Comparative Pharmacokinetics Profileof Vasa Swaras with Vasicineand Vasicinone

Ranjeet Prasad Dash1, Bhupendrasinh F. Chauhan1, Sheetal Anandjiwala2, Manish Nivsarkar1,&

1 Department of Pharmacology and Toxicology, B. V. Patel Pharmaceutical Education and Research Development (PERD) Centre,S. G. Highway, Thaltej, Ahmedabad 380054 Gujarat, India; E-Mail: manishnivsarkar@gmail.com

2 Department of Natural Products, National Institute of Pharmaceutical Education and Research-Ahmedabad, S. G. Highway, Thaltej,Ahmedabad 380054 Gujarat, India

Received: 21 October 2009 / Revised: 10 December 2009 / Accepted: 22 January 2010Online publication: 12 March 2010

Abstract

An RP-LC method was developed and validated for comparing the pharmacokinetics profileof Vasa Swaras (leaf juice of Adhatoda vasica Nees., Fam. Acanthaceae) with that of thepure vasicine and vasicinone (chief marker compounds of A. vasica) upon oral adminis-tration of Vasa Swaras in rats, and also in different animal groups. Significant difference withp < 0.05 was found in the oral bioavailability of vasicine and vasicinone when adminis-tered as Vasa Swaras and as single vasicine and/or vasicinone. Vasicine and vasicinonewere found to be more bioavailable from Vasa Swaras than pure vasicine and/or vasici-none.

Keywords

Column liquid chromatographyLiquid–liquid extractionPharmacokineticsVasicine and vasicinone

Introduction

Adhatoda vasica (L.) Nees (family:

Acanthaceae) commonly known 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 more than 2000 years [1] and

is a well-known drug in Ayurvedic and

the Unani System of Medicine [2]. It is

known as Vasa in Ayurveda. It is an

important drug prescribed for malarial

fever, fever caused by pitta and kapha,

chronic fever, intrinsic hemorrhage,

cough, asthma, leprosy, skin diseases and

piles [3]. It is reported to be expectorant

[4], abortifacient [5], antimicrobial [6, 7]

and antitussive [8]. Important chemical

constituents of the leaf include pyrrolo-

quinazoline alkaloids––vasicine, vasicol,

adhatonine, vasicinone, vasicinol, vasi-

cinolone [9]. Vasicine is reported to have

bronchodilatory, respiratory stimulant

and uterine stimulant effects [10] and

vasicinone is reported to have bron-

chodilatory, weak cardiac stimulant and

anti-anaphylactic action [11]. In Ayurve-

dic preparations, Vasa Swaras is incor-

porated in more than 20 formulations

including Vasarishta,Mahatiktaka ghrita,

Triphala ghrita, Vasavaleha, Vasakasa-

va, Mahatriphalaghrita, Panchatik-

taghritaguggulu and Panchatikta ghrita

[12].

There are numerous reports on the

biological activities of vasicine and

vasicinone as mentioned earlier. There

are reports on the pharmacokinetics of

vasicine in rats [13] and humans [14].

However, there are no reports on the

bioavailability and pharmacokinetics of

vasicine and vasicinone when adminis-

tered as an extract. Liquid chromato-

graphic (LC) methods are widely used

for the determination of drugs in bio-

logical samples. Though, attempts have

been made to develop LC methods

[15–17] using UV detection for the quan-

tification of vasicine and vasicinone,

these methods lack baseline separation

2010, 71, 609–615

DOI: 10.1365/s10337-010-1517-x0009-5893/10/04 � 2010 Vieweg+Teubner Verlag | Springer Fachmedien Wiesbaden GmbH

Original Chromatographia 2010, 71, April (No. 7/8) 609

of vasicine and vasicinone which may

result in impure peaks. Srivastava et al.

[18] developed an LC method using

photo diode array detection to achieve

better resolution and baseline separation

of vasicine and vasicinone than the pre-

viously reported methods. Das et al. [19]

developed HPTLC methods for deter-

mination of vasicine and vasicinone but

these methods are less precise and accu-

rate than LC because of low sensitivity.

However, no method is reported for

simultaneous quantification of vasicine

and vasicinone in biological samples.

Vasa Swaras is used in many traditional

formulations which contains both vasi-

cine and vasicinone. Thus, there is a need

for the development of a method for the

analysis of both vasicine andvasicinone in

plasma. A simple, rapid, economic and

sensitive RP-LC method for the simulta-

neous plasma determination of both vas-

icine and vasicinone has been developed

and validated. The developed method

was applied for the pharmacokinetics

study of vasicine and vasicinone in rats.

Experimental

Materials and Chemicals

Vasicine (99.6% pure) and vasicinone

(99.4% pure), (Fig. 1) were respectively,

purchased from SPIC, Chennai, India

and Natural Remedies, Bengaluru,

India. Metronidazole (99.7% pure),

(Fig. 1) was obtained as gift sample from

J.B. Chemicals, Mumbai, India and used

as an internal standard (IS). All the

solvents and chemicals were of LC grade

and purchased from Qualigen Fine

Chemicals, Mumbai, India. Potassium

dihydrogen orthophosphate (Qualigen

Fine Chemicals) and glacial acetic acid

(Qualigen Fine Chemicals) of analytical

grade were used. tert-Butyl methyl ether

(TBME) was purchased from Merck

Schuchardt, Hohenbrunn, Germany.

Heparin was purchased from Biological

E., Hyderabad, India. Deionized water

for LC was prepared in-house using a

water purifier system (Millipore Elix,

Germany).

Collection and Identificationof the Plant

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 [20].

Then they were shade dried, stored in an

air-tight container and powdered to 40

meshes whenever required. A specimen

of the collected plant was preserved in

the Department of Natural Products,

NIPER-Ahmedabad (Herbarium Speci-

men #: NIPER-A/NP/0808/07).

Preparation of Vasa Swaras

Vasa Swaras was prepared following a

pre-reported method cited in Sarangh-

dhar Samhita [21]. The dried A. vasica

leaf powder weighing 100 g was taken.

200 mL of double distilled water were

added and allowed to macerate for 24 h

at room temperature. The above mixture

was filtered through muslin cloth to ob-

tain a 50 mL filtrate. The amount of

vasicine and vasicinone present in the

aqueous extract were determined by LC–

UV. Freshly prepared Vasa Swaras was

always used for experiment.

Chromatographic Conditions

The LC system consisted of PU-980

intelligent LC pump 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 Grace Vydac ODS

column (4.6 mm i.d. 9 250 mm, 5 lm)

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-1. Samples

were quantified by determining the

response (Peak AreaDrug/Peak AreaIS).

Calibration Curve

1 mg each of vasicine and vasicinonewere

dissolved inmethanol and the volumewas

adjusted to 100 mL with methanol in a

volumetric flask to obtain a stock solution

concentration of 1 lg mL-1. From the

stock solution, a series of working solu-

tions were prepared by dilution with

methanol. Spiked plasma samples con-

taining vasicine and vasicinone at con-

centrations of 5, 10, 100, 200, 400, 600,

800 and 1000 ng mL-1 were prepared

using the stock solutions and working

solutions in order to plot calibration

curves. 25 lL of internal standard to a

concentration of 12 lg mL-1 were added

to each sample. Extraction was carried

Fig. 1. Chemical structures of (a) vasicine (b) vasicinone (c) metronidazole

610 Chromatographia 2010, 71, April (No. 7/8) Original

out as per the procedure described below.

Standard curves were constructed by

plotting ratio of the peak areas of vasicine

to internal standard and vasicinone to

internal standard versus concentration.

The calibration curves were obtained by

least square linear regression analysis

using weight scheme as 1/c (c = concen-

tration) employing Borwin software

Ver. 1.3.

Preparation of Quality Control(QC) Samples

The concentrations of vasicine and

vasicinone in rat plasma were 50, 500,

and 900 ng mL-1 to represent low, mid

and high quality control (QC) samples,

respectively. To prepare QC samples,

appropriate volumes of vasicine and

vasicinone from the stock solution were

transferred to 10 mL stoppered centri-

fuge tubes and solvent was evaporated

under a gentle stream of nitrogen. Then

the required amount of rat blank plasma

was added and mixed. These quality

control samples were stored at -80 �Cand were used for validation and appli-

cation of the method.

Precision and Accuracy

Intra-day precision and accuracy were

calculated by taking five replicates of

quality control samples with vasicine and

vasicinone (50, 500 and 900 ng mL-1) on

one day whereas inter-day accuracy and

precision were calculated by taking five

replicates of concentrations 50, 500 and

900 ng mL-1 from QC samples for three

consecutive days along with the standard

calibration curve. The samples were

extracted as described below and ana-

lyzed by LC. Their concentrations were

calculated from the calibration curve.

Extraction Procedure

25 lL of metronidazole (internal stan-

dard) (12 lg mL-1) were added to 100 lLof rat plasma, and vortexed for 1 min.

pH was adjusted to 9 by adding 50 lLof 0.3% sodium hydroxide solution.

The solution was vortexed for 1 min,

followed by addition of 1 mL of TBME.

The mixture was vortexed for 1 min and

centrifuged at 400 9 g for 10 min at 4 �C.The organic layer (supernatant) was

transferred into a 10 mL conical glass

tube and evaporated under a gentle

stream of nitrogen gas. The residue was

reconstituted in 50 lL mobile phase, of

which 20 lL were then injected into the

LC column.

Recovery

Recovery of the extraction procedure

was calculated by analyzing six extracted

samples of 50, 500 and 900 ng mL-1 and

comparing the peak area ratio of these

samples with those of unextracted vasi-

cine and vasicinone samples.

Stability Studies

During storage and processing the sta-

bility of vasicine and vasicinone in rat

plasma was checked using quality con-

trol 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 and vasicinone for

four replicates was checked for 30 days

at –80 �C.

Quantification of Vasicineand Vasicinone in VasaSwaras

5 mL of Vasa Swaras was taken and

basified up to pH 9 with 5% sodium

hydroxide. It was extracted with chlo-

roform until the aqueous portion

showed negative Dragendroff’s test. The

chloroform fraction were pooled over

sodium sulphate and concentrated. The

amounts of vasicine and vasicinone were

determined by LC with the same chro-

matographic conditions as stated above.

Animals

Male Sprague–Dawley rats, 12–16 weeks

old, weighing 348.44 ± 40.02 g were

obtained from the animal house of the

National Institute of Pharmaceutical

Education and Research, Ahmedabad.

The animals were housed singly per cage

in polypropylene cages and placed in the

experimental room where they were

allowed to acclimatize for a week before

experiment. A 10% air exhaust in the air

Fig. 2. LC chromatogram of blank rat plasma

Original Chromatographia 2010, 71, April (No. 7/8) 611

conditioning unit was maintained along

with a relative humidity of 60 ± 5% and

a temperature of 25 ± 3 �C was stabi-

lized. A 10: 14 h light: dark cycle was

also regulated for the experimental ani-

mals. Amrut certified rodent diet

(Maharashtra Chakan Oil Mill) and tap

water (boiling hot water cooled to room

temperature) was provided ad libitum to

the experimental animals. All experi-

mental protocols were reviewed and

accepted by the Institutional Animal

Ethics Committee (IAEC) prior to initi-

ation of the experiment.

Pharmacokinetics Studyof Vasa Swaras

Twenty four animals were used in the

study. Jugular veins of all the animals

were cannulated for collection of blood

at different sampling time points. The

animals were divided into four groups

each consisting of six animals. The first

group was treated with Vasa Swaras,

the second with vasicine, the third with

vasicinone and the fourth with both

vasicine and vasicinone. Effective dose

of vasicine in rats was 1.25 mg kg-1

[22] based upon which the animals were

dosed with 0.9 mg kg-1 body weight of

vasicine and 0.3 mg kg-1 body weight

of vasicinone both as Vasa Swaras and

as pure vasicine and vasicinone. The

amount of Vasa Swaras given to each

animal was calculated according to

their body weight. The dose of vasicine

and vasicinone were the same in all

four groups. Blood samples of 0.5 mL

were withdrawn at 0, 15, 30 min, 1, 2,

4, 6, 8, 12, 24 and 36 h; collected

into microcentrifuge tubes containing

approximately 10 mg heparin and cen-

trifuged at 1500 9 g for 7 min at 4 �C.The resulting plasma samples were kept

frozen at -80 �C prior to LC analysis.

Data Analysis

The maximum plasma concentration

(Cmax) and the time to reach the maxi-

mum concentration (Tmax) were directly

determined from the plasma concentra-

tion versus time curves. The area under

the curve from 0–t (AUC0–t) was cal-

culated following the linear trapezoidal

rule by summing the area from 0 to t h.

Elimination rate constant (Kel) was

determined by taking the positive slope

of any three points lying on a straight

line of the curve after the Cmax i.e.

during the elimination phase. Elimina-

tion half life (t1/2) was determined using

the relationship 0.693/Kel. The volume

of distribution (Vd) was calculated by

dividing the 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.

Fig. 5. LC chromatogram of rat plasma 45 min after dosing of Vasa Swaras

Fig. 3. LC chromatogram of unextracted vasicine and vasicinone along with the internalstandard

Fig. 4. LC chromatogram of rat plasma spiked with vasicine and vasicinone

612 Chromatographia 2010, 71, April (No. 7/8) Original

Results

A new analytical method for simulta-

neous estimation of vasicine and vasici-

none in rat plasma was developed and

validated. The current method uses

liquid–liquid extraction procedure by

TBME. The method is stable, repro-

ducible and specific. The chromatograms

of blank rat plasma, unextracted pure

standards of vasicine and vasicinone,

plasma spiked with mixed standards of

vasicine and vasicinone at a concentra-

tion of 200 ng mL-1 and plasma sample

from a rat 45 min after administration of

Vasa Swaras are shown in Figs. 2, 3, 4

and 5, respectively. A well-resolved

chromatogram of vasicine and vasici-

none was obtained following the use of

the present LC–UV conditions. Reten-

tion time of vasicine, vasicinone and

metronidazole (internal standard) were

4.19, 7.33 and 5.53 min, respectively.

Total LC run time was 10 min. Vasicine

and vasicinone were unambiguously

identified in the plasma upon compari-

son of the retention times with those of

their respective standards.

Quantification of Vasicineand Vasicinone in VasaSwaras

The amount of vasicine and vasicinone

in Vasa Swaras was quantified by

RP-LC. The amount of vasicine and

vasicinone were found to be 0.223 ± 0.06

and 0.069 ± 0.004 mg mL-1 of Vasa

Swaras, respectively.

Linearity and Lower Limitof Quantification

Standard curves were constructed by

plotting ratio of peak areas of vasicine

and vasicinone to internal standard

versus their respective concentration

and were linear in the range of

5–1,000 ng mL-1. The correlation coef-

ficient was higher than 0.999 and 0.998

(n = 3) for extracted vasicine and vasic-

inone samples and 0.999 and 0.998

(n = 3) for unextracted vasicine and

vasicinone samples, respectively.The lower

limit of quantification was 5 ng mL-1.

Precision and Accuracy

Table 1 shows intra-day and inter-day

precision and accuracy. The intra-day

precision (% CV) of quality control

samples of vasicine prepared to yield

concentrations of low, medium and high

QC samples of vasicine were 11.97,

10.73, and 4.91% and 6.53, 5.81 and

9.51% for vasicinone. Inter-day preci-

sion (% CV) of low, medium and high

QC samples of vasicine were 6.97, 11.68

and 6.91% and 9.19, 8.42 and 10.19%

for vasicinone. Accuracy for vasicine

ranged between 90.63 and 101.79% and

for vasicinone from 89.77 to 91.37%.

Recovery from Plasma

Recovery of vasicine ranged between

81.38 and 82.94% and that for vasicinone

between 81.66 and 83.14%. The recovery

of internal standard was 85.37%.

Stability

Table 2 shows the results of bench top

stability, freeze thaw stability, dry ex-

tract stability and long term stability.

Results showed that vasicine and vasici-

Table 1. Intra-day and inter-day precision and accuracy for vasicine

Nominalconcentration(ng mL-1)

Estimatedconcentration(ng mL-1)

Precision (% CV) Accuracy (%)

Vasicine Vasicinone Vasicine Vasicinone Vasicine Vasicinone

aIntra-day (n = 6)900 885.03 811.83 4.91 9.51 98.34 89.05500 514.48 448.84 10.73 5.81 102.90 89.9550 45.76 46.76 11.97 6.53 90.18 90.18

bInter-day (n = 18)900 848.89 825.35 6.91 10.19 94.21 91.37500 508.95 446.85 11.68 8.42 101.79 89.7950 45.32 45.24 6.97 9.19 90.63 90.38

a Intra-day precision: data expressed as mean (n = 6)b Inter-day precision: data is expressed as mean (n = 18)

Table 2. Stability data of vasicine and vasicinone in rat plasma

QC samples Mean concentrationobserved at0 h (ng mL-1)

Mean concentrationobserved atlast h (ng mL-1)

% Deviation

Vasicine Vasicinone Vasicine Vasicinone Vasicine Vasicinone

Bench top stability (n = 4) (after 6 h)High 878.76 848.45 867.34 836.46 2.60 5.72Low 47.56 46.14 46.68 45.37 0.33 7.76

Freeze thaw stability (n = 4) (three cycles)High 878.76 848.45 859.04 839.04 4.45 6.72Low 47.56 46.14 46.16 45.16 3.59 10.44

Dry extract stability (n = 4) (24 h)High 878.76 848.45 849.67 829.65 2.74 3.72Low 47.56 46.14 46.52 45.52 4.85 4.95

Long- term stability (n = 4) (30 days)High 869.47 837.57 852.73 824.86 5.14 2.14Low 46.59 45.89 45.78 44.89 4.03 6.73

Original Chromatographia 2010, 71, April (No. 7/8) 613

none was stable during processing and

storage up to one month.

Pharmacokinetic Parameters

Pharmacokinetic parameters of the dif-

ferent animal groups are shown in

Table 3. Mean plasma concentration

versus time profiles of vasicine and va-

sicinone in rats are shown inFigs. 6 and 7.

Statistical Analysis

Student’s t test was applied to determine

the significant difference in the bioavail-

ability of vasicine and vasicinone when

administered as Vasa Swaras and as iso-

lated single compound. Significant dif-

ference was found between the

bioavailability of vasicine and vasicinone

administered as Vasa Swaras and as iso-

lated single compound with p < 0.05

calculated fromAUCsofdifferent groups.

Discussion

The results inferred that vasicine and

vasicinone followed first order kinetics

consisting of a rapid absorption phase

followed by a slow elimination phase.

The overall bioavailability of vasicine

and vasicinone was found to be low. The

results also state that vasicine is con-

verted into vasicinone as inferred from

the change in the proportion of dose

administered and the plasma concentra-

tion observed. This is in conjunction to

the fact that vasicine is converted into

vasicinone in vitro [23]. Vasicinone was

not detected when administered alone

(group 3) and may be due to its extensive

metabolism resulting in its low bio-

availability. The reason behind the

detection of vasicinone in groups 1, 2

and 4 may be the conversion of a certain

amount of vasicine into vasicinone.

Profound difference was found in the

bioavailability of vasicine and vasicinone

when administered as Vasa Swaras and

as single vasicine and/or vasicinone.

Vasicine and vasicinone were found to

be more bioavailable from Vasa Swaras

than that administered as single vasicine

and/or vasicinone. 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 and vasici-

none. The unknown compounds may

show more affinity and faster competi-

tion for the drug metabolizing enzymes.

Thus, the metabolism of vasicine and

vasicinone get hindered and show

greater bioavailability upon oral adminis-

tration of Vasa Swaras. Vasicine and

vasicinone as single moiety have no

Fig. 6. Mean (± SD) plasma concentration of vasicine upon oraladministration of Vasa Swaras, pure vasicine and pure vasicine andvasicinone

Fig. 7. Mean (± SD) plasma concentration of vasicinone upon oraladministration of Vasa Swaras, pure vasicine and pure vasicine andvasicinone

Table 3. Pharmacokinetic parameters of vasicine and vasicinone in rats upon oral administration of Vasa Swaras and pure vasicine and vasicinone

Groups Cmax (ng mL-1) Tmax (h) AUC0-t (ng h mL-1)* Kel (h-1) t1/2 (h) CLT (L h-1) Vd (L)

Group 1 (Vasicine) 449.5 ± 16.1 0.45 ± 0.007 67529.38 ± 3694.56 3.635 ± 0.72 0.19 ± 0.004 10.76 ± 1.27 2.96 ± 0.86Group 1 (vasicinone) 214.1 ± 15.6 0.45 ± 0.002 22505.25 ± 2187.54 0.45 ± 0.14 1.54 ± 0.98 1.19 ± 0.23 2.67 ± 0.47Group 2 (Vasicine) 211.7 ± 10.8 0.30 ± 0.005 22237.00 ± 1641.15 0.77 ± 0.12 0.90 ± 0.11 6.89 ± 1.76 8.99 ± 1.89Group 2 (Vasicinone) 126.4 ± 5.2 0.30 ± 0.001 6371.63 ± 257.85 0.86 ± 0.17 0.81 ± 0.08 8.08 ± 1.57 9.42 ± 1.22Group 3 (Vasicinone) Plasma concentration of vasicinone was below the limit of detection in all 6 animalsGroup 4 (Vasicine) 241.3 ± 15.3 0.30 ± 0.002 12863.13 ± 882.47 0.87 ± 0.07 0.80 ± 0.14 13.45 ± 1.22 15.55 ± 1.43Group 4 (Vasicinone) 158.6 ± 24.0 0.30 ± 0.007 7917.00 ± 242.82 0.99 ± 0.13 0.69 ± 0.07 7.54 ± 0.89 7.58 ± 1.07

* p < 0.05 compared to pure vasicine and vasicinone** Results represented as mean ± SD

614 Chromatographia 2010, 71, April (No. 7/8) Original

competitors for the metabolizing enzyme

and thus get preferentially metabolized

exhibiting less oral bioavailability.

Conclusion

A simple, precise, economical and

sensitive method for simultaneous deter-

mination of vasicine and vasicinone in

rat plasma was developed and validated.

This analytical procedure was success-

fully applied to the pharmacokinetic

study of Vasa Swaras as well as of pure

vasicine and vasicinone in male Sprague–

Dawley rats. Significant difference was

observed in the pharmacokinetics profile

of vasicine and vasicinone upon oral

administration of Vasa Swaras and pure

vasicine and vasicinone. The analytical

method developed and pharmacokinetic

parameters obtained are likely to be

helpful for quality control and mecha-

nistic study of Vasa Swaras.

Acknowledgments

The authors wish to acknowledgeNIPER

– Ahmedabad for their grant as Junior

Research Scholarship to Mr. Ranjeet

Prasad Dash to carry out this work and

J.B Chemicals, Mumbai, India for pro-

viding metronidazole as gift sample.

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