Abstract: The combined hepatoprotective effect of Bi-Herbal Ethanolic Extract (BHEE) pretreatment against carbon tetra chloride (CCl4) induced hepatic damage in rats was investigated. Ethanolic extract from the leaves of Melia azedarach and seeds of Piper longum at a dose level of 50 mg kg-1 body weight was administered orally daily once for 14 days, prior to the administration of 0.2 mL of CCl4 kg-1 body weight for 7 days. The substantially elevated serum marker enzymes such as Serum glutamic Oxaloacetic Transaminase (SGOT), Serum Glutamic Pyruvic Transaminase (SGPT), Alkaline Phosphatase (ALP), Acid Phosphatase (ACP), Lactatedehydrogenase (LDH), Gamma glutamyl Transferase (γGT) and 5 Nucleotidase (5NT), due to CCl4 treatment were restored towards normalization in rats pretreated with BHEE. The determination of liver weight and biochemical parameters such as serum bilirubin and blood urea showed a remarkable decrease in rats pre- treated with the BHEE when compared with those administered CCl4 alone. In addition BHEE significantly increased the total protein, total cholesterol and triglyceride also towards normal levels. Silymarin at a dose level of 50 mg kg-1 was used as a standard reference also exhibited significant hepatoprotective activity against CCl4 induced hepatotoxicity. The results of this study strongly indicate that BHEE has got a potent hepatoprotective action against CCl4 induced hepatic damage in rats.
INTRODUCTION
The use of herbal medicine in the treatment of diseases is increasing worldwide. Indeed, the effectiveness of some medicinal herbs in the treatment of diseases has been validated by rough research and clinical studies (Oguntola, 2007). Liver, an important organ actively involved in many metabolic functions, is the frequent target for number of toxicants (Meyer and Kulkarni, 2001). The disorders associated with the organ are numerous and varied (Wolf, 1999). Serum or plasma enzyme levels have been employed as markers for monitoring chemically induced tissue damages (Hukkeri et al., 2002). Carbon tetra chloride is toxic to the liver and its toxicity is dose dependent and time of exposure (Junnila et al., 2000). In the liver, CCl4 is metabolized in to the highly reactive trichloromethyl radical. The free radical generated would lead to auto oxidation of the fatty acids present in the cytoplasmic membrane phospholipids and cause functional and morphological changes in the cell membrane (Pandit et al., 2004).
In spite of tremendous strides in modern medicine, there are hardly any drugs that stimulate liver function and offer protection to the liver from the damage or help in the regeneration of hepatic cells (Guntupalli et al., 2006). In absence of a reliable liver protective drug in the modern medicine, there are number of medicinal preparations in Ayurveda recommended for the treatment of liver disorders (Chatterjee, 2000). A single drug cannot be effective against all types of severe liver diseases (Shahani, 1999). Therefore effective formulations have to be developed using indigenous medicinal plants, with proper pharmacological experiments and clinical trials.
With the above scenario, the Bi-Herbal Ethanolic Extract (BHEE) made up equal quantities of leaves of Melia azedarach and seeds of Piper longum were subjected to various assays in order to evaluate their hepatoprotective effect from mixture of these herbs against CCl4 toxicity in albino rats. Melia azedarach, a member of the family Meliaceae is widely grown as an ornamental tree, is being used against intestinal worms, in skin diseases, stomach ache, intestinal disorders, uterine illnesses, cystitis, as diuretic and febrifuge (Senthil Nathan et al., 2005; Alche et al., 2002). It has got antiviral, antimalarial, anthelmintic and cytotoxic activities (Khan et al., 2001). Piper longum Linn., an important medicinal plant belonging to the family Piperaceae is been used in traditional medicine by many people in Asia and Pacific islands especially in Indian medicine (Guido and David, 1998). Piper longum is a component of medicines reported as good remedy for treating gonorrhea, menstrual pain, tuberculosis, sleeping problems, respiratory tract infections, chronic gut related pain and arthritic conditions (Singh, 1992).
MATERIALS AND METHODS
Plant Material
The leaves of Melia azedarach and seeds of Piper longum
were collected in the month of November 2005 from center for Advanced
Studies in Botany Field Research Laboratory, University of Madras, Chennai,
India and were authenticated by Dr. P.T. Kalaichelvan at the same centre.
The voucher specimen is also available in herbarium file of the Studies
in Botany Field Research Laboratory, University of Madras and Chennai,
India.
Extraction
The leaves of Melia azedarach (1 kg) and seeds of P. longum
(1 kg) were shade-dried and pulverized to a coarse powder. Equal quantities
of the powder was passed through 40 mesh sieve and exhaustively extracted
with 90% (v/v) ethanol in soxhlet apparatus at 60°C (Chattopadhyay,
2003). The extract was evaporated under pressure until all the solvent
had been removed and further removal the of water was carried out by freeze
drying to give an extract sample with the yield of 19.7% (w/w). The extract
was stored in refrigerator; weighed amount was dissolved in Tween-80 and
used for present investigation.
Animals
Adult albino male rats of wistar strain weighing 150-175 g were used
in the pharmacological and toxicological studies. The inbred animals were
taken from animal house in Madras Medical College, Chennai, India. The
animals were maintained in well-ventilated room temperature with natural
12±1 h day-night cycle in the propylene cages. They were with fed
balanced rodent pellet diet from Poultry Research Station, Nandam, Chennai,
India and tap water ad libitum was provided throughout the experimental
period. The animals were sheltered for one week and prior to the experiment
they were acclimatized to laboratory temperature. Acute toxicity studies
were carried out as per up and down or staircase method (Ghosh, 1984).
The protocol was approved by Animal Ethics Committee constituted for the
purpose as per CPCSEA Guideline.
Experimental Groups
The rats were divided into five groups with six animals in each group
and were given dose schedule as:
| Group 1: | Animals were given a single administration of 0.5 mL vehicle (2% (v/v) aqueous Tween- 80) p.o for 14 days. This group served as control. |
| Group 2, 3 and 5: | Animals were given a single dose of 2 mL kg-1, p.o CCl4 (2% (v/v) aqueous Tween-80) daily for 7 days. |
| Group 3: | Animals were treated with 50 mg kg-1, p.o of (BHEE) daily for 14 days. |
| Group 4: | Animals received only 50 mg kg-1, p.o of BHEE daily for 14 days. |
| Group 5: | Animals received 50 mg kg-1 p.o Silymarin in 2 % (v/v) aqueous Tween-80 daily for 14 days and this group served as positive control. |
On the 15th day the animals were sacrificed and various biochemical parameters were analyzed.
Liver Weight and Biochemical Parameters
At the end of the experimental period animals were sacrificed by cervical
decapitation under mild pentobarbitone anesthesia, blood was collected
and the serum was separated by centrifuging at 3,000 rpm for 10 min. Immediately
after the sacrifice, the liver was excised from the animals, washed in
ice-cold saline. The weight of the liver is measured and expressed as
(mg g-1) wet tissue. The biochemical parameter such as total
protein was estimated by the method of Gornall et al. (1949). The
total cholesterol was estimated by the method of Wybenga et al.
(1970). The total bilirubin was estimated by Malloy and Evelyn (1937)
method. Triglyceride was estimated by the method of Fossati and Lorenzo
(1982) and urea concentration was determined by the method of Varley (1967).
Marker Enzymes
The above collected serum was also used for the assay of marker enzymes.
The Glutamate Oxaloacetate Transaminase (GOT) and Glutamate Pyruvate Transaminase
(GPT) were estimated by the method of Reitman and Frankel (1957). Alkaline
Phosphatase (ALP) and Acidphosphatase (ACP) were determined by the method
of Kind and King (1954). The enzyme Lactatedehydrogenase (LDH) was analyzed
by the method of King (1965). The gamma glutamyl transferase (γGT)
enzyme was determined by the method of Szasz (1969) and 5 nucleotidase
(5 NT) enzyme by Luly et al. (1972). All the enzymatic and biochemical
assays were taken at particular nm using Shimadzu spectrophotometer, UV-1601
model.
Statistical Analysis
Values reported are the mean±SEM. The statistical analysis was carried
out using Analysis of Variance (ANOVA) followed by Dunnets t-test. p<0.05
were considered as significant.
RESULTS
Liver Weight and Biochemical Parameters
In the present investigation a significant reduction in the liver
weight (p<0.001) was shown in group 3 BHEE pretreated animals when
compared to that of the group 2 CCl4 intoxicated animals.Table
1 shows that in group 3 there was a significant increase in total
protein (6.1±0.32 g dL-1), total cholesterol (128.16±3.34
mg dL-1) and triglyceride (187.33±7.0 mg dL-1)
levels in the CCl4 intoxicated and BHEE pretreated animals
(p<0.001) when compared with group 2 CCl4 intoxicated animals
which has the total protein (5.25±0.18 g dL-1), total
cholesterol (125.33±2.901 mg dL-1) and
| Table 1: | The average values of weight and biochemical parameters of the liver under different experimental conditions |
| Values are Mean±SEM of 6 animals each in a group. Statistical significant test for comparison was done by ANOVA, followed by Dunnets `t-test (n = 6) Comparison between: a: Group 1 vs Group 2, b: Group 2 vs. Group 3 and 4, c: Group 1 vs Group 5, *p<0.001, NS: Not Significant | |
| Table 2: | The average values of liver marker enzymes under different experimental conditions |
| Values are Mean±SEM of 6 animals each in a group. Statistical significant test for comparison was done by ANOVA, followed by Dunnets t-test (n = 6) Comparison between: a: Group 1 vs Group 2, b: Group 2 vs. Group 3 and 4, c: Group 1 vs Group 5, *p<0.001, NS: Not Significant | |
triglyceride (125±2.01 mg dL-1), respectively. The biochemical
parameters such as serum bilirubin (1.57±0.10 mg dL-1)
and urea (32.16±2.75 mg dL-1) levels were also decreased
in the BHEE (at a dose level of 50 mg kg-1 of body wt) pretreated
group 3 animals, (p<0.001) When compared with the CCl4 intoxicated
group 2 animals which has the total bilirubin and urea (2.47±0.09
and 45.0±2.4 mg dL-1), respectively. Group Comparison
between group 1 and 4 shows no significant variation in liver weight and
biochemical parameter levels indicates no appreciable adverse side effect
due to the administration of Tween-80 and BHEE alone. Group comparison
between group 3 and group 5 shows no significant variation in these parameters
indicating that BHEE has got the same effect as that of the Silymarin,
which was considered as the positive control in this study.
Marker Enzymes Level
A significant increase in the serum GOT (145.50±1.08 U L-1)
and GPT (143.79±4.5 U L-1) levels were seen in the group
II CCl4 intoxicated animals. These enzymes were brought back
to near normal levels such as (75±1.46 U L-1) and (87.30±3.4
U L-1), respectively in BHEE (50 mg kg-1 body weight)
pretreated group 3 animals (p<0.001). Similarly the elevated ALP (172.68±0.64
KA) and ACP (12.25±1.06 KA) enzyme levels in group 2 CCl4
intoxicated animals were also significantly decreased to (121.75±0.72
KA) and (6.76±0.24 KA), respectively in the group 3 BHEE pretreated
and CCl4 intoxicated animals (Table 2).
The enzymes such as LDH (235.0±1.50 U L-1), γ-GT
(20.41±1.04 U L-1) and 5 NT (5.85±0.28
U L-1) were also significantly decreased in the group 3 CCl4
intoxicated and BHEE pretreated, animals when compared with the
group 2 CCl4 intoxicated animals which showed the elevated
enzyme levels of LDH (435.38±1.84 U L-1), γ-GT
(45.03±1.59 U L-1) and 5 NT(7.6±0.4
U L-1) (p<0.001), respectively. Group comparison between
Group 1 control rats and the animals of group 4 which received only BHEE
shows no significant variation in the marker enzyme levels indicating
no adverse side effects due to the administration of Tween-80 and BHEE
alone. All the parameters were under normal limits in the group 5 animals
that acted as a positive control, which were intoxicated by CCl4
and treated by silymarin.
DISCUSSION
It is well established that CCl4 induces hepatotoxicity
by metabolic activation; therefore it selectively causes toxicity in liver
cells maintaining semi-normal metabolic function. CCl4 is bio-transformed
by the cytochrome p450 system in the endoplasmic reticulum to produce
trichloromethyl free radical (CCl3). Trichloromethyl free radical
when combined with cellular lipids and proteins in the presence of oxygen
form trichloromethyl peroxyl radical, which may attack lipids on the membrane
of endoplasmic reticulum faster than trichloromethyl free radical (Reckengel
et al., 1989). Thus, trichloromethylperoxyl free radical leads
to elicit lipid peroxidation, the destruction of Ca2+ homeostasis
and finally, results in cell death (Opuku et al., 2007).
In this present study it was noted that the administration of CCl4 decreased the levels of total protein, total cholesterol and triglycerides. These parameters were brought back to the normal levels in BHEE pretreated group 3 and silymarin treated group 5 animals. BHEE treatment showed a protection against the injuries effects of carbon tetra chloride that may result from the interference with cytochrome p450, resulting in the hinderence of the formation of hepatotoxic free radicals (Cavin et al., 2001). The site-specific oxidative damage in some susceptible amino acids of proteins is now regarded as the major cause of metabolic dysfunction during pathogenesis. (Bandyopadhyay et al., 1999). Attainment of near normalcy in protein, cholesterol and triglyceride levels in CCl4 intoxicated and BHEE treated rats confirms the hepatoprotective effect of the plant extract. It can be said that BHEE had played a crucial role in synthesizing newer cytoplasm thus enabling regeneration of more and more hepatocytes, which in turn will take over the liver function with improved vigor and vitality. (Venukumar and Latha, 2002). It has been reported that the antioxidant activity or inhibition of the generation of free radicals is important in the protection against CCl4 induced liver lesion.
The marked elevation of bilirubin and urea level in the serum of group 2 CCl4 intoxicated group rats were significantly decreased in the group III BHEE pretreated animals. Bilirubin is the conventional indicator of liver diseases (Girish et al., 2004). Bilirubin an endogenous organic anion binds reversibly to albumin and it is transported to the liver, conjugates with the glucouronic acid and excreted in the bile. Hepatobiliary disease is indicated when total bilirubin exceeds the upper limit of normal (Rosen and Keefe, 1998) The bilirubin lowering ability of the extract in pretreated rats further indicate the hepatoprotective nature of the BHEE on hepatocytes when compared with rats administered CCl4 alone.
Estimating the activities of serum GOT, GPT, ALP, LDH, 5 NT, can make assessment of liver damage. These are the enzymes originally present in higher concentration in cytoplasm. When there is hepatopathy, these enzymes leak into the blood stream in conformity with the extent of liver damage (Nkosi et al., 2005). The elevated level of these entire marker enzymes observed in the group 2 CCl4 treated rats in the present investigation corresponded to the extensive liver damage induced by the toxin. These marker enzymes are cytoplasmic in origin and are released into the circulation after cellular damage (Lin et al., 2000). The tendency of these marker enzymes to return towards a near- normalcy in-group III BHEE pretreated rats was a clear manifestation of anti- hepatotoxic effect of BHEE. The reduced concentrations of GOT and GPT as a result of plant extract administration observed during the present study might probably be due to the presence of catechins in the extract (Naidoo et al., 2006). The results were found comparable to Silymarin. Silymarin that is composite name of three flavonoids isolated from milk thistle Silybum marinum and are used as hepatoprotectives against experimental hepatotoxicity of various chemicals including CCl4 (Gadgoli and Mishra, 1999).
In conclusion the Bi herbal ethanolic extract afforded protection from CCl4 induced liver damage. The protections against liver damage by the BHEE were found comparable to silymarin. Possible mechanism that may be responsible for the protection of CCl4 induced liver damage by BHEE may be it could act as a free radical scavenger intercepting those radicals involved in CCl4 metabolism by microsomal enzymes. By trapping oxygen related free radicals the extract could hinder their interaction with polyunsaturated fatty acids and would abolish the enhancement of lipid peroxidative processes (Upadhyay et al., 2001). It is well documented that flavonoids and glycosides are strong antioxidants (Kavithalakshmi et al., 2006). Antioxidant principles from herbal resources are multifaceted in their effects and provide enormous scope in correcting the imbalance through regular intake of a proper diet. Thus, from the foregoing findings, it was observed that BHEE is a promising hepatoprotective agent and this hepatoprotective activity of BHEE may be due to its antioxidant chemicals present in it. Work is in progress here to identify the antioxidant ability of this Bi-herbal extract.