extract addition and encapsulation enhanced phenolic efficiency against lipid

47
oxidation, by preserving the antioxidant activity and oil/water nanoemulsion was

found to be the best among the tested systems [139].

Poiana (2012) studied oxidative stability of SFO induced by GSE, in comparison with

BHT during microwave heating up to 240 min and under frying conditions. Lipid

oxidation was evaluated by measuring PV, CD, CT, p-anisidine value, and TOTOX

value. Significantly inhibitory effect on lipid oxidation was recorded for both the samples

having GSE and BHT, to a different extent. Respective microwave exposure as well as

convective heating, for 240 min of samples having GSE (1000 ppm) caused significant

decrease in investigated indices relative to the control values. In comparison to BHT,

GSE up to 600–800 ppm retarded lipid oxidation. On the basis of findings of this study,

GSE can be preferred as a potential natural extract for increasing oxidative stability of

sunflower oil during thermal applications [140].

Shelf life of sunflower oil was tested under accelerated storage conditions by Iqbal &

Bhanger (2007) using garlic extract. Garlic extracts were prepared in different

solvents and methanolic extract was found having highest antioxidant potential.

Stability of methanolic extract was evaluated at 185 ºC for different intervals, i.e. 0–

80 min followed by evaluation of antioxidant activity of the heated extract in the

linoleic acid system (71.6% inhibition). Methanolic garlic extract at three different

concentrations and BHA and BHT (200 ppm) were used as standard. Effect of garlic

extract on sunflower oil stability was assessed in terms of weight gain (WG),

antioxidant activity index, free fatty acid content, peroxide value, conjugated dienes,

conjugated trienes, and thiobarbituric acid-reactive substance analysis and results

recorded for the selected parameters were found in agreement with each other. Oil

sample having garlic extract up to 1000 ppm inhibited the production of oxidation

products that indicate that garlic extract can resist the process of oxidation in oil in

comparison with synthetic antioxidants i.e. BHA and BHT used in this study [57].

48
Part VI

2.6. Structure-activity Relationship of Selected Phenolic Acids in Order to

Propose Mechanism of Their Antioxidative Action

Erzincan et al., (2015) developed QSAR for some new coumarin derivatives. Thirty

seven new coumarin derivatives were synthesized and for the modification of

structure to enhance their antioxidant activities, initially antioxidant potential was

determined followed by the generation of QSAR. Coumarin derivatives were

optimized by using SPARTAN 10 software and calculation of descriptors was done

by using DRAGON. Multiple linear regression (MLR) models were developed.

Descriptors appeared at the end of model indicated that H-bond donor and lipophilic

character are decisive parameters in antioxidant activity of any molecule [141].

Filipovic et al., (2015) established quantitative structure–activity relationship (QSAR)

of selected hydroxybenzoic acids and antiradical activity was taken as response.

Descriptors related to different structural aspects of free radical scavenging processes

contributed to develop a reliable QSAR models. Weakness in standardization of

antioxidant assays created some doubts in successful use of the QSAR procedures.

However, the authors confirmed that results of radical scavenging potential and

calculation of relevant descriptors, it is possible to generate fair antiradical QSAR

models [142].

Hoelz et al., (2010) reported QSAR for 15 phenolic compounds having promising

antioxidant activity. Two electronic properties such as homolytic dissociation

enthalpy and ionization potential, in addition, two other parameters lipophilicity and

relative lipophilicity, were estimated. The best QSAR model was generated by using

multiple regression analysis, systematic selection of variables which showed a high

statistical significance [143].

49
Nandi et al., (2007) developed QSAR for nine sets of phenolic compounds using ridge

regression and neural networks. Anticancer activity was taken as biological activity

and ridge regression analysis provided better significant correlation compared to

report by Selassie and co‐workers. Artificial neural network were used by the authors

for better understanding of multidimensional rational patterns in complex data sets

[144].`

Rasulev et al., (2005) studied QSAR of the antioxidant activity of 27 flavonoids

belonging to four different groups. In this study, correlation and prediction of the

inhibition of lipids peroxidation was made. For the selection of descriptors, genetic

algorithm and multiple linear regression analysis was used. Correlation models related

to the structural features and biological activities of flavonoids were developed.

Equations obtained at the end were found having one to four descriptors calculated

using DRAGON and quantum‐chemical methods. The results showed that magnitude

of dipole moment, position of the OH groups and geometry of the molecule contribute

significantly in inhibition of lipids peroxidation. The significant QSAR models were

obtained that also confirms the statistical significance of our models [145].

50
 Chapter 3

MATERIALS

AND

METHODS

51
3.1. Chemicals and Reagents

All the chemicals and reagents including Folin-Ciocalteu’s, 2, 2-Azino-bis-(3-

ethylbenzthiazoline-6-sulfonic acid) diammonium salt (ABTS), 2, 2-diphenyl-1-

picrylhydrazyl (DPPH), butylated hydroxytoluene (BHT), butylated hydroxyanisole

(BHA), 2,4,6-tripyridyl-s-triazine (TPTZ), α-tocopherol, thiobarbituric acid and gallic

acid, 2,4,6-tripyridyl-S-triazine (TPTZ), metaphosphoric acid, 2,6-

dichloroindophenol, rutin, aluminium chloride, glacial acetic acid and other chemicals

were purchased from Sigma/Aldrich.

All the solvents (HPLC grade) were purchased from Fisher Scientific, Leicestershire,

UK. During analysis and sample preparation, demineralized water (DMW,

conductivity< 5µS/cm) was used.

3.2. Sample collection

Samples of Artemisia annua L. leaves were collected from peripheral areas of

Islamabad, Pakistan. Leaf samples of selected mulberry species (Morus alba, Morus

nigra and Morus rubra) were collected from hilly areas of Azad Kashmir, Pakistan.

Plants of Periploca aphylla L. were collected from Kallar kahar, Chakwal, Punjab,

Pakistan.

Samples were initially washed with running tap water and then rinsed with DMW. All

the samples were chopped and placed in electric oven at 40 °C to attain constant

weight. The dried samples were ground to powder form and passed through a sieve (1

mm) and were stored at ambient conditions after packing in polyethylene bags.

52
Part I

3.3. Determination of Proximate Composition and Antioxidant Potential of

Selected Medicinal Plants

3.3.1. Determination of proximate composition of selected medicinal plants

Proximate composition of leave samples of mulberry and A. annua, was determined in

terms of ash, alkaloid, crude fibre, lipid, moisture and protein contents following

AOAC procedure [146], while total carbohydrate content by anthrone method [147]

and phytate content was calculated following a method of Wheeler and Ferrel [148].

3.3.1.a. Ash content

For the determination of ash content, samples were ignited in a furnace at 550-600 oC

for 1 h. The remaining ash was cooled and weighed and percent ash was calculated.

3.3.1.b. Moisture

By placing fresh samples in hot-air oven at 60 oC, constant weight was attained and

%age of moisture was calculated using the formula given below:

W1
% Moisture = (1 − ) × 100
W2

W1 = Sample dry weight and W2 = Sample fresh weight

3.3.1.c. Lipid content

Each sample was extracted with petroleum ether (250 mL) using soxhlet apparatus for

6 hours. The solvent was evaporated by rotary evaporator and percentage of lipid

content was calculated according to AOAC.

3.3.1.d. Alkaloid content

Each powdered sample was added in 200 mL acetic acid (20 % solution) and kept for

4 h. The volume of the mixture was reduced to one quarter followed by dropwise

53
addition of ammonium solution (27 %) to the extracts till precipitation occurred. After

filteration, precipitates were dried and weighed [146].

3.3.1.e. Protein content

For estimation of protein, nitrogen content was determined initially using to Kjeldahl

method given by AOCS [146]. Sample (0.2 g) was added in DW and was introduced

in Kjeldahl flask followed by addition of concentrated H2SO4 (5 mL). The resulting

mixture was heated on water bath till frothing ceased and again heated till white

fumes appears and kept on burner for 10 minutes. Mixture was cooled down and

volume was made up to 50 mL. Five milliligter of the resulting solution was taken in

distillation flask and 40 % NaOH (2-3 mL) and distillation was carried out. Ammonia

gas (NH3) was collected for 10 min in which 5 mL H2SO4 (1 N) was added. Distillate

was titrated against NaOH (1 N) using methyl red and methylene blue as indicator.

Nitrogen content was calculated using following equation:

(A − B) × N × 0.014007 × 20 × 100
% Nitrogen =
weight of sample

Where, A = mL of 1 N H2SO4 taken, B = mL of 1 N NaOH used and N = Normality

of NaOH.

3.3.1.f. Total carbohydrate content

Total carbohydrates were determined using anthrone method [147]. Mixture of

sample (100 mg) and 2.5 N HCl (5 mL) was boiled for 3 h. The resulting mixture was

cooled to room temperature and neutralized with solid Na2CO3. After making the

volume up to 100 mL, it was centrifuged with the supernatant collected and 1.5 mL

aliquots used for further analysis. Different standards were prepared and volume 1 mL

was made by adding DMW in all test tubes. Anthrone reagent (4 mL) was added and

solution was heated (8 min) in water bath. Absorbance was measured at λmax ≈ 630

54
nm. After drawing the calibration curve, concentration of total carbohydrate in the

sample was determined.

3.3.1.g. Phytate content

Determination of phytate content was done following method of Wheeler and Ferrel

(1971) [148]. Accurately weighed a ground (40 mesh) sample into an Erlenmeyer

flask (125 mL) and extracted with 50 mL of trichloroacetic acid (3%) (TCA) for 45

min. Centrifuged and collected 10 mL aliquot for analysis. Four mililiter ferric

chloride solutions (2000 ppm ferric iron in 3 % TCA) was added and then heated on

water bath for 45 min. Precipitates were obtained after centrifugation and washed

twice with 20– 30 mL TCA (3 %) solution. In resulting mixture, NaOH (3 mL,1.5 N)

solution was added and mixed well and volume was raised up to ~ 30 mL with DMW

and heated on water bath for 30 min and filtered through Whatman filter paper No. 2.

Precipitates were washed with hot water (60 – 70 mL) and filtrate was discarded.

Precipitates were dissolved in hot HNO3 solution (3.2N, 40 mL) in a 100 mL

volumetric flask. Five milliliter aliquot was transferred to another 100 mL volumetric

flask after cooling and diluted with DW up to 70 mL approximately. Twenty milliliter

of KSCN solution (1.5 M) was added, made up the volume (100 mL) and absorbance

recorded at 480 nm.

For calibration curve of Fe(NO3)3 (434 mg) was dissolved in 100 mL DW in a

volumetric flask. For working solution. stock solution (2.5 mL) was diluted up to 250

mL in a volumetic flask. Working standard were prepared using different

concentrations in 100 mL volumetric flasks. Iron contents were calculated from a

Fe(NO3)3 prepared standard curve. Phytate phosphorus from the iron results were

calculated, assumed iron: phosphorus (4:6 molecular ratio). Micro grams of iron

present in the test sample were determined from the standard curve.

55
3.3.2. Determination of Antioxidant Potential

3.3.2.a. Extraction of Samples

Five grams of powdered leaf sample of each variety (Morus alba, Morus nigra and

Morus rubra) were extracted in ultrasonic bath for 45 min at ambient temperature,

using methanol (50 mL, 80%) as extraction media. After filtration through nylon

membrane filter (0.45 mm), the resulting solutions were dried using rotary evaporator,

to obtain solvent free extracts and were stored at −20 °C [54].

Ground A. annua leaves were loaded in Soxhlet apparatus and extraction was carried

out for 6 h using hexane and same procedure was repeated for other solvents

separately. Each extracts were filtered using nylon membrane filter (0.45 mm) and

using a rotary evaporator solvents were evaporated 45 °C and remaining extract was

stored at −20 °C.

3.3.2.b. Determination of total phenolic content (TPC)

For the determination of TPC Folin-Ciocalteu (FC) reagent assay was employed

[113]. Sample extract (0.2 mL) was added to test tube having FC reagent (7.5 mL). To

make the contents alkaline, Na2CO3 solution (20 %, 1.5 mL) was added. For dilution

of reaction mixture DMW was added up to 10 mL volume in total. Reaction was

allowed to complete by placing reaction mixture in dark for 2 h. Absorbance (λmax =

755 nm) of resulting solution was measured using CICIL double beam

spectrophotometer. TPC was calculated from standard curve and results were

calculated as milligram Gallic acid equivalents per gram of the sample (mg GAE/g).

3.3.2.c. Determination of total flavonoid content (TFC)

Determination of TFC was carried out by using a method reported by Hussain et al.

(2012) [113]. In short, sample extract (1 mL), DMW (5 mL) and NaNO2 solution (0.4

mL, 5 %,) was added to a 10 ml volumetric flask and AlCl3 solution (0.6 mL, 10 %)

56
was added along with NaOH solution (1 M, 2 mL) and the remaining volume was

filled with DMW. After mixing the contents of the flask thoroughly, absorbance of

the reaction mixture was recorded at 510 nm (λmax). TFC of the samples were

determined as microgram epicatechin equivalents per gram (µg ECE/g).

3.3.2.d. Determination of ascorbic acid content (AAC)

Solution of 2, 6-dichloroindophenol (DCIP) was prepared by dissolving 50.0 mg in

DMW (100 mL). Sodium carbonate (405 mg) was then added and dilution of resulting

solution was done by adding DMW (1000 mL). Metaphosphoric acid (7.5 g) and

acetic acid (20 mL) were mixed in 100 mL DMW and mixture was diluted up to 250

mL with DMW. Further, standard ascorbic acid solution (7.00 mL) was pipetted out

in 250-mL Erlenmeyer flask, metaphosphoric acid-acetic acid solution (2 mL) and ~

25 mL of DMW were added in a flask with constant stirring. DCIP solution was filled

in the burette and titrated the sample solution, until a permanent (lasting more than 30

sec) light pink color appeared. This volume of DCIP corresponded to amount needed

to oxidize entire amount of ascorbic acid.

The determination of AA in a sample extract is similar as described for standardizing

of the DCIP solution. Same volume of extract sample (7 mL) was used for the

determination of AA. This procedure was repeated until three values with less than 5

% error were obtained.

F V
mg asorbic acid/mL = (X − B) × ( ) × ( )
E Y

Where

X = mL for sample titration, B = mL for blank titration, F = titer of dye (mg AA

equivalent to 1.0 mL DCIP standard solution), E = mL assayed (2 mL), V = volume

of initial assay solution (7 mL), Y = volume of sample aliquote titrated (7 mL) [149].

57
3.3.2.e. Determination of total tannins content (TTC)

For the estimation of total tannin content, a method as described by Atanassova

(2009) was used. Briefly, 2.5 mL of each extract and indigo solution (indicator) and

75 mL DMW was added in a volumetric flask of 100 mL capacity. The above

contents were titrated against KMnO4 (0.1 N) solution till the change of blue color to

green is observed. Then few drops of KMnO4 solution were till golden yellow color

appeared [150]. The total tannin contents (%) were calculated as follows:

(V − V₀) × 0.004157 × 250 × 100

TTC(%) =
g × 100

Where V = mL used (KMnO4 solution) for sample; Vo = mL used (KMnO4 solution)

for blank; 0.004157 = total tannin equivalent in 1 mL KMnO4 solution; g = mass of

the sample; 250 – volume of volumetric flask; 100 – percent, %.

3.3.3. Determination of Antioxidant Activities

For the evaluation of antioxidant activities, DPPH (2,2-diphenyl-1-picrylhydrazyl)

and ABTS (2,2-Azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt)

assays were used. In addition, ferric ion reducing and ferrous ion chelating activities

of the samples were also assessed.

3.3.3.a. DPPH free radical scavenging activity assay

In order to measure DPPH free radical scavenging potential of the samples, DPPH

stock solution was prepared (2.4 mg of DPPH radical/mL of methanol), which was

further diluted to adjust the absorbance at 0.7 at a wavelength of 515 nm. MeOH

extracts of leaves (5–25 µL) were added to test tubes having 975–995 µL of DPPH•

solution. The reaction mixture was placed in dark for 30 min at room temperature and

absorbance of the resulting solution was taken at 515 nm. DPPH• scavenging

antioxidant capacity was calculated as millimole per liter Trolox equivalent (mM TE)

dry weight of leave samples as average of three concordant readings [151].

58
For some of the samples DPPH• scavenging activity in terms of percent inhibition was

also calculated and an already reported method was used [116]. An aliquot (1 mL) of

extract was mixed with DW (5 mL) and 3.9 mL of DPPH MeOH solution. After

through mixing, the solution was placed in dark for 30 min. The absorbance was

recorded at 515 nm, using methanol without DPPH•. Percentage of inhibition of the

DPPH radical was calculated as:

% inhibition of DPPH = (Abs control - Abs sample/Abs control)*100

where Abs control is the absorbance of DPPH solution without extracts.

3.3.3.b. ABTS radical cation scavenging activity assay

ABTS radical cation scavenging assay was carried out following an earlier method

[152]. ABTS•+ (5 mM) were generated in an aqueous medium by oxidizing ABTS

with MnO2. For dilution, 5 mM phosphate buffered saline (PBS, pH 7.4) was added

to leave extracts, maintained the absorbance (at 734 nm) of the resulting solution at

0.700 (±0.020). Then the extracts treated with buffer (2.5 mL) were added to 7 mL

ABTS•+ solution and after 15 min, absorbance of resulting mixture was measured at

room temperature against PBS as blank. The antioxidant activity of leave extracts in

terms of ABTS•+ scavenging potential was determined as mM TE dry weight of

leaves samples.

For some extracts %age inhibition of ABTS•+ was also evaluated according to the an

assay reported by spigno et al [153], which is based decrease in absorbance (at 734

nm) of extract-ABTS•+ mixture. ABTS radical scavenging potential was calculated as:

% inhibition of ABTS = (Abs control - Abs sample/Abs control)*100

where Abs control is the absorbance of ABTS solution without extracts.

59
3.3.3.c. Ferric Reducing Antioxidant Power (FRAP) Assay

Ferric reducing antioxidant power of the samples was evaluated following a reported

method [66] with slight modifications. Required stock solutions of, acetate buffer

(150 mM), FeCl3·6H2O (10 mM), TPTZ (20 mM) and HCl (5 mM), were prepared

having 500 mL volume each. For the preparation of working solution, Twenty five

milliliter of acetate buffer, TPTZ solution (1.25 mL) and FeCl3·6H2O (1.25 mL) were

mixed followed by heating to 37 °C and pH at 3.6 was maintained. Fifty microliters of

individual sample were mixed with 2 mL of working solution, and absorbance was

recorded (593 nm) against blank for 5 min. The average results of FRAP assay were

calculated as millimole per liter Trolox equivalents in accordance to standard curve of

Trolox.

3.3.3.f. Lipid peroxidation

Measurement of Lipid peroxidation represents the presence of malondialdehyde

(MDA) contents. Measurement of lipid peroxidation was carried out following a

method of Heath and Packer [48]. Briefly, 0.5 mg dried extract was mixed with of 1%

trichloroacetic acid (TCA) and then centrifuged at 15,000 g for 15 min. Supernatant

(0.5 mL) was added in 2 mL of solution having 20% TCA and 0.5% TBA. The

resulting mixture was heated at 96 °C for 20 min, followed by cooling and

centrifugation at 10,000 g for 15 min at 4 °C. Absorbance was recorded at 532 and

600 nm and using extinction coefficient of 155 mM-1cm-1, MDA content was

measured.

60
Part II

3.4. Optimization of Extraction Process for Bioactive Compounds from

Leaves of Morus nigra and Artemisia annua Using RSM

For comprehensive optimization, it was considered necessary to consider the effect of

different drying procedures, solvents and extraction methods simultaneously.

Therefore, samples of A. annua and M. nigra were collected and initially dried using

three dying procedures including shade drying [154], hot air oven drying [155],

microwave-assisted drying [154].

After drying, extracts of the selected samples were prepared using five solvents

(hexane, chloroform, ethanol, methanol and water) of varying polarties and three

extraction methods (Soxhlet, orbital shaker and microwave assisted). Results revealed

that maximum antioxidant potential was exhibited by M. nigra leaves dried under-

shed and extracted using ethanol with orbital shaking extraction technique. For A.

annua, oven drying was found suitable for achieving maximum recovery of

antioxidants from the leaf matrix.

The efficiency of an extraction method is influenced by many other factors including

extraction time, temperature, shaking speed, microwave frequency, pH, solid to

solvent ratio and particle size. Of the many extraction conditions, the focus of this part

of present study was given to the parameters of selected extraction technique that

govern the extraction of antioxidants. Parameters, including time and speed for orbital

shaker assisted extraction, time and microwave intensity for microwave assisted

extraction, were selected with an objective of applying the RSM approach to

optimize.

61
3.4.1. Response Surface Methodology (RSM)

Response surface methodology (RSM) was executed using well known software

(Minitab 17) to find out the optimal conditions. A central composite design was

employed to examine the effects of selected independent variables (X1=Rotation

time/microwave radiation power and X2= rotation speed/microwave radiation time)

on the dependent variables (TPC, TFC, AA, DPPH• & ABTS•+ % inhibition).

Experimental values and coded levels of independent variables used for orbital

shaker-assisted extraction and microwave-assisted extraction were used for statistical

analysis. For example, a two level full factorial design was employed to examine the

effect of extraction parameters and their interactions. The complete design using

aqueous 70 % MeOH was based of 9 experimental points having one center point.

Central Composite Design (CCD) was used to fit the data in a quadratic model. The

regression equation model in un-coded units for each response is as follows:

Y = β₀ − β₁X₁ − β₂X₂ + 𝛽₁₁X² + β₂₂X² + β₁β₂X₁X₂

Where Y = response value, β = constant coefficients, and X = independent variable.

Regression analysis (R2) and ANOVA (p < 0.05) was used to predict validity of

model. Statistical implication of model and its parameters was evaluated at 5%

probability level (α = 0.05). At the end three-dimensional response surface plots, of

three level of a parameter having three different levels, were produced against one

response variable (TPC, TFC, AA, DPPH• & ABTS•+ % inhibition). Independent

variables for orbital shaker-assisted extraction technique were rotation speed and

time, while for microwave-assisted extraction technique were microwave radiation

power and time.

62
3.4.2. Orbital shaker-assisted extraction

Powdered (50 g each) samples was added in 1 L extraction flask. In order to enhance

the efficiency of extraction process, glass beads (~ 5 g/bead) were added in the flask.

A measured quantity (500 mL) of 70 % MeOH were added into the flask. After 5

minutes of addition of the solvent, samples were placed in adjustment position of

orbital shaker. The warm air (35 oC) was allowed to pass through the shaker, while

the rotation speed was kept at 50, 100 and 150 rpm respectively. At each rotation

speed, extracts were collected after 180, 360 and 540 min. Extracted samples were

filtered and centrifuged at 10,000 rpm for 15 min. The extracts were dried using a

rotary evaporator. The collected extracts were kept in ambert colored glass bottles in a

refrigerator (temperature = + 4 oC) [156]. The extracts were analyzed for TPC, TFC,

AAC and %age inhibition of radicals (DPPH• and ABTS•+).

3.4.3. Microwave-assisted extraction

Extracts of powdered (10 g each) sample were prepared with 70 % MeOH (100 mL).

Extraction was carried out at variable microwave oven power (200, 300 and 400

watts) for 3, 6 and 9 min. After filtration, centrifugation of extracts was conducted at

10,000 rpm for 15 min. The extracts were dried using a rotary evaporator. The

extracts were collected in ambert colored glass bottles and stored in a refrigerator

(temperature = + 4 oC) [157]. The extracts were analyzed for TPC, TFC, AAC and

%age inhibition of radicals (DPPH• and ABTS•+).

3.4.4. Determination of antioxidant potential

Antioxidant potential of extracts in terms of TPC, TFC, AAC and percentage

inhibition (DPPH & ABTS) was determined following procedures given in section

3.1.4. and 3.1.5.

63
Part III

3.5. Bioactive-Enriched Fractionation of Periploca Aphylla Crude Extract

Followed by Identification and Quantification of Novel Bioactive

Compounds

3.5.1. Fractionation of Periploca aphylla (PA) crude extract

For fractionation, crude extract of Periploca aphylla (PA) crude extract was prepared

using methanol as solvent. For the purpose, 500 g of powdered plant was extracted

with 2 liter of methanol. Solvent was recovered using rotary evaporator and crude

extract was recovered. The extract was then suspended in DW (500 mL) in a

separating funnel (1000 mL) followed by addition of second solvent i.e. n-hexane

(500 mL). After shaking, the mixture was allowed to stand to achieve the separation

of two solvent layers properly. 1st hexane fraction was recovered and in remaining

aqueous layer chloroform (500 mL) was added and 2nd chloroform fraction was

obtained. In the same way, EtOAc and ButOH fractions were prepared and was

repeated three times for each solvent. At the end aqueous fraction was collected

separately. The process of fractionation was repeated thrice for each solvent and a

volume of 1.5 liter was obtained for each solvent. All the fractions (n-hexane fraction,

Chloroform fraction, EtOAc fraction and ButOH fraction) were concentrated, dried

and then stored at –80°C prior to use for further analysis.

3.5.2. Determination of antioxidant potential

Antioxidant potential of extracts was determined in terms of TPC and antiradical

potential using DPPH• and ABTS•+ assays. (Section 3.1.4. and 3.1.5)

64
3.5.3. Quantification of phenolic acids in Periploca aphylla fractions by HPLC
3.5.3.a. Sample preparation

Identification and quantification of phenolics in different solvent fractions of

Periploca aphylla (PA) was carried out by HPLC method. Twenty five milligrams of

each fraction was dissolved in 6M HCl (5 mL), and then 10 mL of methanol was

added. The resulting solution was incubated at 90°C for 2h. The solution was filtered

with membrane filter (0.2mm Millipore) before injection into HPLC.

3.5.3.b. HPLC analysis

Some well established phenolic acids were quantified in different solvent (n-hexane,

chloroform, EtOAc, ButOH and water) fractions by HPLC analysis. Samples were

injected using Agilent autosampler into an Agilent series HPLC equipped with UV–

VIS Spectra-Focus detector. Phenolic components present in fractions were separated

on Agilent C18 column 20RBAX ECLIPSE, XDB (5μm; 4.6 × 150 mm, Agilent

USA). The solvent system was isocratic mobile phase, (aceto-nitrile 0.05%

phosphoric acid solution (20:3:77, v/v/v)) and a flow-rate of 1mL/min. Prior to use

the mobile phase was filtered through 0.2 mm Millipore membrane filters and

sonication was done in order to dgass the solvent mixture. Separated components

were identified at 280 nm.

3.5.4. Sub-fractionation of PA ButOH fraction

All the fractions were first analyzed for their antioxidant potential following the

protocols given in section 3.5 and the potent fraction i.e. ButOH fraction was further

processed for sub-fractionation.

For sub-fractionation, slury silica gel (200 mesh size, 100 g) was loaded in glass

column with tapping to achieve uniform packing. ButOH fraction was introduced on

silica followed by the addition of 5 gram silica over the sample, so that sample my not

65
get disturb during the addition of solvent. Gradient elution was performed using

mixture of solvent ButOH, EtOH and MeOH. Sub-fractions were analyzed by TLC, at

the end sub-fractions with same TLC profile pooled together and then concentrated in

rotary evaporator.

3.5.5. Determination of bioactive compounds in PA sub-fraction by GC-MS

Analysis of sub-fraction was performed by GC-MS (Agilent technologies 7890A GC

equipped with Agilent 5975 MSD) [158]. Chemstation GC-MS software was used to

run the instrument. DB-5 MS 30 m×0.25 mm×0.25 μm (composed of 5% diphenyl -

95% dimethylpolysiloxane) Agilent column was used for the analysis. Helium

(99.999 %) was selected as carrier gas, while flow rate was maintained at 1 mL/min

constant flow. Electron impact mode was applied to develop all the mass spectra and

ionization was achieved at 70 eV. Ionization kept off during first 1 min, to avoid

solvent overloading. Storage level of ionization was up to 35 m/z, having maximum

ionization time 25,000 μs. Quadropole triple axis MS detector maintained at 250 °C

(transfer line). Mass spectra was used for the identification of bioactive compounds

using NIST and Wiley (main and rep) database library.

Sub-fraction (25 mg) was added to sampling vial having 1 mL of methanol followed

by addition of 5 mg 3-hydroxy benzoic acid (as an internal standard). For

derivatization of the sample components, 300 µL pyridine, 50 µL

trimethylchlorosilane (TMCS) and 150 µL N,O-bis (trimethylsilyl)trifluoroacetamide

(BSTFA) and mixing of contents was carried out. In order to complete derivatization,

sample vial was placed in an oven at 80 oC for 1 h. Samples were cooled and

centrifuged. Supernatant liquid (0.2 µL) was injected using splitless injection system

into GC–MS. Results were obtained in the form of trimethylsilylester. The mass

selective range was 90–550 m/z [159, 160].

66
Part IV

3.6. Stability of Antioxidant Components from Defatted Kenaf (Hibiscus

cannabinus L.) Seed Meal (DKSM) Under Simulated Gastrointestinal pH

Conditions

3.6.1. Preparation of defatted kenaf seed meal (DKSM)

Kenaf seeds (variety V 36) were purchased and then washed with tap water for 10

min, rinsed twice with DW followed by drying in an oven at 50 °C overnight. Then,

crushed kenaf seeds (200 g) were mixed with 400 mL of n-hexane for 15 min at 9500

rpm. Mixture was filtered and the residue was re-extracted twice with same

procedure. In order to remove residual solid, DKSM was placed in an oven at 50 °C

for 3 h. After passing through 30 mesh sieve, DKSM was stored at -20 °C prior

further processing.

3.6.2. Preparation of defatted kenaf seed meal (DKSM) extracts

Extracts of DKSM were prepared according to a previously reported method [161].

Briefly, 10 g of defatted sample was mixed with 150 mL of deionized water followed

by centrifugation (4500 rpm) for 20 min. Supernatants were filtered using Whatman

No. 1 filter paper and stored at 4 ºC. This extract was named as untreated DKSM

extract.

In order to evaluate the fate of phenolics and antioxidant potential under

gastrointestinal conditions, DKSM samples were exposed to simulated

gastrointestinal pH conditions following a previously reported method [162]. Briefly,

10 g of DKSM sample was incubated for 20 min, keeping the pH of medium within

6.5-7.0 followed by decreasing the pH to 2 using HCl (6 N) followed by incubation at

37 °C for 30 min. After incubation, NaOH solution (4 N) was added to the mixture to

67
raise pH up to 6 followed by incubation at 37 °C for further 30 min. Finally, the

treated samples were centrifuged under ambient conditions (7500 g, 15 min). The

resulting mixture was filtered and treated DKSM extract was stored at -20 °C until

used for further analyses.

3.6.3. Determination of antioxidant potential of treated and untreated DKSM

Antioxidant potential of treated and untreated DKSM extracts was determined in

terms of TPC, free radical scavenging potential (DPPH• and ABTS•+), ferric ion

reducing power (FRAP) following the assays discussed in section 3.3.2 and 3.3.3.

In addition, hydroxyl radical scavenging activity of treated and untreated DKSM

extracts were determined. For the purpose, treated and untreated DKSM extracts were

allowed to react with hydroxyl radicals, generated through Fenton reaction. The

reaction mixture consisted of DMPO (40 μL; 400 mM), FeSO4 (37.5μL; 0.6 mM),

EDTA (112.5 μL; 0.1 mM), 60 μL of sample or blank and 150 μL of H2O2 (3 mM).

After 2 min of mixture preparation, measurement was done using electron spin

resonance (ESR) spectroscopic technique at room temperature. The conditions of ESR

measurement were as follows: sweeping field, 338.716 ± 5 mT; sweep time, 30 s;

time constant, 0.1 s; microwave power, 8 mW; mod width, 0.1 mT; and amplitude,

250. Dimethyl sulphoxide (DMSO) was used as standard in this assay and hydroxyl

radical scavenging activity was expressed a µg DMSO equivalent/ g.

3.6.4. HPLC-DAD analysis of major phenolic compounds in treated and

untreated DKSM extracts

HPLC-DAD analysis of treated and untreated DKSM was conducted using HPLC

system of Agilent G1310A pumps (Agilent, Stevens Creek Blvd Santa Clara, USA),

having diode array detector, Agilent Zorbax SB C-18 column (5 μm, 150 × 4.6 mm),

mobile phases, solvent (A): water-acetic acid (94:6, v/v) and solvent (B): methanol-

68
acetonitrile-acetic acid (95:5:1, v/v/v). Twenty microliter of crude phenolic extract

was injected and solvent gradient elution at flow rate of 1 mL/min was performed as:

0 to 5% B in 2 min, 5 to 25% B in 8 min, and 25 to 40% B in 10 min, 40 to 50% B in

10 min, 50 to 100% B in 10 min, 100% B in 5 min and finally 100 to 5% B in 5 min.

Millipore syringe filter (0.22 μm) was used for the filtration of samples and mobile

phases. The analysis was conducted in triplicate. For the identification and

quantification of major phenolic compounds of treated and untreated DKSM, a

comparison was made between retention times of sample components to known

previously injected standards.

3.7. Nano-encapsulation of DKSM Extract to Enhance its Bioavailability and

Antioxidant Activity

3.7.1. Preparation of DKSM extract loaded nanoparticles

Nanoparticles carrying untreated DKSM MeOH extract was prepared according to

previously reported method [129]. For nano-encapsulation, DKSM extract were

prepared as described in part II of this dissertation for the extraction of A. annua and

M. nigra. For the synthesis of DKSM extract loaded nanoparticles, 5 g of DKSM

extract was dissolved in 10 mL MeOH and TPC was determined using FC reagent

method and TPC were recorded as 5.14±0.12 mg/mL. For the synthesis of

nanoparticles, DKSM extract was drop wise added to chitosan solution (1 % w/v)

prepared in acetic acid (0.1%), and magnetic stirring was continued till homogenous

mixture appeared. Solution was maintained at a pH of 4 and TPP (10% w/v TPP in

DMW) was added drop wise to the chitosan- DKSM solution and stirring continue at

1000 rpm, 2hours. Centrifugation (40000 × g for 10 min) of the mixture was carried

for the separation of nanoparticles and after washing with DMW, nanoparticles were

stored at 4 ºC.

69
3.7.2. Characterization of nanoparticles

Scanning electron microscope (MIRA3 TESCAN (FSEM) operating at 20 kV) was

used for the determination of particle size and morphology of nanoparticles was

performed by using. Drops of diluted solution of nanoparticles were spread on the

glass plate and allowed to dry at room temperature. After coating the dried sample

with gold and SEM results were recorded.

3.7.3. Determination of extract loading efficiency

Encapsulation efficiency (EE) of DKSM MeOH extracts was determined by an

already reported method. Initially, chitosan nanoparticles were separated using

centrifugation at 25000×g for 40 min [163]. The amount of free phenolic content in

the supernatant was assessed by FC reagent assay.

Encapsulation efficiency was calculated according to the equation:

EE= (TPC-FPC/TPC)×100

Where TPC= Total phenolic content

FPC= Free phenolic content

3.7.4. In-vitro release of phenolic compounds from DKSM extract loaded

nanoparticles

Extract release profile DKSM extract loaded chitosan-TPP nanoparticles was studied

under different conditions of pH and temperature, using a method as reported by Jain

et al. [164]. Three different pH levels (2.0, 7.4 and 10.0) were selected to examine the

effect of pH on the release of encapsulated extract. Thirty milliliter of HCl solution

was taken in a flask followed by addition of a known quantity of nanoparticles.

Dispersion was carried out at different pH followed by incubation in a water bath at

37 °C± 0.5°C. Determination of TPC was done at different time intervals (0.1, 0.5, 1,

2, 4, 6, 8, 10, 12 h), by withdrawing 1 mL of aliquots from samples under different

70
pH conditions. Same volume of fresh buffer solution was added at the same time to

keep the volume constant. After centrifugation aliquots at 18000 × g for 15 min and

the release of DKSM encapsulated extract into the supernatant was determined in

terms of TPC. The percent of extract release was determined by the following

Equation:

% extract release= (TPCt/TPCn) × 100

Where; TPCn is the total phenolic content loaded and TPCn represents total phenolic

content released at a time t.

To analyze the release profile of phenolic compounds from DKSM extract loaded

nanoparticles at different temperature. Nanoparticles were added in different

Eppendorf tubes and placed at different temperatures (60, 80, and 100°C) having

pH≈7 and stirred in a water bath for 0, 20, 40, 80, 120 and 180 min in the dark. After

completion of selected time intervals, the samples were cooled in an ice bath followed

by determination of TPC using FC reagent assay and % extract release was calculated

as give above.

Part V

3.8. Stabilization Efficiency of Encapsulated and Non-encapsulated A. annua

Leave Extract on Shelf-life of Sunflower Oil

3.8.1. Sample Preparation

In this part of the project stabilization efficiency of Artemisia annua L. leaves

MeOH extract (ALE) was examined. ALE extract (10, 50, 100, 500 and 1000 ppm)

was added to five brown glass bottles having sunflower oil (SFO) (100 g each and

free from any antioxidant). In one bottle separately, SFO sample was administered

with defined amount of ALE encapsulated extract (≈200 mg phenolic content),

71
prepared by the method described in section 3.7 of this dissertation. In other two

bottles, synthetic antioxidants having commercial importance (BHA and BHT) were

added at a level of 200 ppm and considered as standard to compare with stabilization

efficiency of ALE (encapsulated and non-encapsulated). Control samples were

prepared by adding same amount of methanol to oil sample, used to dissolve the

extract. Bottles were sealed and placed an oven at 70 ◦C for 72 h, to maintain

accelerated storage conditions for oxidation. After regular time intervals (5, 10, 15,

20, 25 & 30 days), 20 g sample was taken and analysis was performed to evaluate the

oxidative status of oil, following the procedures given below [57, 58].

3.8.2. Oil Stabilization Analysis

3.8.2.a. Weight gain analysis

For weight gain analysis, each oil sample (in triplicate) was taken in petri dish, and

placed in oven for 6 hours at 35 ºC to remove any moisture content. Weight of sample

was recorded and again stored in the oven at 65 ºC, along with other samples. Weight

gain of oil samples was recorded at 3 days interval [57].

3.8.2.b. Determination of peroxide value (PV)

To determine peroxide value following method was applied. The method is based on

the ISO 3960 (2007) procedure. Sodium thiosulfate solution (0.1M) was prepared and

stored. Oil sample (5 g) was carefully weighed in conical flask with stopper. The

sample was diluted with 30 mL chloroform/acetic acid solution (2:3). After this the

freshly prepared saturated KI solution (0.5 mL) was added and immediately sealed the

flask. Then the sample was stirred gently for 1 min. The flask was placed at ambient

conditions in a dark place. The mixture was then further diluted with 30 mL pure

water and the flask was again sealed and stirred. The reaction mixture was titrated

with 0.1M sodium thiosulfate with continuous and strong agitation till the yellow

72
iodine color almost disappeared. After this, 0.5mL of indicator (starch solution) was

added which provide blue color to the mixture. Titration with sodium thiosulfate

continues till the blue color disappeared. A blank determination was conducted of the

reagents [165].

PV (mEq peroxide kg-1 oil) = CT × (VT – blank) × 1000 / Ws

where

CT = titrant concentration in mol/L

VT = necessary titrant volume in mL

Ws = weighed amount of sample in grams

3.8.2.c. Determination of acid content

Fifty milliliter ethyl alcohol was taken in dry 150 mL flask having 2 mL

phenolphthalein. Place flask in water bath at 60–65°C followed by addition of NaOH

(0.2 M) till pink color appears. Add 56.4 g oil in neutralized alcohol and titrate the

mixture with 0. 1M NaOH. Titration is carried out in association with warming and

shaking, until faint pink color is observed in alcohol. Multiply volume of 0.1M NaOH

used, with 0.05 and report as percent free fatty acids expressed as oleic acid. Free

acids may also be represented in terms of acid value (mg KOH/ g of oil) [165].

3.8.2.d. Determination of p-anisidine value

For the determination of p-anisidine value, weighed oil samples were taken in 25 mL

flasks separately. Iso-octane was added to dilute the samples and absorbance (A1)

was measured at 350 nm in glass cuvettes against a pure iso-octane blank. After this,

p-anisidine reagent (0.5 mL) was added to 2.5 mL diluted sample and placed the

samples in the dark for 10 min and new spectrophotometric reading (A2) was

recorded [165]. Following formula was used for the calculation of P-anisidine value:

AV = 25mL × [1.2 × (AS2 – AB2) – (AS1 – AB1) ] / Ws

73
where, 25mL = volume of iso-octane used to dissolve the sample

1.2 = correction factor

AS1 = Absorbance of oil solution

AS2 = Absorbance of oil-anisidine solution

AB1 = Absorbance of iso-octane

AB2 = Absorbance of iso-octane + anisidine

Ws = weight of sample

3.8.2.e. Conjugated dienes and conjugated trienes

Oil samples were diluted by adding iso-octane and absorbance of the resuting solution

was maintained within limits prescribed in IUPAC method. Specific extinctions at

232 and 268 nm (i.e. conjugated dienes and conjugated trienes, respectively) were

determined using a spectrophotometer and contents of conjugated diene and

conjugated triene were measured. [165].

3.8.2.f. Thiobarbituric acid reactive substances (TBARS) assay

The TBARS value was calculated by a method reported by Ke et al., [166]. Briefly,

10mg of each oil sample was taken into a kimax test tube. Then 5 mL of

Thiobarbituric acid (TBA) was added to each oil sample and then the test tube was

closed tightly. The test tubes were placed in water bath at 5 C for 1 hour with

continuous shaking. To separate the chloroform phase from water, 3mL TCA solution

was added and centrifuged at 2500 rpm for 10 min. The water phase was separated in

QS cuvettes and absorbance was taken against distilled water at 538 nm as a blank. In

final calculations this dilution was considered. A standard curve was constructed of

known concentrations of 0.1 mM TEP (tetraethoxypropane) solution. By using the

given formula the value of TBARS was calculated.

μM TBARS / g oil = (A-b) / (a × m × 1000)

74
 Where, A = absorbance by oil sample

a = slope of standard curve

b = intercept of standard curve
m = amount of oil sample (g.)
1000 = conversion to μM / g.

3.9. Cinnamon Bark Deodorised Aqueous Extract as Potential Natural

Antioxidant in Meat Emulsion System

3.9.1. Preparation of cinnamon bark deodorised aqueous extracts (CinDAE)

The cinnamon barks were cleaned and dried in an oven (Binder, Great River, New

York, USA) at 50 °C to attain constant weight; with the final moisture content being

less than 5 %. The cinnamon barks were then pulverized for 3 min with a stainless

steel blender followed by sieving through a sieve of mesh size 30 before being added

to hot water (100 °C) at ratio of 1:20. Using magnetic stirrer the mixture was stirred

and then filtered. After removing water, the yield of CinDAE was calculated and

CinDAE was kept at −4 °C.

3.9.2. Chicken meatball processing and storage

In brief, the chicken meatball samples consisted of C (control meatballs), T1

(meatballs supplemented with 200 ppm of CinDAE), T2 (meatballs supplemented

with 200 ppm of ascorbic acid) and T3 (meatballs supplemented with 200 ppm of

BHA and BHT combination i.e. 50 %+50 %). The storage quality evaluation of

chicken meatballs was carried out according to Accelerated Shelf Life Testing

(ASLT) protocol as described by Institute of Food Science and Technology [167].

Chicken meatballs were prepared in the laboratory using following ingredients (w/w):

65 % minced chicken breast, 20 % Socfat, 40 palm fat, 6.5 % cold distilled water, 6.0

% potato starch, 1.5 % sunflower seed oil and 1.0 % salt. Firstly, minced chicken

breast meat was mixed well with cold distilled water (<4 °C), potato starch and salt in

75
a mixer or 1.5 min followed by addition of sunflower seed oil and Socfat 40 palm fat

into the batter and mixing again for 3.5 min to form an emulsified batter. After that,

200 ppm CinDAE or BHA/BHT or ascorbic acid were mixed thoroughly for 5 min

into the batter and moulded into balls with 20±5 mm diameter and weighing up to

15±1 g. Subsequently, the chicken meatballs were flash fried at 180 °C for 30 s in 1

kg of palm oil before cooking in the oven (Panasonic, Kadoma, Osaka, Japan) at 250

°C for 4 min. The chicken meatballs were then cooled immediately to 4 °C in a

refrigerator. When the internal temperature of the chicken meatballs dropped to

approximately 12 °C, they were immediately transferred to a plastic container covered

with an oxygen semi-permeable polyvinyl chloride (PVC) film and stored in dark at

8±1 °C for 12 days. Analyses were done regularly after 1, 3, 6, 9 and 12 days.

3.9.3. Oxidative stability assessment

For oxidative stability assessment of meatballs, peroxide value (PV) and TBRAS

values were determined following the methods given in section 3.7.

Part VI

3.10. Structure-activity Relationship of Selected Phenolic Acids in Order to

Propose Mechanism of Their Antioxidative Action

Computer modeling has gained attention of scientist as it auspicious technique to

understand relationship of structure with its certain biological activity. In this

technique, structure of a molecule is usually related with biological activity to

generate a relationship using different computational and statistical programs.

76
3.10.1. Optimization of 3-D molecules of phenolic acids

QSAR for 22 different phenolic acids was developed. Initially, equimolar solutions of

all these phenolic acids were prepared and their antiradical potential was assessed

using DPPH• and ABTS•+ assays. 3-D structures of phenolic acids were generated in

Chemsketch and optimized for energy in Avogadro software using force field “UFF”

and MOPAC 2016 (Molecular Orbital Package) by employing semi empirical PM7

method. Using Winmostar, sdf files were generated which were submitted to

DRAGON program for descriptors calculations.

3.10.2. Artificial neural network (ANN)

In QSAR modeling, Artificial Neural Network (ANN) is considered a valuable tool

for the analysis of data using linear and non-linear mechanisms [168]. Usually this

model is related to human nervous system having several neurons, systematized in the

form of layers. These layers hidden/ output layer are considered as input layers.

Neurons are responsible for the transfer signals from input layer to output layer

through hidden layer. Relationship, between dependent variable and independent

parameters is generated, using interconnected neurons, [169]. Ability of prediction

and accuracy of ANN models is well acknowledged by the scientific community.

In present study, after selection of descriptors from SMLR, were used as continuous

input signal, while output signal was antiradical potential of the selected phenolic

acids. Statistica 10 was applied for automated artificial neural network generation.

77
 Chapter 4

Results and Discussion

78
Part I

4.1. Proximate Composition and Antioxidant Potential of Selected Medicinal

Plants

4.1.1. Proximate Composition and Antioxidant Potential of Leaves from Three

Mulberry Varieties

4.1.1.a. Proximate composition

Table 4.1.1. contain mean values of ash, moisture, lipid, fibre and protein for the

leaves of three mulberry verities and the varieties were found statistically different

(P<0.05) in terms of their proximate composition. The minimum value of ash content

was recorded for M. alba while maximum for M. rubra, M. alba was found haing

high moisture contents as compared to all other species. The values of moisture are

much lower while those for ash are higher as compared to the values of some

vegetables reported previously [170]. Heavy amount of inorganic nutrients may be

expected from the specie having high ash contents [171], whereas roughness of leaves

may appear due to low moisture contents.

Estimation of lipids is one of the key factors to decide about nutritional standard of

any food material [172]. Significant variation in amount of lipids was observed for the

varieties of Morus, M. alba was found containing highest amount of lipids followed

by M. nigra, but the values recorded for the three species were lower than those

reported for Nigerian plants [173]. However, appreciable amount of lipids is present

in leaves of selected species that recommends their use for the preparation of

functional foods.

Dietary fibres, non-starch polysaccharides (anti-nutrient). Fibres cause reduction in

bioavailability and absorption of minerals after binding and accelerating their passage

79
through digestive tract. If fibre interacts with nutrients like phytate, tannin or oxalate,

this phenomenon become more prominent [174]. The species under study were found

significantly (P < 0.05) different in terms of their fibre content. M. nigra was found

having highest fibre contents as compared to other species. However, all the species

were found rich in fibre as compared to leafy vegetables [170].

Leaves of three species were found statistically different (p << 0.05) from one another

as far as protein content are concerned and the trend of protein content was observed

in the following order, M. rubra > M. nigra > M. alba. Mulberry leaves may be

considered as better source of protein, if compared to other leafy vegetables [175]. In

addition, these leaves may be explored as good source antioxidant components [176].

Table 4.1.1. Proximate composition (%age dry mass) of Leaves from Three
Varieties of Morus (Mulberry)
Morus alba L. Morus nigra L. Morus rubra L. P value
Ash 8.91 ± 0.51 9.12 ± 0.41 11.73 ± 1.09 0.005
Moisture 5.3 ± 0.2 6.7 ± 0.3 4.5 ± 0.2 <<0.05
Lipid 6.57 ± 0.23 5.13 ± 0.19 4.24 ± 0.11 <<0.05
Fibre 10.11 ± 0.37 12.32 ± 1.18 8.17 ± 0.89 0.003
Protein 18.41 ± 1.36 19.76 ± 2.12 24.63 ± 0.86 0.006

4.1.1.b. Total phenolic content (TPC)

Disease preventive role of phenolic compounds due to their antioxidant activities, is

now well proven. TPC estimated in leaves of mulberry species were found

statistically different (P = 0.004). TPC was estimated as mg GAE/g of dried leaves

and M. nigra was found having high content of phenolic compounds (Figure 4.4.1).

Mulberry leaves were found rich in phenolic compounds, if compared with palm

leaves and some Iranian [177] medicinal plants.

80
4.1.1.c. Total flavonoid content (TFC)

Free radical scavenging activity of flavonoids due to their structural features is now

well proven [9]. The flavonoid content in leaves of three species of Morus was

estimated as mg rutin equivalent/g of dried leaf samples and results have been

presented in Figure 4.4.1. Leaves of M. rubra were found having significantly (P <

0.05) high flavonoid content as compared to other species. The tested leaf samples

was found rich in flavonoids as compared to Algerian medicinal plants [178].

4.1.1.d. Ascorbic Acid Content (AAC)

Ascorbic acid (vitamin C) is a non-enzymatic natural antioxidant that is water soluble

in nature, which is preferably used as an alternate to synthetic antioxidants [179].

Leaves of different Morus varieties were found significantly different regarding AAC.

Leaves of M. alba were found containing maximum amount of AAC followed by M.

nigra while minimum were recorded in M. rubra (Figure 4.1.1.).

4.1.1.e. DPPH radical scavenging activity

DPPH radical scavenging assay is preferred for the determination of antioxidant

activity of different botanical materials. In this assay, DPPH free radical is allowed to

react with hydrogen donating species i.e. antioxidants obtained from plant material.

The extent of discoloration exhibit the extent of reaction [180] and hence antioxidant

potential of sample under study. The results of DPPH assay for the mulberry leave

extracts have been expressed as mM TROLOX-equivalent/g of dried leaves (Figure

4.1.2). Antiradical potential of three tested species of Morus was found statistically

different. M. nigra exhibited highest radical scavenging potential as compared to

other species. However, DPPH radical scavenging potential calculated for tested

samples recommends that the sample may be considered for the preparation of

functional foods [181].

81
 35

30

Concnetration mg/g
25

20 M. alba L.
15 M. nigra L.
10 M. rubra L.

0
Yield (%) TPC TFC A.A
Yield and antioxidant components

Figure 4.1.1. Yield and contents of Phenolics, Flavonoids and Ascorbic acid in
extracts from leaves of three Morus varieties (P < 0.05)
4.1.1.f. ABTS radical cation scavenging activity

In order to determine radical scavenging potential, ABTS and DPPH assays are used

side by side. The mechanism of both assays is same but due to solubility of ABTS

reagent in both aqueous and non-aqueous mediums, results of ABTS method are

considered more authentic as compare to DPPH method [182]. The leaves from

different mulberry species exhibited significantly different (P < 0.05) antiradical

potential and highest value i.e. 9.89 ± 0.87 milimoles TROLOX-equivalent was

recorded M. nigra (Figure 4.1.2). However, mulberry leaves possess high ABTS

radical scavenging potential than those reported in recent literature [151].

4.1.1.g. Ferric ion reducing antioxidant power (FRAP)

In this assay, antioxidant potential of the extract is evaluated in terms of reducing

ability. In this reduction process single electron transfer mechanism is followed that is

applicable in both aqueous and non-aqueous systems [183]. The extracts of leaves

from three Morus species were analyzed for antioxidant activity using FRAP assay.

Results were calculated as mM TROLOX-equivalent/g of dried leaf samples are

presented in figure 4.1.2. Among all the tested varieties were found having different

(P < 0.05) reducing potential and M. alba leaves appeared with high FRAP value
82
while least was recorded for M. nigra. Ferric reducing antioxidant power of mulberry

leaves is quite higher than many reported species [151, 184].

12

10
mM Trolox equivalent
M. alba L.
8 M. nigra L.
M. rubra L.
6

0
DPPH ABTS FRAP
Antioxidant assays

Figure 4.1.2. Antioxidant activity (mMTE) of leaves from three species of Morus
(Mulberry) against DPPH, ABTS and FRAP assay.

The TROLOX equivalent (TE) values calculated by ABTS assay were three to four

fold higher than calculated from DPPH assay, similar is the case with FRAP assay i.e.

the values of TE are far lower than ABTS and DPPH assays. This difference has

already been reported for barley varieties and may be attributed to reaction kinetics of

different radicals and phenolic constituents of sample or may be due to specific

response towards different radicals by certain phenolics present in different varieties

of biological samples [185]. These findings confirmed the efficiency of mulberry

leaves extracts against ABTS and DPPH rather than FRAP assay.

4.1.1.h. Correlation among antioxidant components and assays

Pearson correlation among results of chemical composition, antioxidant constituents

and antioxidant potential of three varieties of Morus leaves was assessed (Table

4.1.2). Negative correlation of lipids with all other parameters was observed except

AAC (r = 0.996) and DPPH (r = 0.307), whereas it was high with ascorbic acid

83
showing that specie high in lipid content was also rich in AAC. Fibre has shown high

correlation with DPPH (r = 0.999) while intermediate with ABTS (r = 0.690) and

TPC (r = 0.505). It shows that the species found with high fibre content were also

good in TPC and offered high resistance against DPPH, while it was moderate against

ABTS radicals. The r value for protein and flavonoid was high (r = 0.866), revealing

good correlation among these two components in leaves from different species of

Morus.

The correlation of AAC with DPPH (r = 0.227) and FRAP (r = 0.392) was fond very

weak, while it was moderately negative (r = -0.515) with ABTS radical cation

scavenging potential. High correlation of TPC with ABTS (r =0.973) confirms the

role of phenolics, present in leaves from various Morus species, for scavenging of free

radical. On the other hand, TPC having intermediate correlation (r = 0.537)with

DPPH while strong negative (r = -0.932) with FRAP, indicating the presence of other

components such as tocopherols, carotenoids and protein in leave extracts of different

Morus varieties and involvement against antioxidant assays. Similar correlation was

observed among TFC and antioxidant assays i.e. the correlation of TFC with all the

assays was negative except ABTS which was moderate (r = 0.505), it also confirms

the contribution of many other components such as vitamins, anthocyanins and

carotenoids towards antioxidant activity exhibited by the sample extracts [186].

Strong correlation of DPPH with ABTS with value 0.717, confirms the validity of

results for the leaves extracts under study. Whereas the contradiction of FRAP assay

appeared as negative correlation with DPPH and ABTS, indicate that single assay

may not be dependable for the evaluation of antioxidant activity of a sample [187,

188] as different components exhibit their activity under different assays, each

working on different principle. Furthermore, during comparison, antioxidant property

84
of different extracts may not only be attributed to contents of ascorbic acid, phenolics

and flavonoids rather the involvement of many other phytochemicals has been taken

into count by many authors [189].

Table 4.1.2. Pearson correlation among chemical/antioxidant constituents and

scavenging activities for leaves from three varieties of mulberry
Lipid Fiber Protein TPC TFC AAC DPPH ABTS FRAP
Lipid 1
Fiber 0.343 1
Protein −0.899 −0.714 1
TPC −0.636 0.505 0.236 1
TFC −0.997 −0.275 0.866 0.690 1
AAC 0.996 0.264 −0.860 −0.698 −0.999 1
DPPH 0.307 0.999 −0.691 0.537 −0.235 0.227 1
ABTS −0.442 0.690 0.006 0.973 0.505 −0.515 0.717 1
FRAP 0.315 −0.783 0.130 −0.932 −0.381 0.392 −0.805 −0.990 1

4.1.2. Artemisia annua L. Leaves: Chemical Composition and Antioxidant

Potential of Different Solvent Extracts

4.1.2.a. Chemical composition

The percentage values of ash and carbohydrate, fat, fiber, moisture and protein are

calculated on dry weight basis (Table 4.1.3). In samples, nutrients (fat and protein) are

present in lesser quantity as compared to anti-nutrients (phytate and total tannin). The

results of our findings are different as compared to previously reported composition of

A. annua leaves [68], indicates weaker nutritive action of these samples. However,

ash percentage designates high amount of inorganic minerals in A. annua leaves.

Carbohydrate and fat proportions in tested leaf samples may contribute towards their

health promoting efficiency.

Fibre is known as anti-nutrient as it possesses potential to obstructs the utilization of

minerals. In addition, fibre assist phytate, tannins and oxalates and hinder the mineral

85
absorption [170]. High amount of crude fibre in A. annua leaves as compared to

traditional vegetables may restrict their consumption more frequently. However, high

protein content than leafy vegetables [175] signify good antioxidant ability of tested

leaf samples [176].

Table 4.1.3. Chemical composition of Artemisia annua leaves.

Sr. No Contents Amount (% dry
weight basis)
1 Ash 7.5
2 Carbohydrate 8.3
3 Fat 6.07
4 Fibre 14.2
5 Moisture 11.4
6 Protein 24.37
mg/100g
7 Phytate 140.4
8 Total Tannins 0.61
9 Tocopherol 2.74

Phytates are also labeled as anti-nutrients due to their protein binding ability that may

reduce protein’s solubility [190] as well as bioavailability of minerals (Ca & Zn) to

the consumer [191]. Higher concentration of phytates than leaves of Solanum nigrum

and Leonotis leonorus [192] may increase their anti-nutritive behavior.

Tannins are polyphenols, which form insoluble complexes with iron through o-

dihydroxyl groups and results in reduction of iron absorption in gastrointestinal tract

[174]. Whereas, antioxidant activity of tannins is also well known [193]. The amount

of tannins calculated in A. annua leaves may not have any considerable contribution

towards their functional attributes.

Vitamins are mandatory for good health. Tocopherols (vitamin E) available in

different forms (α, β, γ and δ tocopherols; α-tocopherol) and are lipophylic in nature.

86
These vitamins are synthesized by botanical materials and usually stored in green

tissues i.e. leaves [194]. The value of total tocopherols estimated in A. annua leaves

suggests their promising potential for functional food and nutraceutical.

4.1.2.b. Total phenolic Content (TPC)

Antioxidant properties of phenolics are well recognized for their chelating redox-

active metal ions and inactivating free radical chain reactions [195]. Significant

different amount of total phenolics was found in various extracts of A. annua leaves,

ranging from 90.12 ± 2.78 (Hexane) to 134.50 ± 4.37 (MeOH).

In many studies, content phenolics have been found strongly correlated with the

solvent polarity i.e. high polarity exhibit better recovery of phenolic compounds

[196]. Therefore, solvents including acetone, ethanol, EtoAC, MeOH and propanal

have been tested for the extraction of phenolics [116]. The efficiency of tested

solvents for the recovery of phenolics was found in the order of MeOH > Water >

EtOAc > Chloroform > Hexane (Figure 4.1.3). Highest concentration of phenolics in

MeOH extract confirms ability of methanol to recover large amount of phenolic

ingredients of polar nature, present in A. annua leaves. Water demonstrated

comparable potential as that of methanol regarding. The same results were reported

for mulberry (Morus indica L.) leaves [197].

4.1.2.b. Total flavonoid content (TFC)

The potential of various solvents for the recovery of flavonoids was same as discussed

for phenolics (Figure 4.1.3) i.e. flavonoids were efficiently extracted in polar solvent.

The results of our findings are in agreement with that reported by Spigno et al., [153,

198], suggesting that solvents having high polarity may be considered as efficient

extracting media for flavonoids, as solubility of flavonoids increase after conjugating

through glycosides with hydroxyl groups [198].

87
 800 Hexane
700 Chloroform
600 EtOAc
Concentration 500 MeOH
400 Water
300
200
100
0
TPC (mg/g) TFC (mg/100g)
Antioxidant Components

Figure 4.1.3. Variation in contents of total phenolics and total flavonoids in

various solvent extracts of Artimisia annua leaves, with increasing
polarity of solvent
4.1.2.c. Ferric reducing antioxidant power (FRAP) assay

Many researchers have used FRAP assay for the estimation of antioxidant capacity of

number of plant materials successfully [199, 200]. This assay follows single electron

transfer mechanism and assess ferric ion reduction ability of sample [201]. Oxidant

probe i.e. ferric ion accepts an electron from antioxidant, which produce colored

complex on chelating with chromogenic ligand. This ligand can be measured at

593nm which represents the ferric reducing ability of the extract [202].

In more recent studies, the influence of solvent polarity on FRAP has been reported

[203], which is also evident in our current work. The FRAP values for hexane,

chloroform and EtOAc extracts were found to be mutually comparable but clearly

different than those for polar solvent i.e. MeOH extract 11.82 ± 1.12. Maximum

FRAP value was 12.37 ± 1.09 but it is also comparable to MeOH extract, when highly

polar solvent i.e. water was added for extraction (Figure 4.1.4). Beyond the fact that

FRAP assay is inadequate for the determination of hydrophilic (water soluble)

88
antioxidants [204], the reduction of maximum ferric ions in aqueous extracts

confirmed the extraction efficiency of polar solvents.

4.1.2.d. Trolox equivalent antioxidant capacity (TEAC) assay

Trolox equivalent antioxidant capacity assay shows the efficiency of antioxidants in

scavenging long life radicals like ABTS•+ [33]. These radicals generate colored

solution having maximum absorbance at 746nm. Antioxidant species present in the

sample reduce color intensity of solution by neutralizing free radicals, which helps to

estimate accurate value of TEAC [205]. This method is widely preferred due to its

efficiency to evaluate antioxidant capacity of food items and biological matrices

[206].

Trolox equivalent antioxidant capacity of A. annua leaves extracts was found to be in

the range of 8.12 ± 0.21 to 17.59 ± 0.71 for hexane and methanol respectively (Figure

4.1.4). The magnitude of reaction of A. annua leaves extracts, prepared in different

solvents, against ABTS•+ radical was found in the order as: MeOH > Water > EtOAc

> Chloroform > Hexane.

TEAC of polar solvents were found significantly higher than those for non-polar

solvents. Hexane, chloroform, and EtOAc extracts of leaves offered comparable

resistance against this assay but after the addition of polar solvents (MeOH and

Water) for extraction purposes high TEAC was observed. Authors have reported that

TEAC assay may determine true antioxidant potential of the extracts extracted in any

medium due to solubility (aqueous or non-aqueous) of reagents involved in this assay.

Moreover this assay can evaluate the potential of lipohilic (fat-soluble) and

hydrophilic (water soluble) antioxidants [204]. Keeping this in view, it may be

concluded that polar solvents were better for extracting the maximum antioxidants

from A. annua leaves.

89
 20 Hexane
18 Chloroform
16 EtOAc
Concentration 14
MeOH
12
Water
10
8
6
4
2
0
FRAP (mM/g) TEAC (m mol/mg)
Antioxidant Assays
Figure 4.1.4. Ferric reducing antioxidant power and trolox equvilant equvilant
antioxidant capacity of various solvent extracts of Artimisia annua
leaves
4.1.2.e. DPPH radical-scavenging potential

This method has extensively been used for the determination of antioxidant potential

of biological samples. DPPH (2,2-diphenyl-2-picrylhydrazyl hydrate) is a nitrogen

centered radical, which accept hydrogen from antioxidant and 1, 1,-diphenyl-2-picryl

hydrazine is formed [207]. Antioxidants donate hydrogen which quenches free

radicals and terminates oxidation [208]. DPPH radical scavenging potential of MeOH

extract of A. annua leaves was found to be significantly higher than all other extracts.

Efficiency of extracts against DPPH assay was similar to that observed for TEAC

(Figure 4.1.5). The extracts prepared in less polar solvents (hexane, chloroform and

EtOAc) acted against DPPH radical with comparable power but MeOH extracts had

the highest scavenging potential, which indicated the presence of maximum

antioxidant constituents in MeOH extracts and mark methanol as potential solvent for

the extraction of antioxidants from A. annua leaves.

90
 40
Hexane
35
Chloroform

Remaining Percentage
30 EtOAc
25 MeOH
20 Water
15
10
5
0
DPPH
Antioxidant Assay

Figure 4.1.5. DPPH radical scavenging potential of various solvent extracts of

Artimisia annua leaves
4.1.2.f. Lipid peroxidation

Unsaturation in structures of fatty acids can easily be attacked by free radical with the

formation of lipid radicals that cause breakdown of lipids by producing

malondialdehyde. The presence of malondialdehyde in a plant extracts indicate its

protection against lipid peroxidation i.e. the presence of antioxidants in the samples

inhibits lipid peroxidation and the hazardous effects of the free radicals [209].

Aqueous
MeOH
EtOAc
Chloroform
Hexane Lipid Peroxidation %

BHA/BHT 50/50
Tocopherol
Control

0 20 40 60 80 100

Figure 4.1.6. Lipid peroxidation in extracts of Artimisia annua leaves prepared in

different solvents

91
In case of A. annua leaves extracts, highest lipid peroxidation (24.24 ± 1.04 %) was

observed in chloroform extract, while lowest (10.72 ± 0.07) in MeOH extract. The

comparison of lipid peroxidation A. annua leave extracts prepared in different

solvents in presented in Figure 4.1.6. A mixture of two synthetic antioxidants

(BHA/BHT, 50/50) and one natural antioxidant (tochpherol) is used for comparison.

Lipid peroxidation in MeOH extract was even lesser than standard synthetic as well as

natural antioxidants.

4.1.2.g. Statistical analysis

Correlation analysis (Table 4.1.3) exhibited positive and strong correlation of TPC

with TFC, FRAP, TEAC and DPPH. Results of this study confirms the role of

phenolics in antioxidant activity of A. annua leaves. While high negative correlation

of TPC with lipid peroxidation proved that presence of phenolics inhibit the process

of autoxidation.

High positive correlation between FRAP and TEAC assays suggested the efficacy of

these two assays for the evaluation of antioxidant activity of Artemisia annua leaves.

Week correlation of DPPH with FRAP and moderately with TEAC illustrated that if

one extract executes better activity against DPPH then its activity against other assays

may not be so satisfactory.

Table 4.1.4. Correlation coefficient between TPC, TFC, Lipid peroxidation

FRAP, TEAC and DPPH assays

TPC TFC FRAP TEAC DPPH L.P

TPC 1
TFC 0.972 1
FRAP 0.866 0.887 1
TEAC 0.971 0.980 0.949 1
DPPH 0.825 0.698 0.493 0.671 1
L.P -0.832 -0.824 -0.884 -0.845 -0.643 1

92
Part II

4.2. Optimization of Extraction Process for Bioactive Compounds from

Leaves of Morus nigra and Artemisia annua Using RSM

Optimization of extraction procedures for the plant samples selected for current study,

was also carried out. Many extraction parameters including particle size of plant

powder, solvent polarity, extraction time and temperature, and extraction technique

that effect extraction of bioactive compounds from plant matrix. In this work, initially

plant samples i.e. M. nigra and A. annua were dried using three drying methodologies

i.e. shed drying, hot air oven drying and microwave drying. Further these samples

were extracted using solvents of varying polarity. Extraction of these plants was

carried out by using Soxhlet, orbital shaking and microwave methodologies.

The results of preliminary studies found that the suitable drying methods for M. nigra

and A. annua were hot air oven drying and microwave drying, respectively. The best

solvent for recovery of antioxidant compounds appeared to be 70% methanol.

Antioxidant compounds present in these two plants can be better extracted with

orbital shaking and microwave assisted extraction technique. Optimization of the

various parameters in the extraction techniques was then examined. An orbital

shaking method involved varying the shaking speed and time while microwave

extraction depended on microwave intensity and time. The optimized conditions were

then used together with 70% methanol for recovering antioxidants from the botanical

materials selected. For this purpose, response surface methodology was applied with

the results summarized as follows.

93
4.2.1. Experimental design for orbital shaker assisted optimized extraction of

antioxidant compounds from A. annua using response surface

methodology

Codes and the corresponding values used in the analysis using RSM for orbital

shaking extraction (OSE) are given in Table 4.2.1. The coded (independent variable),

measured and predicted values (TPC, TFC, AAC and %age inhibition (DPPH• and

ABTS•+)) are given in Table 4.2.2. Two level full factorial randomized regression

equations are shown in Table 4.2.3 and the validation of the RSM was done using

coefficients determination (R2) and F and P values. The P value of any matter <0.05

with larger F were considered significant [210].

Table 4.2.1. The coded values and corresponding actual values of the
optimization parameters used in Response Surface Analysis
Variables Coded variable levels
Coded units
(OSE) -1 0 +1
Speed (rpm) X1 50 100 150
Time (min) X2 180 360 540
OSE = orbital shaking extraction, rpm = rotation per minute

4.2.2. Optimization of orbital shaker assisted extraction of antioxidants from A.

annua leaves using response surface methodology

4.2.2.a. Total phenolic content

In orbital shaker-assisted extraction, the parameters of extraction that can affect the

recovery of TPC and required optimization of both the time and rotation speed. As

rotation speed increased from 50 to 100 rpm for 380 minutes, there was corresponding

increase in TPC (Figure 4.2.1). However, data suggests that further high rotation

speed for extraction or increase in time may not further enhance the recovery of

phenolic compounds from A. annua leaves.

94
Statistical data (Table 4.2.3) revealed that the effect of speed and time under orbital

shaker-assisted extraction treatment exhibited linear quadratic and interactive effect

was significant because p value is less than 0.05 and results were significant for TPC

recovery. Measured and predicted TPC values (RSM) are given in Table 4.2.2.

Measured values for both response variables are in close agreement with the predicted

values, indicating a satisfactory model. R2 value for orbital shaker assisted extraction

was 0.9849 exhibited that the method was best for the recovery of TPC as shown by

surface plot Figure 4.2.1.

4.2.2.b. Total flavonoid content (TFC)

If we compare responses for the recovery of TFC, different rotation speed and time

were recorded. Statistical data (Table 4.2.3.) revealed that the effect of orbital shaking

speed and time under oven treatment was significant as p value is less than 0.05, the

linear quadratic and interactive effect of speed and time was also significant. In case

of microwave-assisted drying, both time and irradiated power were found significant

for TFC recovery.

Measured and predicted TFC values (RSM) are given in Table 4.2.2. Predicted values

for both response variables are in close agreement with the measured values,

indicating a satisfactory model. R2 value for shade drying was 0.9967 exhibited that

the method may be best fit for the recovery of TFC (Figure 4.2.1).

4.2.2.c. Ascorbic acid content (AAC)

Statistical data (Table 4.2.3) revealed that the effect of speed and time under

extraction treatment was significant because p value is less than 0.05; the linear

quadratic and interactive effect of speed and time was also significant. In case of

microwave-assisted extraction, only radiation power and time was significant for

AAC recovery.

95
Figure 4.2.1. Response Surface plots showing the effect of orbital shaker-assisted
extraction parameters on TPC, TFC, AAC, % inhibition (DPPH• & ABTS•+) of
70 % MeOH extracts of A. annua leaves

96
Table 4.2.2. Measured and predicted TPC, TFC, AAC and % inhibition (DPPH• & ABTS•+) values for A. Annua leaves using orbital
shaker-assisted extraction technique
Design
Independent Response Response Response Response Response
point
variables (TPC, mg GAE/g) (TFC, µg ECE/g) (AAC, mg AAE/g) (DPPH % Inhibition) (ABTS% Inhibition)

Sr.
X1 X2 Measured Predicted Measured predicted Measured predicted Measured predicted Measured predicted
No.
1 0 0 148.79 147.32 7.80 8.00 0.92 1.23 78.16 78.17 73.14 74.34

2 0 -1 150.53 152.34 7.50 7.50 0.80 1.22 75.20 75.87 70.18 71.82

3 0 1 151.53 151.22 8.50 8.30 0.84 0.57 80.02 79.24 75.00 74.84

4 1 1 145.85 148.20 7.85 8.08 1.98 1.83 78.50 77.27 73.48 74.05

5 1 -1 142.07 141.20 7.35 7.27 1.84 1.90 77.54 77.65 72.52 71.91

6 1 0 137.74 140.12 7.32 7.44 1.96 1.59 79.35 80.70 74.33 72.17

7 -1 1 143.49 145.27 7.93 7.90 1.94 1.89 79.30 79.18 74.28 73.30

8 -1 -1 147.09 145.59 7.29 7.37 1.86 1.82 75.58 76.14 70.56 71.57

9 -1 0 150.80 146.64 8.02 7.70 1.82 1.91 80.20 79.63 75.18 74.83

X1= Coded units for speed, X2=coded units for time

97
Table 4.2.3. Polynomial equations and statistical parameters for A. annua leaves
using orbital shaker-assisted extraction technique

Response
2nd order polynomial equation (Orbital shaker-assisted
variables R2 F P
extraction)
A. annua

TPC TPC = 120.0 + 0.268 X1 + 0.1165 X2 - 0.00158 X12 -

0.9849 1.95 0.039
(mg GAE/g) 0.000152 X22 - 0.000027 X1X2

TFC TFC = 7.01 - 0.0084X1+0.01031X2 + 0.000005X12 - 0.000017X22

0.9967 2.35 0.046
(µg ECE/g) + 0.000009X1X2

AAC AAC = 1.16 + 0.0204X1+0.00587X2 - 0.000009X12 -

0.9743 1.95 0.015
(mg AAE/g) 0.000001X22 - 0.000033X1X2

DPPH % Inhibition = 67.00 + 0.1815 X1 + 0.0299 X2 - 0.000991 X12 -

0.9714 2.51 0.039
% Inhibition 0.000039 X22 - 0.000007 X1X2

ABTS % Inhibition = 74.09 - 0.021 X1 - 0.0157 X2 + 0.000313 X12

0.5575 0.75 0.635
% Inhibition + 0.000040 X22 - 0.000077 X1X2

Measured and predicted AAC values (RSM) are given in Table 4.2.2 for applied

drying method. Predicted values for both response variables are in close agreement

with the measured values, indicating a satisfactory model. R2 value for microwave

drying was 0.9743 exhibited that the method was insignificant for the recovery of

AAC as shown by surface plots (Figure 4.2.1).

4.2.2.d. DPPH radical scavenging activity

Statistical data (Table 4.2.3) revealed that the effect of speed and time under orbital

shaker assisted extraction treatment was significant because of p value is less than

0.05 for linear quadratic and two way interactive effects for DPPH. Predicted DPPH

% inhibition values (Table 4.2.2) for both response variables are in close agreement

with the measured values, indicating a satisfactory model. R2 value (Table 4.2.3) for

microwave extraction was 0.9714 exhibited that the method was insignificant for the

antioxidant activity using DPPH % inhibition as shown in Figure 4.2.1 surface plots.

98
4.2.2.e. ABTS radical cation scavenging activity

Statistical data (Table 4.2.3.) revealed that the effect of speed and time under orbital

shaker-assisted extraction treatment was insignificant because of p value is greater

than 0.05 for linear quadratic and two way interactive effects for ABTS. Predicted

ABTS % inhibition values (RSM) are given in Table 4.2.2 for both response variables

are in close agreement with the measured values, indicating a satisfactory model. R 2

value for microwave drying was 0.75 exhibited that the method (Table 4.2.2) was

insignificant for the antioxidant activity using ABTS % inhibition as shown in Figure

4.2.1 surface plots.

Many researchers used alcohol-water mixture for the extraction of phenolics from

different botanical materials [211]. In all the cases, trends as well as inconsistencies

were observed regarding the optimal efficiency of 70 % MeOH. Optimization of

polyphenol recovery from different medicinal plant leaves with different polyphenolic

composition depend upon case investigation. No universal model available that can

describe the optimal conditions for the best recovery of bioactive ingredients from

botanical material. Keeping in view, this study was focused on the effect of extraction

solvent conditions on biologically active compounds (antioxidant potential) of A.

annua leaves. This set of examinations highlighted for the first time that the A. annua

leaves bioactive compounds can be better recovered if leaves are dried with

microwave radiation and extracted with orbital shaking extraction method using set of

optimized conditions.

Leave extracts obtained from A. Annua were measured as % inhibition with high

radical scavenging activity (DPPH % inhibition) and all extractions performed under

same experimental conditions. The equations developed for 70 % MeOH extracts

were significant up to 95% confidence level. The investigation presented here

99
conditions used for optimized extraction of phenolics from A. Annua leaves may vary

significantly. For the extraction of different group of bioactive compounds from A.

Annua leaves requires different set of conditions and extracts enriched in particular

components can be prepared with greater ease.

Table 4.2.4. Optimized conditions to recover maximum antioxidant components

from A. Annua leaves
Antioxidant activity Time (min) Speed (rpm)
TPC (mg GAE/g) = 152.884 380.082 82.1644
TFC = 8.32437 315.306 50.3182
AAC = 1.90774 536.543 149.155
DPPH• % inhibition = 80.7985 382.645 89.5933
ABTS•+ % = 74.7705 537.810 148.942

CCD was constructed and extractions were carried out for different time intervals and

shaking speeds. RSM was employed to find out the optimized conditions (Table

4.2.4) for the maximum recovery of antioxidant components. Two parameters, such as

rotation speed and extraction time were found to have influence the Orbital shaking

extraction process. It was observed that with the change of extraction time and

rotation speed (180 min to 540 min and 50 to 150 RPM) significantly influence the

yields of bioactives from A. Annua if we consider the parameters TPC, TFC AAC and

DPPH radical scavenging activity. A notable increase in the extraction yields of

phenolic and flavonoids compounds was observed, when extraction is carried out

from 180 to 360 min.

100
4.2.3. Experimental design for microwave-assisted optimized extraction of

antioxidant compounds from Morus nigra using response surface

methodology

Codes and the corresponding values used for optimization purposes while extracting

M. nigra leaves by microwave-assisted extraction (MAE) are given in Table 4.2.5.

The coded (independent variable), measured and predicted values (TPC, TFC, AAC

and %age inhibition (DPPH• and ABTS•+)) are given in Tables 4.2.6. Two level full

factorial randomized regression equations are shown in Table 4.2.7 and the validity of

the model was established using the coefficients determination (R2) and F and P

values. The P value of any matter <0.05 with larger F were considered significant

[210].

Table 4.2.5: The coded values and corresponding actual values of the
optimization parameters used in Response Surface Analysis
Variables Coded variable levels
Coded units
(MAE) -1 0 +1
Time (min) X1 3 6 9
Power (watts) X2 100 300 400
MAE = microwave-assisted extraction

4.2.4. Optimization of microwave assisted extraction of antioxidants from M.

nigra leaves using response surface methodology

4.2.4.a. Total phenolic content (TPC)

RSM was used to optimize the extraction of TPC from M. nigra leaves, dried using

mechanical hot air oven. The selected parameters (time and power) of microwave-

assisted extraction technique were found effective in the recovery of TPC, TFC and

AA. As microwave power was increased from 100 to 200 for 3 to 6 min and up to 20

% increase in TPC was recorded. However, data suggests that use of high intensity

radiations may not further increase the content of total phenolics in the extracts.

101
If we compare responses for the recovery of TPC, different power and time were

recorded for microwave-assisted extraction technique under study (Figure 4.2.2).

Maximum quantity of TPC was obtained for the sample dried using hot air oven.

Stability of phenolics was found best for hot air oven drying procedure.

Statistical data (Table 4.2.7) revealed that the effect of time and power under

microwave-assisted extraction technique was linear quadratic and interactive effect of

time and power were significant for TPC recovery because value of p was less than

0.05. Measured and predicted TPC values (RSM) are given in Table 4.2.5. Measured

values for both response variables are in close agreement with the predicted values,

indicating a satisfactory model.

4.2.4.b. Total flavonoid content (TFC)

If we compare responses for the recovery of TFC, different extraction time and

radiation power were recorded for microwave-assisted extraction techniques under

study (Figure 4.2.2). Statistical data (Table 4.2.7) revealed that the effect of radiation

power and time under oven treatment was insignificant because p value is greater than

0.05, the linear quadratic and interactive effect of power and time were insignificant.

Measured and predicted TFC values (RSM) are given in Table 4.2.6, for both

response variables are in close agreement with the measured values, indicating a

satisfactory model.

4.2.4.c. Ascorbic acid content (AAC)

If we compare responses for the recovery of AAC, different radiation power and time

were recorded for microwave-assisted extraction techniques under study. Statistical

data (Table 4.2.7) revealed that the effect of drying time under hot air oven treatment

was significant because p value is less than 0.05; the linear quadratic and interactive

effect of radiation power and time was significant.

102
In case of hot air oven drying, time was significant for AAC. R2 value for hot air oven

drying was 0.9750 indicating that the method was significant for the recovery of AAC

as shown by surface plots (Figure 4.2.2).

Measured and predicted AAC values (with RSM) are given in Table 4.2.6 for applied

drying method. Predicted values for both response variables are in close agreement

with the measured values, indicating a satisfactory model.

4.2.4.d. DPPH radical scavenging activity

Statistical data (Table 4.2.7) revealed that the effect of radiation power and time under

microwave-assisted extraction treatment was found to be significant because of p

value is less than 0.05 for linear quadratic and two way interactive effects for DPPH.

R2 value for hot air oven drying was 0.9920 indicating that the method was significant

for the antioxidant activity using DPPH % inhibition as shown by (Figure 4.2.2)

surface plots. Predicted DPPH % inhibition values (RSM) are given in Table 4.2.5 for

for both response variables are in close agreement with the measured values,

indicating a satisfactory model.

4.2.4.e. ABTS radical cation scavenging activity

Statistical data (Table 4.2.7) revealed that the effect of radiation power and time under

microwave drying treatment was insignificant because of p value is greater than 0.05

for linear quadratic and two way interactive effects for ABTS. R2 value for hot air

oven drying was 0.9920 exhibited that the method was significant for the antioxidant

activity using DPPH % inhibition as shown by surface plots. Predicted ABTS %

inhibition values (with RSM) are given in Table 4.2.7 for both response variables are

in close agreement with the measured values, indicating a satisfactory model.

103
Figure 4.2.2. Response Surface plots showing the effect of microwave-assisted
extraction parameters on TPC, TFC, AA and %age inhibition (DPPH • and
ABTS•+) of 70 % MeOH extracts of M. nigra leaves

104
Table 4.2.6. Measured and predicted TPC, TFC, AAC, %age inhibition (DPPH• and ABTS•+) values for M. nigra leaves extract
prepared using microwave-assisted extraction technique
Design
Independent Response Response Response Response Response
point
variables (TPC, mg GAE/g) (TFC, µg ECE/g) (AAC, mg AAE/g) (DPPH % Inhibition) (ABTS % Inhibition)

Sr. No. X1 X2 Measured Predicted Measured predicted Measured predicted Measured predicted Measured predicted
1 0 0 48.80 46.56 111.8 119.53 1.92 2.24 70.16 70.14 78.16 78.39
2 0 -1 50.54 46.74 124.3 124.23 1.80 1.58 65.26 66.02 75.20 77.08
3 0 1 51.53 52.68 126.2 127.40 1.84 2.16 70.00 70.87 80.02 79.43
4 1 1 45.85 47.09 124.8 131.26 2.98 2.88 68.65 67.80 78.50 76.31
5 1 -1 42.07 s44.88 126.3 126.96 2.84 2.92 66.25 66.59 77.54 77.85
6 1 0 37.74 38.73 126.6 133.80 2.96 2.99 69.30 69.53 79.35 77.97
7 -1 1 43.49 43.07 127.5 119.83 2.94 2.55 69.30 68.20 79.30 79.58
8 -1 -1 47.09 49.75 133.5 125.10 2.86 2.77 65.48 65.57 75.58 77.55
9 -1 0 50.80 48.41 146.8 139.67 2.82 2.89 70.01 69.69 80.01 79.50
X1= Coded units for speed, X2=coded units for time

105
 Table 4.2.7. Polynomial equations and statistical parameters for M. nigra leaves
using microwave-assisted extraction technique, leaves dried under hot air oven

Response
2nd order polynomial equation (Microwave-assisted
variables R2 F P
extraction)
M. nigra

TPC TPC = 10.7 + 4.38 X1 + 0.203 X2 - 0.548 X12 - 0.000444 X22

0.9831 1.74 0.035
(mg GAE/g) + 0.00803 X1X2
TFC TFC = 126.0 + 2.4X1-0.119X2 - 0.206X12 + 0.000205X22 +
0.5039 0.61 0.707
(µg ECE/g) 0.0067X1X2
AAC AAC = 2.76 + 0.161X1+0.0012X2 - 0.0015X12 + 0.000002X22 -
0.9750 2.46 0.025
(mg AAE/g) 0.000900X1X2

DPPH• % Inhibition = 46.24 + 2.01X1 + 0.1079 X2 - 0.2226 X12 -

0.9920 4.96 0.019
% Inhibition 0.000190 X22 + 0.00295 X1X2

ABTS•+ % Inhibition = 97.4 – 2.02 X1 - 0.1021 X2 + 0.129 X12

0.8148 0.43 0.811
% Inhibition + 0.000166 X22 + 0.00115 X1X2

CCD was constructed and extractions were carried out for different time intervals and

intensity of microwaves. RSM was employed to find out the optimized conditions

(Table 4.2.8) for the maximum recovery of antioxidant components. Two parameters,

such as radiation intensity and extraction time potentially influenced the microwave

assisted extraction process. It was observed that with the change of extraction time

and radiation power (6 min to 9 min and 200 to 400 rpm) significantly influence the

antioxidant potential of bioactives from M. nigra. A notable increase in TPC was

recorded with increase in radiation power from 100 to 200 watts. The content of total

phenolics remained the same even after increasing the extraction time, indicating that

target products were not stable. The effect of radiation power on antioxidant potential

was investigated using different times 3, 6 and 9 min; similar results were observed at

3 and 6 min. Based on our experiments, 70 % MeOH was found to be the best

extractive solvent for bioactive compounds recovery from M. nigra leaves when

extraction is carried out according to optimized conditions prescribed in this study.

106
Table 4.2.8. Optimized conditions to recover maximum antioxidant components
from M. nigra leaves
Antioxidant activity Power (watts) Time (min)
TPC (mg GAE/g) = 52.8092 285.319 6.05001
TFC = 139.280 397.055 8.93952
AAC = 2.98322 200.977 3.05190
DPPH % = 71.1618% 339.850 6.68272
ABTS % = 79.5346% 399.116 3.02058

Part III

4.3. Bioactive-Enriched Fractionation of Periploca Aphylla Extracts Followed

by Identification and Quantification of Novel Antioxidant Compounds

4.3.1. Antioxidant potential of different solvent fractions from Periploca aphylla

(PA) crude extract

4.3.1.a. Total phenolic content (TPC)

Total phenolic content in different solvent fractions of crude extract obtained from

whole plant of Periploca aphylla (PA) are given in Figure 4.3.1 According to the

data, high content of total phenolics was found in butanol fraction followed by water

fraction. Least amount of phenolic compounds was found in hexane fraction. All the

fractions contained significantly (p << 0.05) different amount of TPC.

It was observed that high polarity solvents exhibit a strong affinity towards phenolic

compounds which confirms the ability of butanol and water to solubilize a large fraction of

phenolic components in the crude extract. Water demonstrated comparable potential to

methanol in fractionation procedure conducted [212]. Similar results were reported by Lee et

al (2009) for the fractions of olive leaves, it was reported that butanol fraction was found

having high content of phenolic as compared to other fractions [212]

107
 140

120

100

80
TPC GAE/g

60

40

20

0
Hexane Chloroform Ethyl acetate Butanol Water
Fractions

Figure 4.3.1. Total phenolic content in solvent fractions of Periploca aphylla (PA)

4.3.1.b. DPPH radical scavenging activity

90
80
70
%age DPPH inhibition

60
50
40
30
20
10
0
Hexane Chloroform Ethyl Butanol Water
acetate
Fractions

Figure 4.3.2. DPPH radical %age inhibition of solvent fractions from Periploca
aphylla (PA)
DPPH radical %age inhibition of different fractions (Hexane, Chloroform, Ethyl

acetate, Butanol and Water), have been presented in figure 4.3.2 (p << 0.05). It is

clear from the results that highest antiradical potential have been exhibited by butanol

fraction followed by ethyl acetate fraction. Least DPPH radical potential was noted

108
for hexane fraction. The results indicate, as polar solvents recover phenolic

compounds from the crude extract suspended in water. As polar antioxidant molecules

have been recovered by butanol and ethyl acetate.

On the other hand, DPPH have ability to evaluate antioxidant potential in aqueous

medium, so higher antiradical potential of butanol, ethyl acetate and water fractions

might be due to better working of DPPH assay in aqueous medium. However, the

results of current study are compare with those conducted by Le at al (2009) [212].

4.3.1.c. ABTS radical cation scavenging activity

Figure 4.3.3 shows ABTS+ radical scavenging activity of different fractions of

Periploca aphylla as %age inhibition (p >> 0.05). No significant different was

statistically calculated for the different solvent fractions. Highest radical scavenging

activity was observed for butanol fraction followed by methanolic fraction.

90
80
%age ABTS inhibition

70
60
50
40
30
20
10
0
Hexane Chloroform Ethyl acetate Butanol Water
Fractions

Figure 4.3.3. ABTS radical cation %age inhibition of different solvent fraction of
Periploca aphylla (PA)

4.3.1.d. HPLC quantification of phenolic acids from Periploca aphylla

solvent fractions
Phenolic compounds are the largest group of exogenous antioxidants, and well-known

free radicals scavenger due to their hydrogen/electron donating potential [213].

109
Ingestion of phenolic compounds, through daily consumption of food items, has been

reported to reduce the incidence of many degenerative and age-related disorders. Most

of these phenolic compounds are acidic in nature [214], which can be divided into two

main groups; one containing derivatives of benzoic acids, while 2nd includes

derivatives of cinnamic acid. Phenolic acids of both groups, with different

hydroxylation levels, are the major components of fruits, vegetables and various

herbs. Some of the famous hydroxybenzoic acids belonging to 1st group include 4-

hydroxybenzoic (p-hydroxybenzoic acid), vanillic (4-hydroxy-3-methoxybenzoic

acid), syringic (4-hydroxy-3,5-dimethoxybenzoic acid) and Gallic (3,4,5-

trihydroxybenzoic acid) acids [215]. Activities of phenolic acids are strongly

influenced by their structure, substituents or side-chains. It is reported that ethylenic

side-chain plays role in resonance-stabilization of peroxyl radicals, while some

reports disagree with any effect of conjugated double bond on antioxidant potential

[216]. Several botanical materials have been identified as potent sources of phenolic

acids and many authors have reported their significant biological activities besides

pharmacological attributes [217]. Oilseeds contain only a fraction of these phenolic

acids in free state i.e. non-hydrolyzable polyphenols, which can be made easily

bioavailable during the course of digestion. Majority of these phenolic acids exists in

bound form (hydrolyzable polyphenols) i.e. in the form of esters, glycosides or

different complexes [43, 218, 219]. In order to convert them to free form, process of

acid or base catalyzed hydrolysis is required [220], which is provided by GI tract,

where these compounds are extracted by mechanical and chemical means [221].

Hydrolysis is a pH dependent process; therefore, GI pH can be considered as the vital

factor for the extraction of these compounds.

110
In this study, PA solvent fraction was analyzed for phenolic compositions using

HPLC and the results of phenolic acids are summarized in Table 4.3.1. Both hexane

and water fractions were found containing 8 phenolic acids each. Phenolic acids

present in hexane fraction were in minute quantity. However, significant amount of 4-

hydroxy-3-methoxy benzoic acid, ferulic acid and Caffeic acid were found in water

fraction. Chloroform and ethyl acetate fractions had four and six phenolic acids

respectively, however quantity of these phenolic acids was not sufficient enough to

perform any significant antioxidant activity. In the butanol fraction, eight phenolic

acids were recorded and highest amount of phenolic acids was obtained in this

fraction.

Table 4.3.1. Phenolic composition of Periploca aphylla solvents fractions by

HPLC
Phenolic Acids Hexane Chloroform Ethyl Butanol Water
fraction fraction Acetate fraction fraction
(mg/g) (mg/g) fraction (mg/g) (mg/g)
(mg/g)
Quercetin 0.17 0.03 0.15 2.42 0.36

Gallic acid 0.11 0.55 1.58 8.51 1.42

Ferulic acid 0.52 - - 2.18 7.41

Sinapic acid 0.86 - 0.23 - -

Vanilic acid 0.49 0.15 7.25 -

4-hydroxy-3- 0.03 - 6.36 23.94 10.19

methoxy
benzoic acid
Chlorogenic - - - 0.37 -
acid
p-Coumaric 0.48 - 1.27 - 0.44
acid
m-Coumaric - 0.22 - - 0.42
acid

111
 Trans-4- - - 0.81 20.94 1.09
hydroxy 3-
methoxy
cinnamic acid
Caffeic acid - - 1.42 0.34 4.46

Syringic acid 0.33 - - - -

Highest amount of 4-hydroxy-3-methoxy benzoic acid was recorded i.e. 23.94 mg/g

of the butanol fraction, whereas, trans-4-hydroxy 3-methoxy cinnamic acid was 20.94

mg/g. The results of HPLC are in agreement with results of radical scavenging assay

i.e. DPPH and ABTS. Butanol fraction exhibited highest percentage inhibition against

both the free radicals, this activity can be attributed to the presence of high amount of

phenolic acids in butanol fraction. Current study suggests the use of PA butanol

fraction as source of natural antioxidants.

4.3.2. Antioxidant activity of sub-fractions from Periploca aphylla butanol

fractions

4.3.2.a. DPPH radial scavenging potential of sub-fractions of Periploca aphylla

butanol fraction

Sub-fractions prepared from Periploca aphylla butanol fraction, may possibly contain

same class of compounds in each sub-fraction. In order to differentiate between these

sub-fractions for their antioxidant potential, DPPH radical scavenging assay was

performed. Data has been presented in Figure 4.3.4 showing that all sub-fractions

were found having significant different DPPH radical scavenging activity.

According to results presented in figure 4.3.4, sub-fraction 12 and 13 exhibited strong

DPPH radical scavenging potential among all tested sub-fractions. Sub-fraction 12

and13 inhibited free radicals in the system up to 88.16% and 79.14% and exhibited

highest radical scavenging potential. Therefore, sub-fraction, PSF12 can be

112
considered for further isolation and purification of compounds of strong antioxidant

activity.

100
90
80
DPPH %age inhibition

70
60
50
40
30
20
10
0

Fractions
Figure 4.3.4. DPPH radical scavenging activity of sub-fractions from Periploca
aphylla butanol fraction

4.3.2.b. ABTS radical cation scavenging activity of sub-fractions of Periploca

aphylla butanol fraction
90
80
ABTS %age inhibition

70
60
50
40
30
20
10
0

Subfractions

Figure 4.3.5. ABTS radical cation scavenging activity of sub-fractions from

Periploca aphylla butanol fraction
Figure 4.3.5 shows that all sub-fraction exhibited variations in ABTS radical

scavenging activity and it is clear that sub-fractions PSF2, PSF12 and PSF13 are the

113
most potent antioxidants so that these sub-fractions can be considered for isolation

and purification of antioxidant compounds of interest.

4.3.2.c. Determination of bioactive compounds butanol sub-fraction 12 by GC-

MS after derivatization

Thirty milligram of each sample extract of were injected into GC-MS after

derivatization. Total ion chromatogram and their peak detail is shown in Figure 4.3.6

having m/z from 120 to 550 m/z and oven ramping was from 50oC to 150oC @5oC

per min and hold for 2 min at 150oC and then from 150oC to 250oC @ 8oC per min

and hold at 250oC for 5 min and total run time was 39.5 min.
Abundance

TIC: C (DV) 108.D\ data.ms

2.757
8000000
2.830
7500000

7000000

6500000

6000000

5500000

5000000
4.05
8.2
3 51 26.126
4500000
4.262
4000000

3500000

3000000

2500000

2000000 4.011
16.805
1500000 4.166
1000000
7
5.647.163
500000 8.298 23.130
0
5.00 10.00 15.00 20.00 25.00 30.00 35.00
Time-->

Figure 4.3.6. Mass spectrum of butanol fraction

This study showed the existence of many secondary metabolites in the tested butanol

fraction. GC-MS analysis of butanol fraction provided an idea of different classes of

compounds present. This study confirmed that the selected plant extracts have

potential for curing many ailments.

The GC-MS analysis of selected plant extracts showed the presence of different types

of compounds in PA butanol fraction like 3,6-dioxa-2,7-disilaoctane, 2,2,4,7,7-

pentamethyl- is a diol compound. Eman et al reported biological activity

(antimicrobial) of this compound [222]. The second compound detected was 1,13-

bis(trimethlysilyloxy)tridecane, an ester 1, 13-dixhydroxy tirdecane, which also

114
exhibited antioxidant activity. Ethanedioic acid, bis(trimethylsilyl) ester usually found

in alcohols. Lay et al reported the presence of this compound in n distilled beverages

[223].

Another compound 2-propenoic acid, 2-[(trimethylsilyl)oxy]-, trimethylsilyl ester

indicated the presence of carboxylic acid. This compound is water-soluble and is

present in beverages as described above. One the compounds was silane, [(3,7-

dimethyl-6-octenyl)oxy]trimethyl- was an alcohol and JD Wuest et al [224] reported

its presence in alcohols and this compound have also been reported as an effective

antidiabatic agent. Silane, [1-(5-ethenyltetrahydro-5-methyl-2-furanyl)-1-

methylethoxy]trimethyl-, trans- is an ester of long chain alcohol. Ahmad et al reported

its presence in Nigella sativa oil [225].

Silanamine, 1,1,1-trimethyl-N-(1-methyl-2-phenylethyl)-N-(trimethylsilyl)-, (ñ)- was

found in PA butanol fraction have been reported to own certain biological activities.

Many analogous alcohols have also been identified in different plant extracts. These

alcohols are very effective in treating different diseases [226]. Another compound was

identified by the name, 2,6-dimethyl-3,4-bis(trimethylsilyloxymethyl)pyridine is an

ester of 3,4-bis-hydroxymethyl-2,6-dimethyl-pyridine. Some 35 oxidation reduction

reactions in which nicotinamide participates as either of the pyridine nucleotides

acting as two-electron transporters [227]. 7-Trimethylsilyloxytridecane is an ester of

iso alcohol [228] and 1,2-bis(trimethylsiloxy)ethane is an ester of 1,2-

dihydroxyethane or ethylene glycol. Ethylene glycol derivatives were identified from

culture broth of six Macromycetes growing in subantarctic forests in southern Chile.

It was found that these derivatives exhibit biological activity [229, 230].

Trimethylsilyl ether of glycerol is an ester of glycerol. Free glycerol appeared to be

the major precursor of monochloropropane-1,2-diol (MCPD) in leavened dough as

115
described earlier [231] Benzoic acid, 4-[(trimethylsilyl)oxy]-, methyl ester is an ester

of 4-hydroxy benzoic acid. It was also identified in carrot, palm oil, grapes and many

other species [217]. Ortho hydroxyl benzoic acid also own antimicrobial properties

[232]. Benzoic acid, 2-hydroxy-, methyl ester also identified in the leaf of Wedelia

chinensis (Osbeck) Merrill which exhibited antimicrobial activity [233].

147
100 100

147
O Si SO
i OSi
Si O
50 50

133
123 131 139 165 177
0 0
120 130 140 150 160 170 180 120 130 140 150 160 170 180
(mainlib) 1,13-Bis(trimethlysilyloxy)tridecane
(replib) 3,6-Dioxa-2,7-disilaoctane, 2,2,4,7,7-pentamethyl-

147 147
100 100

O
Si O O
O Si
Si O
50 50 O Si
O

217
133 190 219 131
175 189
0 0
120 140 160 180 200 220 120 140 160 180 200 220
(replib) Ethanedioic acid, bis(trimethylsilyl) ester (replib) 2-Propenoic acid, 2-[(trimethylsilyl)oxy]-, trimethylsilyl ester

146 131
100 100

Si
O
O
Si
50 50
O

130

161 185 213 228 129 137 143 147 157

0 0
120 140 160 180 200 220 120 130 140 150 160
(mainlib) Silane, [1-(5-ethenyltetrahydro-5-methyl-2-furanyl)-1-methylethoxy]trimethyl-, trans-
(mainlib) Silane, [(3,7-dimethyl-6-octenyl)oxy]trimethyl-

188 221
100 100

Si O
N Si
Si
50 50
147 206 O
Si
178
149 264 133
121 172 207 266 296
0 0
120 150 180 210 240 270 120 180 240 300 360 420
(mainlib) Silanamine, 1,1,1-trimethyl-N-(1-methyl-2-phenylethyl)-N-(trimethylsilyl)-, (ñ)-
(mainlib) 2,6-Dimethyl-3,4-bis(trimethylsilyloxymethyl)pyridine

116
 187
100
147
100

O Si
50 Si O
O Si
50

191
257
0 0
120 180 240 300 360 420 120 180 240 300 360 420
(mainlib) 7-Trimethylsilyloxytridecane (mainlib) 1,2-Bis(trimethylsiloxy)ethane

209
100
267
100
O O
Si
224 O O

50
50 193
O O 223 282
135 Si 193 Si

149 177 126 149

121 165 177 207
0 0
120 140 160 180 200 220 240 120 160 200 240 280
(mainlib) Benzoic acid, 4-[(trimethylsilyl)oxy]-, methyl ester Benzoic acid, 3-[(trimethylsilyl)oxy]-, trimethylsilyl ester
(replib)

Figures 4.3.7. Structures of compounds identified and quantified in sub

fraction of PA from butanol fraction

117
 Table 4.3.2. GC-MS analysis of Periploca aphylla butanol sub-fraction
Sr.
Compound name
No RT ( ng/ mg of
(formula) Reference
. sub-fraction)
(MW)
3,6-Dioxa-2,7-
disilaoctane, 2,2,4,7,7-
1 pentamethyl- 2.757 (0.79)
(C9H24O2Si2)
(220) [222]
1,13-
Bis(trimethlysilyloxy)tr
2 idecane 2.83 (0.84)
(C19H44O2Si2)
(360)
Ethanedioic acid,
bis(trimethylsilyl) ester
3 4.011 (0.71)
(C8H18O4Si2)
(234)
2-Propenoic acid, 2-
[(trimethylsilyl)oxy]-, [223]
trimethylsilyl ester
4 4.083 (0.07)
(C9H20O3Si2)
(232)

Silane, [(3,7-dimethyl-
6- [224]
5 octenyl)oxy]trimethyl- 4.166 (0.10)
(C13H28OSi)
(228)
Silane, [1-(5-
ethenyltetrahydro-5-
methyl-2-furanyl)-1-
6 methylethoxy]trimethyl 4.262 (0.87) [225]
-, trans-
(C13H26O2Si)
(242)

118
 Silanamine, 1,1,1-
trimethyl-N-(1-methyl- [234]
2-phenylethyl)-N-
7 5.251 (1.16)
(trimethylsilyl)-, (ñ)-
(C15H29NSi2)
(279)
2,6-Dimethyl-3,4-
bis(trimethylsilyloxym [227]
8 ethyl)pyridine 5.647 (0.002)
(C15H29NO2Si2)
(311)
7-
Trimethylsilyloxytridec
9 ane 7.163 (0.03) [228]
(C16H36OSi)
(272)
1,2-
Bis(trimethylsiloxy)eth
10 ane 8.298 (0.02) [230]
(C8H22O2Si2)
(206)
Trimethylsilyl ether of
glycerol [231, 235]
11 16.805 (0.16)
(C12H32O3Si3)
(308)
Benzoic acid, 4-
[(trimethylsilyl)oxy]-,
12 methyl ester 23.13 (0.07)
(C11H16O3Si)
(224)
[232, 236]
-
-
26.126 (5.00)
Benzoic acid, 3-
[used as
13 [(trimethylsilyl)oxy]-,
internal
trimethylsilyl ester
standard]
(C13H22O3Si2)
(282)

119
Part IV

4.4. Stability of Antioxidant Components from Defatted Kenaf (Hibiscus

cannabinus L.) Seed Meal (DKSM) Under Simulated Gastrointestinal pH

Conditions

To get a clearer view about the nutritional efficiency, activity or health benefits of any

dietary item, its in vivo digestibility evaluation is very important. In this study,

digestibility of DKSM is studied by creating gastrointestinal conditions under in vitro

system, which provides results analogous to in vivo system.

4.4.1. Effect of simulated GI pH conditions on extract yield and TPC of DKSM

Extracts of an underestimated agrowaste, i.e. DKSM were prepared and yield was

determined without any treatment (untreated) and after subjecting to gastrointestinal

pH conditions (treated) (Table 4.4.1). Simulated GI pH conditions increased yield of

the extracts significantly, indicating the potential of GI pH for converting bound

constituents to free form from seed matrix. These results confirmed that the

availability of bioactive compounds from plant matrix is strongly dependent upon pH

of the extraction media, and different pH conditions are potent for altering the

solubility of plant components [237].

Seeds of various plant species have been reported to be containing high amount of

phenolics and their extractability is influenced by different extraction conditions

including pH [238]. Value of TPC for treated DKSM decreased to half as compared to

untreated DKSM (Table 4.4.1.). Lower TPC in treated DKSM may be hypothesized to

reduced solubility of DKSM phenolics under GI pH conditions. Findings of the study

may also be understood by the report of Bermudez-soto et al (2007) [239], who

reported that phenolics upon exposure to alkaline conditions may undergo structural

transformations, resulting in formation of different structural compounds with

120
different chemical properties. Though these newly formed compounds may have

antioxidant properties but are usually undetectable by conventional assays of phenolic

content determination. The effect of GI pH conditions on TPC of DKSM is same as

reported for Capparis spinosa L. and Crithmum maritimum L, which were subjected

to two-stage in vitro digestion model [240]. Inspite of all these literature reports

justifying the findings of our work, Folin-Ciocalteu method is only ranked among the

preliminary assays due to number of interferences associated with this, especially

from amino acids and purines. Therefore, major phenolic contents were individually

quantified using HPLC to get the clearer picture.

Table 4.4.1. Extract yield and total phenolic content of defatted kenaf seed meal
as subjected to simulated gastrointestinal pH condition
Defatted Kenaf Seed Extract yield Total phenolic content
Meal (% w/w) (mg GAE/g defatted seed
meal)
Untreated 25.74 ± 0.82a 3.40 ± 0.02a
Treated 29.36 ± 1.31b 1.93 ± 0.01b
a-b: Results are obtained from means of three determinations ± standard deviation. Different alphabet
within the same column indicates significant difference (p < 0.05)

4.4.2. Effect of simulated gastrointestinal conditions on antioxidant potential of

DKSM

4.4.2.a. DPPH radical scavenging potential

Being technically simple and highly sensitive, 2,2-diphenyl-1-picrylhydrazyl (DPPH)

radical scavenging assay has been widely used for the measurement of antioxidant

potential of plant materials. DPPH• is a stable organic nitrogen radical of purple color

having maximum absorption at 517 nm. DPPH radical accepts hydrogen from

antioxidant compounds, which alters the colour of solution, causing a change in

121
absorbance. This is used to calculate antioxidant effect of samples in comparison with

standards such as Trolox [241, 242].

DPPH• scavenging potential of DKSM before and after GI pH treatment is presented

in Figure 4.4.1. DPPH radical scavenging potential of treated DKSM was found to be

lower than the untreated counterpart, however, the difference was not significant.

Such a small difference in DPPH radical scavenging values may be hypothesized to

limitation of this assay in aqueous medium. The suitable medium for DPPH• to

interact with lipophilic and hydrophilic antioxidants is mixture of water and organic

solvents in ratio of 50:50 (v/v). The presence of water content above 60% may

coagulate a part of DPPH•; resulting in error during estimation of scavenging

potential of samples [242].

4.4.2.b. ABTS radical cation scavenging assay

The ABTS radical cation scavenging assay has been extensively employed by many

researchers for studying antioxidant potential of number of herbal products. Results of

this assay are independent of pH and nature of solvent used that is why this assay is

considered more reliable as compared to other assays. The solution of ABTS radical

cation is of blue-green color having maximum absorption at 734 nm. The extent of

decolorization of ABTS•+ upon receiving electron from antioxidant species, present in

the samples, is measured in terms of absorbance. The absorbance of reaction mixture

is compared to standard curve of Trolox and results are expressed as Trolox

Equivalent Antioxidant Capacity [241, 242].

The ABTS radical cation scavenging assay was employed to evaluate the effect of GI

pH on antioxidant potential of DKSM and results are expressed as Trolox equivalent

in Figure 4.4.2. Untreated DKSM showed higher ABTS•+ scavenging potential than

treated DKSM samples, which showed a significant decrease compare to the untreated

122
sample. The anti-radical potential of botanical materials is found to be associated with

their content of bioactive components. In this study, lower scavenging values for

DPPH radical and ABTS radical cation verify low amount of antioxidant compounds

in extracts of treated DKSM samples as compared to its untreated counterpart. This

may be due to possible structural transformations in phenolics and other antioxidative

compounds under gastrointestinal pH conditions, which results in decrease in their

activity. Furthermore, poor scavenging efficacy of treated DKSM against DPPH and

ABTS radicals may be due to phenol-protein binding at GI pH conditions. It may also

be hypothesized that under GI conditions, production of novel compounds, possessing

prooxidant properties may take place which contribute towards decrease in DKSM

antiradical potential.

4.4.2.c. Ferric Ion Reducing Antioxidant Power

This assay interprets the ferric ion reduction capacity of samples under study. Ferric

ion in Fe3+-TPTZ accepts an electron from antioxidant species in the sample and

converts itself to ferrous state under acidic conditions, which on chelating with a

chromogenic ligand (tripyridyltriazine, TPTZ) gives a colored complex having a

maximum absorbance at 593 nm. The reducing ability (antioxidant effect) of plant

extract can be determined by measuring absorbance after formation of Fe2+-TPTZ

complex. The FRAP assay works well in aqueous systems and can also be employed

to test ferric ion reducing potential of lipophilic antioxidants [241]. The extracts of

DKSM prepared in deionized water as such and under simulated gastrointestinal

conditions were evaluated for their FRAP and results are presented in Figure 4.4.1 as

µg Trolox equivalent/g of DKSM. The results of FRAP assay reveal that untreated

DKSM possesses significantly (p < 0.05) higher ferric ion reducing antioxidant power

as compared to treated sample. Lower ferric ion reducing power of DKSM under

123
 gastrointestinal pH conditions indicates a lower suitability of DKSM for edible

purposes. Excessive accumulation of ferric ion usually takes place through drinking

water and ingestion of contaminated food which possesses potential to instigate the

production of free radicals within human body. In this condition, edible use of DKSM

may not be health promoting due to its lower ferric ion reducing potential.

35000
Antioxidant Activity (µg standard equivalent / g

30000
25000 Untreated
20000 Treated
15000
DKSM)

10000
5000
0
DPPH· ABTS· Ferric Hydroxyl Iron
Scavenging Scavenging Reducing Radical Chelating
Activity Activity Power Scavenging Activity
Activity
Antioxidant Activity Assays

Results are obtained from means of three determinations ± standard deviation. TROLOX was used as
standard in DPPH scavenging activity, ABTS scavenging activity and ferric reducing power assays.
Standards for hydroxyl radical scavenging activity and iron chelating activity assays were DMSO and
EDTA, respectively.
Figure 4.4.1. Antioxidant activities of defatted kenaf seed meal as affected by
simulated gastrointestinal pH condition

4.4.2.d. Iron Chelating Potential

Bivalent transition metal ions such as Fe (II) play an important role in proper

functioning of biological systems. Ingestion of iron in body should be within safe

limits, as its excessive amount can cause damage to living cells due to its involvement

in oxidative processes such as Fenton reaction. But excessive accumulation of iron

124
through processed food or contaminated water occurs, which is required to be

removed from the body [243, 244].

Antioxidant compounds in plants have been identified as iron chelators and recently

natural iron chelators have been reported to be exhibiting protective effects against

iron-induced oxidative damage to liver [245]. In this study, treated and untreated

DKSM samples were also studied for their iron chelating potential. The results of iron

chelating potential are presented in Figure 4.4.1, which demonstrates high chelation

potential of untreated DKSM. Under gastrointestinal conditions, the iron chelating

potential of DKSM was found to improve which may be due to modification in

structure of antioxidant compounds. These compounds, especially phenolics form

complex with iron due to strong nucleophilic character of its aromatic ring. Although

structural degradation has been discussed throughout this article, however, changes

induced by GI pH conditions seem to favor Fe-chelating potential of compounds

present in DKSM.

4.4.2.e. Hydroxyl radical scavenging potential

In biological systems, reaction of hydrogen peroxide with Fe(II) is believed to be the

cause of hydroxyl radicals (HO•) production (Fenton reaction). The antioxidant

compounds hinder the generation of HO• either by reacting with H2O2 or chelating

Fe(II). It is difficult to predict the actual target of antioxidant compounds for the

suppression of hydroxyl radical, as these compounds are potent Fe(III) chelators.

Ascorbic acid along with other reagents, in hydroxyl radical scavenging assay, works

as reducing agent for Fe(III) and provides medium for the interaction of antioxidants

and hydroxyl radicals [241, 242]. This assay was employed for the evaluation of

hydroxyl radical scavenging potential of treated and untreated DKSM. The results are

presented in Figure 4.4.1 as µg DMSO equivalent/ g of DKSM, which demonstrate

125
weaker hydroxyl radical scavenging potential of DKSM under simulated GI pH

condition, as compared to untreated DKSM.

Free radicals including superoxide, hydroxyl and DPPH have been analyzed using

electron spin resonance (ESR) spectroscopy and this technique is being widely used

as reliable and powerful tool to analyze free radicals due to its high sensitivity and

short time consumption [246]. ESR spectra of untreated and treated DKSM samples

along with standard (DMSO) are presented in Figure 4.4.2, which revealed noticeable

differences among them both. It is clear from the difference in peaks that hydroxyl

radical scavenging potential of untreated DKSM is greater than the treated

counterpart. It may be due to acidic conditions provided to DKSM, as these

conditions have been reported to alter the compositions and activity of phenolics,

resulting in lower hydroxyl radical scavenging potential of treated DKSM as

compared to its untreated counterpart.

Figure 4.4.2. ESR spectra of hydroxyl radical scavenging activity of treated and
untreated defatted kenaf seed meal

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4.4.2. Effect of simulated GI pH conditions on Phenolic composition of DKSM

Phenolic compounds are the largest group of exogenous antioxidants, and well-known

free radicals scavenger due to their hydrogen/electron donating potential [247].

Ingestion of phenolic compounds, through daily consumption of food items, has been

reported to be reducing the incident of many degenerative and age-related disorders.

Most of these phenolic compounds are acidic in nature [214], which can be divided

into two main groups; one containing derivatives of benzoic acids, while 2nd includes

derivatives of cinnamic acid. Phenolic acids of both groups, with different

hydroxylation levels, are the major components of fruits, vegetables and various

herbs. Some of the famous hydroxybenzoic acids belonging to 1st group include 4-

hydroxybenzoic (p-hydroxybenzoic acid), vanillic (4-hydroxy-3-methoxybenzoic

acid), syringic (4-hydroxy-3,5-dimethoxybenzoic acid) and gallic (3,4,5-

trihydroxybenzoic acid) acids [248]. Activities of phenolic acids are strongly

influenced by their structure, substituents or side-chains. It is reported that ethylenic

side-chain plays role in resonance-stabilization of the peroxyl radicals, while some

reports disagree with any role of conjugated double bond in antioxidant potential

[216]. Several botanical materials have been identified as potent sources of these

phenolic acids and many authors have reported their significant biological activities

besides pharmacological attributes [217]. Oilseeds contain only a fraction of these

phenolic acids in free state i.e. non-hydrolyzable polyphenols, which can be made

easily bioavailable during the course of digestion. Majority of these phenolic acids

exists in bound form (hydrolyzable polyphenols) i.e. in the form of esters, glycosides

or different complexes [238, 249, 250]. In order to convert them to free form, the

process of acid or base catalyzed hydrolysis is required [251], which is provided by

GI tract, where these compounds are extracted by mechanical and chemical means

127
[221]. Hydrolysis is a pH dependent process; therefore, GI pH can be considered as

the vital factor for the extraction of these compounds.

In this study, DKSM samples before and after subjecting to simulated GI pH

conditions were analyzed for selected phenolic acids using HPLC-DAD. The results

of phenolic acids, determined in untreated and treated DKSM are summarized in

Table 4.4.2. Findings of the study showed significant decrease in bioavailable

concentration of all phenolic acids, in DKSM, individually except vanillic acid under

simulated GI pH conditions, which may be attributed to structural modifications with

the change in pH [15]. Only 0.68 and 31.30% for 4-hydroxybenzoic and gallic acids,

respectively, of their amount present in untreated DKSM, could be detected under

simulated GI pH conditions. The effect of GI pH conditions was found less significant

in case of syringic acid, which retained its 72% amount after treatment. These results

suggest better bioavailability and stability of vanillic acid as compared to other

phenolic acids under GI pH conditions. Keeping in view the results of this study, it

may be hypothesized that during transition from neutral to highly acidic conditions,

hydroxyl group of phenol started acting as base and after accepting H+ got converted

to hydronium ion. Although, the addition of NaOH to the mixture helped in making

the pH 7, but it did not help phenol to attain its structure, leaving lesser amount of

phenolics to be detected by the methods of routine use.

Catechin belongs to a subgroup of polyphenols i.e. flavonoids. The hydroxyl group of

flavonoids can scavenge excited free radicals by donating electrons, whereas their

metal complexation potential inhibits metal ion catalyzed production of free radical

species [252]. Untreated DKSM samples were found to be containing high amount of

catechin, but 80% of total available catechin was lost upon treatment. These results

128
 indicate strong negative impact of GI pH conditions on yield of extractable fraction of

catechin present in DKSM.

Table 4.4.2. Phenolic composition of defatted kenaf seed meal as affected by

simulated gastrointestinal pH condition
Sr. Phenolic Compounds Individual Phenolic Content in Defatted Kenaf Seed Meal
No (μg / g defatted seed meal)
Untreated Treated
1 366.41 ±
Gallic acid 1170.52 ± 2.26a 6.24b
2 123.76 ±
Catechin 653.86 ± 5.31a 7.08b
3 4-hydroxybenzoic acid 100.31 ± 0.26a 0.68 ± 0.05b
4 Vanillic acid 24.79 ± 4.02a 20.30 ± 5.85a
5 Syringic acid 50.31 ± 0.54a 36.58 ± 4.24b
Sum 1999.80 547.73
a-b: Results are obtained from means of three determinations ± standard deviation. Different alphabet
within the same row indicates significant difference (p < 0.05)
The results of phenolic acids recorded in this study are well in agreement with those

of Cilla et al (2009) and Tagliazucchi et al (2010), who reported a decrease in amount

of phenolic compounds upon subjecting the samples to simulated GI conditions [221,

253, 254]. Most of the phenolic compounds are stable in acidic environment, while

these compounds may undergo degradation at alkaline pH. Transitions from stomach

to intestinal pH conditions may cause ionization of hydroxyl groups resulting in

structural changes in phenolic compounds or degradation of reactive sites [221].

Furthermore, protein enriched food items may cause a decrease in available fraction

of phenols, because protein may be involved in binding with phenol [255, 256], which

becomes resistant to gastric hydrolysis [257]. Plant proteins are larger molecules as

compared to phenolics, therefore a protein molecule may engage many phenol

molecules with itself, leaving smaller number of free phenolics to be detected by

HPLC method of analysis. Kenaf seeds are rich in protein [77], therefore, protein-

129
phenol binding may be the reason for lower bioavailability of phenolic acids at GI pH

conditions. On the contrary, in some cases, increase in concentration of phenolic acids

under simulated GI conditions has been observed. Authors have designated this

increase in concentration to bio-accessibility and stability of these phenolic acids

under acidic conditions.

4.5. Nano-encapsulation of DKSM Extract to Enhance its Bioavailability and

Antioxidant Activity

4.5.1. Characterization of DKSM extract loaded nanoparticles

4.5.1.a. SEM analysis of nanoparticles
Particle size (Figure 4.5.1) recorded using SEM was almost 150 nm, however the size

and symmetrical. Nanoparticles were of aggregated forms, having rod shape,

Pulicharla et al., Gan et al., [129, 258] found similar results with chitosan-TPP

nanoparticles.

Figure 4.5.1. SEM images of nanoparticles loaded with DKSM extract

4.5.1.b. Encapsulation efficiency and loading efficiency

High encapsulation efficiency (EE) is necessary, as a sufficiently high concentration

of phenolic compounds is required to have their effects in different systems.

Furthermore, EE depends represents composition of the nanoparticles and also

determine release characteristics [259]. In current work, for the production of

nanoparticles, 0.1% chitosan and 0.05% sodium tripolyphosphate at pH 4.5, was used.

Encapsulated material i.e. extract of DKSM was polar in nature as extract was

130
prepared using methanol as solvent. Such extract may be considered suitable for

encapsulation in chitosan particles and this is why EE was recorded up to 73%. The

high EEs obtained indicate that the formulation used may be considered suitable for

hydrophilic compounds. The result of EE is comparable to that reported for

polyphenol encapsulation by Pulicharla et al [129] but less than those reported for the

encapsulation of carotenoids by Silva et al [260].

The loading efficiency (LE) recorded for the current work was 48%. LE is strongly

influenced by physicochemical characteristics of extracts and properties of the

encapsulating material used. Other parameters including hydrogen bonding,

electrostatic and hydrophobic forces that affect the interaction of phenolic compounds

with chitosan polymer [261]. Hydroxyl functional groups of phenolics and chitosan

amine functional groups link with one another hydrogen bonding when pH 4 is

maintained. In addition to this, chitosan also carry hydroxyl groups that have negative

charge that may repels phenolic compounds at the same pH. Hence, repulsion occurs

between water soluble phenolic compounds and chitosan molecules that may explain

the reason for the low loading efficiency. The results of LE in this work is in

agreement with previous studies by Pulicharla et al., [129].

4.5.1.c In-vitro release of phenolic compounds from DKSM extract loaded

nanoparticles

Release profile of DKSM extract from encapsulated material was recorded in terms of

TPC at room temperature under different pH conditions for 24 hours (Figure 4.5.2).

Release of phenolic molecule was found to be pH dependent as shown in Figure 4.5.2.

The highest release of phenolic compounds was observed at pH 7.4 with the lowest

release recorded at pH 10. For different nanoparticles using chitosan, a rapid release

occurred at the start while a decrease in rate of release have already been reported by

131
Alishahi et al., [262]. Acidic pH may increase the ionization of the protonated amine

groups that increases swelling of the chitosan, electrostatic repulsion and water

content that help in swelling of chitosan and make the outer layer viscous. Whereas,

slow penetration of water into nanoparticles takes place at low pH that results in slow

swelling of chitosan shell and slow diffusion of entrapped molecules. Improved

penetration of water can be achieved by increasing the pH of chitosan nanostructures

[263].

Diffusion and elongation of chitosan surface might occur at pH 7.4 and as a result

upper layer becomes rigid and elastic that causes maximum increase in size and

higher release of entrapped polyphenols. The higher solubility of phenolic compounds

at pH 7.4 as compared to pH 2 may also be the factor for high % release from DKSM

extracts loaded nanoparticles.

Release of encapsulated material form polymeric nanoparticles follows several

mechanisms. Initially, desorption of the surface bound drug takes place followed by

diffusion of the inner drug molecules through the polymer matrix, as a result of

polymeric nanoparticles erosion [264]. Nanoparticle formulation displayed different

release of phenolic compounds under different pH conditions. But at every pH,

biphasic pattern of drug release can be observed, initial sudden release of phenolics is

just because of the immediate release of the surface-connected drug. Whereas,

However, release of molecules covalently bonded with chitosan may follow three

mechanisms including desorption, diffusion and biodegradation encapsulation

material [129].

132
 90
80

%age release
70
60
50
pH 2
40
pH 7.4
30
pH 10
20
10
0
0.1 0.5 1 2 4 6 8 10 12
Time (h)

Figure 4.5.2. Percentage release of total phenolic content from encapsulates at

different pH

80
70
%age release

60
50
40 60
30 80

20 100

10
0
20 40 80 120 180
Time (min)

Figure 4.5.3. Percentage release of total phenolic content from encapsulates at

different Temperature
Effect of temperature on percent release of DKSM extract was recorded at pH≈7 and

three different temperatures (60, 80, 100 ºC) separately for a selected time duration

(20, 40, 80, 120 and 180 min) (Figure 4.5.3). At the start of temperature treatment,

rapid release in phenolics from DKSM extract containing nanoparticles was observed

(Fig. 4.5.3.). But with the passage of time the release pattern for different temperature

133
was different. At 60 ºC, %age release of phenolic molecule increase with the passage

of time. On the other hand, samples treated at 80 ºC, release of phenolic molecules

increases up to 120 min then it becomes constant. The release trend observed for

samples at 100 ºC was entirely different. The release of phenolic compounds increases

up to 80 min, then it starts decreasing. It may be due to decomposition of the phenolic

molecules due to long exposure to high temperature.

Part V

4.6.1. Stabilization Efficiency of Encapsulated and Non-encapsulated A. annua

Leave Extract on Shelf-life of Sunflower Oil

4.6.1.a. Weight gain analysis (WGA)

Oil can gain some weight, when atmospheric oxygen react with unsaturated sites in

oils and fats and formation of hydroperoxide takes place. The amount of oxygen

added during this reaction, is usually measured quantitatively and is considered as an

important parameter for evaluation of induction period, extent of oxidation and

contribution of antioxidants towards stability of oils. Weight of oil either remains

constant or a very little change is observed, when oil samples experience no oxidation

or oxidation up to some extent. WG was measured for all the sunflower oil samples

including control as well as oil two samples having ALE (500 & 1000 ppm) and two

oil samples having BHA and BHT (200 ppm each). One oil sample was administered

with chitosan nanoparticles containing almost 1 g of ALE. Weight gain percentage

was recorded at different intervals of 3, 6, 9, 12, 15 and 18 days (Figure 4.6.1).

Weight gain analysis can provide clear picture about the induction period of the

samples under study. Induction period is considered as the time taken to achieve 0.5%

WG. Significant increase in induction period was recorded for all the stabilized

134
samples as compared to control. For sunflower oil having 1000 ppm of ALE gained

weight slowly as compared to other samples and weight gain of less than 2.5% was

recorded. Efficiency of 1000 ppm ALE is comparable with the synthetic antioxidants

used. In addition, encapsulated ALE exhibited better induction period as compared to

non-encapsulated extract. This indicates that encapsulated extract, release antioxidant

molecules in oil system for a long time and exhibit better storage efficiency as

compared to non-encapsulated extract. Iqbal et al., (2007 & 2008) [57, 58] reported

efficacy of natural antioxidants that supports the findings of our study.

4
3.5
3
Weight gain %

SFO Ctrl
2.5
SFO 500
2
SFO 1000
1.5
SFO 200A
1
SFO 200T
0.5 SFO encap
0
3 6 9 12 15 18
Storage period (Days)

Figure 4.6.1. Variation in weight gain of control and stabilized sunflower

oil samples

4.6.1.b. Peroxide value (PV)

Peroxide value determines the degree of oxidation of oils and fats during storage. A

continuous increase in PV was observed for all the samples (Figure 4.6.2), that may

be due to formation of hydroperoxides, i.e. primary oxidation products. Initially, a

slow increase in PV was observed, but a rapid increase was recorded on the 10th day

of storage which increased further till the 30th day while in case of the control the PV

didn’t reached a maximum until the 20th day. Maximum peroxide value observed for

135
SFO containing ALE 500 and 1000 ppm nonencapsulated extract was 131 and 72

meq/kg, while it was 38.13 meq/kg for SFO sample stabilized with encapsulated ALE

and these values are closer to those recorded for SFO samples having BHA and BHT

(200 ppm each).

200
180
160
140 SFO Ctrl
PV (meq/kg)

120
SFO 500
100
SFO 1000
80
60 SFO 200A
40 SFO 200T
20 SFO encap
0
5 10 15 20 25 30
Storage period (Days)

Figure 4.6.2. Variation in peroxide value (PV) of control and stabilized oil
samples
At all the stages of storage period, there was a regular pattern of increase in PV for all

the stabilized samples. Samples containing 500 ppm ALE exhibited highest peroxide

value throughout the study period but less than the control. On the other hand

encapsulated APL extract stabilized sample, increase in PV was not sharp rather lease

peroxide value was observed for this sample during all the storage period. Maximum

PV of SFO having encapsulated ALE after 25 days storage was found to be 38.01

meq/kg, which is far less than reported for sunflower oil stabilized by guava leaves

and garlic extract [265].

4.6.1.c. Free fatty acids (FFA) content

Formation of free fatty acids is also considered as an important parameter to evaluate

rancidity in food items. Formation of FFAs takes place due to hydrolysis of

triglycerides and may get accelerated by the reaction of oil with moisture [266]. It’s a
136
reaction that continue in the presence of moisture and amount of FFA content went on

increasing with the passage of storage time. For the current study, control exhibited

the highest FFA, SFO containing encapsulated ALE exhibited least (Figure 4.6.3).

Initially, significant increase in FFA of stabilized samples was not observed but, after

9 days of storage, an increase was recorded. Efficiency of ALE (encapsulated and

nonencapsulated) added in SFO at a level of 1000 ppm, is comparable with BHT or

even better than BHA and BHT.

0.9
0.8
0.7
Free fatty acid %

0.6 SFO Ctrl

0.5 SFO 500
0.4 SFO 1000
0.3 SFO 200A
0.2 SFO 200T
0.1 SFO encap
0
5 10 15 20 25 30
Storage period (Days)

Figure 4.6.3. Variation in free fatty acid content in control and stabilized
oil samples

4.6.1.d. Conjugated dienes and trienes (CD & CT)

Oxidative stability of the oils can be monitored by measuring CD and CT [267].

Methylene-interrupted dienes or polyenes are the part of lipids that show double bond

shift based oxidation. The molecules exhibit intense absorption at 232 nm; in the same

way conjugated trienes show absorbance at 268 nm. The increase in CD and CT

contents is directly related with the oxygen uptake by the oil samples. Higher amounts

of CD and CT indicate lower oxidative stability of the oils. Iqbal and Bhanger (2007)

[57], Siddiq et al., (2005) [268] reported antioxidant potential of different extracts for

137
the stabilization of sunflower oil, assessed under accelerated conditions, by measuring

CD and CT contents in oil samples.

Figures. 4.6.4.a and 4.6.4.b show relative change in CD and CT contents in SFO

under accelerated storage conditions, as function of time. SFO-Ctrl sample exhibited

the highest content of CD followed by SFO-500, SFO-BHA, SFO-1000 and leased

were observed in sample labelled SFO-encap; while CT content followed the pattern

control > SFO 500 > SFO BHA > SFO-1000 > SFO BHT > SFO encap respectively.

As compared to control, CD and CT of all the stabilized samples are lower that

indicate potential use of ALE (especially encapsulated) as additive to inhibit oxidative

deterioration in oil [269]. CD content of SFO-500 was greater than the other samples

but significantly lower than the control during storage period but its rate increases

tremendously after 20th day of storage.

35
30
25
SFO Ctrl
Conjugated dienes

20 SFO 500
15 SFO 1000
SFO 200A
10
SFO 200T
5
SFO encap
0
5 10 15 20 25 30
Storage period (Days)

Figure 4.6.4.a. Variation in conjugated diene content of control and

treated oil samples
However, rate of increase in CD remained uniform for SFO-1000 and SFO-

encapsulated and comparable with efficiency of BHT. Sample SFO-BHT was found

having lowest CT content followed by the sample containing encapsulated ALE. All

138
the stabilized samples were found having less CT content as compared to control.

Rapid increase in CT content was recorded for SFO BHA, SFO 500, SFO 1000 but

CT content in SFO BHT and SFO encap samples remained almost constant. All these

results are in favor that use of antioxidant extract after encapsulation can increase its

activity due to slow release of the antioxidant molecules. After 20th day nanoparticles

keep releasing antioxidant molecules to the sample that help to prevent the production

of CT. Whereas, in other samples the same quantity of nonencapsulated ALE does not

perform parallel to encapsulated one. It may be due to degradation of antioxidant

molecules under storage condition, whereas encapsulating material protect antioxidant

extract form degradation.

10
9
8
Conjugated trienes

7 SFO Ctrl
6
SFO 500
5
SFO 1000
4
3 SFO 200A
2 SFO 200T
1 SFO encap
0
5 10 15 20 25 30
Storage period (Days)

Figure 4.6.4.b. Variation in conjugated triene content of control and

treated oil samples

4.6.1.e. Thiobarbituric acid reactive substances (TBARS)

For the measurement of secondary oxidation products, TBARS is the most preferred

method. Increase in TBARS was recorded for the samples but this increase was less

as compared to control sample. Results for the measurement of TBARS for ctrl and

stabilized samples are shown in Figure 4.6.5. SFO-1000 and SFO BHA showed

139
similar behavior of increase in TBARS till 30th day. TBARS formation rate was slow

in SFO-BHT, was very slow up to 12th day, and was slightly lower than SFO-BHT

and SOF encap suggesting higher stabilization efficiency of encapsulated ALE as

compared to synthetic antioxidant (BHT).

5
4.5
4
3.5
TBARS µmole/g oil

SFO Ctrl
3
SFO 500
2.5
SFO 1000
2
1.5 SFO 200A
1 SFO 200T
0.5 SFO encap
0
5 10 15 20 25 30
Storage period (Days)

Figure 4.6.5. Variation in TBARS in control and stabilized oil samples

4.6.2. Cinnamon bark deodorised aqueous extract as potential natural

antioxidant in meat emulsion system (Meatballs)

The meatball samples were subjected to peroxide value (PV) and thiobarbituric acid

reactive substances (TBARS) assays to determine the primary and secondary

oxidation products formed, respectively. PV and TBARS values of tested chicken

meatballs samples, i.e. C, T1, T2 and T3 over the entire range of 12 days chilled

storage period (Figure 5.2.1 & 5.2.2). The PV and TBARS values were in the range of

0.878–1.161 meq peroxide/kg sample and 0.041–0.197 mgMDA/ kg sample

respectively on Day 1 of chilled storage. At the end of 12-day storage duration, the

range was 1.076–2.287 meq peroxide/kg sample and 0.070–0.386 mgMDA/kg sample

correspondingly. The increase of both PV and TBARS values mark the

commencement and progression of lipid oxidation in the meatball samples. From

140
Figure 5.2.1, the control sample showed minimal PV changes, which indicate the

occurrence of an induction period (IP) from Day 1 to 3 of chilled storage followed by

sharp rise in PV in the following days; suggesting termination of IP (p< 0.05).

Meatballs supplemented with CinDAE (T1) showed the termination of IP on 9th day

of storage, the difference in shelf-life is too much as compared to control; revealing

appreciable stabilizing potential of CinDAE against lipid peroxidation. The induction

period is a common occurrence during lipid autoxidation, in which the oxidative

changes are minimal. The rate of lipid oxidation increases after the induction period,

causing easier detection of rancidity or a “warmed-over” flavour [270]. On the other

hand, other meatball samples (T2 and T3) did not show the termination of IP, even till

the end of storage period, implying that supplementation of 200 ppm ascorbic acid

and BHA/ BHT can inhibit the formation of hydroperoxide in the meatball samples,

upto much longer periods of time. Throughout the entire storage period, it was found

that meatball samples T1, T2 and T3 demonstrated lower PV compared to control

sample (C). At the end of storage duration, CinDAE (T1), ascorbic acid T2) and

BHA/BHT (T3) were able to reduce the PV value in chicken meatball for

approximately 34.8–53 %, compared to C; in the order given as: ascorbic acid ≥

BHA/BHT ≥ cinnamon bark extract (p>0.05). The lowered PV suggests that less

primary oxidative products were formed in meatball samples, likely caused by the

action of chain-breaking antioxidants such as phenolics and flavonoids present in

CinDAE, which inhibit further lipid peroxidation in the meatball samples. Meanwhile,

Figure 3.2.2 shows that the control sample (C) possessed the highest TBARS value,

i.e. 0.197 mg MDA/ kg sample, on the first day of storage compared to all other

meatball samples (p<0.05). However, the PV for all samples was similar and in the

range of 0.878–1.161 meq peroxide on Day 1 of chilled storage. This indicates that

141
hydroperoxides in the control sample are broken down to form malondialdehyde at a

higher rate than all other samples; at the beginning of storage period. The acceleration

of lipid peroxidation is most likely due to cooking of the meatball samples at 250 °C

before chilled storage. The heating process is one of the main prooxidative processes

of meat products preparation and it might last till the early stages of refrigeration

[271]. However, additions of CinDAE, BHA/BHT and ascorbic acid to the meatball

samples have impeded the progression of lipid oxidation in the meatballs, thus

resulting in lower TBARS values compared to control sample (C).

Samples that were mixed with CinDAE, ascorbic acid and BHA/BHT, i.e. T1, T2 and

T3, exhibited the ability to decrease TBARS values of tested chicken meatballs as

much as 71.7–81 % in comparison to the control sample (C) in the following

sequence: BHA/BHT (T3) > CinDAE (T1) > ascorbic acid (T2) (p<0.05). Reduction

of TBARS values in meatball samples supplemented with CinDAE, ascorbic acid and

BHA/BHT is likely due to lower accumulation of hydroperoxides in T1, T2 and T3 as

mentioned above. In a previous study, Kumar et al. (2011)[272] reported TBARS

value of 0.40–0.45 mg MDA/kg in chicken nuggets formulated with 4 % green

banana and soybean hulls flour on the 15th day of refrigeration. Hence, it can be

inferred that CinDAE may possess hydroperoxide inhibition capability or secondary

antioxidant activity comparable or better than the synthetic antioxidants used in this

study. However, the secondary antioxidant action of CinDAE is not clearly

demonstrated in the present study. Data obtained from PV and TBARS assays suggest

that CinDAE is a good antioxidant with activity comparable to both the reference

antioxidants, i.e. ascorbic acid and BHA/ BHT, at the same concentration of 200 ppm.

An earlier study [273] showed that cinnamon oils and oleoresins have comparable

antioxidant activities with BHA and propyl gallate (PG), as demonstrated by TBARS,

142
PV and linoleic acid systems. These findings are parallel to the present study, which

supports that extracts from cinnamon bark possess high antioxidant activity, thus

ensuring that CinDAE can effectively lower both PV and TBARS values of the tested

meatball samples. In addition, our previous study (data not shown) showed that

CinDAE contains large amounts of phenolic compounds, i.e. gallic acid and other

flavonoids, which might be responsible for the antioxidant activity of CinDAE. As

such, this suggests that the cinnamon extract obtained through hot water extraction

also possessed high antioxidant activity as proven through the meatball model in the

present study.

143
Figure 4.6.6.a. Change in peroxide value of chicken meatballs during storage

Figure 4.6.6.b. Change in TBRAS value of chicken meatballs during storage

144
Part VI

4.7. Structure-activity Relationship of Selected Phenolic Acids in Order to

Propose Mechanism of Their Antioxidative Action

Quantitative structure activity relationship was taken as important tool to study

relationship between structure of phenolic acids and their activity against free radicals

(DPPH• & ABTS•+). In present study we recorded %age radical inhibition of 24

selected phenolic acids.

4.7.1. Descriptors calculation and selection

Initially 1766 descriptors were obtained from DRAGON and Heuristic method was

used to select significant number of descriptors. Initially, reducing step constant or

near constant descriptors were removed step wise. In the 1st step, descriptors having

variance less than 0.0001 were removed and those having 7 or more than 7 zeros were

also removed. In second step, descriptors having weak correlation (r < 0.33 for

ABTS) (r<0.40 for DPPH) with response (%age inhibition) were removed. For ABTS

almost 220 descriptors were left with correlation more than 0.4. For DPPH almost 250

descriptors were obtained with correlation more than 0.3.

Overlapping of information in these descriptors was further reduced by applied the

concept of non-redundant descriptors (NRD). In NRD linear correlation coefficient of

these descriptors was calculated and as a result 22 descriptors were left for ABTS and

08 descriptors for DPPH.

On these descriptors step wise linear regression analysis was performed and seven

best suited descriptors were selected for the further study.

145
4.7.2. QSAR model generation

Step wise multiple linear (SMLR) regression analysis was applied on these remaining

descriptors and finally 5 descriptors were selected which shows best correlation (R-Sq

= 0.913868) with response.

Model based on these five descriptors for prediction of %age inhibition of ABTS•+

ABTS•+ %age inhibition = 18.9 + 11.17 MATS5e + 33.5 Mor31m + 166.2 HATS3p +

0.977 RTe + 4.66 Hy

Model Summary
S R-sq R-sq(adj) R-sq(pred)
9.87655 75.68% 68.92% 58.34%

On the basis of SMLR regression analysis five descriptors were selected that shows

best correlation (R-Sq = 0.913868)

Model based on these five descriptors for prediction of %age inhibition of DPPH•

DPPH percent inhibition = -16.2 + 10.12 MATS5e + 25.5 MATS2p + 27.7 Mor30m +

248 HATS3p + 33.0 R5u

Model Summary
S R-sq R-sq(adj) R-sq(pred)
13.2717 70.79% 49.90% 17.21%

Table 4.7.1. Descriptors selected for modeling purpose (ABTS•+ %age inhibition)
Descriptor Descriptor Name Type
Notation
MATS5e Moran autocorrelation of lag 5 2D autocorrelation
weighted by Sanderson
electronegativity
Mor31m Signal 31 / weighted by mass 3D-MoRSE
descriptors
HATS3p Leverage-weighted autocorrelation of GATEWAY
lag 3 / weighted by polarizability descriptor
RTe R total index / weighted by Sanderson GATEWAY
electronegativity descriptor
Hy Hydrophilic factor Molecular properties

146
Table 4.7.2. Descriptors selected for modeling purpose (DPPH• %age inhibition)
Descriptor Descriptor Name Type
Notation
MATS5e Moran autocorrelation of lag 5 2D autocorrelation
weighted by Sanderson
electronegativity
MATS2p Moran autocorrelation of lag 2 2D autocorrelation
weighted by polarizability
Mor30m signal 30 / weighted by mass 3D-MoRSE
descriptors
HATS3p Leverage-weighted autocorrelation of GATEWAY
lag 3 / weighted by polarizability descriptor
R5u R autocorrelation of lag 5 / GATEWAY
unweighted descriptor

4.7.3. Interpretation of model

4.7.3.a ABTS radical cation scavenging activity

Significant descriptors calculated for model generation for ABTS radical cation

scavenging activity of selected phenolic acids is MATS5e; Moran autocorrelation of

lag 5 weighted by Sanderson electronegativity. This descriptor belong families of 2D

autocorrelation descriptors that interpret certain functions, at interims equal to the lag

d, are correlated. For such descriptor, lag is the topological distance; functions at the

atomic level are correlated. This descriptor describe the distribution of activity under

study along the structure of the selected molecule having polarizable atoms at

topological distance [274]. Another descriptor was found influential is Mor31m;

weighted by mass, that represent a molecular structure based on electron diffraction

[275].

Third descriptor used for the modeling purpose was, HATS3p; leverage-weighted

autocorrelation of lag 3, weighted by atomic polarizabilities. It is a GETAWAY

descriptor that consider molecular geometry in tridimensional manner with certain

chemical facts related to molecule such as mass, polarizability, and electronegativity

[276]. Fourth descriptor used for the modeling is RTe; R total index / weighted by

147
Sanderson electronegativity. It is GETAWAY descriptor and is in coordination with

HATS3p, as both are associated with electronegativity of the molecules.

Last descriptor that was obtained modelling purpose is Hy; Hydrophilic factor, it

indicate the presence of hydrophilic groups present in the molecule. This descriptor is

considered as critically important in anti-radical activity as it indicate that with

increase in hydrophilic groups may cause an increase antiradical potential of the

molecules under study [277].

4.7.3.a DPPH radical scavenging activity

Significant descriptors calculated for model generation for DPPH radical scavenging

activity of selected phenolic acids. First descriptor is same as obtained for ABTS

radical cation scavenging activity and that is MATS5e; Moran autocorrelation of lag 5

weighted by Sanderson electronegativity. This descriptor belong families of 2D

autocorrelation descriptors that interpret certain functions, at interims equal to the lag

d, are correlated. For such descriptor, lag is the topological distance, functions at the

atomic level are correlated. This descriptor describe the distribution of activity under

study along the structure of the selected molecule having polarizable atoms at

topological distance [274]. Second descriptor for the modeling was MATS2p; Moran

autocorrelation of lag 2 weighted by polarizability. It describe the relationship of

polarizability of the molecules with the antiradical activity studied.

Next descriptor used for the modeling is Mor30m; signal 30 / weighted by mass. This

descriptor represents 3D structure of the molecules and is related with volume or mass

of the molecules. It predict that, molecules having bigger substituents may exhibit

high attraction towards free radicals [278].

Third descriptor used for the modeling purpose was, HATS3p; leverage-weighted

autocorrelation of lag 3, weighted by atomic polarizabilities. It is a GETAWAY

148
descriptor that consider molecular geometry in tridimensional manner with certain

chemical facts related to molecule such as mass, polarizability, and electronegativity

[276].

Table 4.7.3. Architecture and specifications used for ANN (ABTS•+ %age
inhibition)
Specifications
No. of neurons in input layer 5
No. of neurons in hidden layer 6
No. of neurons in output layer 1
Training error 0.010835

Test error 0.015046

Hidden activation Gaussian

Output activation Identity

Table 4.7.4. Architecture and specifications used for ANN (DPPH• %age
inhibition)
Specifications
No. of neurons in input layer 5
No. of neurons in hidden layer 6
No. of neurons in output layer 1
Training error 0.024181
Test error 0.022127
Hidden activation Gaussian
Output activation Identity

149
 Table 4.7.5. Experimental and predicted % ABTS•+ inhibition for selected
phenolic acids
Molecule name Molecular Experimental ANN
formula results predicted
results
2-5 Dihydroxybenzoic
82.11 68.38681
acid C7H6O4
3-5-
67.35 71.08419
dihydroxy_benzoic_acid C7H6O4
3,4-
45.01 51.92679
Dihydroxycinnamic acid C7H6O4
2_Hydroxy_benzoic_acid. C7H6O3 58.73 62.88354
3-Hydroxy_benzoic_acid C7H6O3 64.14 54.98147
4-hydroxy_benzoic_acid C7H6O3 59.18 57.26093
Cinnamic_acid C₆H₅CHCHCO₂H 38.21 29.78883
Ellagic_acid C14H6O8 79.14 76.53777
Ferulic_acid C10H10O4 55.82 46.13147
Gallic_acid C7H6O5 83.73 74.91028
Linolenic_acid C18H30O2 78.11 75.96553
p-coumaric_acid. C9H8O3 47.36 52.12843
Picric_acid C6H3N3O7 27.16 41.09469
Sinapic_acid C11H12O5 49.26 42.9968
Syringic_acid C9H10O5 41.43 48.67987
Vanilic_acid C8H8O4 53.49 48.18168
Ascorbic acid C6H8O6 63.37 77.92817
Caffeic_acid C9H8O4 84.42 79.60479
Sorbic_acid C6H8O2 79.81 78.4647
BHT C15H24O 68.41 70.01833

150
Table 4.7.6. Experimental and predicted % DPPH• inhibition for selected
phenolic acids
Molecule name Moleuclar Experimental ANN
formula results Predicted
results
2-5 Dihydroxybenzoic
68 60.57356
acid C7H6O4
3-5-
61 56.90435
dihydroxy_benzoic_acid C7H6O4
3,4-
38 50.29024
Dihydroxycinnamic acid C7H6O4
2_Hydroxy_benzoic_acid. C7H6O3 46 54.23769
3-Hydroxy_benzoic_acid C7H6O3 51 43.9957
4-hydroxy_benzoic_acid C7H6O3 52 38.84006
Cinnamic_acid C₆H₅CHCHCO₂H 34 38.73797
Ellagic_acid C14H6O8 72 59.96626
Ferulic_acid C10H10O4 38 45.71973
Gallic_acid C7H6O5 67 67.05747
linolenic_acid C18H30O2 71 55.37807
p-coumaric_acid. C9H8O3 38 25.99281
picric_acid C6H3N3O7 24 44.27716
Sinapic_acid C11H12O5 40 36.33988
Syringic_acid C9H10O5 29 28.9339
Vanilic_acid C8H8O4 36 42.99013
Ascorbic acid C6H8O6 30 59.0719
Caffeic_acid C9H8O4 82 62.86369
Sorbic_acid C6H8O2 74 61.76546
BHT C15H24O 40 57.06397

151
 Table 4.7.7. Parameters of ANN (ABTS•+ %age inhibition)
Parameters of Hidden Layer Parameters of Output
Layer
Bias 0.939762
Weights of Hidden Layer Weights of Output Layer
Neurons MATS5e Mor31m HATS3p RTe Hy Input bias Neurons
Weights
1 0.000000 0.106711 0.825581 0.1958 0.338825 0.485882 1 -0.494136
2 2
0.335534 0.011628 0.776514 0.0900 0.747954 0.373497 0.036529

3 0.488372 0.489909 1.000000 0.0906 0.536854 0.811535 3 0.130284

4 0.471442 0.289400 0.285870 0.8061 0.093023 0.373497 4 -0.473546
5 0.361206 0.549918 0.295930 0.7590 0.613115 0.445242 5 -0.666095
6 0.346972 0.306931 0.232558 -0.3670 0.943736 0.811535 6 -0.031733

152
 Table 4.7.8. Parameters of ANN (DPPH• %age inhibition)

Parameters of Hidden Layer Parameters of Output

Layer
Neurons Weights of Hidden Layer Bias 0.68351
Weights of Output Layer
MATS5e MATS2p Mor30m HATS3p R5u Input bias Neurons
Weights
1 0.74795 0.26121 0.51892 0.16279 0.37249 0.08338 1 0.00029
2 0.20556 0.49820 0.08140 0.00000 0.90016 0.41361 2 -0.23897
3 0.54595 0.01163 0.65079 0.28587 0.31839 0.08338 3 -0.00033
4 0.09302 0.51852 0.27278 0.84699 0.49369 0.49825 4 -1.71709
5 0.72169 0.73104 0.52705 0.46847 0.30233 0.49825 5 -0.08758
6 0.34697 0.23338 0.42252 0.23256 0.69418 0.28843 6 0.01880

153
 Samples: Train
85

80

75

70
ABTS percent inhibition (Output)

65

60

55

50

45

40

35

30

25

20
20 25 30 35 40 45 50 55 60 65 70 75 80 85 90
ABTS percent inhibition (Target)

Figure 4.7.1. Plot of experimental results vs ANN predicted response (ABTS•+)

154
 Samples: Train
75

70

DPPH percent inhibition (Output) 65

60

55

50

45

40

35

30

25

20
15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90
DPPH percent inhibition (Target)

Figure 4.7.2. Plot of experimental results vs ANN predicted response (DPPH •)

155
 Conclusion

Leaves from different species of Morus were found to be significantly different in context

of all the investigated parameters, revealing the fact that proximate composition,

phenolics, flavonoids and antioxidant activities are considerably affected with variety of

the plant chosen. Due to presence of less anti-nutrient species like fiber while high in

protein and ash contents, M. ruba can be investigated for its nutraceutical applications.

On the other hand, a high amount of phenolics, DPPH radical and ABTS radical cations

scavenging potential suggest the superiority of M. nigra over the other species regarding

their disease preventive potential.

Various solvents having different polarities were employed for the extraction of

antioxidative components present in A. annua leaves, and estimation of antioxidant

activity was done in those extracts using different assays. Our study confirms the

difference in extraction efficiency of various solvents, which suggests the solvent effect

should be taken in to consideration when addressing the antioxidant potential of any

sample. This study points to the benefits of methanol as an extractant for A. annua leaf

nutraceuticals. The evaluation of antioxidant competency of A. annua leaves by means of

different assays recommends the use of these leaves as nutraceuticals.

Optimized extraction of bioactives from leaves of Artemisia annua and Morus nigra was

achieved using RSM. Variation in antioxidant potential was observed as affected by

extraction conditions selected for orbital shaking and microwave-assisted extraction. On

the basis of results conclusion can be drawn that extraction conditions must be taken in to

count, whenever, extraction of bioactives is to be carried out. In addition, statistical tool

156
such as RSM should be employed to recommend optimum extraction conditions as

suggested in this study.

Crude extract of Periploca aphylla (PA) was fractionated and sub-fractionated. Among

different solvent fractions, ButOH fraction exhibited highest antioxidant potential.

Results indicate that, by adopting the procedure of fractionation numerous antioxidant

compounds present in crude extract can be divided in group based on their similar

structure. Butanol was further fractionated using column chromatography and collected

sub-fractions were also tested for their anti-radical potential. Sub-fraction-12 came up

with highest antioxidant potential that represent the class of compounds having promising

group of antioxidants. GC-MS analysis of sub-fraction revealed the presence of different

bioactive compounds but in very small amount. Therefore, it is suggested that

fractionation can be considered rather than sub-fractionation or isolation of any bioactive

compound.

Results obtained in this study clearly demonstrate the effect of simulated gastrointestinal

pH conditions on phenolic composition and antioxidant attributes of DKSM.

Significantly, lesser amount of total phenolics and phenolic acids in treated DKSM

samples as compared to untreated counterparts indicates that bioactive compounds of

potential importance might not be bioavailable after digestion. Furthermore, decrease in

antioxidant potential indicates breakdown of major bioactives or prooxidant activity of

DKSM samples under gastrointestinal pH conditions. Conclusively, keeping in view the

results of our on the basis of findings of this study, the use of defatted konaf seed meal

may not be recommended as it has limited health benefits.

157
Encapsulation of DKSM extract achieved in current study successfully contributed

towards bioavailability enhancement of extract. Encapsulation efficiency and loading

capacity was recorded up to 73% and 48% respectively. Study of release profile under

different pH and temperature conditions confirmed controlled or sustained release of

phenolic content from encapsulated extract. Encapsulation of bioactive extract must be

considered prior use of antioxidants as therapeutic agent or food preservative. On the

basis of results it is concluded that bioavailability bioactive components can be enhanced

using encapsulation technique.

Sunflower oil was administered with ALE, synthetic antioxidants (BHA & BHT) and

encapsulated ALE. Stabilization efficiency was monitored at regular intervals, by

measuring peroxide value, free fatty acids, conjugated diene & trienes and TBRS.

Efficiency of ALE at concentration of 1000 ppm was found comparable with synthetic

antioxidants. It was also recorded that encapsulated extract can contribute towards

enhancement of shelf life in more better way as compared to non-encapsulated extract.

158
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