Fitoterapia 82 (2011) 446–453

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Fitoterapia
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / f i t o t e

Formulation and pharmacokinetic evaluation of hard gelatin capsule

encapsulating lyophilized Vasa Swaras for improved stability and oral
bioavailability of vasicine
Tejas Vyas a, Ranjeet Prasad Dash b, Sheetal Anandjiwala a, Manish Nivsarkar b,⁎
a
Department of Natural Products, National Institute of Pharmaceutical Education and Research-Ahmedabad, S. G. Highway, Thaltej,
Ahmedabad – 380054, Gujarat, India
b
Department of Pharmacology and Toxicology, B. V. Patel Pharmaceutical Education and Research Development (PERD) Centre, S. G. Highway, Thaltej,
Ahmedabad – 380054, Gujarat, India

a r t i c l e i n f o a b s t r a c t
Article history: The oral bioavailability of vasicine (1) was investigated in hard gelatin capsules of lyophilized
Received 24 September 2010
Vasa Swaras (aqueous extract of Adhatoda vasica Nees.,Fam.: Acanthaceae) The rat
Accepted in revised form 9 December 2010
pharmacokinetic profile of lyophilized Vasa Swaras, Vasa Swaras, vasicine (1) (chief marker
Available online 24 December 2010
compounds of A. vasica) and a marketed capsule formulation of A. vasica were compared.
Vasicine (1) was found to be more orally bioavailable from lyophilized Vasa Swaras, with an
Keywords: overall minor conversion to vasicinone (2).
Vasicine
© 2010 Elsevier B.V. All rights reserved.
Vasicinone
RP-HPLC
Capsule
Pharmacokinetic

1. Introduction stimulant and uterine stimulant effects [10]. Although vasicine

Adhatoda vasica (L.) Nees (family: Acanthaceae) known
commonly as Malabar nut tree, is a shrub growing throughout
the Indian peninsula up to an altitude of 1300 m. The plant
has been used in the indigenous system of medicine in India
for over 2000 years and is a well-known drug in both
Ayurvedic, and Unani System of Medicine [1,2]. It is known
as Vasa in Ayurveda. Ethnomedically, the leaves are pre-
scribed for malarial fever, fever caused by pitta (alteration in
body temperature and inflammation) and kapha (congestion
due to phlegm), chronic fever, intrinsic hemorrhage, cough,
asthma, leprosy, skin diseases and piles [3]. The plant is reported
to show expectorant [4], abortifacient [5], antimicrobial [6,7] and
antitussive [8] activities. Important chemical constituents of the
leaf include pyrroloquinazoline alkaloids — vasicine (1), vasicol,
adhatonine, vasicinone (2), vasicinol, and vasicinolone [9].
Vasicine (1) is reported to have bronchodilatory, respiratory
⁎ Corresponding author. Tel.: + 91 7927413219; fax: + 91 7927450449.
E-mail address: manishnivsarkar@gmail.com (M. Nivsarkar).
(1) is not yet available as a single therapeutic formulation,
the aqueous extract of A. vasica (Vasa Swaras) is incorporated in
more than 20 Ayurvedic preparations and formulations includ-
ing Vasarishta, Mahatiktaka ghrita, Triphala ghrita, Vasavaleha,
Vasakasava, Mahatriphalaghrita, Panchatiktaghritaguggulu and
Panchatikta ghrita [11]. The extract of A. vasica is also used in the
preparation of some polyherbal pharmaceutical formulations
(lozenges) intended for treatment and management of coughs
and sore throats [12]. Some of the marketed polyherbal
formulations incorporating A. vasica extract are Diakof ®,
Evecare®, Geriforte®, Koflet®, Lukol®, Styplon®, and Geriforte
Aqua® (doses varying from 15 mg/5 ml of syrup to 30 mg/
capsule as A. vasica extract). The dosages of these formulations
are decided by the physician depending on the severity of the
disease. However, despite of the availability of several study
reports on vasicine (1), it has not yet been approved and/or used
as a single therapeutic molecule. One of the major problems
associated with vasicine (1) is its low bioavailability and stability
which is reflected from its rapid and significant conversion to
vasicinone (2) both in vitro and in vivo [13,14]. Our previous

0367-326X/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.fitote.2010.12.005
 T. Vyas et al. / Fitoterapia 82 (2011) 446–453
study based on determining the pharmacokinetic profile of Vasa
Swaras and its comparison with pure vasicine (1) and vasicinone
(2) inferred that vasicine (1) was found to be more orally
bioavailable from Vasa Swaras and its conversion into vasicinone
(2) was also less as compared to pure vasicine (1) [14]. Hence, in
continuation to our previous study, an attempt was made for
enhancing the stability of vasicine (1).
In the present study, the stability of vasicine (1) in its crude
form-Vasa Swaras, was enhanced by formulating it as lyophi-
lized hard gelatin capsules. The authors have evaluated the
comparative pharmacokinetic profile of the developed formula-
tion (Lyophilized Vasa Swaras), Vasa Swaras (aqueous extract),
vasicine (1) and marketed capsule formulation of A. vasica.
Differences among pharmacokinetic profile of vasicine (1) and
vasicinone (2) upon oral administration of these formulations in
rats are discussed.
2. Materials and methods
2.1. Chemicals and reagents
Vasicine (1) (99.6% pure) and vasicinone (2) (99.4% pure),
(Fig. 1) were respectively purchased from SPIC, Chennai,
India and Natural Remedies, Bengaluru, India. Metronidazole
(3) (99.7% pure), (Fig. 1) obtained as gift sample from J.B.
Chemicals, Mumbai, India was used as an internal standard
(IS). Commercial capsule formulation of A. vasica was
purchased from the local market. All the solvents and
chemicals were of chromatographic grade and purchased
from Qualigen Fine Chemicals, Mumbai, India. Potassium
dihydrogen ortho phosphate (Qualigen Fine Chemicals,
Mumbai, India) and glacial acetic acid (Qualigen Fine
Chemicals, Mumbai, India) were of analytical grade. tertiary-
Butyl methyl ether (TBME) of chromatographic grade was
purchased from Merck, Schuchardt, Hohenbrunn, Germany.
Magnesium stearate (Loba Chemie Pvt. Ltd., India), Avicel PH
101 (Signet Chemical Corporation, India) and Aerosil® 200
(Degussa, Frankfurt, Germany) were used as the pharmaceutical
excipients. Heparin was purchased from Biological E. Ltd.,
Hyderabad, India. Deionized water for HPLC was prepared
in-house using a Milli-Q water purifier system (Millipore Elix,
Germany).
Fig. 1. Chemical structures of (1) vasicine, (2) vasicinone, and (3) metronidazole.
447
2.2. Plant materials
Leaves of A. vasica were collected locally during the month
of August as it is reported to have the highest amount of total
alkaloids during this period [15]. These were shade dried,
stored in an air-tight container and powdered to 40 mesh
whenever required. A specimen of the plant collected was
preserved in the Department of Natural Products, NIPER-
Ahmedabad (Herbarium Specimen #: NIPER-A/NP/0709/07).
2.3. Preparation of Vasa Swaras
Vasa Swaras was prepared following a pre reported
method cited in Saranghdhar Samhita [16], which involves
maceration of dried powdered leaves of A. vasica (100 g) in
double distilled water (200 ml) for 24 h at room temperature.
The resulting mixture was filtered through muslin cloth to
obtain 50 ml of the aqueous extract. The amount of vasicine
(1) and vasicinone (2) in the aqueous extract were quantified
using HPLC–UV method.
2.4. Quantification of vasicine (1) and vasicinone (2) in
Vasa Swaras
Five millilitres of Vasa Swaras was basified upto pH 9 with
5% sodium hydroxide and then extracted with chloroform
until the aqueous portion showed negative Dragendroff's test.
The chloroform fraction were pooled over sodium sulphate
and concentrated. The amount of vasicine (1) and vasicinone
(2) were determined using HPLC with the same chromato-
graphic conditions as stated below.
2.5. Chromatographic conditions
The HPLC system consisted of PU-980 intelligent HPLC pump
(Jasco, Hachioji, Tokyo, Japan) and Intelligent UV-975 UV–
Visible detector (Jasco, Hachioji, Tokyo, Japan), set at 298 nm
and manual injection port. The data were analyzed using Borwin
version 3.1 software. Chromatographic separation was achieved
using a HIQ Sil C18HS column (4.6 mm i.d.×250 mm, 5 μm)
maintained at room temperature. The mobile phase consisted of
acetonitrile:potassium dihydrogen orthophosphate (0.01 M)
(18:82 v/v), pH adjusted to 3.9 with glacial acetic acid. Flow
rate was maintained at 1.0 ml/min. Samples were quantified by
determining the response (Peak AreaDrug / Peak AreaIS).
2.6. Dosage formulation development
2.6.1. Lyophilisation of Vasa Swaras
The aqueous extract (Vasa Swaras) prepared was lyophilized
using lyophilizer (Allied frost, India) at −80 °C for 16–17 h in
order to remove the water content and to obtain the extract in
dry solid form with an objective to enhance the stability and
shelf-life of the aqueous extract.
2.6.2. Differential scanning calorimetry analysis
Thermal characteristics of the vasicine (1) and physical
mixtures of the selected diluents viz. Aerosil® 200, Avicel PH
101 and magnesium stearate with vasicine (1) were
determined by a differential scanning calorimeter (DSC 204,
Netzsch, Germany) using an aluminium sealed pan. The
448 T. Vyas et al. / Fitoterapia 82 (2011) 446–453
scanning rate was 10 °C/min, and the scanning temperature
range was between 25 and 250 °C.
2.6.3. Formulation of hard gelatin capsules
Hard gelatin capsules containing pure vasicine (1) were
prepared by mixing vasicine (1) with the diluents viz. Avicel PH
101 in geometric proportion followed by addition of required
quantity of Aerosil® 200 and magnesium stearate (passed
through sieve no. 80).
Capsules of lyophilized Vasa Swaras were prepared by
mixing lyophilized Vasa Swaras with Avicel PH 101 and
further sifting the mixture through sieve no. 36. The required
quantity of Aerosil® 200 and magnesium stearate was passed
through sieve no. 80, added to vasicine (1) blend and mixed
lightly in a poly bag for 30 s. The quantity of excipients
selected was based on dosage calculation. The resultant
powder blend was filled in hard gelatin capsule shell (size-3)
to obtain the final formulation.
2.7. Physicochemical properties of lyophilized Vasa Swaras
capsules
The developed formulations of vasicine (1) and lyophilized
Vasa Swaras as well as the marketed formulation of A. vasica
were characterized for the following physicochemical para-
meters [17] viz. weight variation, disintegration test, dissolution
test, assay (estimation of drug content), uniformity of content,
moisture content, and stability study.
2.7.1. Weight variation test
Filled capsules (n = 20) of each of the vasicine (1),
lyophilized Vasa Swaras and that of the marketed formulation
were weighed. The weights of the intact capsule and empty
capsule shell were taken, in order to determine the variation in
weight of the prepared formulation.
2.7.2. Disintegration test
Disintegration time of different formulated capsules
(n = 20) were determined according to I.P. disintegration
test for hard gelatin capsules using a disintegration test
apparatus with discs (disintegration media: water; disinte-
gration temperature: 37 ± 0.5 °C). The time taken until no
material from any of the capsule was left on the mesh was
considered as the disintegration time.
2.7.3. Estimation of drug content
The content of vasicine (1) and vasicinone (2) within the
different formulated hard gelatin capsules (n = 20) were
estimated using validated HPLC–UV method. The values of
drug peak area were used for quantitative determination.
2.7.4. Uniformity of content
Uniformity of content of vasicine (1) and vasicinone (2) was
determined from the assay of 20 individual capsules of the
vasicine (1), lyophilized Vasa Swaras and that of the marketed
formulation of the A. vasica. Each sample was analyzed by the
validated HPLC–UV method.
2.7.5. Moisture analysis
The pre-weighed sample of the capsule blend was placed
in an oven at 105 °C and equilibrated. The moisture content of
capsules was determined gravimetrically by reweighing the
capsules after drying (loss on drying) using moisture balance.
2.7.6. Stability study
The developed formulations of vasicine (1), lyophilized
Vasa Swaras and marketed formulation of A. vasica were strip
packed in laminated aluminium foils and subjected to
accelerated stability testing (40 ± 2 °C and 75 ± 5% RH) as
per the ICH guidelines. The formulations were also stored at
ambient temperature. The samples of stability study were
analyzed for water content, dissolution profile and drug
content at specified time intervals. The dissolution profiles of
the stability batches were compared with the initial dissolu-
tion profile of the drug using similarity factor; f2.
2.7.7. In vitro dissolution testing
Dissolution profiles of capsule formulation (n = 20) con-
taining vasicine (1), lyophilized Vasa Swaras and that of
marketed formulation were determined according to USP
Basket method (USP XXI/XXII Model TQT-06, Electrolab,
India) using 900 ml of phosphate buffer (pH 7.4) as dissolution
media (maintained at 37 ± 0.5 °C) at 100 rpm. Aliquots (5 ml)
of dissolution medium collected at different intervals of time
(0, 15, 30, 45 and 60 min) were filtered through 0.45 μm
syringe filters and analyzed using the validated HPLC–UV
method.
2.8. Pharmacokinetic study
2.8.1. Calibration curve
One milligram each of vasicine (1) and vasicinone (2)
were dissolved in methanol (final adjusted volume: 100 ml)
to obtain a stock solution of concentration 1 μg/ml. The
working solutions were prepared from the stock solution by
dilution with methanol. Spiked plasma samples containing
vasicine (1) and vasicinone (2) at concentration of 5, 10, 100,
200, 400, 600, 800 and 1000 ng/ml were prepared using the
stock solutions and working solutions in order to plot
calibration curve. The concentration of internal standard
was 12 μg/ml, from which 25 μl was added to each sample.
Liquid–liquid extraction of the plasma samples were carried
out as per the procedure described below. Standard curves
were constructed by plotting ratio of the peak areas of
vasicine (1) to internal standard and vasicinone (2) to
internal standard versus concentration. The calibration curves
were obtained by least square linear regression analysis using
weight scheme as 1 / c (c = concentration) using Borwin
software Ver. 1.3.
2.8.2. Preparation of quality control (QC) samples
The concentrations of vasicine (1) and vasicinone (2) in
rat plasma were 50, 500, and 900 ng/ml to represent low, mid
and high quality control (QC) samples, respectively. To
prepare QC samples, appropriate volumes of vasicine (1)
and vasicinone (2) from the stock solution were transferred
to 10 ml stoppered centrifuge tubes and solvent was
evaporated under gentle stream of nitrogen. Then required
amount of rat blank plasma was added and mixed. These QC
samples were stored at −80 °C and used for validation and
application of the method.
 T. Vyas et al. / Fitoterapia 82 (2011) 446–453
2.8.3. Precision and accuracy
Intra-day precision and accuracy were calculated by
taking six replicates of QC samples with vasicine (1) and
vasicinone (2) (50, 500 and 900 ng/ml) whereas inter-day
accuracy and precision were calculated by taking six
replicates of concentrations 50, 500 and 900 ng/ml from QC
samples for 3 consecutive days along with the standard
calibration curve. The samples were extracted as per the
procedure described below and analyzed using HPLC–UV.
Their concentrations were calculated from the calibration
curve.
2.8.4. Extraction procedure
Twenty five microlitres of metronidazole (3) (Internal
Standard) (12 μg/ml) was added to 100 μl of rat plasma, and
vortexed for 1 min. pH was adjusted to 9 by adding 50 μl of
0.3% sodium hydroxide solution. The resulting sample was
vortexed for 1 min, followed by addition of 1 ml of TBME. The
mixture was vortexed for 1 min and centrifuged at 2000 rpm
for 10 min at 4 °C. The organic layer (supernatant) was
transferred into a 10 ml conical glass tube and evaporated
under the gentle stream of nitrogen gas. The residue was
reconstituted in 50 μl of mobile phase, of which 20 μl was
then injected to the HPLC column.
2.8.5. Recovery
Recovery of the extraction procedure was calculated by
analyzing six extracted samples of 50, 500 and 900 ng/ml and
then comparing the peak area ratio of these samples with
those of unextracted vasicine (1) and vasicinone (2) samples.
2.8.6. Stability studies of plasma samples
Stability of vasicine (1) and vasicinone (2) in rat plasma
during storage and processing was checked using quality
control samples. For freeze thaw stability, four replicates of
high and low controls were frozen at −80 °C and analyzed for
three freeze thaw cycles, while for bench top stability, four
replicates of high and low controls were analyzed after 0 and
6 h at room temperature. Dry extract stability study was done
for four replicates of high and low controls after storing it at
−80 °C for 24 h. Long-term stability of vasicine (1) and
vasicinone (2) for four replicates was checked for 30 days at
−80 °C.
2.8.7. Animals
Male Sprague–Dawley rats of 12–16 weeks old, weighing
296.69 ± 36.44 g were obtained from the animal house of B.
V. Patel PERD Centre, Ahmedabad. The animals were housed
singly per cage in polypropylene cages and were placed in the
experimental room where they were allowed to acclimatize
for a week before experiment. A 10% air exhaust in the air
conditioning unit was maintained along with a relative
humidity of 60 ± 5% and a temperature of 25 ± 3 °C was
stabilised. A 10:14 h light:dark cycle was also regulated for
the experimental animals. Amrut certified rodent diet
(Maharashtra Chakan Oil Mill Ltd.) and tap water (boiling hot
water cooled to room temperature) was provided ad libitum to
the experimental animals. All experimental protocols were
reviewed and accepted by the Institutional Animal Ethics
Committee (IAEC) prior to initiation of the experiment.
449
2.8.8. Pharmacokinetic study of lyophilized Vasa Swaras, Vasa
Swaras, vasicine (1) and marketed formulation of A. vasica
Twenty four animals (6 animals per group) were used in
the study. The 1st group was treated with vasicine capsules,
the 2nd with lyophilized Vasa Swaras, the 3rd with a
marketed formulation of A. vasica and 4th with Vasa Swaras.
The dosing of the capsule formulations were done by
suspending the capsule content in 0.2% agar. Effective dose
of vasicine (1) in rats is reported to be 1.25 mg/kg [18] based
upon which the animals were dosed with 0.9 mg/kg body
weight of vasicine (1) and 0.3 mg/kg body weight of
vasicinone (2) both as Vasa Swaras and as pure vasicine (1)
and vasicinone (2). The amount of Vasa Swaras given to each
animal was calculated according to their body weight. The
dose of vasicine (1) and vasicinone (2) were same in all the
four groups. Jugular veins of all the animals were cannulated
for collection of the blood at different sampling time points.
The blood samples of 0.5 ml were withdrawn at 15 min, 30 min,
45 min, 1 h, 1.5 h, 2 h, 4 h, 6 h, 8 h, and 12 h, post dose from
each rat; collected into heparinised microcentrifuge tubes and
centrifuged at 4000 rpm for 7 min at 4 °C. The resulting plasma
samples were kept frozen at −80 °C prior to HPLC analysis.
2.8.9. Pharmacokinetic data analysis
The maximum plasma concentration (Cmax) and the time
to reach the maximum concentration (Tmax) were directly
determined from the plasma concentration versus time
curves. The area under the curve from 0 − t (AUC0 − t) was
calculated following linear trapezoidal rule by summing the
area from 0 h to t h. Elimination rate constant (Kel) was
determined by taking the positive slope of the any three
points lying on a straight line of the curve after the Cmax i.e.,
during the elimination phase. Elimination half life (t1/2) was
determined using the relationship 0.693/Kel. The volume of
distribution (Vd) was calculated by dividing amount of drug
in the body to the total plasma concentration. The total
clearance (CLT ) was calculated using the relationship
CLT = 0.693Vd / t1/2.
3. Results and discussion
Hard gelatin capsule of lyophilized Vasa Swaras was
formulated in order to enhance the stability and oral
bioavailability of vasicine (1) and limit its conversion into
vasicinone (2). The developed formulation was characterized
and evaluated for different physicochemical parameters. The
in vivo pharmacokinetic characteristics and bioavailability of
lyophilized Vasa Swaras was investigated and compared with
the pharmacokinetic profile with vasicine (1), Vasa Swaras
and the marketed formulation of A. vasica. A pre-reported
analytical method for simultaneous estimation of vasicine (1)
and vasicinone (2) in rat plasma was re-established and
validated and the same was used for analytical and
bioanalytical study.
3.1. Quantification of vasicine (1) and vasicinone (2) in Vasa
Swaras
The amount of vasicine (1) and vasicinone (2) in
lyophilized Vasa Swaras, Vasa Swaras and marketed formulation
of A. vasica were quantified using RP-HPLC found to be 0.3635±
450 T. Vyas et al. / Fitoterapia 82 (2011) 446–453
0.039 mg/ml and 0.10±0.0287 mg/ml, 0.253±0.05 mg/ml and
0.069±0.004 mg/ml and 0.042±0.009 mg/ml and 0.0102±
0.0025 mg/ml, respectively.
3.2. Differential scanning calorimetry (DSC)
DSC curves were obtained for vasicine (1) and physical
mixtures of the excipients viz. Aerosil® 200, Avicel PH 101
and magnesium stearate with vasicine (1). DSC thermogram
for pure drug obtained on scanning at 20 °C/min gave a sharp
peak at 203.39 °C. The drug retained its peak in all other
combinations with different excipients viz. Aerosil® 200,
Avicel PH 101, magnesium stearate. There was no elimination
of exothermic peak or appearance of new exothermic or
endothermic peaks, nor was there any shift in the position of
the characteristic peak of the drug. Thus, the thermal
analytical technique provides a clear inference of no incom-
patibility existing in the prospective final formulation and
hence, all the excipients were found to be compatible with
vasicine (1).
3.3. Characterization of formulation
Vasicine capsules, lyophilized Vasa Swaras capsules and
marketed formulation of A. vasica were characterized for
various pharmacopoeial tests as per I.P. The results shown in
Table 1 for various parameters viz. weight variation test,
disintegration test and assay (estimation of drug content),
uniformity of content, moisture analysis and stability of the
formulations were found within the prescribed limits as per I.P.
3.3.1. Weight variation test
Table 1 shows variation in weight for 20 capsules
containing vasicine (1), lyophilized Vasa Swaras and mar-
keted formulation of A. vasica which were found to be within
the limits prescribed in I.P.
3.3.2. Disintegration test
Disintegration time was found to be 4.04± 0.06 min, 4.23 ±
0.136 min, and 7.04 ± 0.088 min for vasicine capsules, lyophi-
lized Vasa Swaras capsules and a marketed formulation of
A. vasica, respectively as shown in Table 1.
3.3.3. Estimation of drug content
Table 1 shows the drug content in different formulations.
The content of vasicine (1) was 99.9% in vasicine capsules.
96.47% of vasicine (1) and 24.5% vasicinone (2) were found
within lyophilized Vasa Swaras capsules whereas 97.25% of
Table 1
Characterization parameters for vasicine capsules, lyophilized Vasa Swaras capsules and marketed formulation of Adhatoda vasica.
Parameters Vasicine capsule
Weight variation (n = 20) Within I.P. limit
Disintegration test (n = 20) 4.04 ± 0.06 min
Assay (estimation of drug content) 99.9% vasicine (1)
(n = 20)
Uniformity of content (n = 20) Within I.P. limit
Moisture content (n = 20) 5.39%
Stability study (n = 20) 99.47% vasicine (1)
vasicine (1) and 76.5% vasicinone (2) were found within the
marketed formulation of A. vasica.
3.3.4. Uniformity of content
Uniformity of content of vasicine (1) and vasicinone (2)
from the assay of 20 individual capsules of the vasicine (1),
lyophilized Vasa Swaras and marketed formulation of the
A. vasica are shown in Table 1. The results inferred that all the
values were within the limits prescribed in I.P.
3.3.5. Moisture analysis
Lyophilized Vasa Swaras capsules contain more amount of
moisture compared to vasicine capsules and marketed
formulation of A. vasica. Moisture content was found to be
5.39%, 8.64% and 3.11% for vasicine capsules, lyophilized Vasa
Swaras and marketed formulation of A. vasica, respectively
which are shown in Table 1.
3.3.6. Stability study
The content of vasicine (1) was found to be 99.47%, 105.18%
and 98.3% within vasicine capsules, lyophilized Vasa Swaras
capsules and marketed formulation of A. vasica, respectively as
shown in Table 1. The results indicate that the formulations are
stable in the prescribed storage conditions.
3.3.7. In vitro dissolution testing
Dissolution study of vasicine capsules, lyophilized Vasa
Swaras capsules and marketed formulation of A. vasica was
carried out in USP Type I (Basket type) apparatus using
phosphate buffer (pH 7.4) as the dissolution medium. The
percentage release of vasicine (1) and vasicinone (2) from the
formulations is shown in Table 2. The highest amount of
vasicine (1) was released from lyophilized Vasa Swaras and
found to be 96.74% in 1 h whereas vasicine capsules and the
marketed formulation showed vasicine (1) releases of 80.52%
and 80.19%, respectively in 1 h. Fig. 2a and b shows vasicine
(1) and vasicinone (2) release in all the three formulations,
respectively.
3.4. Pharmacokinetic study
A pre-reported analytical method for performing the
pharmacokinetic study of vasicine (1) and vasicinone (2) in
rat plasma was re-established and validated. A well-resolved
chromatogram of vasicine (1) and vasicinone (2) was
obtained following the use of the present HPLC–UV condi-
tions. Vasicine (1) and vasicinone (2) were unambiguously
identified in the plasma upon comparison of the retention
times with those of their respective standards.
Lyophilized Vasa Swaras capsule Marketed formulation of Adhatoda vasica
Within I.P. limit Within I.P. limit
4.23 ± 0.136 min 7.04 ± 0.088 min
96.47% vasicine (1) 24.5 % vasicinone (1) 97.25% vasicine (1) 76.5 % vasicinone (1)
Within I.P. limit Within I.P. limit
8.64% 3.11%
105.18% vasicine (1) 98.3% vasicine (1)
 T. Vyas et al. / Fitoterapia 82 (2011) 446–453 451

Table 2
Dissolution profile of vasicine and vasicinone from vasicine capsules, lyophilized Vasa Swaras capsules and marketed formulation of Adhatoda vasica.

Sr. Time Vasicine capsule (n = 6) Lyophilized Vasa Swaras capsule (n = 6) Marketed formulation of Adhatoda vasica (n = 6)
no. (min)
%vasicine (1) release %vasicine (1) release %vasicinone (2) release %vasicine (1) release %vasicinone (2) release

1 0 – – – – –
2 15 20.15 28.92 3.84 13.02 51.74
3 30 63.52 77.03 11.32 63.74 66.75
4 45 75.65 91.52 15.05 75.38 70.86
5 60 80.52 96.74 21.08 80.19 80.02

3.4.1. Linearity and lower limit of quantification
Standard curves were constructed by plotting ratio of peak
areas of vasicine (1) and vasicinone (2) to internal standard
versus their respective concentration and which was linear in
the range of 5 to 1000 ng/ml. The correlation coefficient was
found to be more than 0.998 and 0.997 (n = 3) for extracted
vasicine (1) and vasicinone (2) samples and 0.999 and 0.997
(n = 3) for unextracted vasicine (1) and vasicinone (2)
samples, respectively. The lower limit of quantification was
found to be 3 ng/ml.
3.4.2. Precision and accuracy
Table 3 shows intra-day and inter-day precision and
accuracy. The intra-day precision (% CV) of QC samples of
vasicine (1) prepared to yield concentrations of low, medium
and high QC samples of vasicine (1) were 10.94, 2.51, and
2.37%, respectively and 5.57, 4.80 and 3.16% for vasicinone
(2), respectively. Inter-day precision (% CV) of low, medium
and high QC samples of vasicine (1) were 6.23, 8.87 and 1.21%
respectively and were 8.17, 4.30 and 5.56% for vasicinone (2),
respectively. Accuracy for vasicine (1) ranged between 86.03
and 100.01% and for vasicinone (2) ranged from 84.64 to
99.16%.
3.4.3. Recovery from plasma
Recovery of vasicine (1) was found to range between
82.58 and 84.94% and that for vasicinone (2) was found to
range between 80.66 and 84.19%. The recovery of internal
standard was 86.37%.
3.4.4. Stability study of plasma samples
Table 4 shows the results of bench top stability, freeze
thaw stability, dry extract stability and long term stability.
Results showed that vasicine (1) and vasicinone (2) was
stable during processing and storage up to one month.

3.4.5. Pharmacokinetic parameters

Pharmacokinetic parameters of the different animal
groups are shown in Table 5. Mean plasma concentration
versus time profiles of vasicine (1) and vasicinone (2) in rats
are shown in Fig. 3a and b.

3.4.6. Statistical analysis

Fig. 2. Dissolution profile of (a) vasicine (1) from vasicine capsules,
lyophilized Vasa Swaras capsules and from marketed formulation of A. vasica
and (b) vasicinone (2) from marketed formulation of A. vasica and lyophilized
Vasa Swaras capsules.
One way ANOVA was applied to determine the difference
in bioavailability of vasicine (1) and vasicinone (2) from the
formulations. A significant difference was found between the
bioavailability of vasicine (1) and vasicinone (2) administered
as lyophilized Vasa Swaras, vasicine capsule and marketed
formulation of A. vasica with p b 0.05 calculated from AUCs of
different groups. The results are expressed as mean ± SD.
As per our previously reported data, vasicine (1) and
vasicinone (2) followed first order kinetics consisting of a
rapid absorption phase followed by a slow elimination phase.
Furthermore, the overall bioavailability of vasicine (1) and
vasicinone (2) was found to be less. The previous results also
stated that vasicine (1) gets converted to vasicinone (2) as
inferred from the change in the proportion of dose administered
and the plasma concentration observed. Hence, in this study an
attempt was made to improve the oral bioavailability of the
vasicine (1) from Vasa Swaras by developing a formulation
which will impart stability to vasicine (1) and lessen its
conversion to vasicinone (2).
The inference drawn from this study suggests that
significant difference was found in the oral bioavailability of
vasicine (1) and vasicinone (2) when administered as
lyophilized Vasa Swaras, Vasa Swaras, vasicine capsules and
marketed formulation of A. vasica. The order of improved oral
452 T. Vyas et al. / Fitoterapia 82 (2011) 446–453

Table 3
Intra-day and inter-day precision and accuracy for vasicine (1) and vasicinone (2).

Nominal concentration Estimated concentration (ng/ml) Precision (% CV) Accuracy (%)

(ng/ml)
Vasicine (1) Vasicinone (2) Vasicine (1) Vasicinone (2) Vasicine (1) Vasicinone (2)

Intra-day (n = 6) a
900 774.27 842.76 2.37 3.16 86.03 93.64
500 499.4 423.2 2.51 4.80 99.88 84.64
50 44.73 46.73 10.94 5.57 91.18 90.14

Inter-day (n = 18) b
900 783.81 892.44 1.21 5.56 87.09 99.16
500 500.05 440.40 8.87 4.30 100.01 88.08
50 45.74 46.23 6.23 8.17 91.63 89.37
a
Intra-day precision: Data expressed as mean (n = 6).
b
Inter-day precision: Data is expressed as mean (n = 18).

Table 4
Stability data of vasicine (1) and vasicinone (2) in rat plasma.

QC Mean concentration observed at 0 h Mean concentration observed at last % Deviation

samples hour

Vasicine (1) Vasicinone (2) Vasicine (1) Vasicinone (2) Vasicine (1) Vasicinone (2)

Bench top stability (n = 4) (after 6 h)

High 867.67 845.44 857.34 833.46 2.67 6.27
Low 48.65 47.44 47.62 46.67 0.57 7.24

Freeze thaw stability (n = 4) (three cycles)

High 869.74 842.45 860.24 836.24 4.87 5.78
Low 48.23 46.14 47.62 45.34 4.56 9.65

Dry extract stability (n = 4) (24 h)

High 875.27 848.45 849.67 828.65 3.74 2.42
Low 47.53 46.46 46.93 45.23 4.12 5.13

Long- term stability (n = 4) (30 days)

High 872.42 847.57 857.71 833.66 6.14 3.17
Low 46.57 45.54 45.27 44.14 3.93 5.98

bioavailability of vasicine (1) was highest from lyophilized
Vasa Swaras followed by Vasa Swaras, vasicine capsules and
marketed formulation of A. vasica. The mean plasma
concentration obtained for vasicinone (2) was highest from
vasicine capsules followed by Vasa Swaras, marketed formu-
lation of A. vasica and lyophilized Vasa Swaras. The conversion
of vasicine (1) into vasicinone (2) was found to be more upon
administration of vasicine (1) whereas the conversion is less
in case of lyophilized Vasa Swaras. Our previous report
suggested that this variation in bioavailability may be
attributed to the presence of some other compounds in
Vasa Swaras which are likely to inhibit the metabolism of
vasicine (1) and vasicinone (2). The unknown compounds
may show more affinity and faster competition for the drug

Table 5
Pharmacokinetic parameters of vasicine (1) and vasicinone (2) in rats upon oral administration of upon oral administration of lyophilized Vasa Swaras, Vasa
Swaras, vasicine capsule and marketed formulation of Adhatoda vasica.

Groups Cmax (ng/ml) Tmax (h) AUC0 − t (ng h/ml) Kel (1/h) t1/2 (h) CLT (L/h) Vd (L)

Group 1 (vasicine capsule) vasicine (1) 111.05 ± 6.13 0.30 ± 0.001 18293.44 ± 2770.42 0.75 ± 0.08 0.92 ± 0.08 6.92 ± 1.14 12.97 ± 0.0003
Group 1 vasicinone (2) 309.26 ± 137.77 0.15 ± 0.015 28807.05 ± 6850.15 0.79 ± 0.09 0.85 ± 0.12 7.89 ± 0.01 9.67 ± 0.84
Group 2 (lyophilized Vasa Swaras capsule) 245.47 ± 82.02 0.45 ± 0.007 21122.79 ± 5100.64 0.262 ± 0.12 2.64 ± 1.78 2.94 ± 1.42 11.23 ± 0.004
vasicine (1)
Group 2 vasicinone (2) 126.56 ± 108.22 0.45 ± 0.012 12910.26 ± 4187.23 0.315 ± 0.19 2.19 ± 1.52 0.114 ± 0.06 0.36 ± 0.0008
Group 3(marketed formulation) 83.68 ± 18.51 0.45 ± 0.186 15388.20 ± 4692.40 0.314 ± 0.26 2.20 ± 0.94 4.84 ± 4.03 15.42 ± 0.002
vasicine (1)
Group 3 vasicinone (2) 60.60 ± 25.24 0.30 ± 0.002 14279.41 ± 4558.05 0.151 ± 0.07 4.57 ± 0.70 0.0495 ± 0.002 0.327 ± 0.004
Group 4 (Vasa Swaras) vasicine (1) 172.18 ± 11.84 0.45 ± 0.005 20261.33 ± 5600.60 3.10 ± 0.20 0.18 ± 2.99 9.87 ± 2.39 3.45 ± 0.002
Group 4 vasicinone (2) 169.82 ± 118.51 0.45 ± 0.004 13717.40 ± 2550.5 0.491 ± 0.16 1.40 ± 0.47 1.12 ± 0.07 2.51 ± 0.0005
 T. Vyas et al. / Fitoterapia 82 (2011) 446–453
Fig. 3. Mean (±SD) plasma concentration of (a) vasicine (1) and (b) vasicinone
(2), upon oral administration of lyophilized Vasa Swaras, vasicine (1), marketed
formulation of A. vasica and Vasa Swaras capsules.
metabolizing enzymes. Thus, in the process metabolism of
vasicine (1) and vasicinone (2) get hindered and show
greater bioavailability upon oral administration of Vasa
Swaras. Vasicine (1) and vasicinone (2) as single moiety
have no competitors for the metabolizing enzyme and thus
get preferentially metabolized exhibiting less oral bioavail-
ability. Hence, it can be concluded that the lyophilized
formulation improved the stability and oral bioavailability
of vasicine (1).
4. Conclusion
Lyophilisation of Vasa Swaras improved the stability and
oral bioavailability of vasicine (1) as well as reduced its
conversion to vasicinone (2). The pharmacokinetic profiles of
the various formulations inferred that the highest bioavail-
ability of vasicine (1) was obtained from hard gelatin capsules
of lyophilized Vasa Swaras. The formulation approach
developed and pharmacokinetic parameters obtained are
likely to be helpful for quality control, mechanistic study and
clinical research of Vasa Swaras. Furthermore, this formula-
453
tion approach can be adopted for improvement in drug
delivery and enhancement of stability as well as the shelf-life
of various aqueous based herbal medicinal preparations.
Acknowledgements
The authors wish to acknowledge NIPER-Ahmedabad for
their grant as Junior Research Scholarship to Mr. Tejas Vyas to
carry out this work and J.B Chemicals, Mumbai, India for
providing metronidazole (3) as gift sample.
Appendix A. Supplementary Data
Supplementary data to this article can be found online at
doi:10.1016/j.fitote.2010.12.005.
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