Thesis Report

on
“Antioxidant, Cytotoxic and Antibacterial Activities of Various
Fractions of Bombax insigne Leaves Extract and Detection of
Compounds in the Most Cytotoxic Fraction by GC-MS”

GONO BISHWABIDYALAY
Submitted By
Name Exam Roll Registration No.
Sanzida Akter 2207 G.Pharma-2919/18
Prity Saha 2209 G.Pharma-2921/18
Bashira Bashar Supty 2211 G.Pharma-2923/18
Mst. Meskat Jahan 2212 G.Pharma-2924/18
Insana Pappa Koly 2217 G.Pharma-2930/18

Supervised by
Md. Golam Mostofa
Assistant Lecturer
Department of Pharmacy
Gono Bishwabidyalay

B.Pharm (33rd Batch)

A dissertation submitted to the Department of Pharmacy, Gono
Bishwabidyalay in the partial fulfillment of the requirements for the
Award of the Degree, Bachelor of Pharmacy (Honors)

Department of Pharmacy
Gono Bishwabidyalay
Savar, Dhaka-1344.
 DECLARATION BY THE CANDIDATE

We hereby declare that this dissertation entitled “Antioxidant,

Cytotoxic and Antibacterial Activities of Various Fractions of
Bombax insigne Leaves Extract and Detection of Compounds in
the Most Cytotoxic Fraction by GC-MS” is an authentic and
genuine research work carried out by us under the guidance of Md.
Golam Mostofa, Department of Pharmacy, Gono Bishwabidyalay,
Savar, Dhaka-1344.

Declared By

Sanzida Akter
Exam Roll: 2207
Reg. No: G.Pharma- 2919/18

Prity Saha
Exam Roll: 2209
Reg. No: G.Pharma -2921/18

Bashira Bashar Supty

Exam Roll: 2211
Reg. No: G.Pharma -2923/18

Mst. Meskat Jahan

Exam Roll: 2212
Reg. No: G.Pharma -2924/18

Insana Pappa Koly

Exam Roll: 2217
Reg. No: G.Pharma -2930/18
 ENDORSEMENT BY CHAIRMAN

This is to certify that the dissertation entitled “Antioxidant, Cytotoxic

and Antibacterial Activities of Various Fractions of Bombax
insigne Leaves Extract and Detection of Compounds in the Most
Cytotoxic Fraction by GC-MS” is a bona fide research work done by
Sanzida Akter (G.Pharma-2919/18), Prity Saha (G.Pharma-2921/18),
Bashira Bashar Supty (G.Pharma-2923/18), Mst. Meskat Jahan
(G.Pharma-2924/18), Insana Pappa Koly (G.Pharma-2930/18) under
the guidance of Md. Golam Mostofa, Department of Pharmacy, Gono
Bishwabidyalay, Savar, Dhaka.

Dr. Nilay Kumar Dey

Professor and Chairman
Department of Pharmacy
Gono Bishwabidyalay
Savar, Dhaka-1344.
 CERTIFICATE BY THE SUPERVISOR

This is to certify that the dissertation entitled is an authentic and

genuine research work done by Sanzida Akter (G.Pharma-2919/18),
Prity Saha (G.Pharma-2921/18), Bashira Bashar Supty
(G.Pharma-2923/18), Mst. Meskat Jahan (G.Pharma-2924/18) and
Insana Pappa Koly (G.Pharma-2930/18), under my guidance and
supervision. This work has been done in Laboratories of Gono
Bishwabidyalay, Savar, Dhaka-1344. These candidates have fulfilled
all the terms and conditions of the thesis work.

Md. Golam Mostofa

Assistant Lecturer
Department of Pharmacy
Gono Bishwabidyalay
Savar, Dhaka-1344
 ACKNOWLEDGMENT

All praises to Almighty Allah who give us strength, patience, and ability to
perform our research work successfully.

We would like to express our best regards, heartfelt gratefulness, deep

appreciation to our honorable, amicable, dynamic, and beloved supervisor, Md.
Golam Mostofa, Assistant Lecturer, Department of Pharmacy, Gono
Bishwabidyalay for his dedicated supervision, proficient guidance, priceless
suggestions, generous help, encouragement, and co-operation throughout the
entire period of our research work as well as to prepare this dissertation. We are
indebted a lot to him and we feel proud for giving us such an opportunity to
work in close association with him.

We would like to offer extraordinary indebtedness and gratitude to Dr. Nilay

Kumar Dey, Professor and Chairman, Department of Pharmacy, Gono
Bishwabidyalay, for his valuable suggestions, inspiration, and support during
our research work.

We are indeed grateful to all of our Lab Officer and Lab Assistants, Department
of Pharmacy, Gono Bishwabidyalay, for their cordial co-operation during our
research work.

Enormous thanks to the Department of Microbiology, Gono Bishwabidyalay,

for helping us with their expertise in microbial assays.

Finally, we would like to express our indebted gratitude to our parents and
family members who inspired and supported us throughout our research work.
 LIST OF CONTENT

CHAPTER ONE: GENERAL INTRODUCTION 1-32

1.1 Oxidative Stress (OS) 1

1.2 Free Radical 1
1.2.1 Classification of Free Radical 2
1.2.2 Mechanism of Formation of Free Radicals 2-3
1.2.3 Production of Free Radical in The Human Body 3
Mechanism of Disease Formation by Free
1.2.4 4
Radicals
1.3 Leading Cause of Death 5
1.4 Oxidative Stress and Human Disease 6
1.4.1 Oxidative Stress and Cancer 7
1.5 Cancer 7-8
1.5.1 Causes of Cancer 8-9
1.5.2 Oxidative Stress Develops Cancer 10
1.6 Oxidative Stress and Cardiovascular Disease 10-11
1.7 Oxidative Stress and Diabetes 11-12
1.8 Preventing of Oxidative Stress 12-13
1.9 Antioxidant 13
1.9.1 Types of Antioxidants 14
1.9.2 Antioxidant Defense System 14
1.9.3 Mechanism of Action of Antioxidants 15
1.9.4 Plant source of Antioxidant 15-17

I
 1.9.5 Antioxidant and Cancer 17
1.10 Plants as a Source of Drugs in Cancer Therapy 18-20
1.11 Description of Plant Investigated 20
1.11.1 The Plant Family: Bombacaceae 20-21
1.11.2 Botanical Features of Bombax insigne 21-26
1.12 Objective 26-27
1.13 Present Study Protocol 27-28
1.14 Reference 28-32

CHAPTER TWO: LITERATURE REVIEW 33-62

2.1 Chemical Work 33

2.1.1 Steroid 33-34
2.1.2 Flavonoid 34-37
2.1.3 Terpenoid 37-39
2.1.4 Phenolic Compound 39
2.1.5 Glycoside 40-41
2.1.6 Cardiac Glycoside 41-42
2.1.7 Tannin 42-43
2.1.8 Alkaloid 44
2.1.9 Saponin 45
2.1.10 Carbohydrate 46-47
2.1.11 Protein 47

II
 2.1.12 Others 48
2.2 Biological Work 48-49
2.2.1 Anticancer and Cytotoxic Activity 49
2.2.2 Antioxidant Activity 49-50
Antimicrobial, Antibacterial, Antiviral, and
2.2.3 50-51
Antifungal Activity
2.2.4 Antidiabetic Activity 51-52
2.2.5 Antipyretic Activity 52
2.2.6 Antidiarrheal Activity 52
2.2.7 Anti-inflammatory Activity 52
2.2.8 Analgesic Property 53
2.2.9 CNS Activity 53
2.2.10 Anti-Obesity Activity 53
2.2.11 Anti-hypertensive Activity 53
2.2.12 Anti-angiogenic Activity 53-54
2.2.13 Hepatoprotective Activity 54
2.2.14 Others Activity 54-55
2.3 Reference 55-62

CHAPTER THREE: METHODS AND MATERIALS 63-97

3.1 Chemical Work of Bombax insigne 63-67

3.1.1 General Methods 63
Collection and Proper Identification of
3.1.1.1 63
Plant
3.1.1.2 Preparation of Plant Material 63

III
 3.1.1.3 Extraction 63
3.1.1.3.1 Cold Extraction 64
3.1.1.3.2 Hot Extraction 64-65
3.1.1.3.3 Extraction with Solvent 65
3.1.2 Chemical Studies on Bombax insigne 65
3.1.2.1 Collection and Identification of Plant 65-66
3.1.2.2 Preparation of Plant’s Leaves 66
3.1.2.3 Extraction of Plant’s Leaves 67
Solvent-solvent Partitioning of Crude
3.1.2.4 68-69
Extract
3.1.2.4.1 Modified Kupchan Method 69-70
3.1.3 Qualitative Phytochemical Analysis 70
3.1.3.1 Test for Saponins 70
3.1.3.2 Test for Tannins 71
3.1.3.3 Test for Glycosides 71
3.1.3.4 Test for Steroids 71
3.1.3.5 Test for Alkaloids 71
3.1.3.6 Test for Carbohydrate 72
3.1.3.7 Test for Protein 72
3.1.4 Quantitative Phytochemical Analysis 72
3.1.4.1 Determination of Total Phenolics 72
3.1.4.1.1 Principle 72
3.1.4.1.2 Materials and Apparatus 73
3.1.4.1.3 Experimental Procedure 73-74

IV
 3.1.4.2 Determination of Total Flavonoids 74-75
3.1.4.2.1 Principle 74
3.1.4.2.2 Materials and Apparatus 74
3.1.4.2.3 Experimental Procedure 74-75
3.1.4.2.3 GC-MS Analysis of CHF 75-76
3.1.4.3.1 Principle 75
3.1.4.3.2 Reagent and Apparatus 75
3.1.4.3.3 Methodology 76
3.2 In-Vitro Antioxidant Activity 77-82
Determination of Total Antioxidant
3.2.1 77-79
Capacity
3.2.1.1 Principle 77
3.2.1.2 Materials and Apparatus 77-78
3.2.1.3 Experimental Procedure 78
3.2.2 Reducing Power Assessment 78-80
3.2.2.1 Principle 78-79
3.2.2.2 Materials and Apparatus 79
3.2.2.3 Experimental Procedure 79-80
DPPH (1,1-diphenyl-2-picrylhydrazyl)
3.2.3 80-82
Free Radical Scavenging Activity
3.2.3.1 Principle 80-81
3.2.3.2 Materials and Apparatus 81
3.2.3.3 Experimental Procedure 81-82
3.3 In-Vivo Biological Activity 82-87
Evaluation of Cytotoxic Activity by
3.3.1 82
Brine Shrimp Bioassay

V
3.3.1.1 Principle 82-83
3.3.1.2 Materials and Apparatus 83-84
3.3.1.3 Preparation of Brine Water 84
3.3.1.4 Hatching of Brine Shrimp Eggs 84
3.3.1.5 Preparation of the Test Sample 84-85
3.3.1.6 Preparation of Control Groups 85
Preparation of the Positive Control
3.3.1.7 85
Group
Preparation of the Negative Control
3.3.1.8 85-86
Group
3.3.1.9 Application of Brine Shrimp Nauplii 86
3.3.1.10 Counting of Nauplii 86
3.3.1.11 Analysis of the Data 86
3.3.2 Antibacterial Screening 87-
Principle of Disc Diffusion Assay
3.3.2.1 88-89
Method
3.3.2.2 Experimental Procedure 89-94
3.3.2.2.1 Test Materials Used for the Study 89
3.3.2.2.2 Materials and Apparatus 89-90
3.3.2.2.3 Test Organisms 90
3.3.2.2.4 Sterilization Procedure 90-91
3.3.2.2.5 Culture Media 91
3.3.2.2.6 Preparation of Medium 91
3.3.2.2.7 Preparation of Subculture 92
3.3.2.2.8 Preparation of Test Plates 92
3.3.2.2.9 Preparation of Discs and Test Samples 92-94

VI
 Placement of the Discs, Diffusion and
3.3.2.2.10 94
Incubation
3.3.2.2.11 Measurement of Zone of Inhibition 94
3.4 Reference 94-97

CHAPTER FOUR: RESULT 98-123

4.1 Chemical Studies 98-119

4.1.1 Preparation of Crude Extract 98
Solvent-solvent Partitioning of Crude
4.1.2 98-99
Extract
4.1.3 Qualitative Phytochemical Analysis 99
4.1.4 Quantitative Analysis 100-105
4.1.4.1 Determination of Total Phenolics 100-102
4.1.4.2 Determination of Total Flavonoids 103-105
4.1.5 In-vitro Antioxidant Activity 106-116
4.1.5.1 Total Antioxidant Activity 106-109
4.1.5.2 Reducing Power Assessment 109-112
4.1.5.3 DPPH Free Radical Scavenging Assay 112-116
Evaluation of Cytotoxic Activity by Brine
4.1.6 116-119
Shrimp Lethality Bioassay
4.2 GC-MS Analysis of CHF 120-121
4.3 In-Vitro Antibacterial Activity
4.3.1 Determination of Zone of Inhibition 121-122
4.4 Reference 123

VII
 CHAPTER FIVE: DISCUSSION 124-132

5 Discussion 124-125
5.1 Qualitative Phytochemical Analysis 125
5.2 Phenolic and Flavonoid Contents 125-126
5.3 Total Antioxidant Capacity 126-127
5.4 Reducing Power Assessment 127
5.5 DPPH Free Radical Scavenging Activity 128
5.6 Cytotoxic Effect on Brine Shrimp nauplii 128-129
5.7 GC-MS Analysis of CHF 129
5.8 Antibacterial Screening 130
5.9 Reference 130-133

6 CHAPTER-SIX: SUMMARY 134-135

VIII
 LIST OF TABLES

CHAPTER ONE: GENERAL INTRODUCTION

Table 1.1 List of some anticancer drugs obtained from 18-19
plants

CHAPTER THREE: METHODS AND MATERIALS

Table 3.1 List of tested bacteria 92
Table 3.2 Composition of nutrient agar media 93

CHAPTER FOUR: RESULT

Results of qualitative tests of different
Table 4.1 fractions of methanolic extract of B. insigne 99
leaves
Absorbance of GA (standard) at different
Table 4.2 100
concentrations after treatment with FCR
Determination of total phenolic contents of
Table 4.3 CME of B. insigne leaves and its four fractions 101
(NHF, CHF, EAF and AQF)
Absorbance of Quercetin (standard) at
Table 4.4 different concentrations for quantitative 103
determination of total flavonoids
Determination of total flavonoids contents of
Table 4.5 CME of B. insigne leaves and its four fractions 104
(NHF, CHF, EAF and AQF)
Determination of total antioxidant activity of
Table 4.6. CME of B. insigne leaves and AA at different 106
concentrations
Table 4.7 Determination of total antioxidant activity of 108

IX
 four fractions of CME of B. insigne leaves at
different concentrations
Determination of Fe3+ reducing power capacity
Table 4.8 of CME of B. insigne leaves and AA at 110
different concentrations
Determination of reducing power capacity of
Table 4.9 four fractions of CME of B. insigne leaves at 111
different concentrations
Determination of DPPH free radical
Table 4.10 scavenging activity of the CME of B. insigne 113
leaves and BHT at different concentrations
Determination of DPPH free radical
114-
Table 4.11 scavenging activity of four fractions of CME
115
of B. insigne leaves at different concentrations
Effect of VCS (standard) on mortality of Brine
Table 4.12 117
Shrimp nauplii at different concentrations
Effect of CME of B. insigne leaf and its four
118-
Table 4.13 fractions on mortality of Brine Shrimp nauplii
119
at different concentrations
120-
Table 4.14 Detected Compounds by GC-MS
121
Antibacterial activity (zone of inhibition, cm)
of different extracts of B. insigne leaves at 200
Table 4.15 122
μg/Disc and 400 μg/Disc and standard at 50
μg/Disc

X
 LIST OF FIGURES

CHAPTER ONE: GENERAL INTRODUCTION

Mechanism of disease formation by free
Fig 1.1 4
radicals & other chemicals
Trends in the incidence of New Cancer Case
Fig 1.2 8
in Bangladesh:2015-2035
Fig 1.3 Causes of cancer 9
Fig 1.4 Mechanism of development of Cancer by OS 10
Fig 1.5 Mechanism of action of antioxidants 15
Mechanism of free radical scavenging by
Fig 1.6 18
antioxidants.
Fig 1.7a Bombax insigne Wall. 23
Fig 1.7b Bombax insigne Wall. 24

CHAPTER THREE: METHODS AND MATERIALS

Fig. 3.1 Rotary Evaporator 64

Fig. 3.2 Soxhlet Extractor 64

Fig. 3.3 Leaves of B. insigne 66

Fig. 3.4 Powder of B. insigne leaves 66

Fig. 3.5 Extraction process of B. insigne 67

Schematic representation of solvent-solvent
Fig. 3.6 partitioning of the crude methanolic extract 68
of B. insigne leaves.
Solvent-solvent partitioning with different
Fig. 3.7 69
solvents.

XI
 CHAPTER FOUR: RESULT
Quantity (gm) of four fraction of 30 gm
Fig. 4.1 99
CME of B. insigne leaves
Standard curve of GA for the determination
Fig. 4.2 101
of total phenolics.
Determination of total phenolic contents of
Fig. 4.3 CME of B. insigne leaves and its four 102
fractions.
Standard curve of Quercetin for the
Fig. 4.4 104
determination of total flavonoids.
Determination of total flavonoids contents
Fig. 4.5 of CME of B. insigne leaves and its four 105
fractions.
Comparison of total antioxidant activity of
Fig. 4.6 CME of B. insigne leaves and AA at 107
different concentrations.
Comparison of total antioxidant activity of
Fig. 4.7 CME of B. insigne leaves and its four 109
fractions with AA.
Comparison of reducing power capacity of
Fig. 4.8 CME of B. insigne leaves and standard, AA 110
at different concentrations.
Comparison of reducing power capacity of
CME of B. insigne leaves and its four
Fig. 4.9 112
fractions with AA at different
concentrations.
Comparison of DPPH free radical
scavenging activity of CME of leaves and
Fig. 4.10 114
BHT (standard) at different log
concentrations
Comparison of DPPH free radical
scavenging activity of CME of B. insigne
Fig. 4.11 115
leaves and its four fractions with BHT at
different log concentrations.
IC50 (µg/mL) values of different extractives
Fig. 4.12 116
of B. insigne leaves and standard, BHT for

XII
 DPPH free radical scavenging.
Effects of VCS (standard) on the mortality
Fig. 4.13 of Brine Shrimp nauplii at different 117
concentrations.
LC50 (µg/mL) values of different
extractives of B. insigne leaves and
Fig. 4.14 119
standard, VCS for Brine Shrimp lethality
Bioassay.
Fig. 4.15 GC-MS spectra of Chloroform Fraction 121
Antibacterial activity of different fraction at
Fig. 4.16 122
different concentration.

XIII
List of Abbreviations, Acronyms and Symbols
OS Oxidative Stress

ROS Reactive Oxygen Species

WHO World Health Organization

GBD Global Burden of Disease

BBS Bureau of Statistics

RS Reactive Species

CVD Cardiovascular Disease

CAD Coronary Artery Disease

FDA Food And Drug Administration

AA Ascorbic Acid

GAE Gallic Acid Equivalent

CME Crude Methanolic Extract

NHF n-Hexane Fraction

CHF Chloroform Fraction

AQF Aqueous Fraction

EAF Ethyl Acetate Fraction

BHT Butylate Hydroxy Toluene

DPPH 1,1- diphenyl-2-picrylhydrazyl

Conc. Concentration

IC50 Half Maximal Inhibitory Concentration

FCR Folin-coicalteu Reagent

FeCl3 Ferric Chloride

 FeSO4 Ferrous Sulphate

GA Gallic Acid

KCl Potassium Chloride

K2HPO4 Dipotassium Hydrogen Phosphate

KH2PO4 Potassium Dihydrogen Phosphate

B. insigne Bombax insigne

RT Room Temperature

NaCl Sodium chloride

ºC Degree Celsius

STD Standard Deviation

UV Ultraviolet

μL Micro Liter

µg Microgram

ml Milliliter

mM Millimolar
 Abstract

The aim of this study was to find out the potential antioxidative fraction
and to detect compounds in the most cytotoxic fraction of B. insigne
leaves. A detailed study was performed to select the potential cytotoxic
and antioxidative fraction(s) using Brine shrimp lethality bioassay and
standard UV spectroscopic methods, respectively. The detection of
compounds was carried out by GC-MS analysis. The antibacterial activity
of crude methanolic extract (CME) and its different fractions, such as n-
Hexane (NHF), chloroform (CHF), ethyl acetate (EAF), and aqueous
(AQF) fraction was determined by the disk diffusion assay method.

Among all the extracts, EAF had showed the highest phenolic and
flavonoids contents. EAF also had the highest antioxidant compounds and
reducing power. The DPPH free radical scavenging activities of different
extractives of B. insigne leaves and standards with their IC50 (µg/mL)
exhibited the order as EAF > CME > AQF > BHT > NHF > CHF
suggest that the EAF had the profound free radical scavenging activities.

CHF showed the highest cytotoxic activity on Brine shrimp among the
extracts. Therefore, CHF had considered for GC-MS analysis to detect
compounds.

GC-MS data revealed that CHF contained 9 compounds such as 1-

Octadecanesulphonyl chloride (98.175%), Diisooctyl phthalate (0.720%),
Zinc,bis(dipentylcarbamodithioato-S,S')-,(T-4)- (0.627%), L-
Sorbofuranose, pentakis (trifluoroacetate) (isomer 1) (0.157%), 2-
methyltetracosane. Of them, 1-Octadecanesulphonyl Chloride is a
phenolic natural antifungal and antimicrobial compound. 2-
methyltetracosane has free radical scavenging activity.
 Abstract

But among all the extracts of CME, no one had antibacterial activity
against both the tested Gram-positive and Gram-negative bacterial strain
at 200 μg/disc and 400 μg/disc.
Chapter One

General
Introduction
 Chapter One (General Introduction)

1. General Introduction
1.1. Oxidative Stress (OS)
OS is essentially an imbalance between the production of free radicals and the
ability of the body to counteract or detoxify their harmful effects through
neutralization by antioxidant (Subramaniam et al., 2020). OS is harmful because
oxygen free radicals attack biological molecules such as lipids, proteins, and
DNA. Disturbances in the normal redox state of tissues can cause toxic effects
through the production of reactive oxygen species (ROS), such as peroxides and
free radicals that damage all components of the cell. By lipid peroxidation or by
oxidizing DNA or proteins, the severe levels of ROS cause necrosis; this damage
causes ATP depletion, preventing controlled apoptotic death and causing the cell
to simply fall apart (Evans et al., 2015). However, OS also has a useful role in
physiologic adaptation and in the regulation of intracellular signal transduction
(Yoshikawa & Naito, 2002).

1.2. Free Radical

A free radical is as any molecular species capable of independent existence that
contains an unpaired electron in an atomic orbital. The presence of an unpaired
electron results in certain common properties that are shared by most radicals.
Many radicals are unstable and highly reactive. They can either donate an electron
to or accept an electron from other molecules, therefore behaving as oxidants or
reductants (Cheeseman & Slater, 1993). The most important oxygen-containing
free radicals in many disease states are hydroxyl radical, superoxide anion radical,
hydrogen peroxide, oxygen singlet, hypochlorite, nitric oxide radical, and per-
oxy-nitrite radical. These are highly reactive species, capable in the nucleus, and
in the membranes of cells of damaging biologically relevant molecules such as
DNA, proteins, carbohydrates, and lipids (Young & Woodside, 2001). Free

1
 Chapter One (General Introduction)

radicals attack important macromolecules leading to cell damage and homeostatic

disruption. Targets of free radicals include all kinds of molecules in the body.
Among them, lipids, nucleic acids, and proteins are the major targets. Free radical
reactions are expected to produce progressive adverse changes that accumulate
with age throughout the body, such normal changes with age are relatively
common to all. However, superimposed on this common pattern are patterns
influenced by genetics and environmental differences that modulate free radical
damage. These are manifested as diseases at certain ages determined by genetic
and environmental factors. Cancer and atherosclerosis, two major causes of death,
are salient “free radical” diseases (Lobo et al., 2010)

1.2.1. Classification of Free Radical

According to the classification proposed, the radicals may be divided into

I. Primary (superoxide, semi quinones and nitric oxide),

II. Secondary (hydroxyl and lipid radicals) and
III. Tertiary (radicals of antioxidants).

The primary radicals are formed by enzymatic systems and perform biologically
important functions. The secondary radicals are formed from hydroperoxides in
the reactions of divalent iron ions and damage to cell structures. (Vladimirov,
1998)

1.2.2. Mechanism of Formation of Free Radicals

Free radicals are the natural byproducts of chemical processes, such as

metabolism. Food, medicine, air, water can generate free radicals. Free radicals
can be formed by three ways-

2
 Chapter One (General Introduction)

❖ By hemolytic cleavage of covalent bond of normal molecule with

each fragment retaining one paired of electrons
X:Y X* + Y*

❖ By the loss of single electron from normal molecule

X:Y X+ + Y-

❖ By addition of single electron to normal molecule

X:Y X-

A radical might donate its unpaired electron to other molecules. It might take
electron from other molecules in order to pair or it might simply join to the
molecule and thus proceed as a chain reaction and produce a free radical.

1.2.3. Production of Free Radical in The Human Body

Free radicals and other ROS are derived either from normal essential metabolic
processes in the human body or from external sources such as exposure to X-rays,
ozone, cigarette smoking, air pollutants, and industrial chemicals. Free radical
formation occurs continuously in the cells as a consequence of both enzymatic
and non-enzymatic reactions. Enzymatic reactions, which serve as source of free
radicals, include those involved in the respiratory chain, in phagocytosis, in
prostaglandin synthesis, and in the cytochrome P-450 system (Liu et al., 1999;
Lobo et al., 2010). Free radicals can also be formed in non-enzymatic reactions
of oxygen with organic compounds as well as those initiated by ionizing
reactions.

Some internally generated sources of free radicals are

• Mitochondria

• Xanthine oxidase

• Peroxisomes

3
 Chapter One (General Introduction)

• Inflammation

• Phagocytosis

• Exercise

• Ischemia/reperfusion injury

• Some externally generated sources of free radicals are:

• Cigarette smoke

• Environmental pollutants

• Radiation

• Certain drugs, pesticides

• Industrial solvents

• Ozone (Lobo et al., 2010)

1.2.4. Mechanism of Disease Formation by Free Radicals

Fig.1.1: Mechanism of disease formation by free radicals & other chemicals.

(Modification of Sharma 2014)

4
 Chapter One (General Introduction)

1.3. Leading Cause of Death

According to WHO, In the world total 56 million people are died in 2019 for
various disease. Among them CVD is the first leading cause of death. An
estimated 17.9 million people died from CVDs in 2019, representing 32% of all
global deaths. Of these deaths, 85% were due to heart attack and stroke. Over
three quarters of CVD deaths take place in low- and middle-income countries.
Prevalent cases of total CVD nearly doubled from 271 million in 1990 to 523
million in 2019 which is reported by GBD (Roth et al., 2020). According to
GLOBOCAN, Cancer is the second leading cause of death and 10.8 million
people died. Lung cancer remained the leading cause of cancer death, with an
estimated 1.8 million deaths (18%), followed by colorectal (9.4%), liver (8.3%),
stomach (7.7%), and female breast (6.9%) cancers. The global cancer burden is
expected to be 28.4 million cases in 2040, a 47% rise from 2020 (Sung et al.,
2021). For the Respiratory disease 3.97 million, lower respiratory infections 2.49
million, Diabetes 1.55 million and 1.2 million death are responsible for
Alzheimer’s disease (Naghavi et al., 2017). Global Burden of Disease study –
estimates that 8.7 million people die prematurely from tobacco use every year.
As of June 2021, these are the latest estimates and refer to deaths in the year 2019.
7.7 million of those deaths are the result of smoking, 1.3 million are non-smokers
who are dying because they are exposed to second-hand smoke. Cancer and
respiratory diseases are the top two killers in Bangladesh, with two being
responsible for about 25% of all deaths in the country. A total of 8,54,253 people
died due to different causes in 2020 and around 21.1 percent died of cardiac arrest,
according to the Bangladesh Bureau of Statistics (BBS). Some 1,80,408 people
have died of heart attacks, according to the data revealed by the BBS(Heart
Attacks the Most Common Cause of Death in Bangladesh Last Year: BBS | The
Daily Star, 2021). The leading cause of adult male death in Bangladesh was
reported by VA to be ischemic heart disease (25.9%), accounting for about 50%

5
 Chapter One (General Introduction)

more deaths than the next two causes, stroke and chronic respiratory disease.
Collectively, these three diseases (ischemic heart disease, stroke, chronic
respiratory disease) comprise 62% and 60% of all adult deaths in males and
females respectively. Diabetes, chronic kidney disease, cirrhosis and pneumonia
appear in the top 15 causes for both sexes. Road traffic accident (3%), lung cancer
(3%), prostate cancer (2%), esophageal cancer (1%), and TB (1%) feature in the
top 15 leading causes of male death but not for women for whom falls (2%),
diarrhea/dysentery (2%), maternal (2%), breast cancer (2%), and cervical cancer
(2%) matter more (Shawon et al., 2021). Diabetes is linked to an estimated 3% of
all deaths in Bangladesh, making the disease one of the top causes of mortality in
the Asian country. Interestingly, the rate of diabetes cases in Bangladesh recorded
a dramatic increase in the early 21st Century prompting health officials to come
up with measures to contain the spread of the disease. Diabetes has now become
one of the biggest health concerns in Bangladesh (The 10 Leading Causes Of
Death In Bangladesh - WorldAtlas, 2018.).

1.4. Oxidative Stress and Human Disease

Oxidative stress has been life threatening conditions, including cancers,

atherosclerosis, inflammatory condition and the process of aging., ischemic
diseases (heart diseases, stroke, intestinal ischemia), hemochromatosis, acquired
immunodeficiency syndrome, emphysema, organ transplantation, gastric ulcers,
hypertension and preeclampsia, neurological disorder (Alzheimer's disease,
Parkinson's disease, muscular dystrophy) (Lobo et al., 2010).

6
 Chapter One (General Introduction)

1.4.1. Oxidative Stress and Cancer

Reactive species’ (RS) of various types are formed in vivo and many are powerful
oxidizing agents, capable of damaging DNA and other biomolecules. Increased
formation of RS can promote the development of malignancy, and the ‘normal’
rates of RS generation may account for the increased risk of cancer development
in the aged. Indeed, knockout of various antioxidant defense enzymes raises
oxidative damage levels and promotes age-related cancer development in
animals. In explaining this, most attention has been paid to direct oxidative
damage to DNA by certain RS, such as hydroxyl radical (OH •). However,
increased levels of DNA base oxidation products such as (8-hydroxy-2′-
deoxyguanosine) do not always lead to malignancy, although malignant tumors
often show increased levels of DNA base oxidation. Hence additional actions of
RS must be important, possibly their effects on p53, cell proliferation,
invasiveness and metastasis. Chronic inflammation predisposes to malignancy,
but the role of RS in this is likely to be complex because RS can sometimes act
as anti-inflammatory agents (Barry Halliwell, 2007).

1.5. Cancer
Cancer is the second-leading cause of death in the world. Accounting for nearly
10 million deaths in 2020 (International Agency for Research on Cancer; 2020).
According to GLOBOCAN 2020 over 19.3 million new cases of cancer will be
diagnosed annually and will be died over 10.0 million a year (Sung et al., 2021).
Cancer is the malignant growth due to uncontrolled cell division which can invade
nearby parts of the body as well as spread distant parts of the body (known as
metastasis) through blood stream (López-Lázaro, 2018). It is now used as a
general term for over a hundred diseases characterized by the uncontrolled,
abnormal growth of cells. It is also known as malignant neoplasia (Magrath,
1989).The most common in 2020 (in terms of new cases of cancer) were breast

7
 Chapter One (General Introduction)

(2.26 million); lung (2.21 million); colon and rectum (1.93 million); prostate
(1.41 million); skin (non-melanoma) (1.20 million); and stomach (1.09 million).
The most common causes of cancer death in 2020 were lung (1.80 million); colon
and rectum (0.91 million); liver (0.83million); stomach (0.77million); and breast
(0.68 million). Approximately 39.5% of men and women will be diagnosed with
cancer at some point during their lifetimes (based on 2015–2017 data). In 2020,
an estimated 16,850 children and adolescents ages 0 to 19 will be diagnosed with
cancer and 1,730 will die of the disease (International Agency for Research on
Cancer). Based on projections, cancer deaths will continue to rise with an
estimated 11.4 million dying in 2030. Breast, esophageal, and cervical cancers
are the most common cancers by incidence in Bangladesh. However, esophageal,
lung, and pharyngeal cancers account for the highest rates of cancer-related
mortality. The incidence of cancers is expected to rise from 136,719 cases in 2015
to 250,726 cases in 2035 (Are, 2017).

Fig.1.2: Trends in the incidence of New Cancer Case in Bangladesh:2015-

2035 (Source: Cancer on the Global Stage: Incidence and Cancer-Related
Mortality in Bangladesh - The ASCO Post)

1.5.1. Causes of Cancer

Cancer is a complex group of diseases with many possible causes include cancer-
causing chemicals, called carcinogens, such as tobacco smoke, chemotherapies,

8
 Chapter One (General Introduction)

asbestos, benzene, vinyl chloride, and many more, as well as environmental

factors, such as ultraviolet light, pollution, and hepatitis B. Smoking is the most
important preventable cause of cancer in the world (William C. Shiel Jr., 2017).
Overweight and obesity is the second biggest preventable cause of cancer after
smoking. Only 5–10% of all cancer cases can be attributed to genetic defects
(Hahn & Weinberg, 2002), whereas the remaining 90– 95% have their roots in
the environment and lifestyle (Czene & Hemminki, 2002). The lifestyle factors
include cigarette smoking, diet (fried foods, red meat), alcohol, sun exposure,
environmental pollutants, infections, Oxidative stress, obesity, and physical
inactivity. The evidence indicates that of all cancer-related deaths, almost 25–
30% are due to tobacco, as many as 30–35% are linked to diet, 15% of cancers
are due to infections such as Helicobacter pylori, hepatitis B, hepatitis C, human
papillomavirus infection, Epstein–Barr virus and human immunodeficiency virus
(HIV) and the remaining percentage are due to other factors like radiation, stress,
physical activity, environmental pollutants etc. (Anand et al., 2008).

Fig.1.3: Causes of cancer

Source: What are the main causes of cancer? (researchgate.net)

9
 Chapter One (General Introduction)

1.5.2. Oxidative Stress Develops Cancer

Cancer initiation and progression have been associated with oxidative stress
by enhancing DNA mutations or increasing DNA damage, genome variability,
and cell proliferation, and hence antioxidant agents could intervene with
carcinogenesis (Mileo & Miccadei, 2016).

Fig.1.4: Mechanism of development of Cancer by OS

Source:https://www.researchgate.net/publication/40696720_Oxidative_Stre
ss_and_Oxidative_Damage_in_Carcinogenesis.

1.6. Oxidative Stress and Cardiovascular Disease

Cardiovascular disease (CVD) is a class of disease that involved the heart or
blood vessel. CVD includes coronary artery disease (CAD) such as angina and
myocardial infarction (commonly known as a heart attack). Other CVDs include

10
 Chapter One (General Introduction)

stroke, heart failure, hypertensive heart disease, rheumatic heart disease,

cardiomyopathy, abnormal, heart disease, peripheral artery disease,
thromboembolic disease and venous thrombosis (Wang et al., 2016).

Cardiovascular diseases (CVDs) are the leading cause of death globally. An

estimated 17.9 million people died from CVDs in 2019, representing 32% of all
global deaths. Of these deaths, 85% were due to heart attack and stroke. Over
three quarters of CVD deaths take place in low- and middle-income countries.
Out of the 17 million premature deaths (under the age of 70) due to non-
communicable diseases in 2019, 38% were caused by CVDs. Most cardiovascular
diseases can be prevented by addressing behavioral risk factors such as tobacco
use, unhealthy diet and obesity, physical inactivity and harmful use of alcohol
(Mileo & Miccadei, 2016).

1.7. Oxidative Stress and Diabetes

Diabetes mellitus is a group of metabolic disorders that is characterized by
elevated levels of glucose in blood (hyperglycemia) and insufficiency in
production or action of insulin produced by the pancreas inside the body (Maritim
et al., 2003). Insulin is a protein (hormone) synthesized in beta cells of pancreas
in response to various stimuli such as glucose, sulphonyl urea, and arginine
however glucose is the major determinant (Joshi et al., 2007). Long term
elevation in blood glucose levels is associated with macro- and micro-vascular
complications leading to heart diseases, stroke, blindness and kidney diseases.
Sidewise to hyperglycemia, there are several other factors that play great role in
pathogenesis of diabetes such as hyperlipidemia and oxidative stress leading to
high risk of complications.

Human body is continuously exposed to different types of agents that results in

the production of reactive species called as free radicals (ROS/RNS) which by

11
 Chapter One (General Introduction)

the transfer of their free unpaired electron causes the oxidation of cellular
machinery. In order to encounter the deleterious effects of such species, body has
got endogenous antioxidant systems or it obtains exogenous antioxidants from
diet that neutralizes such species and keeps the homeostasis of body. Any
imbalance between the RS and antioxidants leads to produce a condition known
as "oxidative stress" that results in the development of pathological condition
among which one is diabetes. Most of the studies reveal the inference of oxidative
stress in diabetes pathogenesis by the alteration in enzymatic systems, lipid
peroxidation, impaired Glutathione metabolism and decreased Vitamin C levels.
Lipids, proteins, DNA damage, Glutathione, catalane and superoxide dismutase
are various biomarkers of oxidative stress in diabetes mellitus. Oxidative stress
induced complications of diabetes may include stroke, neuropathy, retinopathy
and nephropathy (Asmat et al., 2016).

The global diabetes prevalence in 2019 is estimated to be 9.3% (463 million

people), rising to 10.2% (578 million) by 2030 and 10.9% (700 million) by 2045.
The prevalence is higher in urban (10.8%) than rural (7.2%) areas, and in high-
income (10.4%) than low-income countries (4.0%). One in two (50.1%) people
living with diabetes do not know that they have diabetes. The global prevalence
of impaired glucose tolerance is estimated to be 7.5% (374 million) in 2019 and
projected to reach 8.0% (454 million) by 2030 and 8.6% (548 million) by 2045
(Saeedi et al., 2019).

1.8. Preventing of Oxidative stress

The reduction of oxidative stress could be achieved in three levels: by lowering

exposure to environmental pollutants with oxidizing properties, by increasing
levels of endogenous and exogenous antioxidants, or by lowering the generation
of oxidative stress by stabilizing mitochondrial energy production and efficiency.

12
 Chapter One (General Introduction)

Endogenous oxidative stress could be influenced in two ways: by prevention of

ROS formation or by quenching of ROS with antioxidants (Poljsak, 2011).

1.9. Antioxidant

An antioxidant is a molecule stable enough to donate an electron to a rampaging

free radical and neutralize it, thus reducing its capacity to damage. These
antioxidants delay or inhibit cellular damage mainly through their free radical
scavenging property (B. Halliwell, 1995; Kebede & Admassu, 2019). These low-
molecular-weight antioxidants can safely interact with free radicals and terminate
the chain reaction before vital molecules are damaged. Some of such antioxidants,
including glutathione and uric acid, are produced during normal metabolism in
the body (Kumar, 2014; Shi et al., 1999). Other lighter antioxidants are found in
the diet. Although there are several enzymes system within the body that
scavenge free radicals, the principle micronutrient (vitamins) antioxidants are
vitamin E (α-tocopherol), vitamin C (ascorbic acid), and B-carotene
(Balakrishnan et al., 2014; Levine et al., 1999). The body cannot manufacture
these micronutrients, so they must be supplied in the diet (Kebede & Admassu,
2019).

13
 Chapter One (General Introduction)

1.9.1. Types of Antioxidant

There are two types of antioxidants in the human body that is enzymatic
and non-enzymatic antioxidant. These are as follows

1.9.2. Antioxidant Defense System

Antioxidants act as radical scavenger, hydrogen donor, electron donor, peroxide
decomposer, singlet oxygen quencher, enzyme inhibitor, synergist, and metal-
chelating agents. Both enzymatic and non-enzymatic antioxidants exist in the
intracellular and extracellular environment to detoxify ROS (Lobo et al., 2010).

14
 Chapter One (General Introduction)

1.9.3. Mechanism of Action of Antioxidants

Two principle mechanisms of action have been proposed for antioxidants (Rice-
Evans & Diplock, 1993). The first is a chain- breaking mechanism by which the
primary antioxidant donates an electron to the free radical present in the systems.
The second mechanism involves removal of ROS/RNS initiators (secondary
antioxidants) by quenching chain-initiating catalyst. Antioxidants may exert their
effect on biological systems by different mechanisms including electron
donation, metal ion chelation, co-antioxidants, or by gene expression regulation
(Krinsky, 1992).

Fig. 1.5: Mechanism of action of antioxidants

(Modification of Sharma 2014)

1.9.4. Plant Source of Antioxidant

Synthetic and natural food antioxidants are used routinely in foods and medicine
especially those containing oils and fats to protect the food against oxidation.
There are a number of synthetic phenolic antioxidants, butylated hydroxyl toluene

15
 Chapter One (General Introduction)

(BHT) and butylated hydroxyl anisole (BHA) being prominent examples (Lobo
et al., 2010).

Plant foods are rich sources of antioxidants. They are most abundant in fruits
and vegetables, as well as other foods including nuts, wholegrains and some
meats, poultry and fish.

Good sources of specific antioxidants include:

• Allium sulfur compounds – leeks, onions and garlic

• Anthocyanins – eggplant, grapes and berries
• Beta-carotene – pumpkin, mangoes, apricots, carrots, spinach and parsley
• Catechins – red wine and tea
• Copper – seafood, lean meat, milk and nuts
• Cryptoxanthins – red capsicum, pumpkin and mangoes
• Flavonoids – tea, green tea, citrus fruits, red wine, onion and apples
• Indoles – cruciferous vegetables such as broccoli, cabbage and cauliflower
• Isoflavonoids – soybeans, tofu, lentils, peas and milk
• Lignans – sesame seeds, bran, whole grains and vegetables
• Lutein – green, leafy vegetables like spinach, and corn
• Lycopene – tomatoes, pink grapefruit and watermelon
• Manganese – seafood, lean meat, milk and nuts
• Polyphenols – thyme and oregano
• Selenium – seafood, offal, lean meat and whole grains
• Vitamin A – liver, sweet potatoes, carrots, milk, and egg yolks
• Vitamin C – oranges, blackcurrants, kiwifruit, mangoes, broccoli, spinach,
capsicum and strawberries
• Vitamin E – vegetable oils (such as wheatgerm oil), avocados, nuts, seeds
and whole grains
• Zinc – seafood, lean meat, milk and nuts

16
 Chapter One (General Introduction)

• Zoochemicals – red meat, offal and fish. Also derived from the plants that
animals eat.

1.9.5. Antioxidant and Cancer

Antioxidants can donate electron to free radicals and scavenge free radicals
produced in OS. In OS-induced carcinogenesis, excess free radicals are produced.
These free radicals take electron from DNA and causing mutation. This mutation
can produce abnormal protein that is the causes of many cancers. Antioxidants
donate or share their excess electron to free radicals and reduce the OS induced
carcinogenesis. β-carotene may protect against cancer through its antioxidant
function by suppressing the free radicals that cause genetic damage. Thus, the
photo protective properties of β-carotene may protect against ultraviolet light
induced carcinogenesis. Immuno-enhancement of β-carotene may contribute to
cancer protection. β-carotene may also have anti-carcinogenic effect by altering
the liver metabolism effects of carcinogens (Van Poppel & Goldbohm,
1995).Vitamin C may be helpful in preventing cancer (Glatthaar et al., 1986). The
possible mechanisms by which vitamin C may affect carcinogenesis include
antioxidant effects, blocking of formation of nitrosamines, enhancement of the
immune response, and acceleration of detoxification of liver enzymes. Vitamin
E, an important antioxidant, plays a role in immune competence by increasing
humoral antibody protection, resistance to bacterial infections, cell-mediated
immunity, the T-lymphocytes m necrosis factor production, inhibition of
mutagen formation, repair of membranes in DNA, and blocking micro cell line
formation (Sokol, 1988). Hence vitamin E may be useful in cancer prevention
and inhibits carcinogenesis by the stimulation of the immune system. The
administration of a mixture of the above three antioxidant reveled the highest
reduction in risk of developing cardiac cancer (Ashok & Ali, 1999).

17
 Chapter One (General Introduction)

Fig.1.6: Mechanism of free radical scavenging by antioxidants.

1.10. Plants as a Source of Drugs in Cancer Therapy

The plants are the biggest natural source of drugs for many diseases including
cancer. Some of the compounds that are potential candidates as anticancer drugs
are mentioned next
Table 1: List of some anticancer drugs obtained from plants

Drug/Chemical Action Plant Source

Camptothecin Anticancerous Camptotheca acuminate
Colchiceine Colchicum autumnale
Antitumor agent
amide (autumn crocus)
Colchicum autumnale
Demecolcine Antitumor agent
(autumn crocus)
Podophyllum peltatum
Etoposide Antitumor agent
(mayapple)
Anticancer, antitumor Camptotheca acuminate
Irinotecan
agent
Tabebuia species
Lapachol Anticancer, antitumor
(trumpet tree)
Monocrotaline Topical antitumor agent Crotalaria sessiliflora
Antitumor, anticancer Podophyllum peltatum
Podophyllotoxin
agent (mayapple)

18
 Chapter One (General Introduction)

Taxus brevifolia (Pacific

Taxol Antitumor agent
yew)
Podophyllum peltatum
Teniposide Antitumor agent
(mayapple)
Antitumor, anticancer Camptotheca acuminate
Topotecan
agent
Antitumor, Catharanthus roseus
Vinblastine
Antileukemic agent (Madagascar periwinkle)
Antitumor, Catharanthus roseus
Vincristine
Antileukemic agent (Madagascar periwinkle)

Recently, Food and Drug Administration (FDA) approved seven plant-derived

drugs for cancer treatment. The detailed descriptions of these drugs are given
below:

Taxol/Paclitaxel: A chemical obtained from Taxus brevifolia is now using as the

first choice of drug in the treatment of different tumorous cancers including breast
cancer.

Vinblastine: A chemical obtained from Catharanthus roseus and is the first drug
of choice in many forms of leukemia that has increased the survival rate of
childhood leukemias by 80% (since the 1950's).

Vincristine: Another antileukemic drug obtained from Catharanthus roseus.

Topotecan: Topotecan is an analog of a plant alkaloid obtained from

Camptotheca acuminate which is approved by the FDA for the treatment of
ovarian and small cell lung cancers. At present, it is under investigation clinically
for the treatment of several types of cancer by alone or combination with other
anticancer drugs.

Irinotecan: Irinotectin is a chemical analog of plant alkaloid obtained from

Camptotheca acuminata. It is approved by the FDA for the treatment of

19
 Chapter One (General Introduction)

metastatic colorectal cancer and is currently under clinical trials for a variety of
cancers.

Etoposide: Etoposide is a semisynthetic derivative of a plant chemical,

epipodophyllotoxin, obtained from Podophyllum peltatum.

Teniposide: Teniposide is also a semisynthetic derivative of a plant chemical

obtained from Podophyllum peltatum.

Aforesaid evidences have given us the fact that plants are a valuable source for
potential inhibitors of cancer.

1.11. Description of Plant Investigated

1.11.1. The Plant Family: Bombacaceae

Bombacaceae (Bombax, Baobab or Kapok family) is a small family of flowering

plants which contains about 28 genera and 200 species (Refaat et al., 2013). Plants
of this family are perennial, deciduous and woody trees. They occur naturally
throughout the tropical and subtropical regions of the world especially in tropical
America. Many species grow to become large trees, with Ceiba pentandra L.
Gaertn. the tallest, reaching a height of 70 m. Additionally, some of these plants
have considerable girth, so called "bottle trees" and their trunks are usually with
buttresses at the base. Besides the great significance of Bombacaceae plants as
ornamentals due to their large branches and brightly colored flowers, several
genera are economically and commercially important, producing timber, edible
fruits, vegetable oils or useful fibers, e.g., silk floss trees (Chorisia spp.) and
Kapok (fibers of Ceiba fruits). The family is also noted for some of the softest
hardwoods commercially traded, especially Balsa wood (Ochroma
lagopus Swartz). The Baobabs (Adansonia spp.) are important icons in certain
parts of Africa and Australia, noted for their immensely stout trunk development

20
 Chapter One (General Introduction)

which is a mechanism for enhancing water storage. Moreover, members of

Bombacaceae found several folkloric medicinal uses in many countries due to
their antipyretic, analgesic, anti-inflammatory, astringent, stimulant, diuretic, and
antimicrobial properties (Refaat et al., 2013).

In old classical literature of taxonomy, Bombacaceae has been considered as a

taxon or subfamily under Malvaceae. However, in the majority of the recent
taxonomic works, Bombacaceae has been treated as an independent family of the
order Malvales (Heywood, 2007). In several anatomical and floral characters,
Bombacaceae shows close affinities with Malvaceae. However, many of its
genera show a close relationship with Dilleniaceae on the basis of their stamen
morphology. Bombacaceae differs from Malvaceae in (i) being exclusively
arborescent (woody trees), (ii) often possessing a prickly trunk, (iii) bearing
dithecous anthers (bi-chambered) in some and monothecous in others, and (iv)
always having smooth pollen grains. Additionally, Cronquist (1981) shows a
close relationship of Bombacaceae with Malvaceae, Sterculiaceae, and Tiliaceae
(Refaat et al., 2013).

1.11.2. Botanical Features of Bombax Insigne

Scientific Name: Bombax insigne

Common Name: The Silk Cotton Tree
Vernacular Name: Poola, Kallilavu, Ilavu, Parei-ilavu (Malayalam);
Shamali (Marathi)

Synonymous Name:
● Bombax insigne var. alba Prain
● Bombax insigne var. andamanica Prain
● Bombax insigne var. cambodiense (Pierre) Prain
● Bombax insigne var. polystemon Prain

21
 Chapter One (General Introduction)

● Bombax insigne var. tenebrosum (Dunn) Robyns

● Bombax insigne var. wightii Prain
● Bombax scopulorum Dunn
● Bombax tenebrosum Dunn
● Gossampinus insignis Bakh
● Salmalia insignis (Wall.) Schott & Endl
● Salmalia scopulorum (Dunn) Stearn

(Bombax Insigne Wall. — The Plant List, 2022)

Scientific Classification:
Kingdom: Plantae
Division: Tracheophyta
Subdivision: Spermatophytina
Class: Magnoliopsida
Superorder: Rosanea
Order: Malvales
Family: Bombacaceae
Genus: Bombax
Species: Bombax insigne

(Bombax Insigne Wall. | Species, 2022)

Habit: Tree

Habitat: Rocky areas in evergreen, semi-evergreen and moist deciduous

forests

Shape and Size: Deciduous trees; to 25 m high; bole buttressed, straight armed
with conical prickles.

Plant Part: Leaves, Flower, Fruit, Bark, Seed.

22
Chapter One (General Introduction)

Fig.1.7a: Bombax insigne Wall.

23
 Chapter One (General Introduction)

Fig.1.7b: Bombax insigne Wall.

Leaves: Leaves digitately compound, alternate, crowded at the tip of branchlets,

stipulate; stipules small, lateral; rachis 15-30 cm long, stout, pubescent, swollen
at base, grooved above; leaflets 6-8, whorled; petiolule 3-8 mm long, stout,
pubescent; lamina 7-23 x 2-7 cm, obovate, obovate-oblong, or elliptic-obovate;
base attenuate; apex acuminate or caudate-acuminate; margin entire, glabrous,
chartaceous; lateral veins 15-22 pairs, pubescent, parallel almost perpendicular to
the midrib, prominent, secondary laterals prominent; intercostae reticulate.

Flowers: Flowers bisexual, pale pink or creamy yellow, solitary, axillary; calyx
irregularly lobed, 3-5 cm long, campanulate, densely silky within; petals 5, 8-12
x 2.5 cm, linear-oblong, narrowed at base, curved at apex, tomentose outside;
stamens about 500 in 5 bundles; anthers dorsifixed, reniform; ovary ovoid,
tomentose, superior, syncarpous; ovules numerous on axile placenta; style long
ending in 5-fid stigma.

Fruits: Fruit a capsule, 5-angled, 8-10 x 4-4.5 cm, 5 valved, glabrous.

24
 Chapter One (General Introduction)

Bark: 20-25 mm, grey to greyish-brown, smooth, fibrous; blaze pink, striated
with radial triangular rays; branches whorled, branchlets prickly.

Seeds: Seeds many, 4-5 mm across, subobovoid, brownish-black, embedded in

dense creamy silky fibers.

Distribution:

• Global Distribution:
India and Myanmar

• Indian distribution:
State - Kerala, Districts: Palakkad, Thiruvananthapuram,
Kollam, Thrissur, Idukki, Pathanamthitta, Wayanad.

Flowering and Fruiting: November- March

Cultivation:

Found at elevations up to 900 meters. The plant grows best in areas where-
annual daytime temperatures are within the range 30 - 35°C, but can tolerate
10 - 43°C. It prefers a mean annual rainfall of 1,500 - 2,500mm, but tolerates
1,000 - 3,750mm.

Grows best in a sunny position. Prefers a well-drained, light to medium soil.

Established plants are drought tolerant. Prefers a pH in the range 6 - 6.5,
tolerating 5.5 - 7.

25
 Chapter One (General Introduction)

Propagation: Seed

Uses:

A fiber obtained from the seed floss can be used as a stuffing material or spun.
The whitish wood is very light, soft, but more durable than that of Bombax
malabaricum.
It is used for making packing cases. It is particularly valued for making matches.
It is also good for pulping to make paper, although it is rather difficult to crush.

1.12. Objective

Oxidative stress plays an essential role in the pathogenesis of chronic diseases

such as cardiovascular diseases, cancer, diabetes, and neurodegenerative
diseases. Long-term exposure to increased levels of pro-oxidant factors can cause
structural defects at a mitochondrial DNA level, as well as functional alteration
of several enzymes and cellular structures leading to aberrations in gene
expression. The modern lifestyle associated with processed food, exposure to a
wide range of chemicals, and lack of exercise play an important role in oxidative
stress induction (Sharifi-Rad et al., 2020).

In OS, reactive oxygen species may be involved in carcinogenesis through the

overexpression of oncogenes (e.g., Bcr-Abl, BCL-2, RAS gene, Myc gene, etc.)
and decreased expression of tumor suppressor genes (e.g., BRCA1, BRCA2, Rb,
p53, etc.). The changes of expression of these genes become more extensive if
the cellular antioxidant defense system becomes weakened (Chow 2010).

Therefore, botanical supplements with enriched antioxidants can afford

protection by suppressing OS due to the presence of phytochemicals, such as
polyphenols, carotenoids, vitamin E, and vitamin C (Zhang et al., 2015).

26
 Chapter One (General Introduction)

Historically, plants are highly rich sources of bioactive compounds and offer
drugs for the prevention and treatment of many diseases including cancer.
Besides its traditional use in the management of cancers for many years, several
new plant-derived compounds, such as vinblastine, vincristine, etoposide,
teniposide, taxol, Taxotere, topotecan, etc. have recently been approved for
cancer treatment.

However, these drugs have benefits in cancer but they are not able to cure cancer
and are very costly with severe side effects that are hazardous, even life
threatening. Moreover, most of the cancer patient in our country can’t able to bear
the expenses of this treatment. Therefore, searching for a novel anticancer drug
having a negligible toxic effect on normal cells from plant origin has gained
renewed interest. Hence, this study was designed to identify compounds from the
bioactive part of B. insigne leaves.

1.13. Present Study Protocol:

The present study focused on the chemical and biological investigations of

different parts of Bombax insigne which includes-

1. Collection of leaves of Bombax insigne from Rangpur.

2. Sun-dried and crushed into a fine powder of different parts of Bombax
insigne.
3. Chemical investigation:
• Cold extraction of leaves with methanol.
• Fractionation of crude extract by n-Hexane, chloroform, and ethyl
acetate, successively.
• Qualitative phytochemical analysis of the extract was performed by
Lead acetate test, Frothing test, Libermann-Burchard’s test, Keller-
Killani Test, Colour test.

27
 Chapter One (General Introduction)

• Determination of total phenolic and flavonoid contents.

4. Biological investigation:
• Determination of DPPH radical scavenging activity.
• Determination of ferric reducing antioxidant capacity.
• Evaluation of in vitro cytotoxic effect on brine shrimp nauplii
• GC-MS Analysis
• Antibacterial Study.

1.14. Reference
Anand, P., Kunnumakara, A. B., Sundaram, C., Harikumar, K. B., Tharakan, S.
T., Lai, O. S., Sung, B., & Aggarwal, B. B. (2008). Cancer is a Preventable
Disease that Requires Major Lifestyle Changes. Pharmaceutical Research,
25(9), 2097.
Are, B. C. (2017). Cancer on the Global Stage: Incidence and Cancer-Related
Mortality in Bangladesh - The ASCO Post.
https://ascopost.com/issues/february-25-2017/cancer-on-the-global-stage-
incidence-and-cancer-related-mortality-in-bangladesh/
Ashok, B. T., & Ali, R. (1999). The aging paradox: free radical theory of aging.
Experimental Gerontology, 34(3), 293–303.
Asmat, U., Abad, K., & Ismail, K. (2016). Diabetes mellitus and oxidative stress-
A concise review. Saudi Pharmaceutical Journal, 24(5), 547–553.
Balakrishnan, D., Kandasamy, D., & Nithyanand, P. (2014). A review on
Antioxidant activity of marine organisms. International Journal of
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Chapter Two

Literature
Review
 Chapter Two (Literature Review)

2. Literature Review

2. 1. Chemical Work
The plant Bombax insigne belongs to the Bombacaceae family which is a group
of about 200 species of tropical trees. It is difficult to detect chemical reviews of
all the plants Bombacaceae family. So, an expansive chemical review of the
genus Bombax has been done and discussed that the plants of this genus were
claimed to possess steroid, terpenoid, flavonoid, phenolic, glycoside, cardiac
glycoside, tannin, alkaloid, saponin, essential oil, sugar, protein, and others
(organic compound). Interestingly, no chemical data was found on Bombax
insigne.
Therefore, different search engines like PubMed, Google, Research Gate, Google
Scholar were used to search the presence of the chemicals in the various species
of the Bombax genus. To perform such work different species of Bombax along
with different keywords like phytochemical screening steroid, alkaloid, saponins,
glycosides, tannin, phenolic compound, flavonoid, terpenoid, protein, etc. were
used within March 2022, and a short description of the detailed chemical review
of Bombax genus below:

2.1.1. Steroid

Chaudhary et al., 2014 reported that steroid was found in petroleum ether and
chloroform extract of Bombax ceiba root.

Depani et al., 2019 and Sirohi et al., 2021 showed the presence of steroids in the
acetone-aqueous and chloroform, and hydroalcoholic extract of thron, petal, and
androecium, and flower of Bombax ceiba, respectively.

33
 Chapter Two (Literature Review)

Apinega et al., 2018 revealed that steroid was present in methanolic extract of
Bombax costatum stem bark.

Khurshid Alam et al., 2018 showed that large amounts of steroids were present
in the ethanolic extract of Bombax ceiba young roots.

Akhtar et al., 2019 confirmed the presence of steroids from chloroform extract
of the gum of Bombax malabaricum.

Akuodor et al., 2011 showed that steroid was present in the methanolic extract
of Bombax buonopozense leaf.
Chauhan et al., 2018 confirmed that steroid was present in the petroleum ether
extract of Bombax ceiba stem bark.

Aguoru, 2015 confirmed the presence of steroids from an aqueous extract of

Bombax ceiba leaves.

Hoque et al., 2018 confirmed that steroid was slightly present in petroleum ether
and dichloromethane extract and moderately present in methanolic extract of
Bombax ceiba root.

2.1.2. Flavonoid
Chauhan et al., 2018 confirmed that flavonoid was present in the methanolic
extract of Bombax ceiba stem bark.

Mohammed et al., 2018 confirmed the presence of flavonoids in methanolic

extract of Bombax costatum stem bark.

34
 Chapter Two (Literature Review)

Pankaj H. Chaudhary et al., 2014 reported that flavonoids were found in

methanolic extract and aqueous extract of Bombax ceiba root.

Depani et al., 2019 identified flavonoids in an acetone-aqueous extract of thron,

petal, androecium of Bombax ceiba.

Chakraborty et al., 2010 confirmed that flavonoids were present in ethanol,

acetone, aqueous and methanolic extracts of Bombax ceiba flower.

Shukla et al., 2020 confirmed the presence of flavonoids from ethyl acetate and
ethanolic extract of Bombax ceiba petals.

Hoque et al., 2018 showed that flavonoid was slightly present in petroleum ether
extract, highly present in dichloromethane extract, and moderately present in
methanolic extract of Bombax ceiba root.

Hait & Goswami, 2017 showed that flavonoid was present in ethyl acetate,
ethanol, and methanol extract of Bombax ceiba flower.

Khurshid Alam et al., 2018 showed that flavonoid was present in a large amount
of ethanolic extract of young roots of Bombax ceiba.

Aguoru, 2015 confirmed the presence of flavonoid from an aqueous extract of

Bombax ceiba leaves.

Sharma & Tandon, 2020 confirmed that flavonoid was present in the aqueous
extract of Bombax ceiba leaves.

Joshi et al., 2013 isolated and identified flavonoids including quercetin (1),
quercetin-3-Ο-β-D-glucopyranoside (2), rutin (3), sexangularetin-3-Ο-
sophoroside (4), vitexin (5), isovitexin (6), vicenin 2 (7), kaempferol-3-Ο-

35
 Chapter Two (Literature Review)

rutinoside (8), and kaepferol-3-Ο-β-D-glucopyranoside (9) methanolic extract of

Bombax ceiba flower.

(1) (2)

(3) (4)

(5) (6)

36
 Chapter Two (Literature Review)

(7) (8)

(9)

2.1.3. Terpenoid

Aguoru, 2015 confirmed the presence of terpenoids from an aqueous extract of

Bombax ceiba leaves.

Shukla et al., 2020 confirmed the presence of triterpenoid in ethyl acetate,

chloroform, and ethanolic extract of Bombax ceiba petals.

37
 Chapter Two (Literature Review)

Hoque et al., 2018 showed that terpenoid was slightly present in petroleum ether
extract and moderately present in dichloromethane & methanolic extract of
Bombax ceiba root.

Hait & Goswami, 2017 showed that terpenoid was present in petroleum ether,
ethanol, and methanol extract of Bombax ceiba flower.

Bila et al., 2020 isolated pentacyclic triterpene name lupeol (10) from chloroform
soluble fraction of a methanolic extract of Bombax costatum root bark.

(10)

Shah et al., 2018 confirmed the presence of terpenoid in the methanolic extracts
of stem bark of Bombax ceiba L.

Akuodor et al., 2011 showed that terpene was present in methanolic leaf extract
of Bombax buonopozense.

Chauhan et al., 2018 confirmed that triterpenoid was present in the petroleum
ether and methanolic extract of Bombax ceiba stem bark.

38
 Chapter Two (Literature Review)

Apinega et al., 2018 revealed that terpenoids were present in methanolic extract
of Bombax costatum stem bark.

2.1.4. Phenolic Compound

Pankaj H. Chaudhary et al., 2014 identified a phenolic compound in the

methanolic and water extract of Bombax ceiba root.

Shukla et al., 2020 confirmed the presence of phenolic compound from

chloroform extracts of Bombax ceiba petals.

Depani et al., 2019 confirmed the presence of phenolic compound from an

acetone-water extract of thron, petal, and androecium of Bombax ceiba.

Hait & Goswami, 2017 confirmed that phenols were present in ethyl acetate,
acetone, ethanol, and methanol extract of Bombax ceiba flower.

Khurshid Alam et al., 2018 showed that phenolic compound was present in a
large amount of ethanolic extract of Bombax ceiba young roots.

Sirohi et al., 2021 confirmed the presence of the phenolic compound in the
hydroalcoholic extract of the Bombax ceiba flower.

Sharma &Tandon, 2020 confirmed that phenols were present in the aqueous and
ethanolic extract of Bombax ceiba leaves.

Chakraborty et al., 2010 confirmed that phenols were present in ethanol,

acetone, aqueous and methanolic extracts of Bombax ceiba flower.

39
 Chapter Two (Literature Review)

2.1.5. Glycoside:

Mohammed et al., 2018 confirmed the presence of glycosides in the methanolic

extract of Bombax costatum stem bark.

Faizi et al., 2011 isolated shamiminol (11) from methanolic extract of stem bark
of Bombax ceiba along with the known constituents stigmasta-3,5- diene (12), ±)-
lyoniresinol 2a-O-β-D-glucopyranoside (13), and opuntiol (14) obtained for the
first time from this plant.

(11) (12)

(13) (14)

40
 Chapter Two (Literature Review)

Apinega et al., 2018 revealed that glycoside was present in methanolic extract of
Bombax costatum stem bark.

Kamble et al., 2017 confirmed that anthraquinone glycoside was present in

hydroalcoholic extracts of Bombax ceiba thrones.

Shah et al., 2018 confirmed that glycoside was present in methanolic extracts of
Bombax ceiba L. stem bark.

Sirohi et al., 2021 showed glycoside was present in the hydroalcoholic extract of
the Bombax ceiba flower.

Akhtar et al., 2019 confirmed that glycoside was present in chloroform extract
of the gum of Bombax malabaricum.

Chakraborty et al., 2010 confirmed that glycoside was present in methanolic

extracts of Bombax ceiba flower.

2.1.6. Cardiac Glycoside

Hait & Goswami, 2017 confirmed that cardiac glycoside was present in
ethanolic and methanolic extract of Bombax ceiba flower.

Khurshid Alam et al., 2018 showed that cardiac glycoside was present in a mild
amount of ethanolic extract of Bombax ceiba young roots.

Apinega et al., 2018 revealed that cardiac glycoside was present in methanolic
extract of Bombax costatum stem bark.

41
 Chapter Two (Literature Review)

Shahat et al., 2003 isolated a flavonoid C-glycoside, shamimin (15), and

identified mangiferin (16), a xanthone that was found to be identical shamimin
from the alcoholic extract of Bombax malabaricum leaves.

Shukla et al., 2020 confirmed the presence of cardiac glycoside in the petroleum
ether, chloroform, and ethanolic extract of Bombax ceiba petals.

(15)

(16)

2.1.7. Tannin
Aguoru, 2015 showed that tannin was present in the aqueous and ethanolic
extract of Bombax ceiba leaves.

42
 Chapter Two (Literature Review)

Kamble et al., 2017 confirmed the presence of tannin in a hydroalcoholic extract

of Bombax ceiba thrones.

Abd-Elhaki et al., 2019 confirmed the presence of tannin in the ethanolic extract
of the Bombax ceiba flower.

Hoque et al., 2018 confirmed that tannin was present in petroleum ether,
dichloromethane, and methanolic extract of Bombax ceiba root.

Mohammed et al., 2018 showed the presence of tannin from the methanolic
extract of Bombax costatum stem bark.

Khurshid Alam et al., 2018 showed that a large amount of tannin was present in
the ethanolic extract of Bombax ceiba young roots.

Shah et al., 2018 showed that tannin was present in methanolic extracts of
Bombax ceiba L. stem bark.

Hait & Goswami, 2017 confirmed that phlobatannin was present in ethanolic
and methanolic extract of Bombax ceiba flower.

Apinega et al., 2018 revealed that tannin was present in methanolic extract of
Bombax costatum stem bark.

Sharma & Tandon, 2020 confirmed that tannin was present in the aqueous
extract of Bombax ceiba leaves.

Chauhan et al., 2018 confirmed that tannin was present in the methanolic extract
of Bombax ceiba stem bark.

43
 Chapter Two (Literature Review)

2.1.8. Alkaloid

Hait & Goswami, 2017 confirmed that alkaloid was present in petroleum ether,
chloroform, ethyl acetate, acetone, ethanol, methanol, and aqueous extract of
Bombax ceiba flower.

Kamble et al., 2017 confirmed that alkaloid was present in hydroalcoholic

extracts of Bombax ceiba thrones.

Pankaj H. Chaudhary et al., 2014 identified that alkaloid was present in

methanolic and aqueous extract of Bombax ceiba root.

Aguoru, 2015 confirmed that alkaloid was present in the ethanolic extract, and
aqueous extract of Bombax ceiba leaves, and roots and stem bark, respectively.

Mohammed et al., 2018 confirmed that alkaloids were present in methanolic

extract of Bombax costatum stem bark.

Khurshid Alam et al., 2018 showed that alkaloid was present in ethanolic extract
Bombax ceiba young roots.

Sirohi et al., 2021 showed alkaloid was present in the hydroalcoholic extract of
Bombax ceiba flower.

Hoque et al., 2018 confirmed that alkaloid was in petroleum ether,

dichloromethane, and methanolic extract of Bombax ceiba root.

Chakraborty et al., 2010 confirmed that alkaloid was present in ethanolic,

aqueous, and methanolic extracts of Bombax ceiba flower.

44
 Chapter Two (Literature Review)

2.1.9. Saponin:

Hait & Goswami, 2017 confirmed the presence of saponins in the n-hexane,
chloroform, and aqueous fraction of ethanolic extract of Bombax ceiba flower.

Mohammed et al., 2018 confirmed the presence of saponin in the methanolic

extract of Bombax costatum stem bark.

Hoque et al., 2018 confirmed that saponin was in petroleum ether,

dichloromethane, and methanolic extract of Bombax ceiba root.

Hait & Goswami, 2017 confirmed that saponin was present in petroleum ether,
chloroform, ethyl acetate, acetone, ethanol, methanol, and aqueous extract of
Bombax ceiba flower.

Shah et al., 2018 confirmed that saponin was present in methanolic extracts of
Bombax ceiba L. stem bark.

Aguoru, 2015 confirmed that saponin was present in aqueous extract, ethanolic
extract of leaves and root, and leaves of Bombax ceiba, respectively.

Sharma & Tandon, 2020 confirmed that saponins were present in the aqueous
and ethanolic extract of Bombax ceiba leaves.

Apinega et al., 2018 revealed that saponin was present in methanolic extract of
Bombax costatum stem bark.

45
 Chapter Two (Literature Review)

2.1.10. Carbohydrate

Hoque et al., 2018 confirmed that carbohydrate was present in petroleum ether,
dichloromethane, and methanolic extract of Bombax ceiba root.

Depani et al., 2019 showed the presence of carbohydrates in the acetone-aqueous

and chloroform extract of thron, petal, and androecium of Bombax ceiba.

Pankaj H. Chaudhary et al., 2014 reported that carbohydrate was found in

methanolic and water extract of Bombax ceiba root.

Mohammed et al., 2018 confirmed the presence of carbohydrates in the

methanolic extract of Bombax costatum stem bark.

Abd-Elhaki et al., 2019 confirmed the presence of carbohydrates in the ethanolic

extract of the Bombax ceiba flower.

Hait & Goswami, 2017 confirmed that carbohydrate was present in petroleum
ether, chloroform, ethyl acetate, acetone, ethanol methanol, and aqueous extract
of Bombax ceiba flower.

Chauhan et al., 2018 confirmed that carbohydrate was present in the methanolic
extract of Bombax ceiba stem bark.

Kamble et al., 2017 confirmed that carbohydrate was present in hydroalcoholic

extracts of Bombax ceiba thrones.

Sirohi et al., 2021 showed that carbohydrate was present in the hydroalcoholic
extract of the Bombax ceiba flower.

46
 Chapter Two (Literature Review)

Akuodor et al., 2011 showed that carbohydrate was present in methanolic leaf
extract of Bombax buonopozense.

2.1.11. Protein
Shukla et al., 2020 showed that protein was present in chloroform and ethanolic
extract of Bombax ceiba petals.

Hait & Goswami., 2017 confirmed that protein was present in ethanolic and
methanolic extract of Bombax ceiba flower.

Pankaj H. Chaudhary et al., 2014 reported that protein was found in methanolic
and aqueous extract of Bombax ceiba root.

Depani et al., 2019 identified that sugar was present from the acetone-aqueous
and chloroform extract petal of Bombax ceiba.

Chakraborty et al., 2010 confirmed that protein and amino acid were present in
ethanolic, aqueous, and methanolic extracts of Bombax ceiba flower.

Aguoru, 2015 confirmed the presence of reducing sugar in the aqueous and
ethanolic extract of Bombax ceiba leaves and roots.

Shah et al., 2018 confirmed that sugar was present in methanolic extracts of
Bombax ceiba L. stem bark.

Depani et al., 2019 showed that sugar was present in the acetone-aqueous and
chloroform extract of thron, petal, and androecium of Bombax ceiba.

47
 Chapter Two (Literature Review)

2.1.12. Others

Pankaj H. Chaudhary et al., 2014 reported that fat and oils were found in
methanolic extract of Bombax ceiba root.

Shukla et al., 2020 confirmed the presence of fixed oils and fats from petroleum
ether extract of Bombax ceiba petals.

Chakraborty et al., 2010 confirmed that fixed oils and fat were present in
acetone extracts of the Bombax ceiba flower.

2.2. Biological work

The Bombacaceae family is rich in medicinal plants having a folkloric reputation

to cure different ailments. Various reported biological data of plants in the
different genus of the Bombacaceae family showed that the plants have
cytotoxicity, antioxidant, antimicrobial, antidiabetic, hepatoprotective, and CNS
activity. So, it is not possible to perform a detailed biological review of all the
genus. To overcome this problem, an extensive biological review on a specific
genus Bombax was carried out. Interestingly, no biological published reports
were observed on Bombax insigne. Different search engines such as Google,
PubMed, Research Gate, and Google Scholar were used to search the biological
activities of different species belonging to the genus of Bombax. To perform such
works, different species of Bombax along with different keywords like biological
work, antipyretic, anti-inflammatory, cytotoxicity, antioxidants, anticancer,
antidiabetic, antibacterial, antifungal, etc. were used within March 2022 and a
short description of the detailed biological review of Bombax genus is as follows:

48
 Chapter Two (Literature Review)

2.2.1 Anticancer and Cytotoxic Activity

Ceiba, 2017 showed that chloroform and n-hexane extract of stem wood part of
Bombax Ceiba possessed a significant cytotoxic effect on the THP-1 cell line.

Sichaem et al., 2010 reported that dichloromethane extract of the root of Bombax
anceps possessed a cytotoxic effect on KB (human epidermoid carcinoma) and
HeLa (human cervical carcinoma) cell lines.

Chauhan et al., 2018 exhibited that methanolic and petroleum ether extract of
Bombax ceiba stem bark showed the highest antiproliferative activity against
UMR-106 cell lines.

Yoshimi et al., 2001 reported that mangiferin on tumorigenesis induced by AOM

showed anticancer activity.

Islam et al., 2011 observed that methanolic extract of Bombax ceiba root
possessed anticancer activity.

Vieira et al., 2009 reported that cytotoxic activity was present in methanolic
extract of Bombax ceiba flower in the Vero cell line.

Zhang et al., 2007 observed that methanolic extract of roots of Bombax

malabarica in the A549 lung carcinoma, MCF-7 breast carcinoma, and HeLa
cervical human cancer cell lines.

2.2.2 Antioxidant Activity

Mohammed et al., 2018 reported that the methanol stem bark extract of Bombax
costatum possessed antioxidant properties.

49
 Chapter Two (Literature Review)

Vieira et al., 2009 reported that the antioxidant activity was present in the
methanolic extract of the Bombax ceiba flower.

Sichaem et al., 2010 reported that the antioxidant activity was present in
methanolic extract of Bombax anceps root.

Eugsene et al., 2019 published that the hydroethanolic extract of dried Bombax
costatum flower had the significant antioxidant property.

Jain et al., 2004 showed that methanolic extracts of Bombax ceiba leave
demonstrated strong antioxidant activity.

2.2.3 Antimicrobial, Antibacterial, Antiviral & Antifungal

Activity

Shah et al., 2018 observed a strong antimicrobial activity in methanolic extract

of Bombax ceiba stem bark.

Kamble et al., 2017 reported the strong anti-fungal and anti-bacterial activity of
ethyl acetate extract of Bombax ceiba leaves.

G. K. Wang et al., 2013 showed that ethanolic extract of Bombax ceiba root
possessed anti-hepatitis B Virus (HBV) activity.

S. Rani et al., 2016 showed that methanol and acetone extracts of Bombax ceiba
leaf had potent antibacterial activity.

Maurya et al., 2018 reported that methanolic extract of Bombax ceiba flower
showed potent antiviral activity.

50
 Chapter Two (Literature Review)

P. Rani & Khullar, 2004 showed that Salmonella typhi was found highly
susceptible to methanolic extract of Bombax ceiba antimicrobial activity.

Digge et al., 2015 evaluated those aqueous extracts of the bark of Bombax ceiba
possessed antibacterial activity.

Nagamani et al., 2014 showed that acetone and methanolic extract of Bombax
ceiba seeds exhibited significant antibacterial and antimycotic activity.

2.2.4 Antidiabetic Activity

Bhavsar & Talele, 2013 reported that the ethyl acetate extract of Bombax ceiba
barks had potential antidiabetic activity in STZ-induced diabetic rats and alloxan-
induced diabetic mice.

Khurshid Alam et al., 2018) showed that ethanolic extract of Bombax ceiba
young roots possessed prolonged and potential hypoglycemic activity.

Pankaj Haribhau Chaudhary & Tawar, 2019 showed the hypoglycaemic

effect of methanolic extract of Bombax ceiba leaves on streptozotocin-induced
rats.

Rahman et al., 2019 revealed that crude methanolic extract of Bombax ceiba
roots showed remarkable hypoglycemic activity.

Harikiran et al., 2011 reported that the ethanolic bark extract of Bombax
malabaricum had pronounced antihyperglycemic activity.

De et al., 2010 reported that hydro-methanolic extract of sepals of Salmalia

malabarica showed antidiabetic activity in streptozotocin-induced diabetic rats.

51
 Chapter Two (Literature Review)

2.2.5 Antipyretic Activity

Hossain, Mandal, et al., 2011 reported that the methanolic extract of Bombax
malabaricum leaves showed significant antipyretic activity in rats.

Sharma & Tandon, 2020 investigated that methanolic extract of Bombax ceiba
leaves possesses significant antipyretic activity.

2.2.6 Antidiarrheal Activity

Akuodor et al., 2011 evaluated that methanolic leaf extract of Bombax

buonopozense leaves produced marked antidiarrheal activity.

Shoba & Thomas, 2001 revealed that methanolic extract of Bombax ceiba root
possessed significant anti-diarrheal activities.

2.2.7 Anti-inflammatory activity

Kamble et al., 2017 reported that ethyl acetate extract of Bombax ceiba leaf had
a strong anti-inflammatory activity.

Shoba & Thomas, 2001 revealed that methanolic extract of Bombax ceiba root
possessed a strong anti-inflammatory activity.

Kumar et al., 2010 revealed that the ethyl acetate fraction of Bombax ceiba
gynaceum possessed significant anti-inflammatory properties.

52
 Chapter Two (Literature Review)

2.2.8 Analgesic property

Meena & Chaudhary, 2017 showed that methanolic extract of leaves of Bombax
ceiba possessed significant analgesic activity.

Sharma & Tandon, 2020 showed that methanolic extract of Bombax ceiba
leaves possessed significant analgesic activity.

Dar et al., 2005 observed that mangiferin demonstrated significant analgesic

activity.

2.2.9 CNS activity

Sirohi et al., 2021 showed that hydroalcoholic extract of Bombax ceiba produced
depressant action on CNS.

2.2.10 Anti-Obesity Activity

Gupta et al., 2013 showed that methanolic extract of stem bark of Bombax ceiba
had significant anti-obesity activity.

Meena & Chaudhary, 2017 showed that stem bark extract of Bombax ceiba
possessed significant anti-obesity activity.

2.2.11 Anti-hypertensive Activity

Saleem et al., 1999 reported that shamimin from methanolic extract of Bombax
ceiba leaves had significant hypotensive activity.

2.2.12 Anti-angiogenic activity

You et al., 2003 observed that methanolic extract of the stem bark of Bombax
ceiba exhibit a significant anti-angiogenic activity.

53
 Chapter Two (Literature Review)

Meena & Chaudhary, 2017 observed that methanolic extract of stem bark of
Bombax malabaricum exhibited significant anti-angiogenic activity.

2.2.13 Hepatoprotective Activity

Ravi et al., 2010 showed that methanolic extract of Bombax ceiba L. flowers had
hepatoprotective activity.

Pankaj Haribhau Chaudhary & Tawar, 2019 and Meena & Chaudhary, 2017
reported that methanolic extract of Bombax ceiba flower showed
hepatoprotective activity.

Khurshid Alam et al., 2018 showed that ethanolic extract of Bombax ceiba
young roots possessed hepatoprotective activity.

Chiu et al., 1992 reported that aqueous extract of Bombax malabarica possessed
hepatoprotective activity.

2.2.14 Others Activity

Patel et al., 2011 reported that aqueous flower extract of Bombax ceiba had
cardioprotective activity against acute Adr-induced cardiotoxicity.

Y. C. Wang & Huang, 2005 reported that ethanolic extract of Bombax ceiba root
possessed antiulcer activity.

Meena & Chaudhary, 2017 showed that alcoholic extract of thron and bark of
Bombax ceiba possessed very good antiacne activity.

Jalalpure & Gadge, 2011 reported that aqueous and ethanolic extract of Bombax
ceiba young fruit possessed significant anti-diuretic activity.

54
 Chapter Two (Literature Review)

Y. C. Wang & Huang, 2005 evaluated that ethanolic extracts of mochrus Bombax
ceiba L.showed strong anti-Helicobacter pylori activities.

Chaudhary & Khadabadi, 2011 observed that ethanolic extract of Bombax ceiba
root showed aphrodisiac activity.

Jalalpure & Gadge, 2011 investigated that aqueous and crude ethanolic extracts
of Bombax ceiba L. fruits showed anti-diuretic activity.

Hossain, Rawani, et al., 2011 reported that methanolic extract of Bombax

malabarica leaves possessed anthelmintic activity.

2.3. Reference
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Sha, S. (2019). Protective and Curative Effects of Bombax ceiba Flower and
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Aguoru, C.U. (2015). Qualitative and Quantitative Phytochemical Analysis of the

Leaf, Stem Bark and Root of Bombax Ceiba (red Silk Cotton Tree) in North
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Akhtar, S., Rauf, A., Rehman, S., Zakir Siddiqui, M., & Sada Akhtar, C. (2019).
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Akuodor, G., Muazzam, I., Usman-Idris, M., Megwas, U., Akpan, J., Chilaka, K.,
Okoroafor, D., & Osunkwo, U. (2011). Evaluation of the antidiarrheal
activity of methanol leaf extract of Bombax Buonopozense in rats. Ibnosina

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 Chapter Two (Literature Review)

Journal of Medicine and Biomedical Sciences, 3(1), 15.

Apinega, L. A., Dlama, S., Ladan, Z., Inusa, K. B., Adejoke, A. S., Dauda, G., &
Musa, A. M. (2018). Isolation and characterisation of Lup-20(29)-en-3β-ol
from the methanol stem bark extract of Bombax costatum pv (bombacacea).
Tropical Journal of Natural Product Research, 2(6), 290–292.

Bhavsar, C. J., & Talele, G. S. (2013). Potential anti-diabetic activity of Bombax

ceiba. Bangladesh Journal of Pharmacology, 8(2), 102–106.

Bila, H. A., Ilyas, M., Musa, A. M., Sani, M. Y., Atiku, I., Dauda, G., Husseini,
K., & Mailafiya, M. M. (2020). Isolation of Lupeol From Methanol Extract
of The Root Bark of Bombax costatum. FUW Trends in Science &
Technology Journa, 5(2), 781–783.

Ceiba, L. (2017). Revealing the Cytotoxic Potential of Medicinal Folklore:

Bombax Ceiba L. Journal of Bioresource Management, 4(3), 27-32.

Chakraborty, D. D., Ravi, V., & Chakraborty, P. (2010). Phytochemical

Evaluation and Tlc Protocol of Various Extracts of Bombax Ceiba Linn.
International Journal of Pharmaceutical Sciences and Research, 1(8), 66.

Chaudhary, Pankaj H., Rai, P. D., Deore, S. L., & Khadabadi, S. S. (2014).
Pharmacognostical and phytochemical studies on roots of Bombax ceiba
Linn. Journal of Pharmacy and Pharmacognosy Research, 2(6), 172–182.

Chaudhary, Pankaj Haribhau, & Tawar, M. G. (2019). Pharmacognostic and

phytopharmacological overview on Bombax ceiba. Systematic Reviews in
Pharmacy, 10(1), 20–25.

Chauhan, S., Sharma, A., Upadhyay, N. K., Singh, G., Lal, U. R., & Goyal, R.
(2018). In-vitro osteoblast proliferation and in-vivo anti-osteoporotic activity
of Bombax ceiba with quantification of Lupeol, gallic acid and β-sitosterol
by HPTLC and HPLC. BMC Complementary and Alternative Medicine,

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 Chapter Two (Literature Review)

18(1), 1-12.

Chiu, H. F., Lin, C. C., Yen, M. H., Wu, P. S., & Yang, C. Y. (1992).
Pharmacological and pathological studies on hepatic protective crude drugs
from Taiwan (V): The effects of Bombax malabarica and Scutellaria
rivularis. The American Journal of Chinese Medicine, 20(3–4), 257–264.

Dar, A., Faizi, S., Naqvi, S., Roome, T., Zikr-Ur-Rehman, S., Ali, M., Firdous,
S., & Moin, S. T. (2005). Analgesic and antioxidant activity of mangiferin
and its derivatives: the structure activity relationship. Biological &
Pharmaceutical Bulletin, 28(4), 596–600.

De, D., Chatterjee, K., Ali, K. M., Mandal, S., Barik, B., & Ghosh, D. (2010).
Antidiabetic and antioxidative effects of hydro-methanolic extract of sepals
of Salmalia malabarica in streptozotocin induced diabetic rats. Journal of
Applied Biomedicine, 8(1), 23–33.

Depani, P., Gadhvi, K., & Vyas, S. (2019). Ethnobotanical Potential and
Phytochemical Screening of Bombax ceiba L. European Journal of
Medicinal Plants, 29(4), 1-8.

Eugene, T., Bi, Z., Monnet, T. Y., Ya, K. C., Serge, E., Ekissi, G., & Bedel, J.
(2019). Phytochemicals compound and antioxidant activity study using
different solvents and drying mode of flower Bombax costatum Pellgr and
Vuillet ( Bombacaceae ) from Côte d ’ Ivoire. European Journal of
Biotechnology and Bioscience, 7(6), 38-48.

Faizi, S., Zikr-Ur-Rehman, S., & Ali Versiani, M. (2011). Shamiminol: A new
aromatic glycoside from the stem bark of Bombax ceiba. Natural Product
Communications, 6(12), 1897–1900.

Gupta, P., Goyal, R., Chauhan, Y., & Sharma, P. L. (2013). Possible modulation
of FAS and PTP-1B signaling in ameliorative potential of Bombax ceiba

57
 Chapter Two (Literature Review)

against high fat diet induced obesity. BMC Complementary and Alternative
Medicine, 13, 1-10.

Hait, M., & Goswami, J. (2017). Physicochemical and phytochemical status on

flower of Bombax ceiba. Journal of Medicinal Plants Studies, 189(3), 189–
192.

Harikiran, L., Ch, S., Someshwar, K., Rama, G., Krishna, K., & Srinivas, A.
(2011). Antihyperglycemic Effects of Bombax malabaricum Extracts in
Alloxan-Induced Diabetic Rats. Der Pharmacia Sinica, 2(5), 74–80.

Hoque, N., Rahman, S., Jahan, I., Afroze Shanta, M., Sultana Tithi, N., & Nasrin,
N. (2018). A Comparative Phytochemical and Biological Study between
Different Solvent Extracts of Bombax ceiba Roots Available in Bangladesh.
Pharmacology & Pharmacy, 09(02), 53–66.

Hossain, E., Mandal, S. C., & Gupta, J. K. (2011). Phytochemical screening and
in-vivo antipyretic activity of the methanol leaf-extract of Bombax
Malabaricum DC (Bombacaceae). Tropical Journal of Pharmaceutical
Research, 10(1), 55–60.

Hossain, E., Rawani, A., Chandra, G., Mandal, S. C., & Gupta, J. K. (2011).
Larvicidal activity of Dregea volubilis and Bombax malabaricum leaf
extracts against the filarial vector Culex quinquefasciatus. Asian Pacific
Journal of Tropical Medicine, 4(6), 436–441.

Islam, M. K., Chowdhury, J. A., & Eti, I. Z. (2011). Biological Activity Study on
a Malvaceae Plant: Bombax ceiba. Journal of Scientific Research, 3(2), 445–
450.

Jain, A., Katewa, S. S., Chaudhary, B. L., & Galav, P. (2004). Folk herbal
medicines used in birth control and sexual diseases by tribals of southern
Rajasthan, India. Journal of Ethnopharmacology, 90(1), 171–177.

58
 Chapter Two (Literature Review)

Jalalpure, S. S., & Gadge, N. B. (2011). Diuretic Effects of Young Fruit Extracts
of Bombax Ceiba L. in Rats. Indian Journal of Pharmaceutical Sciences,
73(3), 306.

Joshi, K. R., Devkota, H. P., & Yahara, S. (2013). Chemical analysis of flowers
of Bombax ceiba from Nepal. Natural Product Communications, 8(5), 583–
584.

Kamble, M. A., Mahapatra, D. K., Dhabarde, D. M., & Ingole, A. R. (2017).

Pharmacognostic and pharmacological studies of Bombax ceiba thorn
extract. Journal of Pharmacy and Pharmacognosy Research, 5(1), 40–54.

Khurshid Alam, A. H. M., Sharmin, R., Islam, M.-, Hasan Joarder, M. H.,
Alamgir, M. M., & Mostofa, M. G. (2018). Antidiabetic and
Hepatoprotective Activities of Bombax ceiba Young Roots in Alloxan-
Induced Diabetic Mice. Journal of Nutritional Health & Food Science, 6(5),
1–7.

Kumar, V., Goel, R., Chawla, R., Silambarasan, M., & Sharma, R. K. (2010).
Chemical, biological, radiological, and nuclear decontamination: Recent
trends and future perspective. Journal of Pharmacy And Bioallied Sciences,
2(3), 220.

Maurya, S. K., Verma, N. K., & Verma, D. K. (2018). Bombax ceiba Linn . : A
review of its phytochemistry and pharmacology. Current Research Journal
of Pharmaceutical and Allied Sciences, 2(3), 14-23.

Meena, V., & Chaudhary, A. K. (2017). Shalmali (Bombax ceiba): Versatility in

its therapeutics. International Journal of Green Pharmacy, 11(3), S401–
S406.

Mohammed, N., Yaro, A. H., & Nazifi, A. B. (2018). Bombax costatum pellegr.
and vuillet stem bark extract prevents paracetamol and carbon tetrachloride-

59
 Chapter Two (Literature Review)

induced liver injury in rats. Tropical Journal of Natural Product Research,

2(5), 220–226.

Nagamani, J. E., Vidya, S. D., & Banu, S. H. (2014). A study on antioxidant and
antimicrobial properties of Bombax ceiba pentandra seed extract. World
Journal of Pharmacy and Pharmaceutical Sciences, 3(12), 692–706.

Patel, S. S., Verma, N. K., Rathore, B., Nayak, G., Singhai, A. K., & Singh, P.
(2011). Cardioprotective effect of Bombax ceiba flowers against acute
adriamycin-induced myocardial infarction in rats. Revista Brasileira de
Farmacognosia, 21(4), 704–709.

Rahman, M. H., Rashid, M. A., & Chowdhury, T. A. (2019). Studies of Biological

Activities of the Roots of Bombax ceiba L. Bangladesh Pharmaceutical
Journal, 22(2), 219–223.

Rani, P., & Khullar, N. (2004). Antimicrobial evaluation of some medicinal

plants for their anti-enteric potential against multi-drug resistant Salmonella
typhi. Phytotherapy Research, 18(8), 670–673.

Rani, S., Rahman, K., & Sultana, A. (2016). Ethnomedicinal and pharmacological
activities of Mochrus (Bombax ceiba Linn.): An overview. Tang [Humanitas
Medicine], 6(1), 2.1-2.9.

Ravi, V., Sharan Patel, S., & Kumar Verma, N. (2010). Hepatoprotective Activity
of Bombax ceiba Linn against Isoniazid and Rifampicin-induced Toxicity in
Experimental Rats. International Journal of Applied Research in Natural
Products, 3(3), 19–26.

Saleem, R., Ahmad, M., Hussain, S. A., Qazi, A. M., Ahmad, S. I., Qazi, M. H.,
Ali, M., Faizi, S., Akhtar, S., & Husnain, S. N. (1999). Hypotensive,
hypoglycaemic and toxicological studies on the flavonol C-glycoside
shamimin from Bombax ceiba. Planta Medica, 65(4), 331–334.

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Digge, V.G., Sonali, S.K., Maheshwar, S.H., Poul, B.N., & Jadge, R.D. (2015).
Screening of Antibacterial Activity of Aqueous Bark Extract of Bombax
ceiba against some Gram Positive and Gram Negative Bacteria. American
Journal of Phytomedicine and Clinical Therapeutics, 3(7), 551–555.

Shah, S. S., Shah, S. S., Iqbal, A., Ahmed, S., Khan, W. M., Hussain, S., & Li, Z.
(2018). Phytochemical screening and antimicrobial activities of red silk
cotton tree (Bombax ceiba L.). Pakistan Journal of Pharmaceutical Sciences,
31(3), 947–952.

Shahat, A. A., Hassan, R. A., Nazif, N. M., Van Miert, S., Pieters, L., Hammuda,
F. M., & Vlietinck, A. J. (2003). Isolation of Mangiferin from Bombax
malabaricum and Structure Revision of Shamimin. Planta Medica, 69(11),
1068–1070.

Sharma, U., & Tandon, D. (2020). Phytochemical Profile, Antimicrobial,

Antioxidant Activities of Medicinal Plants in Local Area of Himachal
Pradesh. International Journal of Science and Research, 9(10), 177–192.

Shoba, F. G., & Thomas, M. (2001). Study of antidiarrhoeal activity of four

medicinal plants in castor-oil induced diarrhoea. Journal of
Ethnopharmacology, 76(1), 73–76.

Shukla, R. K., Nandan, K., Shukla, A., & Kaur, A. (2020). Phytochemical
Analysis and Nutritive Value of Bombax Ceiba Linn. (Petals). Plant
Archives, 20(1), 1201–1206.

Sichaem, J., Siripong, P., Khumkratok, S., & Tip-Pyang, S. (2010). Chemical
constituents from the roots of Bombax anceps. Journal of the Chilean
Chemical Society, 55(3), 325–327.

Sirohi, B., Arya, H., Jaiswal, J., Sen, Y., Sen, R., & Dobra, F. (2021). CNS
activity of hydroalcohalic extract of Bombax ceiba flower. International

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Journal of Pharmaceutics & Drug Research, 9(1), 16–26.

Vieira, T. O., Said, A., Aboutabl, E., Azzam, M., & Creczynski-Pasa, T. B.
(2009). Antioxidant activity of methanolic extract of Bombax ceiba. Redox
Report, 14(1), 41–46.

Wang, G. K., Lin, B. Bin, Rao, R., Zhu, K., Qin, X. Y., Xie, G. Y., & Qin, M. J.
(2013). A new lignan with anti-HBV activity from the roots of Bombax ceiba.
Natural Product Research, 27(15), 1348–1352.

Wang, Y. C., & Huang, T. L. (2005). Screening of anti-Helicobacter pylori herbs

deriving from Taiwanese folk medicinal plants. FEMS Immunology and
Medical Microbiology, 43(2), 295–300.

Yoshimi, N., Matsunaga, K., Katayama, M., Yamada, Y., Kuno, T., Qiao, Z.,
Hara, A., Yamahara, J., & Mori, H. (2001). The inhibitory effects of
mangiferin, a naturally occurring glucosylxanthone, in bowel carcinogenesis
of male F344 rats. Cancer Letters, 163(2), 163–170.

You, Y. J., Nam, N. H., Kim, Y., Bae, K. H., & Ahn, B. Z. (2003). Antiangiogenic
activity of lupeol from Bombax ceiba. Phytotherapy Research, 17(4), 341–
344.

Zhang, X., Zhu, H., Zhang, S., Yu, Q., & Xuan, L. (2007). Sesquiterpenoids from
Bombax malabaricum. Journal of Natural Products, 70(9), 1526–1528.

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Chapter Three

Methods
and
Materials
 Chapter Three (Methods and Materials)

3.1. Chemical Work of Bombax insigne

3.1.1. General Methods
Generally, the following are three major steps in the chemical investigation of a
plant:
Collection and proper identification
Preparation of plant sample
Extraction

3.1.1.1. Collection and Proper Identification of Plant

The whole plant or plant parts must be collected from an authentic source and
identified by an expert taxonomist. A voucher specimen ought to be submitted to
the national herbarium for future reference.

3.1.1.2. Preparation of Plant Material

Usually, the plant part is collected and washed with tap water in fresh condition.
It is cut into small pieces, sun-dried and then dried in an oven at 35-400C, if
required to make it suitable for grinding purpose. The coarse powder is stored in
an airtight container for future use.

3.1.1.3. Extraction
Extraction can be done in two ways, such as:
A. Cold Extraction and
B. Hot Extraction.

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 Chapter Three (Methods and Materials)

3.1.1.3.1. Cold Extraction

Organic compounds present in plant

material can be isolated using suitable
solvent or solvent systems in which the
desired compounds are soluble. Selection
of solvent or solvents actually depends on
the type of compounds required to
isolate. It is supposed to be assumed that
polar solvents are likely to dissolve polar
compounds and non-polar solvents are
likely to dissolve non-polar compounds. For cold extraction, the plant materials
are dipped into the appropriate solvent at Fig. 3.1: Rotary Evaporator
room temperature (RT) and allowed to
stand for several days with occasional shaking. When maximum concentration of
the compounds in the solvent is achieved, the solvent is separated from the
powdered plant materials by filtration. Evaporation of the solvent in vacuum
affords a crude mixture of the soluble compounds in that particular solvent.
Separation is then performed using various techniques of chromatography
(Harwood et al., 1989).

3.1.1.3.2. Hot Extraction

In hot extraction process, the sample can be extracted at

elevated temperature (usually reflux pump). In actual
practice, the extraction from solids is often tedious and
requires thorough contact and heating with the solvent.
This is done in a special apparatus, the Soxhlet
Extractor. The apparatus ensures maximum extraction Fig. 3.2: Soxhlet Extractor

64
 Chapter Three (Methods and Materials)

with a limited quantity of solvent. Evaporation of the solvent under reduce

pressure affords a crude mixture of the compound dissolve in those particular
solvents and proceeds for separation using various techniques of
chromatography.

3.1.1.3.3. Extraction with Solvent

The process of removing organic compounds from its aqueous solution by

shaking with a suitable organic solvent is termed as extraction. It is always better
to extract two or three times with smaller quantities of the solvent than once with
the bulk of the solvent provided. This is done in a separating funnel.

Extraction is the process by which separation of the organic compound dissolved

in aqueous phase affected using organic solvent, which is not miscible with water.
This is solvent-solvent extraction. The compounds are partitioned between
aqueous and organic phase. The organic layer is separated from aqueous layer
using a separating funnel. For maximum extraction, the process is repeated
several times (not less than three times) with not more than 10 minutes for each
separation.

3.1.2. Chemical Studies on Bombax insigne

3.1.2.1. Collection and Identification of Plant

Leaves of Bombax insigne were collected from Rangpur, Bangladesh, in 15 th
September, 2021 and were identified by an expert taxonomist at Bangladesh
National Herbarium where a voucher specimen (DACB Accession Number
65589) is deposited.

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 Chapter Three (Methods and Materials)

Fig.3.3: Leaves of B. insigne

3.1.2.2. Preparation of Plant’s Leaves

Collected leaves were washed properly with fresh tap water to remove dirty
materials and were shade dried for several days with occasional sun drying. These
were then dried in an oven for 24 hours at considerably low temperature for better
grinding. The dried materials were ground into coarse powder by a grinding
machine in the Department of Pharmacy, Gono Bishwabidyalay, Bangladesh and
stored at RT for future use.

Fig.3.4: Powder of B. insigne leaves

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 Chapter Three (Methods and Materials)

3.1.2.3. Extraction of Plant’s Leaves

Extraction was performed according to Alam et al., 2002. About 800 gm of
powdered leaves of Bombax insigne was taken in an amber colored extraction
bottle (2.5 liters capacity) and soaked the leaves with 100% methanol (1.5 L × 3
times). The sealed bottle was occasional shaking and stirring. The final extract
was filtered separately through cotton and then Whatman No.1 filter papers
followed by concentrated with a rotary evaporator under vacuum pressure at 40°C
to afford 45.41 gm leaves extract. From 45.41 gm leaves extract we were used 30
gm for Kupchan method.

Powder is wetting in
amber glass bottle
Leaves are in Powder form Filtration

Leaves of Bombax insigne Rotary Evaporator

Extract of Bombax insigne

Fig. 3.5: Extraction process of B. insigne

67
 Chapter Three (Methods and Materials)

3.1.2.4. Solvent-Solvent Partitioning of Crude Extract

An aliquot (30 gm) of the concentrated methanolic leaves extract was fractionated
by modified Kupchan method (Vanwagenen et al., 1993). 5 gm CME was
dissolved in 90 McLaughlin methanol and 10 mL Distilled water. This solution
was partitioning 6 times with 100 mL n-Hexane in each time. The upper layer
was n-Hexane fraction and lower layer was aqueous methanolic fraction. The
aqueous methanolic solution was mixed with 18 mL distilled water and
partitioning it 3 times by adding 100 mL chloroform in each time. The upper layer
was aqueous methanolic fraction and lower layer was chloroform fraction. The
aqueous methanolic solution was again mixed with 16 mL distilled water and
partitioning 3 times by adding 100 mL ethyl acetate in each time. The upper layer
was ethyl acetate fraction and lower layer was aqueous fraction.

3.1.2.4.1. Modified Kupchan Method

Extract (5 gm)

Methanol (90 mL)

Distilled Water (10 mL)

Aq. Extract Solution

n- Hexane (100 mL × 6)

Aq. Fraction n-Hexane Fraction

Water (18 mL × 1)

Chloroform (100 mL × 3)

68
 Chapter Three (Methods and Materials)

Aq. Fraction Chloroform Fraction

Water (16 mL × 1)
Ethyl Acetate (100 mL × 3)

Aq. Fraction Ethyl Acetate Fraction

Fig. 3.6: Schematic representation of solvent-solvent partitioning of the

crude methanolic extract of B. insigne leaves.

Fraction with n-Hexane

Fraction with chloroform

Fraction with ethyl acetate

Fig. 3.7: Solvent-solvent partitioning with different solvents.

69
 Chapter Three (Methods and Materials)

3.1.3. Qualitative Phytochemical Analysis

3.1.3.1. Test for Saponins

Frothing test: Ten (10) mg of each extract was taken in different test tubes and
shaken vigorously with 5 mL of water. Production of persistent foam indicates
the presence of saponins.

3.1.3.2. Test for Tannins

Lead acetate test: About 5 mL of an aqueous solution of different fractions was
taken in different test tubes and few drops of 1% solution of lead acetate were
added to the test tube. A yellow or red precipitate was the indication for the
presence of tannins.

3.1.3.3. Test for Glycosides

Keller-Killani Test: About 2 mL of each extract was taken in different test tubes
followed by 1 mL of glacial acetic acid containing trace amount of FeCl 3 and 1
mL of concentrated H2SO4 were added to the extract carefully. A reddish-brown
colour is formed at the junction of two layer and the upper layer turns bluish green
in presence of glycosides.

3.1.3.4. Test for Steroids

Libermann-Burchard’s Test: Each extract was dissolved in 1 mL of
chloroform. 2 mL acetic anhydride and 1 mL concentrated sulfuric acid were
added. Formation of greenish color solution indicates the presence of steroids.

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3.1.3.5. Test for Alkaloids

Mayer’s Test: 1 mL of extract was taken and placed into a test tube. Then 1 mL
of potassium mercuric iodide solution (Mayer’s reagent) was added and shaken.
Emergence of whitish or cream precipitate implies the presence of alkaloids.

3.1.3.6. Test for Carbohydrate

Benedict’s Test: To 0.5 mL of the filtrate, 0.5 mL of Benedict’s reagent was
added. The mixture was heated on a boiling water bath for 2 minutes. A
characteristic red color precipitate indicates the presence of sugar.

3.1.3.7. Test for Protein

Ninhydrin Test: To 1 mL of extract few drops of Ninhydrin reagent was added
and heated in a boiling water bath. A purple blue color indicates the presence of
proteins.

3.1.4. Quantitative Phytochemical Analysis

3.1.4.1. Determination of Total Phenolics

The content of total phenolics of different extractives, such as CME, NHF, CHF,
EAF and AQF of leaves of B. insigne were determined according to Wolfe et al.,
2003 in which Folin-ciocalteu reagent (FCR) was used as oxidizing agent and
Gallic Acid (GA) was used as standard.

3.1.4.1.1. Principle
The content of total phenolic compounds of different extractives of the plant was
determined by FCR. The FCR actually measured a sample’s reducing capacity.
The exact chemical nature of the FCR is not known, but it is believed to contain

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heteropolyphosphotunstates-molybdates. Sequences of reversible one or two-

electron reduction reactions lead to blue species, possibly (PMoW 11O40)4. In
essence, it is believed that the molybdenum is easier to be reduced in the complex
and electron-transfer reaction occurs between reductants and Mo (VI):

3.1.4.1.2. Materials and Apparatus

a. Folin-ciocalteu reagent
b. Sodium carbonate
c. Methanol
d. Gallic acid
e. Micropipette (10-100 µL)
f. Micropipette (100-1000 µL)
g. Pipette (1-10 mL)
h. UV-spectrophotometer

3.1.4.1.3. Experimental Procedure

1. 0.4 mL of plant extract or standard of different concentrations were
taken in different test tubes.
2. 2.0 mL of FCR (Diluted 10 times with de-ionize water) reagent
solution was added into each test tube.
3. 3.0 mL of sodium carbonate (7.5%) solution was added into each
test tube.
4. The test tubes were incubated for 30 minutes at 250C to complete the
reaction.
5. Then the absorbance of the solution of each test tube was measured
at 760 nm using a spectrophotometer against blank.
6. A typical blank solution contained all reagents except plant extracts
and/or standard solution.

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7. The total content of phenolic compounds in methanolic extracts and

its four fractions was calculated as gallic acid equivalent (GAE) by
the following formula:
𝑥×𝑉
C=
𝑀

Where,

C = total content of phenolic compounds as mg GAE in each

gram of dried extract.

x = GAE concentration in mg/mL present in that particular

sample concentration.

V = Final volume of the solution in mL.

M = Mass of the sample in final solution in gm.

3.1.4.2. Determination of Total Flavonoids

The content of total flavonoids of different extractives, such as CME, NHF, CHF,
EAF and AQF of leaves of B. insigne were determined by aluminum chloride
colorimetric method according to Zhisen et al., 1999. Quercetin was used as
standard and the total flavonoids content of the extractives were expressed as mg
of QE/gm of dried extract.

3.1.4.2.1. Principle
The content of total flavonoids in different extractives was determined by the
well-known aluminum chloride-colorimetric method. In this method, aluminum
chloride formed complex with hydroxyl groups of flavonoids present in the
samples. This complex has the maximum absorbance at 510 nm.

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3.1.4.2.2. Materials and Apparatus

• Aluminum chloride
• Sodium nitrate
• Sodium hydroxide
• Methanol
• Quercetin
• Micropipette (10-100 µL)
• Micropipette (100-1000 µL)
• Pipette (1-10 mL)
• UV-spectrophotometer

3.1.4.2.3. Experimental Procedure

1. 0.5 mL of plant extract or standard of different concentrations were
taken in different test tubes.
2. 150 µL of 5% sodium nitrate and 2.5 mL of distilled water were added
in each test tube.
3. After 5 min, 0.3 mL of 10% AlCl3 was added in each test tube.
4. At 6 min, 1 mL of 4% NaOH was added to the mixture.
5. The test tubes were then incubated at room temperature for 15 minutes
to complete the reaction.
6. Then the absorbance of the solution of each test tube was measured at
510 nm using a spectrophotometer against blank.
7. A typical blank solution contained all reagents except plant extract
and/or standard solution.
8. The total content of flavonoid compounds in methanolic extract and its
four fractions was calculated as Quercetin equivalent (QE) by the
following formula equation:

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 Chapter Three (Methods and Materials)
𝑥×𝑉
C=
𝑀

Where,
C = total content of flavonoid compounds as mg QE in each gram
of dried extract.
x = QE concentration in mg/mL present in that particular sample
concentration.
V = Final volume of the solution in mL.
M = Mass of the sample in final solution in gm.

3.1.4.3. GC-MS Analysis of CHF

3.1.4.3.1. Principle
The GC works on the principle that a mixture will separate into individual
substances when heated. The heated gases are carried through a column with an
inert gas (such as helium). As the separated substances emerge from the column
opening, they flow into the MS. Mass spectrometry identifies compounds by the
mass of the analyte molecule.

3.1.4.3.2. Reagent and Apparatus

• Injector
• Column
• Electronic assemblies
• Vacuum Pump
• Mass Analyzer
• Ion Detector

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 Chapter Three (Methods and Materials)

3.1.4.3.3. Methodology
GC-MS is a unique analysis technique used for identification and quantification
which is limited to analytes that are not only volatile and thermally labile but can
also withstand the harsh partitioning conditions of the gas chromatograph (Al
Mansur et al., 2017) A representative spectral output of all the ascertainable
compounds from the empirical sample is displayed by this technique. The Gas-
chromatography device has an injection port from where the process is initiated
by injecting the sample to that port. After this, evaporation and separation of the
components take place one by one and finally this equipment identifies the
components present in the corresponding sample. A specific spectral pick is
produced for each component which is recorded on a paper chart electronically.
In our present study, SRM was analyzed by Electron Impact Ionization (EI)
method on a GC-17A gas chromatograph which was coupled to a MS 2010 plus
mass spectrometer. The temperature of fused silica capillary column was kept
40°C with carrier gas helium at a constant pressure of 49.5 kPa. Sample was
injected by splitting with the split ratio 50. The sample was dissolved in
chloroform. The operating conditions were as follows: name of column-SH-Rxi-
5Sil MS, diameter 0.25mm, length 30m, column flow rate 1 mL/min temperature
of the column-initial temperature 40°C (for 1 min), injector temperature- 250° C,
holding time 10 min, column packing was done with 10% diethylene glycol
succinate on 100-200 mesh diatomic CAW, splitting samples were injected by
splitting with split ratio 50, carrier gas - helium gas at constant pressure 90 kPa,
sample dissolved in methanol and range of linear temperature increase 10°C per
min, and the range of m/z ratio was 50-800. The compounds of the extract were
determined by comparing M/Z ratio with Wiley and NIST Libraries (Afrin NS et
al., 2019).

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3.2. In-Vitro Antioxidant Activity

3.2.1. Determination of Total Antioxidant Capacity

The total antioxidant capacity of different extractives, such as CME, NHF, CHF,
EAF and AQF of leaves B. insigne was determined according to Prieto et al.,
1999 with some modifications.

3.2.1.1. Principle
The phosphomolybdenum method usually detects antioxidants, such as ascorbic
acid, some phenolics, α-tocopherol and carotenoids. The phosphomolybdenum
method was based on the reduction of Mo (VI) to Mo (V) by the antioxidant
compounds and subsequent formation of a green phosphate/Mo (V) complex at
acidic ph. In essence, it is believed that the molybdenum is easier to be reduced
in the complex and electron-transfer reaction occurs between reductants and Mo
(VI) and the formation of a green phosphate/Mo (V) complex with a maximal
absorption at 695 nm.

Mo (VI) + e → Mo (V)

3.2.1.2. Materials and Apparatus

• Sulphuric acid
• Sodium phosphate
• Ammonium molybdate
• Ascorbic acid
• Methanol
• Water bath
• Micropipette (10-100 µL)
• Micropipette (100-1000 µL)

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 Chapter Three (Methods and Materials)

• Pipette (1-10 mL)

• UV-spectrophotometer

3.2.1.3. Experimental Procedure

1. 0.5 mL of plant extract or standard of different concentrations was taken
in different test tubes.
2. 3 mL of reaction mixture containing 0.6 M sulphuric acid, 28 mm sodium
phosphate and 1% ammonium molybdate was added into each test tube.
3. The test tubes were incubated at 950C for 10 minutes to complete the
reaction.
4. Then the absorbance of the solution of each test tube was measured at
695 nm using a spectrophotometer against blank after cooling at room
temperature.
5. A typical blank solution contained the same solution mixture without
plant extracts and/or standard and it was incubated under the same
conditions as the rest of the sample solution.

3.2.2. Reducing Power Assessment

The Fe3+ reducing power of different extractives, such as CME, NHF, CHF, EAF
and AQF of leaves of B. insigne was evaluated by the method of Oyaizu 1986.

3.2.2.1. Principle
In this assay, the yellow color of the test solution changes to various shades of
green and blue depending on the reducing power of antioxidant samples. The
reducing capacity of a compound may serve as a significant indicator of its
potential antioxidant activity. The presence of reductants, such as antioxidant
substances in the samples causes the reduction of the Fe3+-ferricyanide complex

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 Chapter Three (Methods and Materials)

to the ferrous form by donating an electron. The amount of Fe2+ complex can then
be monitored by measuring the formation of Perl’s Prussian blue at 700 nm.

Fe3+-ferricyanide + e Fe2+-ferricyanide

3.2.2.2. Materials and Apparatus

• Potassium ferricyanide
• Trichloro acetic acid, TCA
• Ferric chloride
• Phosphate buffer
• Ascorbic acid
• Water bath
• Centrifuge machine
• Micropipette (10-100 µL)
• Micropipette (100-1000 µL)
• Pipette (1-10 mL)
• UV spectrophotometer

3.2.2.3. Experimental Procedure

1. 0.25 mL of plant extract or standard of different concentrations was
taken in different test tubes.
2. 0.625 mL of potassium buffer (0.2 M, pH 6.6) and 0.625 mL of 1%
potassium ferricyanide [K3Fe(CN)6], solution was added into each test
tube.
3. All the test tubes were incubated for 20 minutes at 50°C to complete the
reaction.
4. 0.625 mL of TCA, 10% solution were added into each test tube.
5. All the test tubes were centrifuged at 3000 rpm for 10 min.

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6. 1.8 mL supernatant was withdrawn from the mixture of each test tubes
and mix with 1.8 mL of distilled water.
7. 0.36 mL of 0.1% ferric chloride (FeCl3) solution was added to each
diluted reaction mixture.
8. Then the absorbance of the solution of each test tube was measured at
700 nm using a spectrophotometer against blank.
9. A typical blank solution contained the same solution mixture without
plant extracts and/or standard and it was incubated under the same
conditions as the rest of the sample solution.

3.2.3. DPPH (1,1-diphenyl-2-picrylhydrazyl) Free Radical

Scavenging Assay
DPPH was used to evaluate the free radical scavenging activity of various
compounds and medicinal plants (Blois 1958; Desmarchelier et al., 1997).

3.2.3.1. Principle
The 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) has been widely used to
evaluate the free radical scavenging capacity of antioxidants. DPPH free radical
is reduced to the corresponding hydrazine when it reacts with hydrogen donors.
DPPH can make stable free radicals in aqueous or methanol solution. With this
method, it is possible to determine the antiradical power of an antioxidant activity
by measurement of the decrease in the absorbance of DPPH at 517 nm. Resulting
from a color change from purple to yellow, the absorbance decreased when the
DPPH was scavenged by an antioxidant, through donation of hydrogen to form a
stable DPPH molecule. In the radical form, this molecule had an absorbance at
517 nm which disappeared after acceptance of an electron or hydrogen radical
from an antioxidant compound to become a stable diamagnetic molecule.

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 Chapter Three (Methods and Materials)

3.2.3.2. Materials and Apparatus

• DPPH
• Methanol
• Butylated hydroxy toluene (BHT)
• Micropipette (10-100 µL)
• Micropipette (100-1000 µL)
• Pipette (1-10 mL)
• UV spectrophotometer

3.2.3.3. Experimental Procedure

1. 1.6 mL of methanol solution of plant extracts or standard at different
concentrations were taken in different test tubes.
2. 2.4 mL of methanol solution of 0.1 mm DPPH was added into each test
tube.
3. The test tubes were incubated at RT for 30 minutes in dark place to
complete the reaction.
4. Then the absorbance of the solution of each test tube was measured at
517 nm using a spectrophotometer.
5. A typical blank solution contained the same solution mixture without
plant extracts and/or standard and it was incubated under the same
conditions as the rest of the sample solution.

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 Chapter Three (Methods and Materials)

6. The percentage (%) scavenging activity of DPPH radicals was

calculated from the following equation
% I = {(Ac-As)/Ac} ×100

Where,
Ac= Absorbance of the control

As= Absorbance of the extract/ standard

7. Then all the % scavenging were plotted against concentration, and from
the graph IC50 was calculated.

3.3. In-Vivo Biological Activity

3.3.1. Evaluation of Cytotoxic Activity by Brine Shrimp

Lethality Bioassay

3.3.1.1. Principle
Brine shrimp lethality bioassay is a recent development in the bioassay for the
bioactive compounds (McLaughlin et al., 1992; Meyer et al., 1982; Persoone et
al., 1980; McLaughlin et al., 1988; Chatterjee et al., 1975). Natural products
(extracts, fractions and pure compounds) can be tested for their bioactivity by this
method. Here the simple zoological organism (brine shrimp nauplii) is used as a
convenient monitor for screening and fractionation in the discovery of new
bioactive natural products. This bioassay indicates cytotoxicity as well as a wide
range of pharmacological activities of the compounds as cytotoxic property may
be due to the presence of phytochemicals such as saponins, triterpenes, tannins
and polyphenolic compounds in the extract (Armania et al., 2013) and this
phytochemical compounds exist in plants exhibited anti-tumorigenic effects via
multiple anticancer pathways such as by interaction with key enzymes in cellular

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signaling pathways, cell cycle, apoptosis and metastasis etc (Zhiyu et al., 2012;
Man et al., 2010; Lamoral-Theys et al., 2010; Kuttan et al., 2007). It is reported
that polyphenolic compounds have antioxidative properties and cytotoxic activity
are correlated with the antioxidant properties. As multiple mechanisms account
for polyphenol-induced cytotoxicity but SARs between polyphenols and
cytotoxicity are not well understood (Mitsuhashi et al., 2008).

The brine shrimp assay has advantages of being rapid (24 hours), inexpensive and
simple (as no aseptic technique is required). It easily utilizes a large number of
organisms for statistical validation and requires no special equipment and
relatively small amount of sample is sufficient. Moreover, it does not require
animal serum, as it is needed for the determination of cytotoxicity.

In previous test all the extracts of B. insigne showed the remarkable phenolic
compounds and antioxidant properties. Therefore, all the extracts of B. insigne
were further investigating whether it has capability to show cytotoxic effect
against brine shrimp nauplii or not.

3.3.1.2. Materials and Apparatus

• Artemia salina leach (brine shrimp eggs)

• Sea salt (non-ionized, NaCl) (Merck, India)

• Small tank with perforated dividing dam to hatch the shrimp

• Lamp to attract the nauplii

• Pipette (1 mL and 5 mL)

• Micropipette (10-100 μl adjustable)

• Glass vials (5 mL)

• Magnifying glass
• Test samples of the experimental plant

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 Chapter Three (Methods and Materials)

• Micropipette (1-10 μl adjustable)

• Vincristine sulphate

• DMSO (Dimethyl sulphoxide)

3.3.1.3. Preparation of Brine Water

38 grams of sea-salt (non-ionized NaCl) was dissolved in 1 liter of sterilized
distilled water and then filtered off to get clear solution.

3.3.1.4. Hatching of Brine Shrimp Eggs

1 liter of seawater (brine water) was taken in the small tank, and 1.5 gm of shrimp
eggs were added to one side of the divided tank. Constant oxygen supply at
constant temperature (around 37°C) was carried out during the hatching time. The
eggs were allowed for two days to hatch and mature as nauplii (larvae). The
hatched shrimps were attracted to the lamp on the other side of the divided tank
through the perforated dam. These nauplii were taken for this bioassay.

3.3.1.5. Preparation of the Test Sample

Samples that were taken for the operation were the methanolic leaves extract of
B. insigne and its four fractions such as, EAF, AQF, NHF, CHF. All the samples
(2 mg each) were initially dissolved in 400 μl DMSO to get a concentration of 5
μg/μl for each of the samples, which was used as stock solution. Seven doses
(6.25, 12.5, 25, 50, 100, 200 and 400 μg/mL) of each sample were used for the
cytotoxicity test of brine shrimp nauplii. With the help of a micropipette 1.25,
2.5, 5, 10, 20, 40 and 80 μl of each sample were transferred from the stock
solution in 7 different test tubes. Brine water was added to each test tube making
the volume up to 5 ml. The final concentration of the samples in these test tubes
becomes 6.25, 12.5, 25, 50, 100, 200 and 400 μg/mL. For each concentration,

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control experiment was done. The concentration of DMSO in this test tube should
not exceed 50 μg/mL of brine as because above this concentration cytotoxicity
due to DMSO may arise.

3.3.1.6. Preparation of Control Groups

The control groups are used in cytotoxicity study to validate the test method and
ensure that the results obtained are only due to the activity of the test agents and
the effects of the other possible factors are nullified. Usually, two types of control
groups are used.

i) Positive control (Vincristine Sulfate)

ii) Negative control (DMSO only)

3.3.1.7. Preparation of the Positive Control Group

In cytotoxicity study, a widely accepted cytotoxic agent Vincristine Sulfate was
used as positive control and the result of the test agents (samples) was compared
with the result obtained for the positive control. In the present study, Vincristine
Sulfate was used as the positive control. 1 mg of Vincristine Sulfate were
dissolved in 200 μl of DMSO to get a concentration of 5 μg/μl. This was used as
stock solution of Vincristine Sulfate. The final concentration of Vincristine
Sulfate in the test tube were adjusted at 0.157, 0.313, 0.625, 1.25, 2.5, 5 and 10
μg/mL concentrations using the same method used for the samples with the help
of micropipette.

3.3.1.8. Preparation of the Negative Control Group

In the negative control group 2.5, 5, 10, 20 and 30 μl of DMSO were added to
each of the remarked glass test tube containing 5 ml of simulated sea water, and
10 shrimp nauplii were used as control groups. If the brine shrimp nauplii in this

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test tube show a rapid mortality rate, then the test is considered as in valid as the
nauplii died due to some reason other than the cytotoxicity of the samples.

3.3.1.9. Application of Brine Shrimp Nauplii

Hundred vials were taken for eight samples and two control groups in different
concentrations. With the help of a dropper, 10 living nauplii were transferred to
each of the test tube containing seawater up to 5 ml. Then with the help of
micropipette, specific volume of samples transferred from the stock solution to
the vials to get final concentration of 6.25, 12.5, 25, 50, 100, 200 and 400 μg/mL.
The concentration of DMSO in this test tube should not exceed 50 μg/mL of brine
as because above this concentration cytotoxicity due to DMSO may arise. A
magnifying glass was used for convenient counting of the nauplii. As counting of
10 nauplii was not being possible accurately, therefore a range of 9-12 nauplii
were taken for this study.

3.3.1.10. Counting of Nauplii

After 24-hours of incubation, the test tubes were observed using a magnifying
glass and the numbers of survivors in each test tube were counted and the results
were noted. From this data, the percentage of mortality of the nauplii was
calculated at each concentration by the following formula:
% Mortality = (Dead/Number)×100

3.3.1.11. Analysis of the Data

The dose of mortality was analyzed by using graphical method. The percentage
of mortality was plotted against respective concentrations used and from the
graph effective dose, IC50 was calculated. This represents the concentration at
which 50% of the nauplii died after a certain exposure time.

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3.3.2. Antibacterial Screening

The antibacterial screening of an agent is essential to ascertain its spectrum
against various types of pathogenic organisms. Antibacterial activity of any plant
can be detected by observing the growth response of various microorganisms to
the plant extract, which is placed in contact with them. Antibacterial activity was
observed against both Gram-positive and Gram-negative bacteria. In general,
antibacterial screening is undertaken in two phases.

A primary qualitative assay to detect the presence or absence of activity and a

secondary assay which quantities the relative potency, expressed as the minimum
inhibitory concentration (MIC) value.

The primary assay can be done in three ways such as-

A. Disc diffusion assay method

B. Dilution method
C. Bioautographic method
Among these methods, the disc diffusion assay method (Bauer et al., 1966) is
widely acceptable for the preliminary evaluation of antibacterial activity. It uses
different concentrations of the agents absorbed on sterile filter paper discs.

There is no standardized method for expressing the results of antibacterial

screening. Some investigators use the diameter of the zone of inhibition or the
minimum weight of extract that inhibits the growth of a microorganism. Disk
diffusion is essentially a qualitative or semi quantitative test indicating the
sensitivity or resistance of microorganisms to the test material. However, no
distinction between bacteriostatic and bactericidal activity can be made by this
method (Ronald 1982). The principal factors that determine the size of the zone
of inhibition are

a. Intrinsic antimicrobial susceptibility of the test sample

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b. Growth rate of the test organisms

c. Diffusion rate of the test sample
d. Concentration of test organisms inoculated in the medium
e. Concentration of test sample per disc and
f. Thickness of the medium in the petri dishes.

3.3.2.1. Principle of Disc Diffusion Assay Method

Disc diffusion assay method is based on the ability of antibiotics to diffuse from
a confined source through the nutrient agar gel and create a concentration
gradient. If the agar is seeded or streaked with a sensitive organism, a zone of
inhibition will result where the concentration exceeds the MIC for the particular
organism.

In this method, measured amount of the test samples is dissolved in definite

volumes of solvent to give solutions of known concentrations (g/mL). Then
sterile filter paper discs having 5 mm in diameter are impregnated with known
amounts of the test substances and dried. These test material discs as well as
standard antibiotic discs are placed on plates containing a suitable medium
(nutrient agar) seeded with the test organisms. These plates are kept at 4 0C for 24
hours to allow maximum diffusion. A number of events take place simultaneously
which includes:

a. The dried discs absorb water from the agar medium, and the material
under test is dissolved.
b. The test material diffuses from the surrounding medium according
to the physical law that controls the diffusion of molecules through
agar gel.

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c. There is a gradual change of test material concentration in the agar

surrounding each disc.
The plates are then kept in an incubator at 37 0C for 12-18 hours to allow the
growth of the organisms. If the test material has antibacterial activity, it will
inhibit the growth of microorganisms, giving clear, distinct zone called “Zone of
Inhibition”. The antibacterial activity of the test agent is determined by measuring
the diameter of the zone of inhibition in term of mm (The World of Plants, 1980).

3.3.2.2. Experimental Procedure

3.3.2.2.1. Test Materials Used for the Study

In our present study, the antibacterial activity of the CME & its different fractions
such as NHF, CHF, EAF & AQF of leaves of B. insigne was investigated in
comparison with a standard antibiotic cefuroxime (5 g/discs).

3.3.2.2.2. Materials and Apparatus

a. Filter paper disks (5 mm in diameter)
b. Test tubes
c. Petri dishes (120 mm in diameter)
d. Sterile forceps
e. Sterile cotton
f. Inoculating loop
g. Bunsen burner
h. Micropipette (10-100 l)
i. Laminar air flow unit
j. Autoclave
k. Incubator
l. Punch machine

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m. Beakers
n. Nutrient agar media
o. Alcohol (95%)
p. Methanol
q. Vials

3.3.2.2.3. Test Organisms

Both Gram-positive and Gram-negative bacterial strains taken for the test were
listed in the Table-3.1. These organisms are available in the Microbiological
Research Laboratory at the Department of Pharmacy. The pure culture of which
was previously collected from the Microbiology Department, Gono
Bishwabidyalay, Savar, Bangladesh.

Table 3.1: List of tested bacteria

Gram-positive Gram-negative

1. Bacillus subtilis 1. Escherichia coli

2. Staphylococcus aureus 2. Pseudomonas aeruginosa

3. Klebsiella sp

3.3.2.2.4. Sterilization Procedure

Antibacterial screening was done in laminar hood and all types of other
precautions were highly maintained to avoid any contamination of the organisms
under test. UV light has switched on before one hour working in laminar hood to
avoid any accidental contamination. Petri dishes and other glassware were
sterilized by autoclaving at 1210C and a pressure of 15-lbs./sq. inch for 20 mins.
Blank discs were first kept in a coverer petri dish and then subjected to dry by

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heat sterilization at 180 0C for 1 hour. Later they were kept in laminar hood under
UV light for 30 mins.

3.3.2.2.5. Culture Media

The following media are usually used to demonstrate the antibacterial activity
and to make subculture of the test organisms.

a. Nutrient agar medium

b. Nutrient broth medium
c. Mueller - Hinton medium
d. Tryptic soy broth medium
e. Trypticase soy agar medium
Among these, the nutrient agar medium is most frequently used and the
composition of nutrient agar medium is given below in Table 3.2.

Table 3.2: Composition of nutrient agar media

Ingredients Amount
Bacteriopeptone 0.5 gm
Sodium chloride 0.5 gm
Bacto yeast extract 1.0 gm
Bacto agar 2.0 gm
Distilled water 100 mL
pH 7.2 ± 0.1 at 250C

3.3.2.2.6. Preparation of Medium

Prepared nutrient agar medium (composition mentioned above) was collected
from the store and was used for antibacterial screening. To prepare required
volume of this medium, 28 gm of the prepared medium was dissolved in distilled
water (q.s. to 1000 mL) as directed on the level of the pack. It was then heated in
water bath to dissolve the agar until a transparent solution was obtained.

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3.3.2.2.7. Preparation of Subculture

The media prepared were then dispensed in 20 mL and 5 mL, to prepare plates
and slants, respectively in a number of clean test tubes. The slants were used for
making fresh culture of microorganisms, which in turn was used for sensitivity
tests. The tubes were then plugged with cotton and sterilized in an autoclave at a
temperature of 1210C and pressure of 15 lbs./sq. inch for 15 mins. With the help
of an inoculation loop, the test organisms were transferred from the pure culture
to the agar slants in a laminar airflow unit. The inoculated slants were then
incubated at 370C for 24 hours to assure the growth of test organisms. This culture
was used within one week.

3.3.2.2.8. Preparation of Test Plates

The test organism was transferred from the subculture to the test tube containing
20 mL autoclaved medium with the help of an inoculating loop in an aseptic area.
The test tube was shaken by rotation to get a uniform suspension of the organism.
The bacterial suspension was immediately transferred to the sterile petri dishes in
an aseptic area and was rotate several times, first clockwise and then anti-
clockwise to assure homogeneous distribution of the test organisms. The depth of
media into each petri dish (120 mm diameter) was approximately 4 mm. After
that the medium was cooled at RT, and then it was stored in a refrigerator at 40C.

3.3.2.2.9. Preparation of Discs and Test Samples

Preparation of Discs

Three types of discs were prepared for antibacterial screening. These are

a. Sample discs
b. Standard discs
c. Control/ Blank discs

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 Chapter Three (Methods and Materials)

a. Sample discs: Sterilized filter paper discs having 5 mm in diameter were

prepared with the help of punch machine and were taken in a blank Petri
dish. Sample solution of desired concentration was applied on the discs
with the help of a micropipette in an aseptic condition.
b. Standard discs: These are used to compare the antibacterial activity of
test materials. In our study, cefuroxime (50 g/discs) standard disk was
used as a reference standard.
c. Blank discs: These were used as negative control to ensure that the
residual solvent and the filter paper were not active themselves.

➢ Preparation of test samples

a. Test sample CME: 5 mg of dried CME were dissolved in 1 mL

methanol in different vial giving the concentration of 200 µg/Disc &
400 µg/Disc, respectively.

b. Test sample NHF: 5 mg of dried NHF were dissolved in 1 mL

methanol in different vial giving the concentration of 200 µg/disc &
400 µg/disc, respectively.

c. Test sample CHF: 5 mg of dried CHF were dissolved in 1 mL

methanol in different vial giving the concentration of 200 µg/disc &
400 µg/disc, respectively.

d. Test sample EAF: 5 mg of dried EAF were dissolved in 1 mL

methanol in different vial giving the concentration of 200 µg/disc &
400 µg/disc, respectively.

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 Chapter Three (Methods and Materials)

e. Test sample AQF: 5 mg of dried AQF were dissolved in 1 mL

methanol in different vial giving the concentration of 200 µg/disc &
400 µg/disc, respectively.

3.3.2.2.10. Placement of the Discs, Diffusion and Incubation

The sample discs impregnated separately with the test materials and standard
antibiotic discs were placed gently on the solidified agar plates, freshly seeded
with the test organisms with the help of a sterile forceps to assure complete
contact with the surface of the medium. The discs were placed in such that the
disc was no closer than 15 mm to the edge of the plate and far enough apart to
prevent overlapping the zones of inhibition. The plates were then inverted and
kept in a refrigerator for about 24 hours at 40C. This is sufficient time for the
material to diffuse a considerable area of the medium. Finally, the plates were
incubated at 370C for 12-18 hours.

3.3.2.2.11. Measurement of Zone of Inhibition

After 12 hours of incubation, the antibacterial activity of the test agents was
determined by measuring the diameter of the zones of inhibition in cm with a
transparent scale and compared with the standard disc.

3.4. Reference
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extracts and GC-MS analysis of n-Hexane soluble part of crude chloroform
extract of Cuscuta reflexa (Roxb.). Journal of Pharmacognosy and
Phytochemistry. 8(2), 560-564.

Alam, AHMK., Rahman, M.A.A., Baki, M.A., Rashid, M.H., Bhuyan, M.S.A.,
& Sadik, M.G. (2002). Antidiarrhoeal principle of Achyranthes ferruginea

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Roxb. and their cytotoxic evaluation. Bangladesh Pharmaceutical Journal.

12, 1–4.

Al-Mansur, M.A., Siddiqi, M.M., Akbor, M.A., & Saha, K. (2017).

Phytochemical Screening and GC-MS Chemical Profiling of Ethyl Acetate
Extract of Seed and Stem of Anethum sowa Linn. Dhaka University Journal
of Pharmaceutical Sciences. 16(2), 187- 94.

Armania, N., Yazan, L., Musa, S.N., Ismail, I.S., Foo, J.B., Chan, K.W., Noreen,
H., Hisyam, A.H., Zulfahmi, S., & Ismail, M. (2013). Dillenia suffruticosa
exhibited antioxidant and cytotoxic activity through induction of apoptosis
and G2/M cell cycle arrest. Journal of Ethnopharmacology. 146, 525-535.

Bauer, A.W., Kirby, W.M., Sherris, J.C., & Turck, M. (1966). Antibiotic
susceptibility testing by a standardized single disk method. American
Journal of Clinical Pathology. 45(4), 493-496.

Blois, M.S., (1958). Antioxidant determinations by the use of a stable free radical.
Nature. 1199–1200.

Chatterjee, K.D., (1975). Parasitology in relation to clinical medicine. 10th ed. 54-
59.

Desmarchelier, C., Bermudez, M.J.N., Coussio, J., Ciccia, G., & Boveris, A.
(1997). Antioxidant and prooxidant activities in aqueous extract of
Argentine plants. International Journal of Pharmacognosy. 35, 116–120.

Harwood, Laurence, M., Moody, & Christopher J. (1989). Experimental organic

chemistry: Principles and Practice (Illustrated ed.). 47–51.

Kuttan, G., Kumar, K.B., Guruvayoorappan, C., & Kuttan, R. (2007). Antitumor,
anti-invasion, and antimetastatic effects of curcumin. Advances in
Experimental Medicine and Biology. 595, 173-184.

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Lamoral-Theys, D., Pottier, L., Dufrasne, F., Neve, J., Dubois, J., Kornienko, A.,
Kiss, R., & Ingrassia, L. (2010). Natural polyphenols that display anticancer
properties through inhibition of kinase activity. Current Medicinal
Chemistry. 17, 812-825.

Man, S., Gao, W., Zhang, Y., Huang, L., & Liu, C. (2010). Chemical study and
medical application of saponins as anti-cancer agents. Fitoterapia. 81, 703-
714.
McLaughlin, J.L. (1988). Brine shrimp and crown gall tumors: Simple bioassays
for the discovery of plant antitumor agents. Proceedings, NH workshop,
bioassay for discovery of antitumor and antiviral agent from natural
sources. Bethesda. 534, 112-137.

McLaughlin, J.L. (1991). Bench-top bioassay for the discovery of bioactive

compound in higher plants. Brenesic. 34, 1-14.

Meyer, B.N., Ferringni, N.R., Putnam, J.E., Lacobsen, L.B., Nichois, D.E., &
Mclaughlin, J.L. (1982). Brine shrimp: a convenient general bioassay for
active constituents. Planta Medica. 45(5), 31-34.

Mitsuhashi, S., Saito, A., Nakajima, N., Shima, H., & Ubukata, M. (2008).
Pyrogallol Structure in Polyphenols is Involved in Apoptosis-induction on
HEK293T and K562 Cells. Molecules. 13, 2998-3006.

Oyaizu, M. (1986). Studies on products of browning reactions: antioxidant

activities of products of browning reaction prepared from glucose amine.
The Japanese Journal of Nutrition and Dietetics. 44, 307–315.

Persoone, G. (1980). Proceeding of the international symposium on brine shrimp,

Artemia salina. Universa press, Witteren, Belgium. 1-3.

Prieto, P., Pineda, M., & Aguilar, M. (1999). Spectrophotometric quantitation of

antioxidant capacity through the formation of a phosphomolybdenum

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complex: specific application to the determination of vitamin E. Analytical

Biochemistry. 269, 337–341.

Ronald, R. (1982) Antibiotics-An Introduction. F. Hoffman La Roche & Co.

Basle, Switzerland. 70-71.

Vanwagenen, B.C., Larsen, R., Cardellina, J.H., Randazzo, D., Lidert, Z.C., &
Swithenbank, C. (1993). Ulosantoin, a potent insecticide from the sponge
Ulosa ruetzleri. Journal of Organic Chemistry. 58(2), 335-337.

Wolfe, K., Wu, X., & Liu, R.H. (2003). Antioxidant activity of apple peels.
Journal of Agricultural and Food Chemistry. 51(3), 609–614.

Zhishen, J., Mengcheng, T., & Jianming, W. (1999). The determination of

flavonoid contents in mulberry and their scavenging effects on superoxide
radicals. Food Chemistry. 64, 555–559.

Zhiyu, W., Yue, C., Neng, W., Mei, W.D., Wei, L.Y., Feng, H., Gang, S.J., Po,
Y.D., Yuan, G.X., & Jian-Ping, C. (2012). Dioscin induces cancer cell
apoptosis through elevated oxidative stress mediated by downregulation of
peroxiredoxins. Cancer Biology & Therapy. 13(3), 138-147.

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Chapter Four

Results
 Chapter Four (Results)

4.1. Chemical Studies

4.1.1. Preparation of Crude Extract

Leaves of B. insigne was collected from Rangpur and washed with fresh tap water
to remove dirty materials and were shade dried for several days with occasional
sun shade drying. These were then dried in an oven for 24 hours at considerably
30˚C temperature for better grinding. The dried materials were ground into coarse
powder by a grinding machine in the Department of Pharmacy, Gono
Bishwabidyalay, Dhaka, Bangladesh and stored in air tight container at RT for
future use.

About 800 gm of powdered leaves of B. insigne was taken in an amber colored

extraction bottle (2.5 liters capacity) and soaked the materials with 100%
methanol (1.5 L × 3 times) for 3 days. After extraction of it, 45.41 gm crude
methanolic leaves extract was obtained.

4.1.2. Solvent-Solvent Partitioning of Crude Extract

An aliquot (30 gm) of the concentrated methanolic leaves extract was fractionated
by modified Kupchan method with n-Hexane, chloroform and ethyl acetate
successively (Vanwagenen et al., 1993) and the resultant soluble fractions, such
as NHF (n-Hexane Fraction), CHF (chloroform fraction), EAF (ethyl acetate
fraction) and AQF (aqueous fraction) were obtained and the amounts of each
fraction were given in Fig. 4.1.

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 Chapter Four (Results)

7.22 9.36 NHF

CHF
EAF
7.14 AQF
6.31

Fig. 4.1: Quantity (gm) of four fractions of 30 gm CME of B. insigne leaves

4.1.3. Qualitative Phytochemical Analysis

The preliminary phytochemical screening of different extractives was done to
ascertain the presence or absence of bioactive components. The presence or
absence of saponins, tannins, glycosides, steroids, alkaloids, carbohydrate and
protein has been shown qualitatively in the Table 4.1.

Table 4.1: Results of qualitative tests of different fractions of methanolic

extract of B. insigne leaves.

Phytochemical Test CME NHF CHF EAF AQF

Saponins + - - ++ ++
Tannins + - ++ +++
Glycosides + +++ - + +
Steroids - - - - -
Alkaloids - - - + ++
Carbohydrate + + - ++ +++
Protein - - - - -
Here,
+ = Present in mild amount +++ = Present in large amount
++ = Present in moderate amount - = Not present

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 Chapter Four (Results)

4.1.4. Quantitative Analysis

4.1.4.1. Determination of Total Phenolics

The content of total phenolics of CME of B. insigne leaves and its four fractions
were determined using FCR. The content of phenolics of the extractives was
calculated on the basis of the standard curve for GA (Gallic Acid) as shown in
Table 4.2 and in Fig. 4.2. The results were expressed as mg of GAE/gm of dried
extractives.

Table 4.2: Absorbance of GA (standard) at different concentrations after

treatment with FCR

Conc. Absorbance
(Mean ± STD)
(µg/ml) a b c
3.125 0.109 0.089 0.107 0.108 ± 0.009
6.25 0.132 0.108 0.124 0.128 ± 0.01
12.5 0.226 0.218 0.21 0.22 ± 0.005
25 0.354 0.347 0.349 0.359 ± 0.007
50 0.618 0.567 0.592 0.605 ± 0.021
100 1.104 0.986 1.127 1.116 ± 0.062
200 2.187 1.993 2.174 2.181 ± 0.089

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 Chapter Four (Results)

Standard Curve of Gallic Acid

Absorbance at 760 nm 2.5
y = 0.0106x + 0.0596
2 R² = 0.9987
1.5

0.5

0
0 50 100 150 200
Concentration (µg/ml)

Fig. 4.2: Standard curve of GA for the determination of total phenolics.

The results of phenolics content of CME of B. insigne leaves and its four fractions
were shown in Table 4.3.

Table 4.3: Determination of total phenolic contents of CME of B. insigne

leaves and its four fractions (NHF, CHF, EAF and AQF).

mg GAE/gm of dried mg GAE/gm of

Sample Conc. Absorbance
sample dried sample
Name (µg/ml)
a b c a b c (Mean± STD)
CME 100 0.469 0.465 0.471 131.026 125.897 133.59 130.171 ± 3.198

NHF 100 0.462 0.458 0.465 122.051 116.923 125.897 121.624 ± 3.676

CHF 100 0.433 0.433 0.444 84.872 84.872 98.974 89.573 ± 6.648

EAF 100 0.595 0.597 0.612 292.564 295.128 314.359 300.684 ± 9.726

AQF 100 0.383 0.385 0.397 20.769 23.333 38.718 27.607 ± 7.926

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 Chapter Four (Results)

Total Phenolic Contents of CME and Its

Four Fractions
350 300.684
mg GAE/gm of dried sample

300
250
200
130.171
150 121.624
89.573
100
27.607
50
0
CME NHF CHF EAF AQF

Fig. 4.3: Determination of total phenolic contents of CME of B. insigne

leaves and its four fractions.

Here, NHF, CHF, EAF and AQF are representing as n-Hexane fraction,
chloroform fraction, ethyl acetate fraction and aqueous fraction of crude
methanolic extract (CME) of B. insigne leaves, respectively. Values are mean of
triplicate experiments and represented as (Mean  STD).

The highest phenolic content was found in EAF (300.684 ± 9.726 mg of GAE
/gm of dried extract) at a concentration of 100 µg/mL followed by CME (130.171
± 3.198 mg of GAE / gm of dried extract), NHF (121.624 ± 3.676 mg of GAE /
gm of dried extract), CHF (89.573 ± 6.648 mg of GAE /gm of dried extract), and
AQF (27.607 ± 7.926 mg of GAE / gm of dried extract) at same concentrations.
To compare the phenolic content among the extractives, it was observed that EAF
had a large amount of phenolic content than that of others.

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 Chapter Four (Results)

4.1.4.2. Determination of Total Flavonoids

The content of total flavonoids of CME of B. insigne leaves with its four fractions,
such as NHF, CHF, EAF and AQF were determined using well known aluminum
chloride colorimetric method using Quercetin as standard. The flavonoids content
of the extractives was calculated on the basis of the standard curve for Quercetin
(shown in Table 4.4 and in Fig. 4.4) and the results were expressed as mg of
QE/gm of extractives.

Table 4.4: Absorbance of Quercetin (standard) at different concentrations

for quantitative determination of total flavonoids.

Conc. Absorbance Absorbance

(µg/ml) a b c (Mean ± STD)
3.125 0.115 0.095 0.009 0.073 ± 0.046

6.25 0.111 0.099 0.025 0.078 ± 0.038

12.5 0.147 0.119 0.103 0.123 ± 0.018

25 0.207 0.206 0.202 0.205 ± 0.002

50 0.391 0.356 0.462 0.403 ± 0.044

100 0.746 0.675 0.827 0.749 ± 0.062

200 1.379 1.405 1.694 1.493 ± 0.143

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 Chapter Four (Results)

Standard Curve of Quercetin

1.6
Absorbance at 510 nm
y = 0.0073x + 0.0345
1.4 R² = 0.9996
1.2
1
0.8
0.6
0.4
0.2
0
0 50 100 150 200
Concentration (µg/ml)

Fig. 4.4: Standard curve of Quercetin for the determination of total

flavonoids.

Table 4.5: Determination of total flavonoids contents of CME of B. insigne

leaves and its four fractions (NHF, CHF, EAF and AQF).

Absorbance mg QE/gm of dried sample mg QE/gm of

Sample Conc.
dried sample
Name (µg/ml) a b c a b c (Mean± STD)
CME 200 0.137 0.137 0.138 70.205 70.205 70.89 70.433 ± 0.323

NHF 200 0.133 0.136 0.137 67.466 69.521 70.205 69.064 ± 1.164

CHF 200 0.169 0.148 0.156 92.123 77.74 83.219 84.361 ± 5.927

EAF 200 0.256 0.264 0.229 151.712 157.192 133.219 147.374 ± 10.256

AQF 200 0.115 0.114 0.122 55.137 54.452 59.932 56.507 ± 2.438

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 Chapter Four (Results)

Total Flavonoids Contents of CME and Its

Four Fractions
180
147.374
mg QE/gm of dried sample

160
140
120
100 84.361
70.433 69.064
80 56.507
60
40
20
0
CME NHF CHF EAF AQF

Fig. 4.5: Determination of total flavonoids contents of CME of B. insigne

leaves and its four fractions.

The flavonoids content of CME and its four fractions, such as NHF, CHF, EAF
and AQF (shown in Table 4.5) were 70.433 ± 0.323, 69.064 ± 1.164, 84.361 ±
5.927, 147.374 ± 10.256, 56.507 ± 2.438 mg of QE/gm of dried extractives at a
concentration of 200µg/mL, respectively. Comparing the total flavonoids content
among different fractions, it was observed that EAF contained highest amounts
of flavonoids. CME and NHF had almost similar amounts of flavonoids, whereas
CHF had significantly higher than others but less than the EAF. AQF contained
less amount compared to others.

Here, CME = Crude methanolic extract, NHF = n-Hexane fraction, CHF =

Chloroform fraction, EAF = Ethyl acetate fraction, AQF = Aqueous fraction, QA
= Quercetin. Values are mean of triplicate experiments and represented as (Mean
 STD).

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 Chapter Four (Results)

4.1.5. In-vitro Antioxidant Activity

4.1.5.1. Total Antioxidant Activity

The total antioxidant activity of different extractives was assessed by
phosphomolybdenum method, based on the reduction of Mo (V1) to Mo (V) by
the standard and the formation of green phosphate/ Mo (v) complex with a
maximal absorption at 695 nm. Total antioxidant activity of CME of B. insigne
leaves and standard (AA) was depicted in Table 4.6 and in Fig. 4.6.

Table 4.6: Determination of total antioxidant activity of CME of B. insigne

leaves and AA at different concentrations.

Absorbance Absorbance
Sample Conc. (µg/mL)
a b c (Mean ± STD)
6.25 0.15 0.157 0.125 0.144 ± 0.014
12.5 0.258 0.286 0.162 0.235 ± 0.053
AA 25 0.545 0.571 0.504 0.54 ± 0.028
50 1.143 1.296 1.222 1.22 ± 0.062
100 2.461 2.45 2.494 2.468 ± 0.019
6.25 0.113 0.094 0.106 0.104 ± 0.008
12.5 0.186 0.179 0.195 0.187 ± 0.007
CME 25 0.409 0.41 0.392 0.404 ± 0.008
50 0.793 0.735 0.697 0.742 ± 0.039
100 1.514 1.573 1.523 1.537 ± 0.026

The absorbance of CME and AA at a concentration of 100 (µg/mL) were 1.537

 0.026 and 2.468 ± 0.019, respectively. Comparing the results, it is observed
that CME had considerable total antioxidant activity between CME and AA.

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 Chapter Four (Results)

Total Antioxidant Capacity of CME and AA

3

2.5
Absorbance at 695 nm

1.5
AA
1 CME

0.5

0
0 20 40 60 80 100
Concentration (µg/mL)

Fig. 4.6: Comparison of total antioxidant activity of CME of B. insigne

leaves and AA at different concentrations.

The total antioxidant activity of four fractions of CME, such as NHF, CHF, EAF
and AQF were showed in Table 4.7 and Fig. 4.7. Among the fractions, EAF had
the highest total antioxidant activity (absorbance of 1.964 ± 0.089) followed by
AQF (absorbance of 0.861 ± 0.046), NHF (absorbance of 0.598 ± 0.013) and CHF
(absorbance of 0.287 ± 0.123) at a concentration of 100 µg/mL. Comparing the
results, it was found that the EAF had almost similar antioxidant activity with that
of the standard, AA. Our results demonstrated that all the extractives of B. insigne
leaves had appreciable antioxidant activity, which was concentration dependent.
The antioxidant activity of different extractives of B. insigne leaves and AA
exhibited the following order:

AA >EAF > CME > AQF > NHF > CHF

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 Chapter Four (Results)

Table 4.7: Determination of total antioxidant activity of four fractions of CME

of B. insigne leaves at different concentrations.

Absorbance Absorbance
Sample Conc. (µg/mL)
a b c (Mean ± STD)
6.25 0.075 0.112 0.112 0.1 ± 0.017
12.5 0.111 0.115 0.111 0.112 ± 0.002
NHF 25 0.167 0.151 0.151 0.156 ± 0.008
50 0.324 0.287 0.287 0.299 ± 0.017
100 0.58 0.607 0.607 0.598 ± 0.013
6.25 0.084 0.067 0.052 0.068 ± 0.013
12.5 0.097 0.071 0.085 0.084 ± 0.011
CHF 25 0.117 0.083 0.115 0.105 ± 0.016
50 0.21 0.121 0.147 0.159 ± 0.037
100 0.45 0.152 0.259 0.287 ± 0.123
6.25 0.128 0.103 0.134 0.122 ± 0.013
12.5 0.203 0.219 0.235 0.219 ± 0.013
EAF 25 0.471 0.462 0.435 0.456 ± 0.015
50 0.942 1.103 0.876 0.974 ± 0.095
100 2.085 1.872 1.936 1.964 ± 0.089
6.25 0.073 0.081 0.075 0.076 ± 0.003
12.5 0.143 0.142 0.148 0.144 ± 0.003
AQF 25 0.235 0.281 0.271 0.262 ± 0.02
50 0.408 0.423 0.437 0.423 ± 0.012
100 0.815 0.843 0.924 0.861 ± 0.046

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 Chapter Four (Results)

Total Antioxidant Capacity

3
Absorbance at 695 nm

2.5

2 AA
CME
1.5
NHF
CHF
1
EAF
0.5 AQF

0
0 20 40 60 80 100
Concentration (µg/mL)

Fig. 4.7: Comparison of total antioxidant activity of CME of B. insigne leaves

and its four fractions with AA.

Here, CME = Crude methanolic extract, NHF = n-Hexane fraction, CHF =

Chloroform fraction, EAF = Ethyl acetate fraction, AQF = Aqueous fraction, AA
= Ascorbic Acid.

4.1.5.2. Reducing Power Assessment

The Fe3+ reducing power of the different fractions of CME of B. insigne leaves
and standard, AA were determined by the method described by Oyaizu (1986)
with slight modification. The reductive capabilities of different extractives and
AA were shown in Table 4.8 and 4.9, and in Fig. 4.8 and 4.9. The CME showed
quiet similar reducing activity as that of AA, a reference standard antioxidant.

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 Chapter Four (Results)

Table 4.8: Determination of Fe3+ reducing power capacity of CME of B. insigne

leaves and AA at different concentrations.

Absorbance Absorbance
Samples Conc. (µg/mL)
a b c (Mean ± STD)
6.25 1.486 1.765 1.517 1.589± 0.125
12.5 3.155 3.128 3.273 3.185± 0.063
AA 25 3.698 3.657 3.504 3.62± 0.083
50 3.769 3.742 3.718 3.743± 0.021
100 3.842 3.791 3. 869 3.817± 0.026
6.25 0.586 0.668 0.771 0.675± 0.076
12.5 0.976 1.114 1.121 1.07± 0.067
CME 25 1.489 1.658 1.784 1.644± 0.121
50 2.397 2.747 2.961 2.702± 0.232
100 3.654 3.576 3.764 3.665± 0.077

Reducing Power Capacity of CME and AA

4.5
4
Absorbance at 700 nm

3.5
3
2.5
2 AA
1.5 CME
1
0.5
0
0 20 40 60 80 100
Concentration (µg/mL)

Fig. 4.8: Comparison of reducing power capacity of CME of B. insigne leaves

and standard, AA at different concentrations.

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 Chapter Four (Results)

The ferric reducing capacity of four fractions of CME, such as NHF, CHF, EAF
and AQF was also investigated.

Table 4.9: Determination of reducing power capacity of four fractions of

CME of B. insigne leaves at different concentrations.

Absorbance
Samples Conc. (µg/mL) Mean ± Deviation
a b c
6.25 0.093 0.099 0.094 0.095 ± 0.003
12.5 0.124 0.105 0.116 0.115 ± 0.008
NHF 25 0.316 0.331 0.325 0.324 ± 0.006
50 0.592 0.588 0.599 0.593 ± 0.005
100 1.098 1.112 1.101 1.104 ± 0.006
6.25 0.165 0.126 0.153 0.148 ± 0.016
12.5 0.345 0.352 0.364 0.354 ± 0.008
CHF 25 0.626 0.659 0.657 0.647 ± 0.015
50 1.314 1.237 1.123 1.225 ± 0.078
100 2.352 2.325 2.356 2.344 ± 0.014
6.25 0.821 0.871 0.993 0.895 ± 0.072
12.5 1.369 1.246 1.545 1.387 ± 0.123
EAF 25 1.987 1.899 1.932 1.939 ± 0.036
50 2.896 2.976 2.985 2.952 ± 0.04
100 3.761 3.674 3.742 3.726 ± 0.037
6.25 0.423 0.457 0.441 0.44 ± 0.014
12.5 0.645 0.651 0.666 0.654 ± 0.009
AQF 25 1.112 1.225 1.141 1.159 ± 0.048
50 1.885 1.912 1.976 1.924 ± 0.038
100 3.215 3.317 3.431 3.321 ± 0.088

Among the fractions, the absorbance (3.726 ± 0.037) of EAF was close to
absorbance (3.817± 0.026) standard, AA at a concentration of 100 µg/mL and
EAF show the highest reducing capacity. Other fractions, such as AQF
(absorbance 3.321 ± 0.088), and CHF (absorbance 2.344 ± 0.014) show moderate
reducing activity compared to AA at a concentration of 100 µg/mL. These results
demonstrated that CME of leaves of B. insigne and its fractions had significant

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 Chapter Four (Results)

iron reducing capacity, which was concentration dependent. The reducing power
of different extractives and AA showed the following order:

AA > EAF > CME > AQF > CHF > NHF

Reducing Power Capacity

4
3.5
Absorbance at 700 nm

3
AA
2.5
CME
2
NHF
1.5
CHF
1
EAF
0.5
AQF
0
0 20 40 60 80 100
Concentration (µg/mL)

Fig. 4.9: Comparison of reducing power capacity of CME of B. insigne leaves

and its four fractions with AA at different concentrations.

Here, CME = Crude methanolic extract, NHF = n-Hexane fraction, CHF =

Chloroform fraction, EAF = Ethyl acetate fraction, AQF = Aqueous fraction, AA
= Ascorbic acid. Values are mean of triplicate experiments and represented as
(Mean  STD).

4.1.5.3. DPPH Free Radical Scavenging Assay

The antioxidant activity of the extractives of B. insigne leaves was evaluated by
the widely used and most reliable DPPH radical scavenging assay method
developed by Blois (1958). The method is based on the ability of the extractives
to scavenge the stable DPPH radical that contains an odd electron. This radical

112
 Chapter Four (Results)

gives absorbance at 517 nm and decolorizes after neutralization by the

antioxidants. The activity is increased by increasing the concentration of the
sample extractives.

The results of DPPH radical scavenging assay by CME and standard BHT
(butylated hydroxytoluene) were given in Table 4.10 and in Fig. 4.10. The
scavenging activity of the CME was almost similar to that of BHT (standard) but
the IC50 values of BHT (31.343 g/mL) were higher than that of CME (14.346
g/mL).

Table 4.10: Determination of DPPH free radical scavenging activity of the

CME of B. insigne leaves and BHT at different concentrations.

% of Scavenging % of
Sample IC50
Conc. (µg/ml) Scavenging
Name a b c (µg/mL)
(Mean ± STD)
6.25 21.462 20.498 22.569 21.51±0.846
12.5 32.675 31.214 33.438 32.442±0.923
BHT 25 46.345 45.738 45.897 45.993±0.257 31.343
50 62.587 60.397 61.016 61.333±0.922
100 67.385 68.587 68.178 68.05±0.499
6.25 30.443 31.157 31.201 30.934±0.347
12.5 45.962 47.779 44.99 46.244±1.156
CME 25 64.226 63.726 65.012 64.321±0.529 14.346
50 76.843 75.471 75.239 75.851±0.708
100 85.375 84.659 83.987 84.674±0.567

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 Chapter Four (Results)

DPPH Free Radical Scavenging Activity

90
80
% of Scavenging

70
60
50
40 BHT
30 CME
20
10
0
0 0.5 1 1.5 2
Log Concentration (µg/mL)

Fig. 4.10: Comparison of DPPH free radical scavenging activity of CME of

leaves and BHT (standard) at different log concentrations.

The scavenging activity of four fractions of CME, such as EAF, AQF, NHF and
CHF were also determined. The results of DPPH radical scavenging assays of
these four fractions and BHT were given in Table 4.11 and in Fig. 4.11.

Table 4.11: Determination of DPPH free radical scavenging activity of four

fractions of CME of B. insigne leaves at different concentrations.

% of Scavenging % of
Sample IC50
Conc. (µg/ml) Scavenging
Name a b c (µg/mL)
(Mean ± STD)
6.25 17.662 15.986 16.827 16.825±0.684
12.5 28.895 26.745 27.625 27.755±0.883
NHF 25 39.532 38.432 37.908 38.624±0.677 50.827
50 51.432 52.148 53.343 52.308±0.788
100 61.764 62.238 60.879 61.627±0.563
6.25 9.865 9.475 10.897 10.079±0.6
12.5 18.921 17.827 17.358 18.035±0.655
CHF 25 32.569 30.534 31.679 31.594±0.833 116.038
50 43.675 42.485 41.372 42.511±0.94
100 52.583 51.865 51.365 51.938±0.5

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 Chapter Four (Results)

% of Scavenging % of
Sample IC50
Conc. (µg/ml) Scavenging
Name a b c (µg/mL)
(Mean ± STD)
6.25 38.324 37.876 39.523 38.574±0.695
12.5 54.248 57.213 56.103 55.855±1.223
EAF 25 70.952 69.77 68.911 69.878±0.837 10.688
50 84.452 81.985 85.768 84.068±1.568
100 90.325 89.573 88.987 89.628±0.548
6.25 25.785 24.331 26.273 25.463±0.825
12.5 41.442 40.132 42.102 41.225±0.819
AQF 25 56.532 55.328 54.897 55.586±0.692 19.709
50 70.654 71.197 69.576 70.476±0.674
100 76.632 75.436 75.879 75.982±0.494

DPPH Free Radical Scavenging Activity

100
90
80
% of Scavenging

70
CHF
60
BHT
50
CME
40
NHF
30
AQF
20
EAF
10
0
0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2
Log Concentration (µg/mL)

Fig. 4.11: Comparison of DPPH free radical scavenging activity of CME of

B. insigne leaves and its four fractions with BHT at different log
concentrations.

Among the fractions, the highest scavenging activity was found in CHF with IC50
value 116.038 g/mL and NHF with IC50 value 50.826 g/mL than that of

115
 Chapter Four (Results)

standard BHT with IC50 31.343 g/mL. On the other hand; EAF and CME showed
moderate scavenging activity with IC50 10.688 g/mL and 14.364 g/mL,
respectively. Our observation demonstrated that all the extractives of leaves of B.
insigne possessed DPPH free radical scavenging activity. The activity of different
extractives of B. insigne leaves and BHT exhibited the following order:

EAF > CME > AQF > BHT > NHF > CHF

DPPH Free Radical Scavenging Activity

140
120 116.038
IC50 Value(µg/mL)

100
80
60 50.826
40 31.343
14.364 19.709
20 10.688
0
BHT CME NHF CHF EAF AQF

Fig. 4.12: IC50 (g/mL) values of different extractives of B. insigne leaves and
standard, BHT for DPPH free radical scavenging.

4.1.6. Evaluation of Cytotoxic Activity by Brine Shrimp

Lethality Bioassay
The cytotoxicity of all the extractives of B. insigne was studied by using Brine
Shrimp lethality bioassay (McLughilin et al., 1992; Meyer et al., 1982; Persoone
et al., 1980; McLaughlin et al., 1988; Chatterjee et al., 1975). The cytotoxicity of
CME, NHF, CHF, EAF and AQF studied by using Brine Shrimp lethality
bioassay. Here the effect of each extractive at different concentrations (6.25-400
µg/ml) on the mortality of nauplii was measured. The effect of different

116
 Chapter Four (Results)

concentrations of VCS (standard) and extractives on nauplii mortality is

presented in Table 4.12 and 4.13 and in Fig. 4.13 and 4.14. The VCS showed
mortality of nauplii when the concentration was lowered gradually from 10 g/ml
(100% mortality) to 0.157 g/ml (20% mortality). The LC50 of VCS was found
to be 1 g/ml.

Table 4.12: Effect of VCS (standard) on mortality of Brine Shrimp nauplii

at different concentrations

LC50
Sample Conc. (µg/mL) Number Dead Live % Mortality
(µg/mL)
0.157 10 2 8 20
0.313 12 3 9 25
0.625 10 4 6 40
VCS 1.25 11 5 6 45.45 1
2.5 10 7 3 70
5 10 8 2 80
10 11 11 0 100

Vincristine Sulfate
100 y = 45.129x + 49.959
80
% Mortality

60
40
20
0
-1 -0.5 0 0.5 1 1.5
Log Concentration (µg/mL)

Fig. 4.13: Effects of VCS (standard) on the mortality of Brine Shrimp

nauplii at different concentrations.

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 Chapter Four (Results)

Table 4.13: Effect of CME of B. insigne leaf and its four fractions on
mortality of Brine Shrimp nauplii at different concentrations.

Conc. % LC50
Sample Number Dead Live
(µg/mL) Mortality (µg/mL)
6.25 10 3 7 30
12.5 10 7 3 70
25 10 8 2 80
CME 50 11 8 3 72.73 14.86
100 10 8 2 80
200 11 10 1 90.91
400 11 10 1 90.91
6.25 11 3 8 27.27
12.5 10 5 5 50
25 10 4 6 40
NHF 50 10 7 3 70 22.34
100 12 9 3 75
200 10 8 2 80
400 10 9 1 90
6.25 11 4 7 36.36
12.5 10 5 5 50
25 12 7 5 58.33
CHF 50 11 7 4 63.64 14.42
100 10 8 2 80
200 10 9 1 90
400 12 11 1 91.67
6.25 10 3 7 30
12.5 10 4 6 40
25 11 5 6 45.45
EAF 50 10 5 5 50 26.38
100 10 6 4 60
200 11 7 4 63.64
400 10 7 3 70

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 Chapter Four (Results)

Conc. % LC50
Sample Number Dead Live
(µg/mL) Mortality (µg/mL)
6.25 10 3 7 30
12.5 11 4 7 36.36
25 10 4 6 40
AQF 50 10 5 5 50 50
100 12 6 6 50
200 11 7 4 63.64
400 10 8 2 80

Among the extractives, the CME showed most potent activity with LC 50 value
14.86 g/ml. The LC50 values of other extractives, such as EAF, NHF CHF, and
AQF were 26.38, 22.34, 14.42 and 50 g/ml, respectively. The lower LC50 means
higher toxicity. Our results demonstrated that all the extractives of B. insigne had
significant cytotoxic activity. The cytotoxic activity of different extractives of B.
insigne and VCS exhibited the following order:

VCS > CHF > CME > NHF > EAF > AQF

Brine Shrimp Lethality Bioassay

60
50
50
LC50 (µg/mL)

40

30 26.38
22.34
20 14.86 14.42
10
1
0
VCS CME NHF CHF EAF AQF

Fig. 4.14: LC50 (g/mL) values of different extractives of B. insigne leaves

and standard, VCS for Brine Shrimp lethality Bioassay.

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 Chapter Four (Results)

4.2. GC-MS Analysis of CHF

GC-MS of the chloroform extract of B. insigne leaves presented in table 4.14.
Mass spectra of the chloroform extract of the leaves are depicted in fig.4.15. The
fragmentation patterns of the mass spectra were compared with those of the
known compounds stored in the National Institute of Standard and Technology
(NIST) research library. In the GC-MS analysis, 9 compounds were detected. The
identification of Phytochemical compounds was based on peak area, molecular
weight, molecular formula.

Table 4.14: Detected Compounds by GC-MS

Name of Molecular Retentio Conc.

Structure M.W.
Compound Formula n Time (%)

1-
Octadecanesulp
353.0 C18H37ClO2S 23.354 98.175
honyl chloride

2-
methyltetracosa
ne
352.7 C25H52 24.053 0.021

Zinc,bis(dipenty
lcarbamodithioa
to-S,S')-,( T-4)-
530.24 C22H44N2S4Zn 25.085 0.627

Diisooctyl
phthalate 390.6 C24H38O4 26.243 0.720

2-
methyltetracosa
ne
352.7 C25H52 26.552 0.055

Acetamide,2,2’-
(CH3)2C(CH2
[(2,2-dimethyl- 104.15 OH)2
27.505 0.097
1,3-propanediol

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 Chapter Four (Results)

2-Tridecenal,
(E)- 196.33 C13H24O 27.711 0.092

L-
Sorbofuranose,
pentakis
(trifluoroacetate
660.2 C16H7F15O11 28.315 0.157
) (isomer 1)

Hexadecanoic
acid, 2-bromo- 335.32 C16H31BrO2 29.350 0.058

Fig.4.15: GC-MS spectra of Chloroform Fraction

4.3. In-Vitro Antibacterial Activity

4.3.1. Determination of Zone of Inhibition

The antibacterial potential of plant extracts was evaluated according to their zone
of inhibition against various pathogens and the results (zone of inhibition) were
compared with the activity of the standards; Cefuroxime (50 μg/disc). No extract
showed activity at 200 µg/disc and 400 µg/disc against both the tested Gram-
positive and Gram-negative bacterial strain is shown in table 4.15.

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 Chapter Four (Results)

Table 4.15. Antibacterial activity (zone of inhibition, cm) of different extracts

of B. insigne leaves at 200 μg/disc and 400 μg/disc and standard at 50 μg/disc.

Name of
Standard CME NHF CHF EAF AQF
Organism
Bacillus subtilis 1cm - - - - -
Staphylococcus
2.2cm - - - - -
aureus
Klebsiella sp. 2cm - - - - -
Escherichia coli 2cm - - - - -
Pseudomonas
- - - - - -
aeruginosa

Fig 4.16: Antibacterial activity of different fraction at different

concentration.

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 Chapter Four (Results)

4.4. Reference
Blois, M.S. (1958). Antioxidant determinations by the use of a stable free radical.
Nature. 181,1199–1200.

Chatterjee, K.D. (1975). Parasitology in relation to clinical medicine. 10th ed. 54-
59.
McLaughlin, J.L. (1988). Brine shrimp and crown gall tumors: Simple bioassays
for the discovery of plant antitumor agents. Proceedings, NH workshop,
bioassay for discovery of antitumor and antiviral agent from natural
sources. Bethesda. 534, 112-137.

McLughilin, J.L. (1991). Bench-top bioassay for the discovery of bioactive

compound in higher plants. Brenesic. 34, 1-14.

Meyer, B.N., Ferringni, N.R., Putnam, J.E., Lacobsen, L.B., Nichois, D.E., &
Mclaughlin, J.L. (1982). Brine shrimp: a convenient general bioassay for
active constituents. Planta Medica. 45(5), 31-34.

Oyaizu, M. (1986). Studies on products of browning reactions: antioxidant

activities of products of browning reaction prepared from glucose amine.
The Japanese Journal of Nutrition and Dietetics. 44, 307–315.

Persoone, G. (1980). Proceeding of the international symposium on brine shrimp,

Artemia salina. Universa press, Witteren, Belgium. 1-3.

Vanwagenen, B.C., Larsen, R., Cardellina, J.H., Randazzo, D., Lidert, Z.C., &
Swithenbank, C. (1993). Ulosantoin, a potent insecticide from the sponge
Ulosa ruetzleri. Journal of Organic Chemistry. 58(2), 335-337.

123
Chapter Five

Discussion
 Chapter Five (Discussion)

5. Discussion
Accumulating evidence suggests that many dangerous pathophysiological
processes, such as cancer, diabetes, cardiovascular and neurodegenerative
diseases, are associated with the accumulation of unstable free radicals due to
lacking of enough antioxidants and higher oxidative stress (Gilgun-Sherki et al.,
2002; Sharifi-Rad et al., 2020). These unstable radicals have the tendency to
become stable through electron pairing with biological macromolecules such as
proteins, lipids, and DNA in healthy human cells, thus causing protein and
DNA damage (Gilgun-Sherki et al., 2002). Therefore, dietary intake of
antioxidants is imperative to protect cells from damage by scavenging the
unstable free radicals (Rahman et al., 2015).

Plants have long been a source of exogenous (i.e., dietary) antioxidants (ARA
Rima et al., 2021; Halliwell, 2007). It is believed that two-thirds of the world's
plant species have medicinal importance, and almost all of these have excellent
antioxidant potential (Krishnaiah et al., 2011). For assessment of antioxidant
potential of compounds, a single assay method is not sufficient. Furthermore,
different antioxidant assays vary in terms of assay principle and experimental
conditions. For instant, some methods use organic radical producers e.g., DPPH
and some use metal ions for oxidation e.g., FRAC assay technique. The time
factor associated with their chemical reactions to produce free radicals by
oxidation reaction also different from each other. Since the procedure and
experimental conditions are different for different techniques, the various
antioxidants are considered as a control for different assay techniques according
to their rate and time of scavenging. In addition, antioxidants could be polar
e.g., phenolics, flavonoids etc. or non-polar e.g., vitamin E in nature and they
can act as radical scavenger by electron donating mechanism or by hydrogen
donating mechanism. Therefore, different control antioxidants (e.g., BHT,

124
 Chapter Five (Discussion)

Quercetin, Ascorbic Acid) were used for different antioxidant assays (Rahman
et al., 2015).

5.1. Qualitative Phytochemical Analysis

Our preliminary phytochemical screening of the different fractions of CME of
B. insigne leaves showed the presence of saponins, tannins, glycosides,
alkaloids and carbohydrate. Saponins and tannin were present in the CME, EAF
and AQF. Glycoside and carbohydrate were present in CME, NHF, EAF, AQF.
Alkaloid were present in EAF and AQF and but steroids and protein were
absent in all the extract. These biologically active constituents provided us
beneficial health effect by different mechanisms. Plants possessing saponins
have anti-inflammatory, antifungal, antibacterial, anti-parasitic, anti-cancer, and
antiviral activities (Mugford & Osbourn, 2013; Podolak et al., 2010; Sparg et
al., 2004). Tannins exert several pharmacological effects, including antioxidant
and free radical scavenging activity as well as antimicrobial, anti‐cancer,
anti‐nutritional, antibacterial, antidiarrheal, anti-inflammatory and
cardio‐protective properties. They also seem to exert beneficial effects on
metabolic disorders and prevent the onset of several oxidative stress‐relate
(Smeriglio et al., 2017; Sparg et al., 2004).

5.2. Phenolic and Flavonoid Contents

Polyphenols are naturally occurring compounds found largely in the fruits,
vegetables, cereals and beverages. Over 8,000 polyphenolic compounds and
more than 6,000 flavonoids were reported in various plants (Amaral et al.,
2019). Polyphenols are generally involved in defense against ultraviolet
radiation or aggression by pathogens. Polyphenols may be classified into
different groups as a function of the number of phenol rings. The main classes
include phenolics, flavonoids, stilbenes and lignans. Polyphenols present in

125
 Chapter Five (Discussion)

plants offered some protection against development of cancers, cardiovascular

diseases, diabetes, osteoporosis and neurodegenerative diseases (Pandey &
Rizvi, 2009).

Our results showed that CME and various fractions of B. insigne contain
significant amount of phenolics. The higher amount of phenolics (mg of GAE /
gm of dried extract) was found in the EAF (300.684 ± 9.726 mg), CME
(130.171 ± 3.198 mg), NHF (121.624 ± 3.676 mg), CHF (89.573 ± 6.648 mg),
and AQF (27.607 ± 7.926 mg) at a concentration of 100 µg/ml (Table 4.3 and
Fig. 4.3). These data indicated that EAF of B. insigne leaves is a potential
source for phenolic compounds.

Flavonoids, another class of bioactive polyphenols, are reported to have potent

antioxidant potential (Raj et al., 1999). The flavonoids content (mg of
Quercetin/gm dried extract) of CME was (70.433 ± 0.323 mg) at a
concentration of 200 µg/mL. Among the fractions (Table 4.5, Fig no. 4.5), the
highest amount was found in EAF (147.374 ± 10.256 mg) followed by CHF
(84.361 ± 5.927 mg) and AQF (56.507 ± 2.438 mg) at the same concentration
(200 µg/mL). This result demonstrated that the B. insigne leaves is a potent
source of antioxidants.

5.3. Total Antioxidant Capacity

The antioxidant potential of the different fractions of CME of B. insigne leaves
was estimated from their ability to reduce the reduction of Mo (VI) to Mo (V)
by the antioxidant-enriched fractions and subsequent formation of a green
phosphate/Mo (V) complex at acidic pH. Antioxidant activity depends on the
presence of its bio-active compounds mainly polyphenols, carotenoids, and
vitamin E and C (Oktay et al., 2003). This suggests that the concentration of the
bioactive compounds present in the extract is important to showing antioxidant
activity. Thus, higher concentration of bioactive compounds in the extracts

126
 Chapter Five (Discussion)

shows higher antioxidant activity. In this study, the antioxidant capacity (in
terms of absorbance at 695 nm) of the extractives was in the range of 0.052–
2.494 at the concentration ranges from 6.25-100 µg/mL. All the extracts showed
good antioxidant activities that gradually increase with increasing concentration
(Table 4.7 and Fig 4.7). Interestingly, EAF contained the highest antioxidant
activity, which was similar to the standard (AA). Our results suggest that the
antioxidant capacity can be attributed to the extractive’s chemical composition
and polyphenol contents.

5.4. Reducing Power Assessment

Reducing power is also widely used in evaluating antioxidant activity of plant
polyphenols. The reducing power is generally associated with the presence of
reductants, which exert antioxidant action by breaking the free radical chains by
donating a hydrogen atom. In this assay, the presence of reductants in the
antioxidant sample reduces Fe3+/ferricyanide complex to the Fe2+/ferrous form.
Thus, the reducing power of the sample can be monitored by measuring the
formation of Perl’s Prussian blue at 700 nm (Oktay et al., 2003). In this study,
the iron reducing capacity of the CME, NHF, CHF, EAF and AQF was
estimated from their ability to reduce the Fe3+/ferricyanide complex to the
ferrous form by donating an electron. The reducing ability of the extracts (in
terms of absorbance at 700 nm) was in the range of 0.093 to 3.869 at the
concentration ranges from 6.25 to 100 µg/mL. All the extracts showed a good
reducing power capacity, which was concentration-dependent (Table 4.9 and
Fig 4.9). Interestingly, EAF possessed the highest reducing activity among all
the extractives. We assumed that the antioxidant activity and the reducing
power capacity of the extracts was likely due to the presence of polyphenols,
which can act as free radical scavenger by donating an electron.

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 Chapter Five (Discussion)

5.5. DPPH Free Radical Scavenging Activity

The effect of antioxidants on DPPH is thought to be due to their hydrogen
donating ability (Baumann et al., 1980). Radical scavenging activities are very
important to prevent the deleterious role of free radicals in different diseases,
including cancer. DPPH free radical scavenging is an accepted mechanism for
screening the antioxidant activity of plant extracts. In the DPPH assay, violet
color DPPH solution is reduced to yellow colored product, diphenylpicryl
hydrazine, by the addition of the extract in a concentration dependent manner.
This method has been used extensively to predict antioxidant activities because
of the relatively short time required for analysis. Among the fractions, the
highest scavenging activity was found in EAF (IC50 value of 10.688 g/mL)
when compared to standard BHT (IC50 of 31.343 g/mL) (Table 4.11 and Fig
4.11). It has been reported that polyphenol contents scavenge the DPPH radicals
by their hydrogen donating ability (Baumann et al., 1980; Huang et al., 2005)
The results obtained in this study suggest that all the extracts of B. insigne
showed radical scavenging activity by their electron transfer ability. It was
reported that the total polyphenols content and radical scavenging antioxidant
activity are highly correlated (Huang et al., 2005).

5.6. Cytotoxic Effect on Brine Shrimp

The Cytotoxicity of CME of B. insigne and its four fractions NHF, CHF, EAF
and AQF was evaluated by brine shrimp lethality bioassay. All the extracts
showed cytotoxic effect. The LC50 values of CME, NHF, CHF, EAF, and AQF
were found to be 14.86, 22.34, 14.42, 26.38 and 50 μg/ml, respectively (Fig.
4.13). Among these five extractives, CHF showed the most cytotoxic activity.
The LC50 value of standard VCS was found to be 1 μg/ml. The lower LC50
means higher toxicity.

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 Chapter Five (Discussion)

On the basis of our results obtained from the present study, it can be concluded
that B. insigne plant extracts possessed remarkable cytotoxic activities and it
reported that cytotoxic property may be due to the presence of phytochemicals
such as saponins, carbohydrate, tannins and polyphenolic compounds in the
extract (Kumar et al., 2021) and this phytochemical compounds exist in plants
exhibited anti-tumorigenic effects via multiple anticancer pathways such as by
interaction with key enzymes in cellular signaling pathways, cell cycle,
apoptosis and metastasis etc. (Manzione et al., 2020). Present work was a
preliminary effort which will require further detailed investigation, including
characterization of active compounds.

5.7. GC-MS Analysis of CHF

CHF is the most cytotoxic fraction among all the fractions of methanolic extract
of B. insigne leaves. GC-MS data of CHF explored the chemical constituents
and its proportion in CHF. 1-Octadecanesulphonyl Chloride was present as
98.175% of CHF which was reported as phenolic natural antifungal and
antimicrobial compound (Sundaram & Prabhakaran, 2017).

Diisooctyl phthalate (0.720%) inhibited the growth of food-borne harmful

(Yang et al., 2020). 2-methyltetracosane (0.076%) of CHF has been reported for
free radical scavenging activity (Ramya et al., 2015). 2-Tridecenal,(E)- was
present as 0.092% of CHF which is known for its antibacterial activity
(Orumwensodia et al., 2021).

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 Chapter Five (Discussion)

5.8. Antibacterial Screening

Antibacterial activity was studied using disk diffusion assay method. The four
fractions of CME of B. insigne leaves of the CHF, EAF, NHF, and AQF had no
antibacterial activity against both the tested Gram-positive and Gram-negative
bacterial strain at 200 μg/disc and 400 μg/disc (Table 4.15 and Fig 4.16).

5.9. Reference
Amaral, R. G., Santos, S. A. dos, Andrade, L. N., Severino, P., & Carvalho, A.
A. (2019). Natural Products as Treatment against Cancer: A Historical and
Current Vision. Clinics in Oncology, 4(1562), 1–5.
ARA Rima, R., Ara Rima, R., Islam Sagor, S., & Anjum, A. (2021). In vitro
antioxidant activities of the roots of Coffea benghalensis B Heyne ex
Schult. Growing in Bangladesh. Journal of Pharmacognosy and
Phytochemistry, 10(2). www.phytojournal.com

Rmya, B., Malarvili, T., & Velavan, S. (2015). GC-MS Analysis of bioactive
compounds in Bryonopsis laciniosa fruit extract. International Journal of
Pharmaceutical Sciences and Research, 6(8), 3375–3379.

Baumann, J., Wurm, G., & Bruchhausen, F. V. (1980). [Prostaglandin

synthetase inhibition by flavonoids and phenolic compounds in relation to
their O2--scavenging properties (author’s transl)]. Archiv Der Pharmazie,
313(4), 330–337.

Fuchs-Tarlovsky, V. (2013). Role of antioxidants in cancer therapy. Nutrition

(Burbank, Los Angeles County, Calif.), 29(1), 15–21.

Gilgun-Sherki, Y., Rosenbaum, Z., Melamed, E., & Offen, D. (2002).

Antioxidant therapy in acute central nervous system injury: Current state.
Pharmacological Reviews, 54(2), 271–284.

Halliwell, B. (2007). Oxidative stress and cancer: have we moved forward?

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 Chapter Five (Discussion)

Biochemical Journal, 401(1), 1–11.

Huang, D., Boxin, O. U., & Prior, R. L. (2005). The Chemistry behind
Antioxidant Capacity Assays. Journal of Agricultural and Food
Chemistry, 53(6), 1841–1856.

Krishnaiah, D., Sarbatly, R., & Nithyanandam, R. (2011). A review of the

antioxidant potential of medicinal plant species. Food and Bioproducts
Processing, 89(3), 217–233.

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Summary
 Chapter Six (Summary)

6. Summary
The main purpose of this study was to find out the potential antioxidative fraction
and to detect compounds from the most cytotoxic fraction of B. insigne leaves.
The leaves of B. insigne were collected from Rangpur and identified by an expert
taxonomist of National Herbarium of Bangladesh. The leaves were washed with
fresh tap water, dried, grinding into coarse powder and soaked into methanol for
better extraction (cold extraction method). The dried crude methanolic extract
(CME) was then successively fractionated into different fractions depending on
the polarity with n-Hexane, chloroform, ethyl acetate by modified Kupchan
methods to get four fractions: n-Hexane fraction (NHF), chloroform fraction
(CHF), ethyl acetate fraction (EAF) and lastly aqueous fraction (AQF).

Phytochemical screening of different fractions confirmed the presence of

saponins, tannins, glycosides, Alkaloids and carbohydrate in the leaves of B.
insigne. The polyphenolic and flavonoid content, antioxidant and free radical
scavenging activity of the different extractives were determined using standard
UV-spectroscopic method. The phenolic and flavonoid contents of CME, NHF,
CHF, EAF and AQF were 130.171, 121.624, 89.573, 300.684 and 27.607 mg
GAE/gm of dried sample and 70.433, 69.064, 84.361, 147.374 and 56.507 mg
QE/gm of dried sample, respectively. The order of polyphenolic and flavonoid
contents of different extractives were EAF > CME > NHF > CHF > AQF and
EAF > CHF > CME> NHF > AQF, respectively. The total antioxidant activity of
different extractives and standard, AA showed the order as AA > EAF > CME >
AQF > NHF > CHF. The reducing power of different extractives and standard
AA showed the order as AA > EAF > CME > AQF > CHF > NHF. The DPPH
scavenging activity of different extractives of B. insigne and standards with their
IC50 (µg/mL) exhibited the order as EAF > CME > AQF > BHT > NHF > CHF.
In the evaluation of In-Vitro cytotoxic effect on brine shrimp nauplii showed that

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the order of cytotoxicity of four extractives and VCS of was VCS > CHF > CME
> NHF > EAF >AQF. In GC-MS analysis, 9 compounds were detected in CHF.
Among 9 compounds 1-Octadecanesulphonyl Chloride was present as 98.175%
which was reported as phenolic natural antifungal and antimicrobial compound.
The four fractions of CME of B. insigne leaves had no antibacterial activity
against both the tested Gram-positive and Gram-negative bacterial strain at 200
μg/disc and 400 μg/disc.

Finally, we can summarize our findings that it is clear that the EAF is a good
source of antioxidants and had possessed maximum polyphenolics, antioxidant
activity and free radical scavenging activity. And also noticed that among all the
extractives, CHF had the most cytotoxic effect.

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