A C Suthar et al.

/ Journal of Pharmacy Research 2009, 2(12),1893-1899

Research Article
ISSN: 0974-6943 Available online through
http://jprsolutions.info
Quantitative estimation of Vasicine and Vasicinone in Adhatoda vasica by HPTLC
A C Suthar 1*, K V Katkar 1, P S Patil 2, P D Hamarapurkar 2, Mridula G 3, V R Naik 1, G R Mundada 1, V S Chauhan 1
1
Herbal Deptt, Piramal Life Sciences Ltd., 1, Nirlon Complex, Off. Western Express Highway,
Goregaon (East), Mumbai – 400 063. Maharashtra, India
2
Pharmaceutical Analysis, Principal K. M. Kundnani College of Pharmacy, Jote Joy Building,
Rambhau Salgaoankar Marg, Cuffe Parade, Mumbai 400 005. India.
3
Pharmacognosy Deptt., Manipal College of Pharmaceutical Sciences, Manipal University, Manipal, Karnataka, India.
Received on: 22-09-2009; Accepted on:05-11-2009
ABSTRACT
The present paper deals with development and standardization of HPTLC method used for quantification of vasicine and vasicinone in
Adhatoda vasica commonly known as Vasaka, a plant of the family Acanthaceae. Today, vasaka is the most widely known herb and widely
used in the treatment for a variety of respiratory ailments including cough, bronchitis and asthma. Both vasicine and vasicinone are two major
alkaloids of A. vasica, which are known to possess biological activities like respiratory stimulant, bronchodilator and hypotensive activity.
Hence, a need to develop and standardize a chromatographic method for quantification of vasicine and vasicinone, for better standardization
of Adhatoda vasica. An attempt has been made to quantify vasicine and vasicinone in A. vasica raw herbs, extracts and formulations by
HPTLC method. The lowest detectable limit of vasicine and vasicinone was found up to 1 ng and 25 ng respectively with good resolution and
separation of vasicine and vasicinone from other constituents of A. vasica. Further, recovery values of vasicine and vasicinone were found
to be about 95-102 %, which shows the reliability and suitability of the method. The validated HPTLC method was found to be simple,
reproducible, accurate and precise. The structure of isolated vasicine and vasicinone was characterized and confirmed by various advanced
spectroscopic methods.

Keywords: HPTLC, Adhatoda vasica, Vasicine, Vasicinone

Peak & UV spectra
of Vasicine
INTRODUCTION
Adhatoda vasica Nees, indigenous to India, is a member of the
Acanthaceae family. It is a well -known plant drug in Ayurvedic and
Unani medicine. A. vasica is one of the commonest shrubs distrib-
uted throughout India and especially in hedges, wastelands and graz-
ing grounds and also over hilly tracts upto a height of 4000 feets
above sea level 1. A number of different principles including, alka- Peak & UV spectra of
loids: vasicine, vasicinone, vasinol, essential oil: betane, vitamins: Vasicinone
vitamin C, b-carotene, a non-crystalline steroid: vasakin and a mixture
of fatty acids have been identified as contributing to the observed
medicinal effects of the plant 2. Vasaka is mentioned as an herbal
remedy for treating cold, cough, whooping cough and chronic bron-
chitis and asthma, as sedative expectorant, tuberculosis, Inflamma-
tion, anti-allergic, antispasmodic and anthelmintic 3-6. The drug is
employed in different forms such as fresh juice, decoction, infusion HPLC 13-14, available for estimation of vasicine and vasicinone. The
and powder; also given as alcoholic extract and liquid extract or syrup limitation of reported HPTLC methods are use of ammonia in solvent
7
. system, which is hazardous, higher limit of detection & quantification
and linear range, which demonstrates lower sensitivity and precision
Vasicine and vasicinone are reported to have bronchodialatory and and use of water in mobile phase with lower linear range. Similarly, in
respiratory stimulation effects and hence regarded as biological mark- various HPLC methods, use of buffer may lead to detoriation of sta-
ers for standardization of A. vasica extract 8-9. Few chromatographic tionary phase and lower sensitivity. Hence, the proposed HPTLC
methods are available and reported, such as HPTLC 10-12 and method was attempted for fast, precise, sensitive and reproducible
method with good recoveries for standardization of extracts of
*Corresponding author. Adhatoda vasica.
Dr. Ashish C. Suthar
Herbal Deptt, Piramal Life Sciences Ltd., Materials and methods
1, Nirlon Complex, Off. Western Express Highway, The vasaka leaves were collected from two different locations in
Goregaon (East), Mumbai – 400 063. Maharashtra, India Maharashtra (coded as AD-1 and AD-2) and used as samples for
quantification of vasicine and vasicinone. Similarly, a syrup formula-
Journal of Pharmacy Research Vol.2.Issue 12.December 2009 1893-1899
 A C Suthar et al. / Journal of Pharmacy Research 2009, 2(12),1893-1899
tion – in-house preparation (coded as IN-1) was also evaluated for per spot. Reproducibility, linearity and validation of the proposed
the content of vasicine and vasicinone. method was checked along with correlation coefficient, coefficient of
variance and the linearity curve for both markers.
Pure vasicine and vasicinone were isolated from the dried leaves of A.
vasica. Purity and structure of isolated compounds (vasicine and Procedure: -2
vasicinone) were confirmed by HPTLC, HPLC and spectral analysis
like NMR, IR and MS 15. These isolated compounds vasicine and
(Calibration curve using extract spiked with vasicine and vasicinone)
vasicinone were used as working standards for quantification in dried
raw herbs, extracts and formulations. The content of vasicine and vasicinone in aqueous and methanolic
extracts was determined by comparing with the calibration curve of
Preparation of the extract the working standard of vasicine and vasicinone. The aqueous ex-
The 100 gms of Adhatoda vasaka leaves dried powder was extracted tract, which showed lowest content of vasicine and vasicinone, was
with 500 ml solvent (methanol and water) by stirring at 500C for 1 hr. then used as blank. This blank was then used to spike extract with the
The filtered extract was concentrated under reduced pressure to re- working standard of vasicine and vasicinone. Different samples with
varying amount of standard vasicine in range of 25 µg / ml to 75 µg /
move the solvent. The extraction carried out for two times with the
ml and vasicinone in range of 250 µg / ml to 750 µg / ml were spiked
above-mentioned protocol. The extract was obtained by drying the separately in 10 mg of blank extract with pre-determined vasicine and
concentrated pooled extract under vacuum. These extracts were used Vasicinone. Procedure for sample preparation was followed as men-
for estimation and comparison of vasicine and vasicinone content.
tioned above. In each sample preparation, 10 µl of spiked solution
was then subjected to HPTLC with 10 µl of blank solution for com-
Equipment
A Camag HPTLC system equipped with a sample applicator Linomat parison. The percent recovery of vasicine and vasicinone standard
V, twin trough plate development chamber, TLC Scanner III and was calculated. Reproducibility, precision and validation of the method
Reprostar, Wincats and integration software 4.02 (Switzerland). were achieved by analyzing six replicate spiked sample solutions.
Correlation coefficient, coefficient of variance was calculated.
Sample preparation
Accurately weighed 20 - 50 mg of aqueous extract and methanol ex- Linearity along with limit of detection and limit of quantitation
tract of vasaka, 1 gm of dried raw herb or 10 gms cough syrup contain- For a long-term use of the analytical method, a rigorous valida-
ing A. vasica were separately extracted with methanol (10 ml x 3) by tion is indicated and requires the following procedures. For the
vortexing and allowed to stand for 5 min. at room temperature. The preparation of calibration curve, the stock solution was diluted
methanol extract was then filtered through Whatmann no.42 filter freshly with methanol to obtain a set of 11 calibration standards.
paper; extracts were pooled and concentrated to dryness under These standards were measured and the integrated peak areas
vacuum. Final volume was made to 10 ml with methanol in volumetric were plotted against the corresponding concentrations of the stan-
flask. Vasicine and Vasicinone content were then analyzed after sub- dards. The complete procedure was repeated on three consecu-
jecting to HPTLC. tive days. The so obtained three calibration curves were used to
calculate a mean calibration graph. The limit of detection was
HPTLC method obtained by analyzing signal to noise ratio and limit of quantifica-
Silica gel 60 F254 precoated plates (20 x 10 cm) were used with Chloro- tion was defined as the lowest concentration of linear range.
form: Methanol (90:10) as solvent system. 1-10 µl of working standard
of vasicine, vasicinone as well as test samples were spotted on pre- Intraday and interday analysis using vasicine and vasicinone
coated HPTLC plates. The bandwidth applied on plate was 6 mm and Three different concentrations using a different stock solution of
ascending mode was used for development of thin layer chromatog- vasicine (25, 50 and 75 ng / spot) and vasicinone (250, 500 and 750 ng
raphy. Saturation time was 20 mins along with humidity level - 65% ± / spot) were spotted. For the determination of the intraday precision
5% RH and room temperature - 25°C ± 2°C. TLC plates were devel- and accuracy, three replicates of the standard solutions were ana-
oped upto 8 cm. The TLC plates were scanned at 280 nm for quantifi- lyzed at the same day in triplicate. The precision and the accuracy of
cation purpose. the interday analysis were determined by analyzing the standard
solution on 3 different days in triplicate.
Procedure: -1
Stability
(Calibration curve of standard vasicine and vasicinone) Vasicine (1 mg), vasicinone (1 mg) and vasaka methanolic extract (10
10 milligram of working standards vasicine and vasicinone were dis- mg) were extracted in 10 ml methanol as per the method described in
solved separately in 10 ml of methanol to yield stock solutions of 1 sample preparation. The sample solutions were kept at 4°C in dark
mg/ml concentration. Calibration curve was obtained by spotting 2 and analyzed on consecutive days (24, 48, 72 and 96 hrs) to observe
ng to 100 ng of vasicine per spot and 25 ng to 1000 ng of vasicinone the stability of standard as well as sample solution.
Journal of Pharmacy Research Vol.2.Issue 12. December 2009 1893-1899
 A C Suthar et al. / Journal of Pharmacy Research 2009, 2(12),1893-1899
Robustness and Ruggedness studies Fig. II: TLC Chromatogram of vasicine and vasicinone in Adhatoda
Robustness and ruggedness parameters were applied by making vasica methanolic extract
small deliberate changes of the conditions (mobile phase composi- Peak & UV
spe ctra of
tion, mobile phase volume, saturation time, time from application to Vasicine

chromatography, time from chromatography to scanning and ana-

lyst) to validate the method.

RESULTS & DISCUSSION Peak & UV

spe ctra of
Mixture of working standards of vasicine and vasicinone showed Vasicinone

peak in HPTLC chromatogram along with their respective UV spec-

tra. (Fig I). HPTLC chromatogram of methanolic extract showing the
presence of two marker compounds such as vasicine and vasicinone
along with their respective UV spectra is shown in fig. II. Spot of
vasicine and vasicinone are visible with Rf ≈ 0.1 - 0.11 and 0.45 – 0.48
at 254 nm as shown in photograph 1. The calibration curve of working
standards vasicine and vasicinone was obtained by spotting stan-
dard vasicine and vasicinone on HPTLC plate after scanning at 280
nm as shown in figure III and IV. Various samples including raw herb,
extracts and formulations of Adhatoda vasica were analysed by the
proposed method and the data are recorded in table I.
Table I: Percentage of Vasicine and Vasicinone in different samples Photograph 1: HPTLC Photograph of methanolic extract of Adhatoda
of Adhatoda vasica by measuring area in HPTLC method vasica along with std. vasicine and vasicinone
Sr. Sample Vasicine content Vasicinone content
name (% w/w) (% w/w)

1 AD-1 0.05 0.015

2 AD-2 0.07 Not detected
3 Aqueous extract 0.3 0.12
4 Methanolic extract 0.75 0.45
5 IN-1 0.40 mg/ 5 ml of syrup 0.25 / 5 ml of syrup Vasicinone

Fig.I: TLC Chromatogram of working standard vasicine and

vasicinone in mixture along with UV spectra
Vasicine

1 2 3 4 5 6 7 8

Peak & UV spectra 1 - 2: Methanol extract of Adhatoda vasica ; 3 – 5: Vasicine; 6 – 8: Vasicinone

of Vasicine
Chromatographic precision and recoveries from spike sample solution
Specificity
It was observed that the other phytoconstituents present in the ex-
tracts did not interfere with the peak of vasicine and vasicinone. There-
fore the method was specific and helps in separation of vasicine and
vasicinone from other constituents of herb and hence, helps to get
Peak & UV spectra the exact content of both vasicine and vasicinone. Test sample of
of Vasicinone
aqueous and methanolic extract of Adhatoda vasica extract showed
separated peak of vasicine and vasicinone along with other
phytoconstituents as obtained in HPTLC chromatogram (Figure II).

Limit of Detection
By applying the proposed method, the minimum detectable limit of
vasicine was found to be 1 nanogram / spot at 280 nm and vasicinone
was found to be 25 nanogram / spot at 280 nm.
Journal of Pharmacy Research Vol.2.Issue 12.December 2009 1893-1899
 A C Suthar et al. / Journal of Pharmacy Research 2009, 2(12),1893-1899
Limit of Quantification Accuracy and precision
By applying the proposed method, the minimum quantification limit The method was applied to determine concentration of spiked vasicine
of vasicine was found to be 5 nanogram / spot at 280 nm, the lowest and vasicinone in test sample with the range of 25 – 75 ng / spot for
concentration in linear range of vasicinone was found to be 50 nano- assessing the accuracy & precision of the procedure. Table II repre-
gram / spot at 280 nm sents the mean values and coefficient variance (C.V.) results indicate
the levels in the above range can be estimated with accuracy and
Linearity precision. (Table II & III)
The linearity of the method was checked with working standards
vasicine with the calibration curve in the concentration range of 5 – Table II: Precision & accuracy of the method applied to spiked vasicine
100 ng / spot based on a 0.5 - 10 µl sample volume. The regression samples
equations (Y = 39.932* X + 157.96) and correlation coefficient were Amount Amount found Precision / Mean
obtained with 6 replicate analysis for each concentration. Correlation added (µg /spot) Reproducibility Recovery
(µg / spot) (Mean ± S.D., n=6) (C.V.) (%)
coefficients were obtained in the range of 0.9965-0.9986 indicated
25 24.74 ± 1.11 3.09 98.96
excellent linearity of the procedure for working standard vasicine ana- 50 46.44 ± 3.26 2.26 95.33
lyzed. Calibration curve of working standard vasicine is shown in fig 75 73.75 ± 4.18 3.46 98.33
III.
Table III: Precision & accuracy of the method applied to spiked
V a s ic ine L ine a r ity vasicinone samples
Amount Amount found Precision / Mean
5000 added (µg /spot) Reproducibility Recovery
(µg / spot) (Mean ± S.D., n=6) (C.V.) (%)
4000
250 255.45 ± 18.41 5.25 102.18
3000 500 475.84 ± 35.00 1.83 95.16
2000 750 713.30 ± 50.13 1.64 95.10
y = 39.932x + 157.96 Intraday and interday analysis using vasicine and vasicinone
1000
R2 = 0.9986
0 Furthermore the precision and accuracy of the intraday and
0 20 40 60 80 100 120 interday analysis were investigated on the basis of a set of stan-
dard solution. The results given in Table IV & V stands for a quite
Fig.III: Calibration curve of working standard vasicine with respect good trueness of the proposed method particularly considering
to the area under curve at various concentrations. interday and intraday analysis.
The linearity of the method was checked with working standards Table IV: Intraday and interday precision & accuracy of the method
vasicinone with the calibration curve in the concentration range of 50 applied to vasicine
– 1000 ng / spot based on a 0.5 - 10 µl sample volume. The regression
Amount Intraday Interday
equations (Y = 5.3032 * X + 301.78) and correlation coefficient were added
obtained with 6 replicate analysis for each concentration. Correlation (µg / spot) Precision Accuracy Precision Accuracy
(R.S.D., %) (%) (R.S.D., %) (%)
coefficients were obtained in the range of 0.9941 - 0.9954 indicated
excellent linearity of the procedure for working standard vasicinone 2 5 4.89 94.39 4.28 95.23
50 3.24 96.32 3.92 95.62
analyzed. Calibration curve of working standard vasicinone is shown 7 5 1.32 98.46 4.26 104.9
in fig IV.
Table V: Intraday and interday precision & accuracy of the method
Vasicinone linearity
applied to vasicinone
Amount added Intraday Interday
6000
(µg / spot)
5000 Precision Accuracy Precision Accuracy
(R.S.D., %) (%) (R.S.D., %) (%)
4000
3000 250 3.43 96 4.01 95.87
y = 5.3022x + 301.78 500 0.73 100.83 3.13 96.82
2000 750 0.71 100.77 2.09 102.42
R2 = 0.9954
1000
0 Stability
0 200 400 600 800 1000 1200 In the current assay, analyses of stability samples in methanol on
consecutive days (24, 48, 72 and 96 hr) revealed that the major con-
Fig. IV: Calibration curve of working standard vasicinone with re- stituents, vasicine and vasicinone either in standard solution or in
spect to the area under curve at various concentrations. the methanolic extract of Adhatoda vasica are stable in solution
Journal of Pharmacy Research Vol.2.Issue 12. December 2009 1893-1899
 A C Suthar et al. / Journal of Pharmacy Research 2009, 2(12),1893-1899
form with relative standard deviation (RSD (%)) - 3.87 and 2.52 (n = 6)
and for vasicine and vasicinone at 4°C respectively.
Robustness and Ruggedness studies 6.
The method was found to be re-producible from one analyst to an-
other. The low values of R.S.D. (2.97% - 3.54%) obtained after small
deliberate changes of the conditions (mobile phase composition, 7.
mobile phase volume, saturation time, time from application to chro-
matography, and time from chromatography to scanning) used for the
method indicated its robustness. 8.
CONCLUSION 9.
The lowest detectable limit of vasicine was found upto 1 ng / spot and
of vasicinone was found to be 25 ng / spot provides good resolution
and separation of vasicine and vasicinone from other constituents of 10.
Adhatoda vasica. Further, recovery values of vasicine and vasicinone
were found to be about 95 – 102 %, which shows the reliability and
suitability of the method. The proposed HPTLC method is simple, 11.
rapid, reproducible, accurate and precise for quantitative monitoring
of vasicine and vasicinone in Adhatoda vasica samples.
REFERENCES:
1. Bagchi, G.D.; Haider, F.; Dwivedi, P.D.; Srivastava, D.; Srivastava,
S.; Chattopadhyay, S.K. (2003) Seasonal variation in vasicine
content in Adhatoda species grown under north Indian plain
conditions. J Med & Aroma Plant Sci. 25(4): 1043 - 45.
2. Anonymous, Wealth of India, 1989. Raw Materials. Council Sci.
Ind. Res., New Delhi, India.
3. Dorsch, W.; Wagner, H. (1991) New antiasthmatic drugs from
traditional medicine. Int Arch Allergy Appl Immunol 94: 262 - 65.
4. Grange, J.M.; Snell, N.J. (1996) Activity of bromhexine and
ambroxol, semi-synthetic derivatives of vasicine from the Indian
shrub Adhatoda vasica, against Mycobacterium tuberculosis
in vitro. J Ethnopharmacol 50: 49 - 53.
Source of support: Nil, Conflict of interest: None Declared
Journal of Pharmacy Research Vol.2.Issue 12.December 2009
5. Chakraborty, A.; Brantner, A.H. (2001) Study of alkaloids from
Adhatoda vasica Nees on their antiinflammatory activity.
Phytother Res 15: 532 -34.
Wagner, H. (1989) Search for new plant constituents with poten-
tial antiphlogistic and antiallergic activity. Planta Med 55: 235 -
41.
Dhuley, J.N. (1999) Antitussive effect of Adhatoda vasica extract
on mechanical or chemical stimulation-induced coughing in ani-
mals. J Ethnopharmacology 67: 361-365.
Gupta, O.P.; Sharma, M.L.; Ghataka, B.J.; Atal, C.K. (1977) Potent
uterine activity of alkaloid vasicine. Ind J Med Res 66: 680-91.
Bhide, M.B.; Naik, P.J.; Ghooi, R.B. (1976) Comparative studies
on the pharmacological evaluation of vasicine and vasicinone.
Bull Haffkin Inst 4: 45-50.
Das, C.; Poi, R.; Chowdhary, A. (2005) HPTLC Determination of
vasicine and vasicinone in Adhatoda vasica. Phytochemical
Analysis 16: 90-92.
Soni, S.; Anadjiwala, S.; Patel, G.; Rajani, M. (2008) Validation of
different methods of preparation of Adhatoda vasica leaf juice
by quantification of total alkaloids and vasicine. Ind J Pharma
Sci. 70 (1): 36-42.
12. Narayana, D.B.A.; Agarwal, S.; Luthra, S.K.; Srinivas, N. (1995) A
HPTLC method for the quantitative analysis of vasicine in
Adhatoda vasica. Indian Drugs 32 (12): 583-86.
13. Chauhan, S.; Kimothi, G.; Singh, B.; Agarwal, S. (1999) Develop-
ment of HPLC method for vasicine and vasicinone in Adhatoda
vasica (Nees). Ind J Nat Products 15 (1): 21-24.
14. Parikh, K.; Doshi, V.; Salunkhe, J.; Kamat, R. (1989) High Perfor-
mance Liquid Chromatographic determination of vasicine and
vasicinone from pharmaceutical formulations. Indian Drugs 27(1):
64-70.
15. Joshi, B.S.; Bai, Y.; Puar, M.S.; Dubose, K.K.; Pelletier, S.W. (1994)
1
H and 13C – NMR assignments for some pyrrole [2,b] –
quinazoline alkaloids of A. vasica. J Nat Prod 57: 953 – 62.
1893-1899