TLC Densitometric Quantification of vasicine, vasicinone and embelin

Chapter 8

TLC Densitometric Quantification of Vasicine,

Vasicinone and Embelin from Adhatoda zeylanica
leaves and Embelia ribes fruits
8.1 INTRODUCTION
With the global increase in the demand for plant derived medicine, there is a need to
ensure the quality of the herbal drugs. Plants are a complex mixture of a variety of
chemical constituents that can vary considerably depending on genetic and
environmental factors, method of cultivation, time of collection, post-harvest
processing, etc. This inherent variability in the chemistry may adversely affect the
efficacy of medicinal plants. Hence it is a pre-requisite to ensure that the plants used for
therapy or for research purposes are of a quality that gives the desired efficacy and that
the quality is maintained at each re-collection of the plant materials. To meet this
requirement it is essential to establish qualitative and quantitative chemoprofiles of the
samples.
The present chapter describes the TLC fingerprint profile of the methanolic extract of
Adhatoda vasica leaf and Embelia ribes fruit, co-chromatography with the bioactive
compounds vasicine, vasicinone and embelin respectively and its quantification using
TLC densitometry.

8.2. EXPERIMENTAL
8.2.1 Chemicals
All solvents and chemicals used were of analytical grade.
8.2.2 Apparatus
(a) Spotting device CAMAG Linomat V Automatic Sample Spotter
(b) Syringe 100 L (Hamilton)
(c) TLC Chamber CAMAG glass twin trough chamber (20 10 4 cm)
(d) Densitometer CAMAG TLC Scanner 3 linked to winCATS software

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(e) TLC plates precoated silica gel 60 F254 TLC plate (E. Merck, Cat. no.
1.05554.0007) (0.2 mm thickness)
8.2.3 Plant material collection and authentication
The leaves of Adhatoda zeylanica and fruits of Embelia ribes were collected from
Gujarat, India. Their authenticity was confirmed by the taxonomist Dr. Sheetal
Anandgiwala of our department and a voucher specimen was deposited at the
Department of Pharmacognosy and Phytochemistry, B. V. Patel Pharmaceutical
Education & Research Development (PERD) Centre, Ahmedabad, India. The material
was stored in air tight containers at room temperature until use.
8.2.4 TLC analysis of Adhatoda zeylanica leaves
8.2.4.1 TLC fingerprint profiling

Preparation of sample solution

1 g of the powdered plant material was extracted with methanol (25 ml 2) under
reflux on a water bath for 30 min. The extracts were filtered, pooled, concentrated and
the volume was made up to 25 ml with methanol.
Preparation of standard solutions
2 mg of vasicine (purity 98.5 %) standard was dissolved in methanol and the volume
was made up to 25 ml in a volumetric flask.
2 mg of vasicinone (purity 97.5 %) standard was dissolved in methanol and the volume
was made up to 25 ml in a volumetric flask.
Mobile phase
Ethyl acetate: Methanol: Ammonia (8: 2: 0.2)
Procedure
10 l each of the sample solution and the standard solutions were applied on a
precoated silica gel 60 F254 TLC plate (E. Merck) of uniform thickness (0.2 mm) and
the plate was developed in a twin trough chamber containing mobile phase Ethyl
acetate: methanol: ammonia (8: 2: 0.2) up to a distance of 8 cm at 25 2C temperature
and 40 % relative humidity. The plate was dried at room temperature and scanned at
292 nm for vasicine and 233 nm for vasicinone.

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TLC Densitometric Quantification of vasicine, vasicinone and embelin

The plate was observed under UV light at 254 nm and the plate was derivatized with
modified Dragendorffs reagent (Wagner and Bladt, 1996). The Rf and colour of the
resolved bands were noted.
8.2.4.2 Estimation of vasicine and vasicinone using TLC densitometric method

Preparation of sample solution

1 g of the powdered plant material was extracted with methanol (25 ml 2) under
reflux on a water bath for 30 min. The extracts were filtered, pooled, concentrated and
the volume was made up to 25 ml with methanol.
Preparation of standard solutions of vasicine and vasicinone
A) Standard solution of vasicine- 5 mg of vasicine standard was dissolved in 25 ml of
methanol in a volumetric flask. From this stock solution standard solutions of 320 - 960
g/ml were prepared by transferring aliquots (1.6 to 4.8 ml) of stock solution to 10 ml
volumetric flasks and adjusting the volume to 10 ml with methanol.
B) Standard solution of vasicinone - A stock solution of vasicinone was prepared by
dissolving 2 mg of accurately weighed vasicinone in methanol and making up the
volume to 25 ml with methanol. From this stock solution standard solutions of 80-480
g/ml were prepared by transferring aliquots (0.5 to 3 ml) of stock solution to 10 ml
volumetric flasks and adjusting the volume with methanol.
Calibration curve for vasicine and vasicinone
10 l of each of the standard solutions of, vasicine and vasicinone were applied in
triplicate on TLC plate. The plates were developed in a solvent system of Ethyl acetate
: Methanol : Ammonia (8 :2 :0.2 v/v) at 252 C temperature and 40 % relative
humidity, upto a distance of 8 cm. After development, the plate was dried in air and
scanned at 292 nm for vasicine and 233 nm for vasicinone. The peak areas were
recorded. Calibration curves of vasicine and vasicinone were prepared by plotting peak
areas vs concentration.
Quantification of vasicine and vasicinone in the sample
10 L of sample solution was applied in triplicate on a TLC plate. The plate was
developed and scanned as described above. The peak areas were recorded. The amount

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TLC Densitometric Quantification of vasicine, vasicinone and embelin

of vasicine and vasicinone in sample was calculated using the linear regression
equation derived from the calibration curve of vasicine and vasicinone respectively.
8.2.4.3 Method validation

International Conference on Harmonization (ICH) guidelines were followed for the

validation of the analytical procedures (CPMP/ICH/281/95 and CPMP/ICH/381/95).
The method was validated for precision, repeatability and accuracy. Instrumental
precision was checked by repeated scanning of the same spot of vasicine (400 ng) and
vasicinone (160 ng) seven times and was expressed as coefficient of variance (% CV).
The repeatability of the method was affirmed by analyzing 400 ng/spot of standard
solution of vasicine and 160 ng/spot of standard solution of vasicinone after application
on the TLC plate (n = 6) and was expressed as % CV. Variability of the method was
studied by analyzing aliquots of standard solution of vasicine (320, 400, 480 ng/spot)
and vasicinone (160, 240, 320 ng/spot) on the same day (intra-day precision) and on
different days (inter-day precision) and the results were expressed as % CV. Accuracy
of the method was tested by performing recovery studies at three levels (50 %, 100 %
and 125 % addition). The percent recovery and the average percent recovery were
calculated. For the determination of limit of detection and limit of quantification
different dilutions of the standard solutions of vasicine and vasicinone were applied
along with methanol as the blank and determined on the basis of signal to noise ratio.
8.2.5 TLC analysis of Embelia ribes fruit
8.2.5.1 TLC fingerprint profiling

Preparation of sample solution

1 g of the powdered plant material was extracted with methanol (25 ml 2) under
reflux on a water bath. The extracts were filtered, pooled, concentrated and the volume
was made up to 25 ml with methanol.
Preparation of standard solutions
2 mg of embelin (purity 95 %) standard was dissolved in methanol and the volume was
made up to 25 ml in a volumetric flask.
Mobile phase
n Propanol : n Butanol : Ammonia (7 : 1 : 2)

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TLC Densitometric Quantification of vasicine, vasicinone and embelin

Procedure
10 l each of the sample solution and the standard solutions were applied on a
precoated silica gel 60 F254 TLC plate (E. Merck) of uniform thickness (0.2 mm) and
the plate was developed in a twin trough chamber containing mobile phase n Propanol
: n Butanol : Ammonia (7 : 1 : 2) up to a distance of 8 cm at 25 2C temperature and
40 % relative humidity. The plate was dried at room temperature and scanned at 333 nm
for embelin.
The plate was observed under UV light at 254 nm and the Rf of the resolved bands were
noted.
8.2.5.2 Estimation of embelin using TLC densitometric method

Preparation of sample solution

1 g of the powdered plant material was extracted with methanol (25 ml 2) under
reflux on a water bath for 30 min. The extracts were filtered, pooled, concentrated and
the volume was made up to 25 ml with methanol.
Preparation of standard solution of embelin
A stock solution of embelin was prepared by dissolving 2 mg of accurately weighed
embelin in methanol and making up the volume to 25 ml with methanol. From this
stock solution standard solutions of 80-480 g/ml were prepared by transferring
aliquots (0.5 to 3 ml) of stock solution to 10 ml volumetric flasks and adjusting the
volume with methanol.
Calibration curve embelin
10 l of the standard solution embelin was applied in triplicate on TLC plate. The plates
were developed in a solvent system of n Propanol : n Butanol : Ammonia (7 : 1 : 2)
at 252 C temperature and 40 % relative humidity, up to a distance of 8 cm. After
development, the plate was dried in air and scanned at 333 nm for embelin. The peak
areas were recorded. Calibration curve of embelin was prepared by plotting peak areas
vs concentration.
Quantification of embelin in the sample
10 L of sample solution was applied in triplicate on a TLC plate. The plate was
developed and scanned as described above. The peak areas were recorded. The amount
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TLC Densitometric Quantification of vasicine, vasicinone and embelin

of embelin in sample was calculated using the linear regression equation derived from
the calibration curve of embelin.
8.2.5.3 Method validation

International Conference on Harmonization (ICH) guidelines were followed for the

validation of the analytical procedures (CPMP/ICH/281/95 and CPMP/ICH/381/95).
The method was validated for precision, repeatability and accuracy. Instrumental
precision was checked by repeated scanning of the same spot of emeblin (320 ng) was
expressed as coefficient of variance (% CV). The repeatability of the method was
affirmed by analyzing 320 ng/spot of standard solution of embelin after application on
the TLC plate (n = 6) and was expressed as % CV. Variability of the method was
studied by analyzing aliquots of standard solution of embelin (160, 240, 320 ng/spot)
on the same day (intra-day precision) and on different days (inter-day precision) and the
results were expressed as % CV. Accuracy of the method was tested by performing
recovery studies at three levels (50 %, 100 % and 125 % addition). The percent
recovery and the average percent recovery were calculated. For the determination of
limit of detection and limit of quantification different dilutions of the standard solutions
of vasicine and vasicinone were applied along with methanol as the blank and
determined on the basis of signal to noise ratio.

8.3. RESULTS AND DISCUSSION

8.3.1 TLC fingerprint profiling of Adhatoda zeylanica leaves
The constituents other than the biomarkers may be contributing to the therapeutic effect
of the plant either by synergizing or adding to the effect of the active ingredients, or by
antagonizing their adverse effects, or by potentiating their bioavailability (Hamburger
and Hostettman, 1991) Therefore, as far as the quality of a medicinal plant drug is
concerned, a systematic consideration of all its phytoconstituents is as important as the
quantification of the active constituents present in it. TLC fingerprint profile is a
systematic documentation of all the constituents of a sample extract resolved in a given
chromatographic system. It provides a semi-quantitative sketch of the chemoprofile of
the plant extract.
The methanolic extract of Adhatoda zeylanica leaf was used for TLC fingerprint
profiling and co-TLC with vasicine and vasicinone. The methanolic extract showed the

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TLC Densitometric Quantification of vasicine, vasicinone and embelin

best possible resolution of its constituents on precoated silica gel TLC plates, when
developed in mobile phase containing Ethyl acetate : Methanol : Ammonia (8 :2 :0.2
v/v). The developed TLC plate, when observed under UV light showed bands at 254
nm and Rf value of which are mentioned in (Table 8.1; Figure 8.1).
Table 8.1: TLC details of
sample
solution
of
Adhatoda zeylanica leaf.
Under UV light at
254 nm
(Rf value)
0.13
0.27
0.62 (Vasicine)
0.72
0.81 (Vasicinone)
0.92

1,1

Figure 8.1 TLC profile of Adhatoda

zeylanica leaf. 1: Vasicine standard
(0.8g) ; 2: Vasicinone standard
(0.8g); 3,4,5: Sample solution (0.4g,
0.8g, 1.6g)

8.3.2 Quantification of biomarkers vasicine and vasicinone

As far as the analysis of herbal raw materials and herbal preparations is concerned,
HPTLC has an edge over other instrumental analytical techniques because it is simple,
economical and requires minimum sample clean-up. That is why HPTLC has emerged
as an efficient tool for the phytochemical evaluation of herbal drugs (Rajani et al.,
2001; Quality Standards of Indian Medicinal Plants, 2003). A densitometric technique
scanned at 298 nm and 233 nm was therefore adopted for the quantification of vasicine
and vasicinone respectively in the leaf of Adhatoda zeylanica leaf using HPTLC.
Of the various solvent systems tried, the one containing Ethyl acetate: Methanol:
Ammonia (8 : 2 : 0.5 v/v) gave the best resolution of vasicine (Rf = 0.62) and vasicinone
(Rf = 0.81) in the presence of other compounds in the sample extract.
The methods were validated in terms of precision, repeatability and accuracy (Table
8.2). The linear relationship between peak area and amount of vasicine applied was
found only within the range of 320 - 960 ng/spot with correlation coefficient 0.999 and
standard deviation 3.62. The linear relationship between peak area and amount of
vasicinone applied was found only within the range of 80-480 ng/spot with correlation
coefficient 0.999 and standard deviation 3.62.The intra-day and inter-day precision

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TLC Densitometric Quantification of vasicine, vasicinone and embelin

expressed as % CV (Table 8.3) indicate that the proposed method was precise and
reproducible. The limit of detection for vasicine was found to be 40 ng and the limit of
quantification was found to be 80 ng. The average of percentage recovery at three
different levels was found to be 99.14 % (Table 8.3). The limit of detection for
vasicinone was found to be 20 ng and the limit of quantification was found to be 40 ng.
The average of percentage recovery at three different levels was found to be 98.45 %
(Table 8.4).
The amount of vasicine and vasicinone in Adhatoda zeylanica leaf was found to be 1.25
% (w/w) and 0.039 % (w/w) respectively as quantified by the proposed method.
A

Figure 8.2: A. TLC densitometric scan at 298 nm of Adhatoda zeylanica leaf methanolic extract
(blue line) along with vasicine standard (purple line). B. Overlay of UV absorption
spectra of the vasicine in the sample track with the vasicine standard

A
4444444444444444444444444444444444444

Figure 8.3: A. TLC densitometric scan at 233 nm of Adhatoda zeylanica leaf methanolic extract
(orange line) along with vasicinone standard (pink line) B. Overlay of UV absorption
spectra of the vasicinone in the sample track with the vasicinone standard

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TLC Densitometric Quantification of vasicine, vasicinone and embelin

Table 8.2: Method validation parameters for the quantification
of vasicine and vasicinone by the proposed method
Parameters

Vasicine Vasicinone

Instrumental precision

0.39

1.55

0.13

1.62

99.14

98.45

Limit of detection

40 ng

20 ng

Limit of quantification

80 ng

40 ng

Specificity

Specific

Specific

Linearity (Correlation coefficient)

0.999

0.999

Range (ng/spot)

320 - 960

80 480

(% CV, n = 7)
Repeatability of standards
(% CV, n = 6)
Accuracy
(average % recovery)

Table 8.3: Intra-day and Inter-day precision of vasicine and vasicinone

Marker compound

Concentration
-1

(ng spot )
Vasicine

Vasicinone

Intra-day

Inter-day

Precision

Precision

320

0.57

0.42

400

0.47

0.30

480

0.68

0.25

160

1.51

1.55

240

0.92

1.55

320

0.65

1.07

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TLC Densitometric Quantification of vasicine, vasicinone and embelin

Table 8.4 Recovery study of vasicine and vasicinone by the proposed TLC
densitometric method
Marker
compound

Vasicine

Vasicinone

Amount
present in
the sample
(g)

Amount
added
(g)

Recoverya
(%)

Amount
founda (g)

1250

625

1855.582.22

98.960.46

1250

1250

2480.85.99

99.231.07

1250

1562.5

185214.13

99.240.69

39

19.5

47.900.01

98.250.54

39

39

76.850.01

98.521.01

39

48.75

86.790.02

98.900.73

Average
recovery
(%)
99.14

98.45

Mean Standard deviation, (n = 3).

8.3.3 TLC fingerprint profiling of Embelia ribes fruits

The methanolic extract of Embelia ribes fruit leaf was used for TLC fingerprint
profiling and co-TLC with embelin. The methanolic extract showed the best possible
resolution of its constituents on precoated silica gel TLC plates, when developed in
mobile phase containing n-propanol : n-butanol : Ammonia (7 : 1 : 2 v/v). The
developed TLC plate, when observed under UV light showed bands at 254 nm and Rf
value of which are mentioned in (Figure 8.4).

Table 8.5: TLC details of

sample solution of Embelia
ribes fruit
Under UV light at
254 nm (Rf value)
0.35 (Embelin)
0.40
0.62
0.68

Figure 8.4: TLC profile of

Embelia ribes fruit.
1: Embelin; 2: Sample
solution

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TLC Densitometric Quantification of vasicine, vasicinone and embelin

8.3.4 Quantification of biomarker embelin

Of the various solvent systems tried, the one containing n-propanol: n-butanol:
Ammonia (7 : 1 : 2 v/v) gave the best resolution of embelin (Rf = 0.35) in the presence
of other compounds in the sample extract.
The methods were validated in terms of precision, repeatability and accuracy (Table
8.6). The linear relationship between peak area and amount of embelin applied was
found only within the range of 320 - 960 ng/spot with correlation coefficient 0.993 and
standard deviation 4.12. The intra-day and inter-day precision expressed as % CV
(Table 8.7) indicate that the proposed method was precise and reproducible. The limit
of detection for embelin was found to be 20 ng and the limit of quantification was found
to be 40 ng. The average of percentage recovery at three different levels was found to be
99.47 % (Table 8.8).
The amount of embelin in Embelia ribes fruit was found to be 3.21 % w/w as quantified
by the proposed method.
A

Figure 8.5 A. TLC densitometric scan at 337 nm of Embelia ribes fruit methanolic extract
(blue line) along with embelin standard (purple line). B. Overlay of UV absorption spectra
of the embelin in the sample track with the embelin standard.

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TLC Densitometric Quantification of vasicine, vasicinone and embelin

Table 8.6: Method validation parameters for the
quantification of embelin by the proposed method
Parameters

Embelin

Instrumental precision

0.83

(% CV, n = 7)
Repeatability of standards

0.46

(% CV, n = 6)
Accuracy

99.47

(average % recovery)
Limit of detection

20 ng

Limit of quantification

40 ng

Specificity

Specific

Linearity (Correlation coefficient)

0.993

Range (ng/spot)

80 480

Table 8.7 Intra-day and Inter-day precision of embelin

Marker
compound

Concentration

Intra-day

Inter-day

(ng spot-1)

precisiona

precisiona

Embelin

160

1.27

1.59

240

0.45

1.11

320

0.55

0.93

Table 8.8 Recovery study of embelin by the proposed TLC densitometric

method
Amount
founda (g)

Recoverya
(%)

1605

4793.5 2.22

99.55 0.46

3210

3210

6381.9 5.99

99.40 1.07

3210

4012.5

7184.2 4.13

99.46 0.69

Marker
compound

Amount
present in
the sample
(g)

Amount
added
(g)

Embelin

3210

Average
recovery
(%)
99.47

Mean Standard deviation, (n = 3)

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TLC Densitometric Quantification of vasicine, vasicinone and embelin

8.4 CONCLUSION
The developed HPTLC methods for the quantification of vasicine, vasicinone and
embelin were found to be simple, precise, specific and sensitive. The amount of
vasicine and vasicinone in Adhatoda zeylanica leaf was found to be 1.25 and 0.039 %
(w/w) respectively. The amount of embelin in Embelia ribes fruit was found to be 3.21
% (w/w).

144