Hepatoprotective Activity of Gloriosa superba on Liver Damage Caused by CCL4 in Rats

 

Pradeep Kumar Samal

SLT Institute of Pharmaceutical Sciences, Guru Ghasidas Vishwavidyalaya, Bilaspur Chhattisgarh

*Corresponding Author E-mail: samalpharmacology@rediffmail.com

 

ABSTRACT:

The objective of this study was to investigate the hepatoprotective activity of alcoholic extract of Gloriosa superba leaves against carbon tetrachloride (CCl4) induced hepatotoxicity. The plant material were dried in shade then powdered and extracted with alcohol. Preliminary phytochemical tests were done. Alcoholic extract showed presence of phenolic compound and flavanoids. The hepatoprotective activity of the alcoholic extract was assessed in CCl4 induced hepatotoxic rats. Alteration in the levels of biochemical markers of hepatic damage like SGOT, SGPT, ALP, Billirubin and Protein were tested in both CCl4 treated and untreated groups. CCl4 (1ml) has enhanced the SGOT, SGPT, ALP and Total Billirubin where decrease in total protein level in liver. Treatment of alcoholic extract of Gloriosa superba (200mg/kg) has brought back the altered levels of biochemical markers to the near normal levels in the dose dependent manner. Our findings suggested that Gloriosa superba alcoholic leaf extract possessed hepatoprotective activity. Moreover, it prevented CCl4 induced prolongation in pentobarbital sleeping time confirming hepatoprotectivity and validates the traditional use of this plant against liver damage.

 

KEYWORDS: Gloriosa superba , Hepatoprotective, Silymarin, CCl4, alcohol

 

 


1.      INTRODUCTION:

Liver is the key organ of metabolism and excretion is constantly endowed with the task of detoxification of xenobiotics, environmental pollutants and chemotherapeutic agents. Thus, disorders associated with this organ are numerous and varied1. Liver disease has become a global concern worldwide. Liver is often abused by environmental toxins, poor eating habits, alcohol and over-the-counter drug use, that damage and weaken the liver leading to important public health problems like hepatitis, cirrhosis and alcoholic liver diseases2. The conventional drugs used in the treatment of liver diseases viz., corticoasteroids, antiviral and immunosuppressant agents are sometimes inadequate and may lead to serious  adverse effects. In India, numerous medicinal plants and their formulations are used for liver disorders in traditional systems of medicine. Some of these plants are evaluated for their hepatoprotective actions against hepatotoxins.

 

However, the readily available hepatoprotective herbal drugs are not sufficiently active to effectively combat severe liver disorders. In view of lack of synthetic agents for the treatment of hepatic disorder, there is a growing focus to evaluate traditional herbal medicines for hepatoprotective activity.3 Therefore; there is a need to develop satisfactory hepatoprotective drugs. Gloriosa superba (Colchicacea) is a large, evergreen shurb growing up to 1.5 – 4 m tall. Leaves are 10-20 cm long oblanceolate to obovate, entire, tip narrow with short stalks. Its bark is used in Arthritis, Rheumatism where as its leaf is used in Fabrifuge generally healing, stomach troubles.

 

2.      MATERIALS AND METHODS: -

2.1 Plant Materials:

The leaves of Gloriosa superba (Colchicacea) were collected from Thakur Chedilal Barristor Agriculture College and Research Centre, Bilaspur, India, in the month of Oct 2012, and air dried at room temperature after wash with tape water. The Plant identification was done by Dr. H. B. Singh Chief Scientist Head of the Raw Materials Herbarium and Museum, NISCAIR, New Delhi (NISCAIR/ RHMD/Consult/-2012-13/2164/170).


 

Table No. –1 Effect of the Gloriosa superba leaves extracts on pentobarbital Induced sleeping time in CCl4 induced hepatotoxic rats.

Group

Treatment

Dose

Mean Sleeping time (min)

I

Solvent control

5ml/kg p.o

145±4

II

Vehicle+CCl4

1 ml/kg p.o(2 ml/kg, s.c.)

229±8***

III

Silymarin+CCl4

50mg/kg p.o(2 ml/kg, s.c.)

177±5***

IV

AEGS+CCl4

100mg/kg p.o(2 ml/kg, s.c.)

199±4***

V

AEGS+CCl4

200mg/kg p.o(2 ml/kg, s.c.)

189±3***

 

 

 

 

 

 

 

Values expressed as mean ± SEM, from six observations, ***p<0.001 when compared with normal control group. Using one-way ANOVA followed by Tukey Kramer’s post hoc test.

 


 

2.2 Drugs and Chemicals:

Analytical grades Chemical were used in this study. Silymarin (Micro labs, Bangalore) was purchased from local market. Chemical like ethanol (CDH, Mumbai), anaesthetic ether (CDH, Mumbai), CCl4 (Ranbaxy, Delhi) and other phytochemical reagents were obtained from Institute. For estimation of biochemical parameter; biochemical kits like SGOT, SGPT, ALP, albumin, total protein, direct bilirubin and total bilirubin were obtained from Span Diagnostics ltd. Surat, India were procured from Matushri Trading Company, Bilaspur.

 

2.3 Animals:

Each experiment had separate set of animals and care was taken to ensure that animals used for one response were not employed elsewhere. Animals were habituated to laboratory conditions for 48 hours prior to experimental protocol to minimize if any of non-specific stress. The approval of the Institutional Animal Ethical Committee (IAEC) of SLT Institute of Pharmaceutical Sciences, Bilaspur (Chhattisgarh) was taken prior to the experiments (Reference No. 25/IAEC/Pharmacy/2013 dated 15/06/13). All the protocols and the experiments were conducted in strict compliance according to ethical principles and guidelines provided by Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA).

 

2.4 Preparation of plant Extracts:

About 300 g of the leaves powder of Gloriosa superba was extracted with 1.2 L of alcohol using Soxhlet apparatus for 72 hrs at 40-50°C. The extract was concentrated to Ό of its original volume by distillation as it was adapted to recover the solvent, which could be used again for extraction.4

 

2.5 Acute toxicity study (AOT):

Acute toxicity study was performed according to the procedure OECD guideline no. 425.5  AOT was performed on Swiss albino mice and the animal were kept fasting for overnight providing water ad libitum, after which the alcoholic extract of Gloriosa superba (AEGS) was administered orally 2000 mg/kg and observed the mortality of animals.

 

2.6 Preliminary Phytochemical Analysis:

The extracts obtained were subjected to various chemical tests to detect the chemical constituents present in extracts of Gloriosa superba .6-9

 

2.7 Assessment of liver function:

The alcoholic extract of Gloriosa superba was evaluated for their hepatoprotective activity by using CCl4 induced acute hepatotoxicity model. Either sex of Wistar rats, weighing (180-220) was divided into 5 groups consisting of 6 animals in each group. Group 1 received distilled water (6 ml/kg, p.o.) for 7 days. Group 2 were treated with vehicle (0.5% of Tween 40, 1 ml/kg, p.o.) for 7 days. Group 3 received silymarin (50 mg/kg, p.o.) for 7 days. Group 4, 5 pretreated with alcoholic extract of Gloriosa superba 100 mg and 200 mg/kg body weight respectively for 7 days. Food was withdrawn 16 hrs before administration to enhance the acute liver toxicity. Group 2, 3, 4 and 5 were treated with CCl4 was administered (2 ml/kg, s.c.) diluted in olive oil (1:1) was administered on 7th day after 1 hrs of extracts treatment and sacrificed 24 hours after administration of CCl4.5 Animals were anesthetized using anesthetic ether and blood sample were collected by cardiac puncture method and serum was used for estimation of AST, ALT, ALP, albumin, total protein, total and direct bilirubin. Immediately after the collection of blood, their livers were separated. The liver was washed by normal saline were preserved in 10% formalin for histopathological studies.

 

2.8 Statistical analysis:

The experimental results were expressed as the Mean ± SEM for six animals in each group. The biochemical parameters were analysed statistically using one-way ANOVA followed by Tukey Kramer’s post hoc test. P value of < 0.05 was considered as statistically significant.

 

3. RESULTS:

Preliminary phytochemical studies with extract revealed the phytoconstituents like cardiac glycoside, carbohydrates, phytosterols, saponins, phenolics and tannins. Different doses of alcoholic extract of Gloriosa superba leaves (AEGS) was screened in albino mice for their acute oral toxicity. No mortality was recorded till 2000 mg/kg body weight. Hence the extract was found to be safe up to the dose levels of 2000 mg/kg. So 1/10th and 1/20th of these dose i.e. 200 and 100 mg/kg body weight of AEGS for oral dose was select as therapeutic dose for pharmacological activity screening. Effect of plant extract on pentobarbital sleeping time was studied in rats and the results are shown in Table: 1 Pentobarbital at a dose of 75 mg/kg. i.p. in normal control( group-1) caused sleep in rats for a period of 145 ± 4 min (mean ± SEM. n = 6). Whereas the sleeping time in the CCl4 induced toxic control group (group-2) of animals was found to 229 ± 8 min.


 

Table No. –2 Effect of the Gloriosa superba leaves extracts on serum enzyme in CCl4 induced hepatic damage in rats.

Group

Treatment

SGOT(IU/L)

SGPT(IU/L)

ALP(IU/L)

I

Solvent control

96.38±4.02

48.28±3.63

107.45±8.98

II

Vehicle+CCl4

436.16±7.45

196.48±2.88

283.10±3.12

III

Silymarin+CCl4

218.40±2.77***

69.46±2.75***

186.23±9.85***

IV

AEGS+CCl4

389.27±9.56***

116.14±4.89 ***

231.12±11.26

V

AEGS+CCl4

283.10±3.52***

94.41±3.781 ***

205.16±8.46***

 

 

 

 

 

 

 

Values expressed as mean ± SEM, from six observations, **p<0.01, ***p<0.001 when compared with CCl4 control group. Using one-way ANOVA followed by Tukey Kramer’s post hoc test.

 

Table No. –3 Effect of the Gloriosa superba leaves extracts on serum biochemical in CCl4 induced hepatic damage in rats.

Group

Treatment

Dose

Albumin

(mg/dl)

Total protein(mg/dl)

Direct bilirubin

(mg/dl)

Total bilirubin

(mg/dl)

I

Solvent control

5ml/kg p.o

4.67±0.29

14.78±0.54

0.28±0.01

0.39±0.01

II

Vehicle+CCL4

1 ml/kg p.o100mg/kg s.c

2.47±0.0.27

7.089±0.15

0.81±0.06

1.09±0.05

III

Silymarin+CCL4

50mg/kg p.o100mg/kg s.c

3.94±0.18**

13.92±0.41***

0.391±0.026***

0.59±0.01***

IV

AEGS+CCL4

250mg/kg p.o100mg/kg s.c

3.59±0.18*

11.86±0.57 ***

0.5±0.01*

0.75±0.02***

V

AEGS+CCL4

500mg/kg p.o100mg/kg s.c

4.04±0.062***

13.21±0.24***

0.42±0.031**

0.6±0.01***

 

Values expressed as mean ± SEM, from six observations, **p<0.01, ***p<0.001 when compared with CCl4 control group. Using one-way ANOVA followed by Tukey Kramer’s post hoc test.

 


When sleeping time of toxic control group of animals was compared with test groups, the higher dose of AEGS (200mg/kg body wt) (Group-V) was highly significant189±3 min (P<0.001) which is very nearer to that of silymarine induced standard (Group-3) (177±5). The effects of AEGS on Serum glutamate oxaloacetate transaminase (SGOT), Serum glutamate pyruvate transaminase (SGPT), Alkaline phosphatase (ALP), Serum direct bilirubin(DBIL), Sreum total bilirubin(TBIL), Serum albumin(ALB) and Serum total protein(TLP) levels in CCl4 induced liver damage in rats are summarized in Table - 2 and 3. Administration of CCl4 (2 ml/kg, s.c.), after 24 hours of intoxication resulted a significant (P<0.05) elevation of hepatospecific serum enzymes markers like SGOT, SGPT and ALP and serum biochemicals markers like DBIL and TBIL in CCl4 treated groups, while seum biochemicals markers like albumin and total protein were found to be decreased in comparison with the normal control group. On administration of AEGS (Group IV and V) and Silymarin at the dose of 50mg/kg (Group III) the level of these enzymes and biochemicals were found retrieving towards normalcy. The hepatoprotective effect offered by AEGS (200 mg/kg p.o.) was found to be significantly greater than AEGS (100 mg/kg p.o.).

 

4. DISCUSSION:

In recent years, many studies have been undertaken with traditional medicines, in an attempt to develop new drugs for hepatitis.10 In the present study alcoholic  extract of Gloriosa superba  leaves were evaluated for the hepatoprotective activity using CCl4 induced hepatotoxicity in rat model and find out the therapeutically better efficacious extract. CCl4 is a well-known hepatotoxic agent and the preventive action of liver damage by CCl4 has been widely used as an indicator of liver protective activity of drugs in general.11 The changes associated with CCl4-induced liver damage are similar to that of acute viral hepatitis.12 CCl4 is biotransformed by Cytochrome P-450 system to produce trichloromethyl free radicals. These free radicals may again react with oxygen to form trichloromethyl peroxyl radicals, which may attack lipids on the membrane of endoplasmic reticulum to elicit lipid peroxidation, finally resulting in cell necrosis and consequent cell death.13 As well known, phenolic antioxidants, such as flavonoids and tannins, are considered promising therapeutic agents for free radical pathologies due to their scavenging ability with ROS.14

 

In the present study, CCL4 was found to cause significant prolonged the pentobarbital induce sleeping time.  In spite of a large biological variation in sleep time, it was observed that sleep time was reduced in the treated group. In the group of animals administered with AEGS the sleeping time was decreased as compared to CCL4 treated group and nearly restored back to the initial sleeping time.

 

SGOT is found in the liver, cardiac muscles, skeletal muscles, pancreas, lungs, kidney, brain, etc., whereas SGPT concentrationis highest in the liver and therefore, it appears to be a more sensitive test to hepatocellular damage than SGOT.15 Leakage of large quantities of enzymes into the blood stream is often associated with massive necrosis of the liver.16 CCl4 is known to cause marked elevation in serum enzymes (SGOT and SGPT). In the present study, a significant increase in the activities of SGOT and SGPT within 24 hr exposure to CCl4 was observed, indicating considerable hepatocellular injury. Our results indicated that Gloriosa superb (200 mg/kg) administration significantly alleviated the increased serum enzyme activity induced by CCl4, indicating improvement of the functional status of the liver. The recovery towards normalization of serum enzymes and liver histological architecture caused by Gloriosa superba was almost similar to that caused by silymarin, in the present study. Similar results have been reported.17 Silymarin is a known hepatoprotective compound. It is reported to have a protective effect on the plasma membrane of hepatocytes.18

 

In conclusion the possible hepatoprotective effect of Gloriosa superba in CCl4 induced liver injuries may be due to: (1) inhibiting Cytochrome P- 450 activity, (2) preventing the process of lipid peroxidation, (3) stabilizing the hepatocellular membrane and (4) enhancing protein and glycoprotein biosynthesis. However the exact hepatoprotective mechanism of Gloriosa superba is still unknown. Further studies are warranted to isolate the active components.

 

5. ACKNOWLEDGEMENTS:

The authors wish to thank Prof. J.S. Dangi, Head of the Institute for facilities and Mr. Karteek Patra for technical assistance.

 

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14.     Halliwell, B., Gutteridge, J.M., 1984. Lipid peroxidation, oxygen radicals, cell damage, and antioxidant therapy. Lancet 1, 1396–1397.

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Received on 30.10.2013       Accepted on 22.11.2013     

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Asian J. Pharm. Res. 3(4): Oct. - Dec.2013; Page 194-197