Hepatoprotective Activity Of Aqueous Extract Of Fruit Pulp Of Cassia Fistula (AFCF) Against Carbon Tetrachloride (CCL4) Induced Liver Damage In Albino Rats.
Correspondence Address :
Department of Pharmacology,
Assam Medical College & Hospital
Dibrugarh, Assam, Pin: 786002,
Objective: To evaluate Hepatoprotective activity of aqueous extract of fruit pulp of Cassia fistula (AFCF) against Carbon tetrachloride (CCL4) induced liver damage in albino rats and compared to standard drug silymarin.
Materials And Methods: Healthy albino rats of either sex weighing 150-200gm were divided into four groups of six animals each.
Group A (Normal control) â€“ 3% gum acacia (2ml/kg/day) orally and olive oil s.c.
Group B (Exp.Control) â€“ 3% gum acacia orally and CCL4 and olive oil (1:1 v/v) s.c.
Group C (Test) â€“ AFCF (200mg/kg/day) orally and CCL4 s.c.
Group D (Standard) - Silymarin (100mg/kg/day) and CCL4 s.c.
Hepatic injury was induced to animals belonging to group B, C and D by giving CCL4 & olive oil mixture s.c on 2nd and 3rd day of experiment. Standard and test drugs were administered for 5 days. Blood samples were collected on 6th day for determination of enzyme markers viz, aspartate transaminase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), total bilirubin (TB) and total protein (TP). Histopathological examinations of liver tissues were also performed.
Results: One way ANOVA followed by Dunnettâ€™s multiple comparison test were used for statistical analysis. Values of p < 0.01 were considered significant. There was significant (p<0.01) increase in all serum marker enzymes and total bilirubin and significant (p<0.01) decrease in total protein in group B. The AFCF and Silymarin resulted in significant (p<0.01) reduction in serum levels of AST, ALT, ALP, TB and increase in TP as compared to group B. Fatty changes, necrosis and fibrosis were observed in group B on histopathology, while in group C and D it was near normal.
Conclusion: As revealed by the study aqueous extract of fruit pulp of Cassia fistula possesses significant hepatoprotective activity.
silymarin, carbon tetrachloride, hepatotoxicity, Cassia fistula.
Native to India, Amazon and Sri Lanka, Cassia fistula Linn. (Leguminosae-Caesalpinoideae) a semi-wild Indian Labernum also known as Golden Shower, has become extensively diffused in various countries including Mauritius, India, South Africa, Mexico, China, West Indies, East Africa and Brazil as an `ornamental tree for its beautiful bunches of yellow flowers (1) . It is found throughout India in all deciduous forests and hilly tracts(2). In Sanskrit it is known as Savarnangah(2). It is commonly known as Sonaru in Assamese (3). In the Indian literature, this plant has been reported useful against skin diseases, liver troubles, tuberculous glands and in treatment of haematemesis, pruritus, leucoderma and diabetes(4).Its antifungal, antibacterial, laxative and antitussive properties have been established(5). Butanol extract of residue of Cassia fistula from 70% alcohol fraction has been shown to have antiviral effect, while hot water extracts have proved useful in treatment of uterine, menstrual disorders and fever (6). Other uses of this plant include antidiarrhoel and anti dysentery (7) effects.Its antitumor(8), antifertility (9), and antioxidant (10) properties have been reported. Hepatoprotective activity has been evaluated recently (11) and very few studies have been done on this property. In view of this, the present study was aimed at evaluating the hepatoprotective activity of aqueous extract of fruit pulp of Cassia fistula (AFCF) against carbon tetrachloride (CCL4) induced hepatotoxicity in albino rats.
The compounds isolated from the pods are :-- rhein; 1, 8-dihydroxy-3-anthraquinone carboxylic acid(12); free rhein complexed with sennidin like compounds(13); fistulic acid; anthraquinone acid(14); 3-formyl-1-hydroxy-8-methoxy anthraquinones(15); flavan-3-ols and proanthracyanidins like catechin, epicatechin, procatechin, procyanidin B-2 and epiafzelechin(16); diterpene, 3β-hydroy-17-norpimar-8(9)-en-15-one(17); sugars; volatile oils(5), 5-nonatetracontanone, 2-hentriacontanone, tricontane,16-hentriacontanol and β-sitosterol(2).
Healthy Wister albino rats of either sex weighing 150-200 g were used for experiments, which were taken from the Central Animal House, Assam Medical College & Hospital, Dibrugarh, Assam.. The animals were acclimatized to laboratory conditions for 5 days prior to experiments and standard animal diet was maintained with bengal gram, wheat, maize and carrot in sufficient quantities daily. Before commencing the study, permission from the Institutional Animal Ethical Committee (Regd. No. 634/02/a/CPCSEA) was obtained.
Ripe fruits of Cassia fistula were collected in month of May-June,2007 from Assam Medical college campus, Dibrugarh and authenticated by Department of Botany, Dibrugarh University. Fruits were peeled off and seeds were separated from fruits. About 250 gm of pulp material was boiled in distilled water for 30 min, kept for 3 days with intermittent shaking, filtered and concentrated using rotary flash evaporator to obtain the aqueous extract. Extract was dried in a desiccator and yield was 30% w/w(18).
Acute oral toxicity test :Healthy Wister albino rats of either sex weighing 150-200 g maintained under standard laboratory conditions were used for acute toxicity test according to OECD guidelines 425 (OECD guideline, 2000).A total of five animals were used which received a single oral-dose (2000mg/kg, body weight) of AFCF. Animals were kept overnight fasting prior to drug administration of AFCF. After administration of AFCF, food was withheld for further 3-4 h. Animals were observed individually at least once during first 30 min after dosing, periodically during first 24 h (with special attention during the first 4 h) and daily thereafter for a period of 14 days. Observations were done daily for changes in skin and fur, eyes and mucus membrane (nasal), respiratory rate, circulatory signs(heart rate and blood pressure), autonomic effects(salivation, lacrimation, perspiration, piloerection, urinary incontinence and defecation) and central nervous system (ptosis, drowsiness, gait, tremors and convulsion) changes(19). None of the mentioned toxic signs and symptoms or mortality were observed in the animals at above mentioned dose. So one tenth of this dose i.e., 200 mg/kg, p.o. of AFCF was selected for evaluation of antihepatotoxic activity.
The study of hepatoprotective activity was carried out as described by Jalalpure SS et al(20). Healthy Wister albino rats of either sex weighing 150-200 g were divided into four groups with six animals in each group (n=6 in each group). Group A (normal control) and group B (CCL4- treated control) were given 5% gum acacia (2ml/kg, b.w) for 5 days. Group C and D were pretreated with AFCF (200 mg/kg, p.o.) and silymarin (100 mg/kg, p.o.) respectively for 5 days. Liver damage was induced in all groups (except group A) with 1:1 (v/v) mixture of CCL4 and olive oil (1 ml/kg, s.c) injected on days 2 and 3 while olive oil (0.5ml/kg,s.c) was injected to group A. The animals were killed under light ether anaesthesia after 48 h of CCL4 treatment, that is, on sixth day. Blood was withdrawn from the carotid artery, allowed to coagulate at 37 degree C for 30 min, serum separated by centrifugation at 2500 rpm for 10 min and biochemical analysis were carried out to asses liver function viz., serum transaminases [aspartate transaminase (AST) , alanine aminotransferase (ALT) (21), alkaline phosphatase (ALP) (22), total bilirubin(23) and total protein (TP)(24).
A portion of liver tissue of all animal groups were excised and then washed with normal saline. They were fixed in 10% buffered neutral formalin for 48 h and then with bovine solution for 6 h and then processed for paraffin embedding. By using a microtome, sections of 5 mm thickness were taken, processed in alcohol-xylene series and were stained with alum-haematoxylin and eosin (25) and subjected to histopathological examination.
The CCL4 treated group showed significant (p<0.01) increase in serum hepatic enzyme levels viz., AST, ALT, ALP and TB; and a significant (p<0.01) decrease in TP levels compared to normal control group indicating liver injury. Whereas in animals pretreated with AFCF and silymarin there was a significant (<0.01) decrease in serum hepatic enzymes and total bilirubin and an increase in total protein as compared to experimental control showing that AFCF has hepatoprotective activity (Table/Fig 1).
Histopathological examination of normal control group showed normal hepatocytes (Table/Fig 2). CCL4 treated rat liver revealed fatty degeneration, necrosis, and fibrosis (Table/Fig 3). Administration of AFCF preserved the histological structure of liver to near normal though there was congestion and regeneration of liver tissue (Table/Fig 4). Sections of liver taken from Silymarin treated group showed hepatic architecture similar to that of normal control group (Table/Fig 5)
CCL4 is one of the most commonly used hepatotoxin in experimental study of liver diseases(26) . CCL4 is biotransformed by cytochrome p-450 in liver to produce highly reactive trichloromethyl free radical. This, in presence of oxygen generated by metabolic leakage from mitochondria causes lipid peroxidation of membrane lipid. This leads to loss of integrity of cell membranes and damage of hepatic tissue(27) which is evidenced by increased levels of serum marker enzymes, namely AST, ALT and ALP and TB. AFCF significantly reduced these liver enzyme levels. Further, AFCF increased the levels of total protein which indicates hepatoprotective activity comparable with standard drug silymarin. Stimulation of protein synthesis accelerates regeneration process and production of liver cells(28).
Histopathological studies showed that CCL4 caused fatty degeneration, necrosis and fibrosis of liver tissue. Pre-treatment with AFCF showed protection of liver tissue, which confirmed the results of biochemical studies.
The bioactive actions ascribed to polyphenols are almost certainly mediated partly by their free radical scavenging and antioxidant actions(29), their ability to decrease localised oxygen concentration and to decompose peroxides(30) .Total phenolics and particularly flavin 3-ol derivatives are known to be potential antioxidant prophylactic agents (31) and it has already been mentioned earlier that antioxidant activity is important in protection against CCL4 induced liver lesion (32). So, hepatoprotective activity of AFCF may be probably due to presence of these antioxidants though it has to be confirmed yet.
The present study revealed that Cassia fistula possesses significant hepatoprotective effect. However further studies on other models and clinical trials are required to confirm these results and to establish the exact mechanism of action and active principles involved in hepatoprotective effect.
Cassia fistula has been used for a variety of diseases in traditional medicine. Very few studies have been done, so this study was done to evaluate its hepatoprotective activity.
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