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1. IOSR Journal of Pharmacy
ISSN: 2250-3013, www.iosrphr.org
‖‖ Volume 2 Issue 5 ‖‖ Sep-Oct. 2012 ‖‖ PP.38-43
Hepatoprotective effect of amomum subulatum roxb seeds on
carbon tetrachloride-induced liver damage in rats
Mihir Y. Parmar*1, 2, Purvi A. Shah2, Vaishali T. Thakkar2,
Salim S. Al-Rejaie1, Tejal R. Gandhi2
1
Department of Centre for Experiment on Animal, College of Pharmacy, King Saud University,
P.O. Box 2457, Riyadh 11451, Saudi Arabia.
2
Anand Pharmacy College, Shree Ram Krishna Seva Mandal, Opp. Town Hall, Sardar Patel
University, Anand-388001, Gujarat, India.
Abstract
Aims of study: - The aim of the present study is to evaluate the protective effect of Amomum subulatum seeds
against experimentally induced liver injury.
Materials and methods: - The methanolic extract of seeds of Amomum subulatum (MEAS) was evaluated for
the hepatoprotective effect against carbon tetrachloride (CCl4) induced hepatotoxicity in rats. Various
biochemical parameters like alanine amino transferase (ALT), aspartate amino transferase (AST), alkaline
phosphatase (ALP), total bilirubin (TB) and total protein (TP) levels were estimated in serum as well as the
glutathione (GSH) and malondialdehyde (MDA) levels in the liver were determined. Histopathological
changes in the liver of different groups were also studied.
Results:- The pre-treatment of MEAS at dose levels of 100 and 300 mg/kg/d, p.o. for 7 d had controlled the
raise of AST, ALT, ALP, TB and MDA levels and the effects were comparable with standard drug (silymarin
200 mg/kg/d, p.o. for 7 d). The GSH and TP levels were significantly increased in the animals received
pretreatment of the extract. The animals received pre-treatment of the extract shown decreased necrotic zones
and hepatocellular degeneration when compared to the liver exposed to CCl 4 intoxication alone. Thus the
histopathological studies also supported the protective effect of the extract.
Conclusion: - This study demonstrates the hepatoprotective effect of Amomum subulatum seeds and thus
scientifically supports the usage of this plant in various Ayurvedic preparations and traditional medicine for
treatment of liver disorders.
Keywords: Amomum subulatum, hepatoprotective effect, CCL4
I. INTRODUCTION
Liver is the organ for metabolism and detoxification of various components enter into the body. It is
involved in wide range of functions and hence it is exposed to toxic substances and drugs absorbed from the
intestine. Apart from the toxins and drugs (Marina, 2006), viral infections (hepatitis A, B, C, D, etc.) and
microbial infections of Entamoeba histolytica (Sharma and Ahuja, 1997) also cause damage to the hepatocytes.
Globally, plant based drugs like Silybum marianum (Scott Luper, 1998), Picrorhiza kurroa (Chander et al.,
1992), Phyllanthus emblica (Gulati et al., 1995), etc. are widely and successfully used in the treatment of liver
disorders.
Amomum subulatum (Zingiberaceae) is commonly known as greater cardamom, is a perennial herb
which grows widely in moist tropical countries. It is one of the plants mentioned in literature having claims of
activity against liver disorders (Sharma et al., 2002). It contains a wide variety of phytoconstituents includes
flavonoids, terpenoids, glycosides and volatile oils (Parmar et al., 2009).
Plant derived natural products such as flavonoids (Defeudis et al., 2003) terpenoids (Takeoka et al.,
2003), glycosides (Quyang et al., 2003), volatile oils (Hikino et al., 1986), steroids (Banskota et al., 2000),
saponins (Yohikawa et al., 2003), alkaloids (Janbaz et al., 2000) and tannins (Gilani et al., 1995) have received
considerable attention in recent years due to their diverse pharmacological properties including antioxidant and
hepatoprotective activity. There has been a growing interest in the analysis of above compounds stimulated by
intense research into their potential benefits to human health. Antioxidants play an important role in inhibiting
and scavenging radicals, thus providing protection to humans against infection and degenerative diseases.
Realizing the fact, this research was carried out to evaluate the hepatoprotective effect of A. subulatum seeds
against CCL4- induced liver damage in rats.
38

2. Hepatoprotective effect of amomum subulatum roxb seeds on carbon tetrachloride…
II. MATERIALS AND METHODS
Plant material and extraction:
A. subulatum fruits were purchased from a local market of Anand, India, during July 2007. The fruits
were identified and authenticated by Dr. A. S. Reddy, Department of Biosciences, Sardar Patel University,
Vallabh Vidyanagar, Gujarat, India where a voucher specimen (No. MP-2: 28/7/2007) was kept for future
reference. The seeds were dried at room temperature and mechanically powdered to obtain a coarse powder;
defatted with petroleum ether (60-80°C) and cold extracted with methanol. The methanol crude extract (9.5 %
yields) was obtained by evaporation using Rotavapour ® (BÜCHI, Switzerland) under reduced pressure.
Animals:
Studies were carried out using either sex Wistar albino rats (200-220 g). They were obtained from the
animal house, Anand pharmacy college (APC), Anand, India. The animals were grouped and housed in
polyacrylic cages (38 × 23 × 10 cm) with not more than six animals per cage and maintained under standard
laboratory conditions; temperature (22 ± 2°C), relative humidity (55 ± 5 %) with dark and light cycle (12/12 h).
They were allowed free access to standard pellet diet (Amrut feed, Sangli, India) and water ad libitum. The rats
were acclimatized to laboratory condition for 10 days before commencement of experiment. Animal studies
were approved by the Committee for the Purpose of Control and Supervision of Experiments on Animals
(CPCSEA) and conducted according to the regulations of Institutional Animal Ethics Committee (Protocol no.
7004 dated 07/08/07).
Drugs and Chemicals:
CCL4 was purchased from S. D. Fine Chem. Ltd. (Mumbai). Silymarin was obtained as a gift sample
from Micro labs, Bangalore, India. SGOT, SGPT, ALP, Bilirubin kits were procured from Span Diagnostics,
Surat, India. LDH, GGT kits were procured from Coral Clinical Systems, Goa, India. Trichloroacetic acid was
purchased from Merck India Ltd. (Mumbai). Thiobarbituric acid and Di thiobis Nitro Benzoic Acid were
purchased from Himedia, Mumbai, India. All other chemicals and reagents used were of analytical grade.
Acute Toxicity Studies:
Healthy adult Wistar albino rats of either sex weighing between 200 to 220 g were subjected to acute
toxicity studies as per guidelines (AOT no. 425) suggested by the Organization for Economic Cooperation and
Development (OECD, 2001) using stair case method. The mice were observed continuously for 2 h for
behavioral, neurological and autonomic profiles for any lethality or death for the next 48 h.
Carbon tetrachloride-induced liver damage in rats: (Shenoy AK et al., 2001)
Animals were randomly divided into five groups six of each. All animals except normal control group
were intoxicated with CCL4 (0.5 ml/kg/d, i.p. for 7 d). Group I (Normal control) received only distilled water
and Group II (CCL4 control) received CCL4 (0.5 ml/kg/d, i.p. for 7 d). Group III (Test-1) and Group IV (Test-2)
received methanol extract of A. subulatum at 100 mg/kg and 300 mg/kg/d, p.o for 7 d. respectively while Group
V (Standard control) received silymarin (200 mg/kg/d, p.o for 7 d). The blood was withdrawn 24 h after the
administration of last dose under anaesthesia using thiopentone sodium (35 mg/kg, i.p.). The blood is allowed to
stand for 30 min at room temperature and then centrifuged to separate the serum. The liver was quickly removed
and perfused immediately with ice-cold saline (0.9% NaCl). A portion of the liver was homogenized in chilled
sodium phosphate buffer (0.1M, pH 7.4) using a Potter Elvehjehm Teflon homogenizer. The homogenate
obtained was centrifuged in a cooling centrifuge at 12,000×g for 30 min at 4°C to obtain a post-mitochondrial
supernatant (PMS) which was used for enzyme analysis. A portion of PMS was centrifuged at 105,000×g for 1h
at 4°C. The pellet was first washed and then suspended in sodium phosphate buffer (0.1M, pH 7.4).
Estimation of biochemical parameters
The separated serum was estimated for various biochemical parameters like ALT, AST (Reitman and
Frankel, 1957) ALP (Kind and King, 1954), TB (Malloy and Evelyn, 1937) and TP (Lowry OH et al.,1951)
levels. The contents of glutathione (GSH) and malondialdehyde (MDA) in the liver were determined by the
methods of Ellman (1959) and Ohkawa et al. (1979), respectively.
Histopathological studies
A portion of the liver was cut into two to three pieces of approximately 6mm3 size and fixed in
phosphate buffered 10% formaldehyde solution. After embedding in paraffin wax, thin sections of 5 μm
thickness of liver tissue were cut and stained with haematoxylin–eosin. The thin sections of liver were made into
permanent slides and examined (Valeer, 2003) under high-resolution microscope with photographic facility and
photomicrographs were taken.
III. STATISTICAL ANALYSIS
All values are expressed as mean ± SEM. Statistical analysis were performed by one-way Analysis of
Variance (ANOVA) and individual comparisons of the group mean values were done using Dunnet’s t-test, with
the help of Graph Pad prism 5.0 software. The value of P lower than 0.05 were considered as significant (P is
probability) (Dunnet, 1964; Osel et al., 1975).
39

3. Hepatoprotective effect of amomum subulatum roxb seeds on carbon tetrachloride…
RESULTS
Acute toxicity studies
The acute toxicity study exhibited no mortality up to a dose level of 2000 mg/kg/d, p.o. As per the
ranking system European Economic Community (EEC) for acute oral toxicity, the LD50 dose of 2000 mg/kg
and above is categorized as unclassified (EC Directive 83/467/EEC, 1983).
Evaluation of hepatoprotective activity
The results observed in pre-treatment of MEAS with respect to induction of hepatotoxicity using CCl 4
were given in Table 1. Rats treated with CCl4 developed significant (P < 0.001) liver damage and it was well
indicated by elevated levels of hepatospecific enzymes like ALT, AST and ALP in serum. A marked elevation
in TB level was observed in the group treated with CCl4 and they were significantly high when compared with
the normal values. The TP levels decreased considerably (P < 0.001) in the toxic group. The MDA and GSH
levels in the liver homogenate were also significantly altered in the group received CCl 4 alone.
Table 1. Hepatoprotective effect of A. Subulatum seeds on CCl4 induced-hepatotoxicity
in rats.
Group AST ALT ALP TBL TP MDA GSH
(IU/L) (IU/L) (KAU/dl) (mg/dl) (mg/ml) (µg/mg (µg/mg protein)
protein)
Normal 33.5 ±4.7 28.17±2.1 7.18 ±0.7 0.36±0.04 7.69±0.8 0.26±0.02 12.19±1.6
control
CCl4 168.3 ±14.9# # 185.00±14.9# 95.90± 5.8# #
1.68±0.14 # #
3.02±0.4# # 1.81±0.20 # # 6.53±1.02 #
control
(0.5 ml/kg)
Test-1 68.83±3.9** 65.67±5.9** 26.11±1.7** 0.58±0.04** 7.07±0.6** 0.59±0.05** 11.61±1.59**
(100
mg/kg)
Test-2 62.33±2.5** 58.67±3.8** 18.40 ±2.1** 0.49 ±0.05** 7.20±0.5** 0.45±0.07** 11.99±1.08**
(300
mg/kg)
Standard 51.67±4.4** 58.33±3.1** 15.13±1.6** 0.46 ±0.1** 7.50±0.6** 0.44±0.06** 12.07±1.09**
Control
(200
mg/kg)
Values are expressed as mean ± SEM; n=6 rats in each group; # P<0.05 and ##
P<0.001 compared with Normal
control; * P<0.05, ** P<0.001 compared with CCl4 control.
The groups received the pre-treatment of MEAS at dose levels of 100 and 300 mg/kg significantly
controlled the change in the biochemical parameters. The extract at a dose level of 300 mg/kg exhibited a sharp
decrease in the serum enzyme levels and the effect was comparable with the standard group treated with
silymarin (200 mg/kg). The TP and GSH levels were significantly (P < 0.001) increased in groups received
MEAS at dose levels of 100 and 300 mg/kg. The increase in dose levels of MEAS exhibited an increase in
efficacy which was reflected in the values of biochemical parameters. The histopathological studies also
supported the protective properties of MEAS. The areas of necrosis and ballooning degeneration of hepatocytes
were observed in the toxic group. The group of animals pre-treated with MEAS showed a marked protective
effect with decreased necrotic zones and hepatocellular degeneration. The photomicrographs of the liver
sections were given in Fig. 1.
40

4. Hepatoprotective effect of amomum subulatum roxb seeds on carbon tetrachloride…
A B
C
D
E
Fig. 1. Photomicrographs (original magnification 45×) of histopathological studies of livers of various
groups stained with haematoxylin and eosin (A). Normal architecture of rat liver (B). Necrosis and
hepatocellular degeneration of CCl4 intoxicated (C, D & E). Lesser damage of hepatocytes and low index of
necrosis in MEAS (100, 300 mg/kg) and Silymarin pre-treated group.
41

5. Hepatoprotective effect of amomum subulatum roxb seeds on carbon tetrachloride…
IV. DISCUSSION
The hepatotoxic agent CCl4 induces selective toxicity to the liver cells due to metabolic activation and
this maintains them with semi normal metabolic function. It also causes functional and morphological changes
in the cell membrane which may lead to cell death (Recknagel et al., 1989). The hepatic cells consist of higher
concentrations of AST and ALT in cytoplasm and AST in particular exists in mitochondria (Wells, 1988). Due
to the damage caused to hepatic cells, the leakage of plasma (Zimmerman and Seef, 1970) causing an increased
levels of hepatospecific enzymes in serum.
The elevated serum enzyme levels like AST and ALT are indicative of cellular leakage and functional
integrity of cell membrane in liver (Drotman and Lawhorn, 1978). The hepatoprotective index of a drug can be
evaluated by its capability to reduce the injurious effects or to preserve the normal hepatic physiological
mechanisms, which have been induced by a hepatotoxin.
The measurement of serum AST, ALT and ALP levels serve as a means for the indirect assessment of
condition of liver. The pre-treatment of the animals with MEAS with respect to intoxication with CCl4
controlled the AST, ALT and ALP levels when compared with the toxic group. A high concentration of
bilirubin in serum is an indication for increased erythrocyte degeneration rate (Singh et al., 1998). Due to the
liver injury caused by the hepatotoxin, there is a defective excretion of bile by the liver which is reflected in
their increased levels in serum (Rao, 1973). The oral administration of MEAS at 100 and 300 mg/kg effectively
reduced the serum TB levels.
The TP levels will be depressed in hepatotoxic conditions due to defective protein biosynthesis in liver
(Clawson, 1989). The CCl4 intoxication causes disruption and disassociation of polyribosomes on endoplasmic
reticulum and thereby reducing the biosynthesis of protein.
The pre-treatment of MEAS well restored the proteins synthesis by protecting the polyribosomes. The
increase in MDA or decrease in GSH levels indicates the lipid peroxidation. MDA is one among the end
products produced by the decomposition of ω3 and ω6 poly-unsaturated fatty acids (Parola et al., 1999). GSH
play a vital role in the defence mechanism of tissue against the reactive oxygen species (Kosover and Kosover,
1976). The groups received the CCl4 treatment alone were prone to high lipid peroxidation, whereas the groups
received the pre-treatment of MEAS exhibited significant protection.
This also suggests the defence mechanism against the reactive oxygen species and thereby the
antioxidant potential of MEAS. The histopathological studies are direct means for assessing the protective effect
of the drug. The groups received CCl4 alone, the damage of cells around the central vein were well evident.
Whereas, the intensity of damage was found lesser in the studies involved pre-treatment of MEAS. The results
of the histopathological studies supported and well correlated with data obtained from evaluation of the
biochemical parameters.
V. CONCLUSION
The methanolic extract of could effectively control the AST, ALT, ALP and TB levels and increased
the protein levels in the protective studies. The protective effect of MEAS may be attributed due to the reduced
lipid peroxidation and improved defence of the hepatocytes against the reactive oxygen species. The
histopathological studies also substantiate the activity of the drug. Therefore the study scientifically supports the
usage of this plant in various Ayurvedic preparations and traditional medicine for treatment of liver disorders
and as a tonic.
VI. ACKNOWLEDGEMENT
The authors express sincere thanks to Dr. A. S. Reddy, Professor, BRD School of biosciences, S. P.
University, V. V. Nagar for the authentication of the plant. The authors also thank Dr. Prera Desai, Ayurvedic
practitioner for her interest and encouragement.
The authors extend their appreciation to the Deanship of Scientific Research at King Saud University
for funding the work through the research group project No RGP-VPP-179.
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