Indian Journal of Experimental Biology

Vol. 54, July 2014, pp. 705-711

Evaluation of hepatoprotective activity of vasicinone in mice

Chaitali Sarkara, Sankhadip Bose

a & Sugato Banerjee

b*

aGupta College of Technological Sciences, College of Pharmacy, Asansol 713 301, India bDepartment of Pharmaceutical Sciences, Birla Institute of Technology, Mesra, Ranchi 835 215, India

Received 8 January 2013; revised 4 April 2014

Justicia adhatoda (vasaka) leaves have long been used in Indian Ayurvedic system of medicine as antitussive. Its crude

extract has been previously reported to have hepatoprotective activity. Vasicinone was isolated from leaves of J. adhatoda,

column purified and characterized using, TLC UV, FT-IR and 1H NMR. The isolated vasicinone was evaluated for

hepatoprotective activity using (CCl4)-induced acute hepatotoxicity model in mice. CCl4 treatments lead to significant

increase in SGOT, SGPT, ALP levels. Pre-treatment with vasicinone and silymarin (25 mg/kg/day for 7 days) significantly

decreased these enzyme levels. Histopathology of the livers from vasicinone and silymarin pre-treated animals showed

normal hepatic cords and absence of necrotic changes suggesting pronounced recovery from CCl4 induced liver damage.

Both vasicinone and silymarin significantly decrease the CCl4 mediated increase in pentobarbital indiced sleeping time in

experimental animals, thus indicating recovery of liver function. Based on the above results it can be concluded that

vasicinone may act as hepatoprotective in mice and warrants further investigation on human volunteers.

Keywords: Hepatoprotective, Justicia adhatoda, Mice, Vasicinone

Liver is one of the largest organs in human body and the primary site for intense metabolism and excretion. It has a major role in the maintenance, performance and regulation of physiological homeostasis. It is involved in almost all the biochemicalpathways involved in growth, immunity, supply of nutrients, energy provision and reproduction

1. The major

functions of the liver are carbohydrate, protein and fat metabolism, detoxification, secretion of bile and storage of vitamin. Thus, a healthy liver is crucial for overall health and well being. However it is continuously and variedly exposed to environmental toxins, alcohol and prescribed/over-the-counter drugs which can eventually lead to various liver ailments like hepatitis, cirrhosis and alcoholic liver disease

2,3.

Modern medicine has little to offer for alleviation of hepatic diseases and it is chiefly the plant based preparations which are employed for the treatment of liver disorders. CCl4-induced hepatotoxicity, which is mainly a free radical mediated cytotoxic model, is widely used for the study of hepatoprotective effects of drugs and plant extracts

4.

Justicia adhatoda (L) Willd. (Acanthaceae),

with the common name ‘vasaka’, is a perennial shrub,

1-2.5 m in height, widespread throughout the

tropical regions of Southeast Asia (Nepal, Pakistan,

Myanmar) including India, up to an altitude of

1300 m in the sub-himalayan regions. It is commonly

known as basak (Bengali), aradusi, adosa (Gujrati),

arusa, bansa, adulsa (Hindi), bansa, basuti, bhekkar

(Punjabi) swetavasa, vasa and vasaka (Sanskrit) and

malabar nut (English)5. The leaves mainly consist of

pyrroquanazoline alkaloids like visicine, vasicinone,

vasicol, preganine along with other minor constituents

like adhatonine, vasicinol and vasicinolone6.

Extract of J. adhatoda leaves has been used for the

treatment of various diseases and disorders in Ayurved

and Unani medicine7. It has been used as an herbal

remedy for allergen induced bronchial obstruction8,

asthma and tuberculosis9. Vasicinone alone has also

been reported to have bronchodilatory, weak cardiac

stimulant and antianaphylactic action10,11. Crude

vasaka leaf extract is a known antioxidant and has also

been reported to possess hepatoprotective activity12,13

.

The present study has been undertaken to we explore

the hepatoprotective action of isolated vasicinone from

the leaves of Justicia adhatoda in mice.

Materials and Methods

Plant materials—The leaves of Justicia adhatoda

(L) Willd were collected from Sripur road, Kulti,

——————

*Correspondent author

Mobile: 7250001584

Fax: 91-0651-2275290

E-mail: banerjeesugato1@gmail.com; sbanerjee@bitmesra.ac.in

INDIAN J EXP BIOL, JULY 2014

706

(23.7/86.8) India during September. The plant

material was authenticated by Shibpur Botanic

Garden, Botanical survey of India, Howrah, West

Bengal, India for authentication. Authentication No.

CNH/29/2012/Tech.II/710.

Isolation of phytoconstituents—Leaves of

J. adhatoda were shed dried in room temperature and

ground to powder (500 g). The powder was soaked

with ethyl alcohol (600 mL) for 24 h and the extract

was filtered and concentrated. The concentrated

material was treated with 5% acetic acid (200 mL),

warmed for 15 min and filtered. The filtrate was then

defatted with hexane and basified with NH3 (pH 9),

followed by extraction with chloroform12

. After

qualitative chemical tests, to confirm the presence of

alkaloids, thin layer chromatography was performed

using chloroform, methanol and ethyl acetate

(85:15:10 and 60:20:10) as solvent system. The crude

alkaloids were passed through a glass column (Length

32cm, diameter 2.5cm and flow rate 35 drops/min)

over silica gel by using chloroform: methanol: ethyl

acetate (85:15:10 and 60:20:10) with increasing

polarity to get pure form of vasicinone14

.

Characterization—Vasicinone was characterized

by comparing UV15

, FT-IR16

and 1H NMR spectra of

isolated compound with standard vasicinone spectra17

.

Experimental animals—Male Swiss Albino mice (20-

25 g) were obtained from animal house of Gupta

College of Technological Sciences. The animals were

housed under standard environmental condition (25 °C,

12:12 h L: D cycle) and fed with standard diet (Tetragon

Chemie Private limited, Bangalore, India) and water ad

libitum. All animal experiments were carried out in

accordance with the guidelines of Committee for the

Purpose of Control and Supervision of Experiments on

Animals (CPCSEA), Ministry of Forests and

Environment, Government of India and the study was

approved by the Institutional Animal Ethics Committee.

Acute toxicity studies—The isolated vasicinone

was administered at the doses of 200, 400 and

600 mg/kg, orally to different groups of mice, as per

OECD test guidelines (425) for oral acute toxicity

study and mortality was observed for up to 7 days17

.

Experimental protocol—Mice (30) were randomly

allocated into following five groups of 6. All animals

were made to fast for 24 h before the experiment.

Gr I: Control group. The animals received corn oil,

po (vol equivalent to vol of corn oil required

administration of 25 mg/kg vasicinone; Vijaya

Enterprise, Mumbai, India) for 7 days.

Gr II: Hepatotoxic control. The animals received corn

oil for 7 days and a single dose of CCl4 (1 mL/kg body

weight, po in corn oil, Merck, India Ltd) on day 818

.

Gr III: The animals were treated with vasicinone in

corn oil (10 mg/kg body weight, po) for 7 days and a

single dose of CCl4 on day 8.

Gr IV: The animals were treated with vasicinone in

corn oil (25 mg/kg body weight, po) for 7 days and a

single dose of CCl4 on day 8.

Gr V: The animals received silymarin (25 mg/kg

body weight, po; Micro Lab Inc, Bangalore India) for

7 days17

and a single dose of CCl4 on day 818

.

On day 9 (24 h after application of CCl4), the

animals were used for behavioural experiments

(pentobarbital-induced sleeping time) after which they

were sacrificed for biochemical and histopathological

studies.

Measurement of pentobarbital-induced sleeping

time—On day 9 of the experiment, pentobarbital

sodium 30 mg/kg, ip (Taj Pharmaceuticals Limited,

Mumbai, India) was injected to control (Gr I), CCl4-

intoxicated (Gr II), vasicinone pre-treated CCl4-

intoxicated (Gr III and IV) and silymarin pre-treated

CCl4-intoxicated groups (Gr V). The onset of sleep

and total sleeping time were recorded for all the

animals of each group20

.

Biochemical estimation—The animals were

anaesthetized using ether and blood was collected by

cardiac puncture. The Serum Glutamate Oxaloacetate

Transaminase (SGOT), Serum Glutamate Pyruvate

Transaminase (SGPT), Alkaline Phosphatase (ALP)

level was estimated in serum by using commercial

kits19

(Span Diagnostics Ltd., India).

Histopathological studies—After blood collection

for biochemical studies (day 10) the animals were

sacrificed by cervical dislocation and livers was

excised, washed with phosphate buffer and dried with

tissue paper. The histopathological studies were

carried out as per Sharma et al2. The liver was

weighed by using electronic balance and transferred

to a 10% formalin fixative solution for 48 hr. The

liver tissues were processed for paraffin embedding

and sections of 5 µm thickness were cut in a

microtome. After staining with haematoxylin

and eosin, slides were examined under (100X)

light microscope (Nikon Eclipse E100, Nikon

Corporations) for histopathological changes21

.

Statistical analysis—The data were expressed as

mean ± SE. The data were evaluated by one-way

ANOVA followed by Tukey's multiple comparison

SARKAR et al.: HEPATOPROTECTIVE ACTIVITY OF VASICINONE IN MICE

707

tests. P < 0.05 was considered statistically significant.

All statistical analysis was carried out using Graph

Pad Prism 4.0 software (GraphPad Software Inc, San

Diego CA, USA).

Results

Isolation of phytoconstituents—Crude alkaloids

(4.98 g) were obtained from the 500 g leaves of

J. adhatoda. As the Rf value (0.61) of isolated crude

alkaloid was same as that of standard vasicinone22

,

it can be concluded that the isolated alkaloid may be

vasicinone. After collecting all the eluents of all

85 test tubes from column, test tube number 1-28 and

46-81 gave a sharp single spot in TLC with Rf

value 0.61. Next, this compound was used for

further characterization. Melting point of isolated

vasicinone was determined and it was found to

be 110-112 ºC, which was comparable to standard

vasicinone23

.

Characterization of isolated vasicinone—The

structure of isolated compound was confirmed by

interpretation of UV, FT-IR spectra16

and 1H-NMR.

λmax of the isolated vasicinone was found to be 236 nm

(Fig. 1) and the IR (KBr) spectrum of the compound

(Fig. 2) showed the characteristic absorption at 1636

cm-1 (due to C=N bond); 1656, 1684 cm

-1 (due to

stretching vibration of C=O); 2343 cm-1

(due to C≡N);

3411 cm-1

(due to stretching vibration of O-H group).

The proton nuclear magnetic resonance (1H NMR)

spectra of vasicinone in CDCl3 exhibited four singlets

between δ 7.53-8.11 resonances which were attributed

to the four aromatic protons. One proton triplet at

5.033 due to methane. Two multiplet at δ 2.32 and

2.65 due to methylene and two multiplet at δ 3.92 and

4.93 due to methylene. By comparing with values of

standard vasicinone16

it was concluded that the above

product was vasicinone.

Fig. 1—UV-spectrum of vasicinone

Fig. 2—FTIR spectrum of vasicinone

INDIAN J EXP BIOL, JULY 2014

708

Acute toxicity studies—The isolated vasicinone did

not show any mortality up to a dose of 600 mg/kg po

for 7 days in mice.

Pharmacological studies—Crude vasaka leaf

extract has been reported to possess hepatoprotective

activity at 50 mg/kg in rats13

, in the present study the

hepatoprotective potential of isolated vasicinone was

explored at lower doses of 10 and 25 mg/kg in CCl4

induced acute liver damage.

Measurement of pentobarbital-induced sleeping

time—Pentobarbital-induced sleeping time is an

established indicator for functional integrity of the

liver20

. Both vasicinone (P<0.01) and potent

hepatoprotective, silymarin (P<0.001) were found to

significantly decrease the CCl4 mediated increase in

pentobarbital-induced sleeping time (Fig. 3) thus

providing evidence for restoration of liver function by

vasicinone.

Estimation of SGOT, SGPT and ALP level—Single

dose of CCl4 treatment led to elevated levels of serum

SGOT, SGPT and ALP (Table 1, P<0.001) which

were significantly reduced upon both vasicinone

(25 mg/kg P<0.05) and silymarin pre-treatment

(25 mg/kg P<0.01). The above biochemical results

show hepatoprotective action of vasicinone.

Histopathological observation—Histology of liver

section of control animal (Fig. 4A) exhibited normal

hepatic cells each with well defined cytoplasm,

prominent nucleus and well brought out central vein,

whereas that of CCl4 treated group (Fig. 4B) showed

total loss of hepatic architecture with centrilobular

hepatic necrosis, inflammatory changes and crowding

of central vein. Liver sections of the mice pre-treated

with vasicinone (25 mg/kg) for 7 days followed

by CCl4 treatment (Fig. 4C) showed minimal

inflammation, the normal cellular architecture was

retained which was comparable with the potent

hepatoprotective agent silymarin (7 day pre-treatment

at 25 mg/kg/day) + CCl4 treated group (Fig. 4D).

This suggests the reparative quality and maintenance

of structural integrity of hepatocytic cell membrane

by vasicinone.

Discussion In the present study vasicinone was isolated from

leaves of J. adhatoda, characterized using UV, FT-IR

and 1H-NMR spectra. The alkaloid vasicine is known

to be the main active constituent of J. adhatoda,

however it may be easily oxidized in presence of light

and moisture21

to its more stable form vasicinone

which may account for more than 30% of crude

vasaka extract22,24

. Some studies even claim

vasicinone to be more efficacious that vasicine26

.

Isolated vasicinone

was evaluated for

hepatoprotective activity in CCl4 induced liver injury.

Vasicinone pre-treatment significantly reversed

CCl4 mediated liver damage in mice. CCl4 is one of

the most commonly used hepatotoxin in animal

models of liver diseases. CCl4 like other halogenated

alkanes (i.e., chloroform, dichloromethane,

bromotrichloromethane, etc.) undergoes cytochrome

P-450 catalyzed reductive dehalogenation and

liberates trichloromethyl (•CCl3)

5. Reaction of CCl3

with poly-unsaturated fatty acids in membrane lipids

Table 1—Effect of Vasicinone on serum SGOT, SGPT and ALP levels in CCl4 induced hepatotoxicity

[Values are mean ± SE from 6 animals in each group]

Group Treatment SGOT (U/L) SGPT (U/L) ALP (U/L)

I Corn oil 40±3.21 50±2.88 81±1.6

II CCl4 (1 ml/kg) 99.3±6.96*** 96.6±4.40*** 151.3±4.48***

III Vasicinone (10 mg/kg) + CCl4 87±1.15 89±2.08 138.4±4.41

IV Vasicinone (25 mg/kg) + CCl4 76.6±4.05* 74±5.78* 115.7±2.3*

V Silymarin (25 mg/kg) + CCl4 52.3±4.33** 61.6±0.88** 98.5±1.6**

P values: ***<0.001; **<0.01, *<0.05

Fig. 3—Effect of vasicinone on pentobarbital induced sleeping

time. Values are Mean ± S.E from 6 animals in each group.

SARKAR et al.: HEPATOPROTECTIVE ACTIVITY OF VASICINONE IN MICE

709

releases lipid free radicals (•R) which along with

reactive oxygen species (ROS) initiates the process of

lipid peroxidation as indicated by increased levels of

malondialdehyde. The process is exacerbated by

reduction in the levels of antioxidant enzymes like

superoxide dismutase, catalase, glutathione and

glutathione peroxidase27

. Free radicals lead to auto-

oxidation of the fatty acids present in the cytoplasmic

membrane phospholipids and cause functional and

morphological changes in the hepatocyte membrane

leading to its loss of integrity. This was evidenced in

the present study with an increase in the levels of

serum hepatic enzymes SGOT, SGPT and ALP in

CCl4 treated animals. Elevation of liver enzymes

especially SGPT is a clear indication of liver injury.

Vasicinone pre-treatment led to a significant decrease

in the levels of CCl4 mediated elevated liver enzymes.

Increase in pentobarbital induced sleeping time is

another indication of reduced liver function in

experimental animals. Pentobarbital is metabolized by

the hepatic microsomal drug metabolizing enzymes

(MDME) to inactive metabolites and any drug or

chemical which inhibits MDME or cause hepatic

damage is likely to prolong pentobarbital-induced

sleeping time20

. The duration of pentobarbital-induced

sleeping time in intact animal is considered to be a

reliable index for the activity of hepatic MDME. In

the present study we show significant decrease in

CCl4 mediated increase in pentobarbital-induced

sleeping time in vasicinone pre-treated animals

indicating normalization of liver function.

Histopathological studies provided additional evidence

for the hepatoprotective actions of vasicinone.

CCl4 induced liver damage being a highly

oxidative process is usually reversed by naturally

occurring antioxidants28

. This may be due to

stabilization of plasma membrane and or repair of

hepatic tissue damage by these compounds. Various

studies have previously shown the hepatoprotective

actions of alkaloids due to their antioxidant

properties29-31

. Crude vasaka extract as well as

extract containing 30% vasicinone are known for

Fig. 4—Effect of vasicinone on liver [A: Control animal exhibiting normal hepatic cells each with well defined cytoplasm, prominent

nucleus and well brought out central vein; B: CCl4 treated group showing total loss of hepatic architecture with centrilobular hepatic

necrosis, inflammation, dilated sinusoids and crowding of central vein; C: Mice pre-treated with vasicinone ( 25 mg/kg) for 7 days

followed by CCl4 treatment showing normalization of cellular architecture with minimal inflammation; D: Hepatocytes from animals

pre-treated with silymarin (7 days at 25 mg/kg) followed by CCl4 treatment showing normal cells with well defined central vein.

Representative image from each treatment group stained with Haematoxylin & Eosin observed under 100 X magnification.]

INDIAN J EXP BIOL, JULY 2014

710

their antioxidant activity24,26

. Thus antioxidant

character of vasicinone may be responsible for its

hepatoprotective function.

To conclude vasicinone was successfully isolated

and characterized from the leaves of J. adhatoda.

Significant decrease in pentobarbital induced sleeping

time, SGOT, SGPT, ALP levels and protection of

hepatic cells from CCl4 induced liver damage as

evidenced by histopathological observation provide

strong evidence that vasicinone may act as

hepatoprotective in mice. Since vasaka extract which

mainly contain vasicine and vasicinone is known for

its pharmacological activities against cough, asthma

and tuberculosis its hepatoprotective action may add

to the benefits of individuals on vasaka therapy.

Acknowledgement Thanks are due to Dr. Kalyan Kumar Sen,

Principal, Gupta College of Technological Sciences,

Asansol, Prof. Debesh Chandra Majumdar, Chairman,

Trinity Trust and all the faculty members of Gupta

College of Technological Sciences, Asansol for

constant support and encouragement and to Dr. Arun

Chandra Karmakar Sr. Vice President, IPCA

Laboratories Ltd for NMR studies.

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