Indian Journal of Experimental Biology

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

Evaluation of hepatoprotective activity of vasicinone in mice

Chaitali Sarkara, Sankhadip Bosea & Sugato Banerjeeb*
a

Gupta College of Technological Sciences, College of Pharmacy, Asansol 713 301, India
Department 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 reproduction1. 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 disease2,3.
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 extracts4.
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.

*Correspondent author
Mobile: 7250001584
Fax: 91-0651-2275290
E-mail: banerjeesugato1@gmail.com; sbanerjee@bitmesra.ac.in

Materials and Methods

Plant materialsThe leaves of Justicia adhatoda
(L) Willd were collected from Sripur road, Kulti,

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INDIAN J EXP BIOL, JULY 2014

(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
phytoconstituentsLeaves
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.
CharacterizationVasicinone was characterized
by comparing UV15, FT-IR16 and 1H NMR spectra of
isolated compound with standard vasicinone spectra17.
Experimental animalsMale Swiss Albino mice (2025 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 studiesThe 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 protocolMice (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
timeOn day 9 of the experiment, pentobarbital
sodium 30 mg/kg, ip (Taj Pharmaceuticals Limited,
Mumbai, India) was injected to control (Gr I), CCl4intoxicated (Gr II), vasicinone pre-treated CCl4intoxicated (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 estimationThe 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 studiesAfter 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 m 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 analysisThe 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

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 phytoconstituentsCrude 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 C, which was comparable to standard
vasicinone23.
Characterization of isolated vasicinoneThe
structure of isolated compound was confirmed by
interpretation of UV, FT-IR spectra16 and 1H-NMR.
max 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

707

stretching vibration of C=O); 2343 cm-1 (due to CN);

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 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 2.32 and
2.65 due to methylene and two multiplet at 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. 1UV-spectrum of vasicinone

Fig. 2FTIR spectrum of vasicinone

INDIAN J EXP BIOL, JULY 2014

708

Acute toxicity studiesThe isolated vasicinone did

not show any mortality up to a dose of 600 mg/kg po
for 7 days in mice.
Pharmacological studiesCrude 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
timePentobarbital-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 levelSingle
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

Fig. 3Effect of vasicinone on pentobarbital induced sleeping

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

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

show hepatoprotective action of vasicinone.
Histopathological observationHistology 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 1Effect 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

I
Corn oil
II
CCl4 (1 ml/kg)
III
Vasicinone (10 mg/kg) + CCl4
IV
Vasicinone (25 mg/kg) + CCl4
V
Silymarin (25 mg/kg) + CCl4
P values: ***<0.001; **<0.01, *<0.05

SGOT (U/L)

SGPT (U/L)

ALP (U/L)

403.21
99.36.96***
871.15
76.64.05*
52.34.33**

502.88
96.64.40***
892.08
745.78*
61.60.88**

811.6
151.34.48***
138.44.41
115.72.3*
98.51.6**

SARKAR et al.: HEPATOPROTECTIVE ACTIVITY OF VASICINONE IN MICE

709

Fig. 4Effect 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.]

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 autooxidation 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

710

INDIAN J EXP BIOL, JULY 2014

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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