EFFECT OF AQUEOUS EXTRACT OF BITTER LEAF (Vernonia ...



Effect of aQUEOUS EXTRACT OF BITTER LEAF (Vernonia amygdalina del) ON CARBON TETRA-CHLORIDE (CCl4) INDUCED LIVER DAMAGE IN ALBINO WISTAR RATS.

BY

Arhoghro, E.M., Anosike, E.O. and (Ibeh, G.O.

Department of Biochemistry,

University of Port Harcourt,

P.M.B. 5323,

Port Harcourt

Nigeria

ABSTRACT

The effect of aqueous extract of the leaves of Vernonia amygdalina del on CCl4-induced liver damage was investigated in experimental rats. Separate groups of rats were administered orally with 2.5 ml/kg body weight of 5, 10 and 15% aqueous leaf extracts of V. amygdalina for 21 days after treatment with CCl4. Another group of rats were administered (5, 10 and 15%) extract before treatment with CCl4. The latter showed less hepatotoxcity, than the former, as measured by serum alkaline phosphatase (ALP), alanine aminotransferase (ALT) and aspartate aminotransferase activities. The difference was significant (P < 0.05). Rats administered extract after treatment with CCl4 showed decrease in ALP, ALT and AST activities. The effect of the extract was dose-dependent, Histopathological study also showed significant reduction and even reversal of liver damage in the rats. The results of this study show that aqueous leaf extract of V. amygdalina might have a potent hepatoprotective action against CCl4-induced liver damage in rats.

Key words: Vernonia amygdalina (v.a), aqueous extract, carbon tetrachloride, Hepatoprotective action, histopathological study.

INTRODUCTION

The use of herbal products for medicinal benefits has played an important role in nearly every culture on earth. Herbal medicine was practised by ancient people of Africa, Asia, Europe and the Americas (Wargovich et al, 2001). Over 50% of all modern clinical drugs are of natural product origin and natural products play an important role in drug development programs of the pharmaceutical industry.

The consumption of a variety of local herbs and vegetables by man is believed to contribute significantly to the improvement of human health, in terms of prevention, and or cure of diseases because plants have long served as a useful and rational source of therapeutic agents (Roberts and Tyler, 1999).

Vernonia amygdalina is a widely used local plant in Nigeria for both therapeutic and nutritional purposes.

V. amygdalina grows in a range of ecological zones in Africa and produces large mass forage and is drought tolerant (Bonsi et al, 1995). The bitter taste is due to anti-nutritional factors such as alkaloids, saponins tannins and glycosides (Butter and Bailey, 1973). The macerated leaves of the plants are consumed as vegetables and condiments while the water extract serves as tonic for the prevention of certain illnesses.

V. amygdalina has been reported for its use by wild chimpanzee for the treatment of parasite related diseases in Tanzania (Huffman and Seifu, 1989). Philipson et al, (1993) reported the antiplasmodial effects of sesquiterpene and steroidal constituents of V. amygdalina and some were also effective against plasmodium falciparium in vitro. Uhegbu and Ogbuehi (2004) reported the antidiabetic effects of aqueous extract of leaves of Vernonia amygdalina.

Recently, Izevbigie (2003) isolated some peptides (edotides) from the aqueous extract of V. amygdalina. The peptides were shown to be potent inhibitor of nitrogen activated protein kinases (NAPK) which are crucial for breast tumour growth and also represents a key regulatory point for tumour growth. The anti-estrogen breast cancer drug (Tamoxiten) has also been shown to modulate MAPK activity (Atanaskova et al, 2002; Mandelekar and Kong 2001), this indicates that edotides from V. amygdalina may be considered as alternative to tamoxiten.

Teas containing bitter leaf (vernonia amygdalina) are also used throughout West Africa for the management of diabetes and other metabolic diseases associated with the liver. The liver is an organ of paramount importance, which plays an essential role in the metabolism of foreign compounds entering the body. Human beings are exposed to these compounds through environmental exposure, consumption of contaminated food or during exposure to chemical substances in the occupational environment. Liver disease is a world wide problem. Conventional drugs used in the treatment of liver diseases are sometimes inadequate and can have serious adverse effects. It is therefore necessary to search for alternative drugs for the treatment of liver disease to replace currently used drugs of doubtful efficacy and safety. In this publication, we report the effects of aqueous leaf extracts of vernonia amygdalina on carbon tetrachloride-induced liver damage in rats.

MATERIALS AND METHODS

Animals

Wistar Albino rats (100-150g) of both sexes about three months old bred in the animal house of Biochemistry department, University of Port Harcourt were used in this study. They were randomly selected and kept in 6 groups of not less than three rats per group. Each group was kept in a separate cage. All animals were fed with commercially formulated rat feed and water was given ad libitum. Their cages were cleaned daily and food and water changed daily. The animals were allowed to acclimatize for 2 weeks.

Chemicals

All chemicals used were of analytical grade.

Preparation of Aqueous Extract:

The leaves of Vernonia amygdalina were collected from Sagbama in Bayelsa State and were identified at the Department of Plant Science and Biotechnology, University of Port Harcourt, Nigeria. The leaves were sun dried, pulverized and then sieved. Twenty-five (25) grams of the powdered leaf was weighed out and dissolved in 250ml of distilled water to make 10% extract. From the 10% stock of bitter leaf extract 5%, 10% and 15% (v/v) solutions in distilled water were prepared.

Experimental procedure:

In the experiment, a total of 19 rats (13 CCl4-induced liver damaged rats, 3 normal rats, 3 vegetable oil treated rats) were used. The rats were assigned to one of 6 groups each of not less than 3 rats. The first group was not treated with CCl4 and served as normal control (C). The second group was treated with 0.5ml veg. oil/kg rat. CCl4 was dissolved in vegetable oil and given intraperitonally (ip) to rats in group 3, (disease control) and those in groups 4, 5 and 6 (test) at a dose of 0.5ml CCl4/kg body weight in not more than 0.5ml of vegetable oil. 5, 10 and 15% (v/v) bitter leaf extracts were given 2.5ml/kg body weight orally by intubation to rats in groups 4, 5 and 6 respectively. At the end of treatment at 7, 14 and 21 days respectively, blood sample were collected by direct cardial puncture and the serum was used for the assay of marker enzymes, aspartate aminotransferase (AST), alanine aminotransferase (ALT), and alkaline phosphatase (ALP). The liver was removed and part of the right lobe was sliced, fixed in 10% buffered formaldehyde solution and used for histological examination.

Assessment of Liver Function

Biochemical analysis of the serum enzymes for ALT and AST was by the method of Reitman and Frankel, (1957). ALP was assayed according to the method of REC (1972).

Statistical Analysis

Results of Biochemical estimations are reported as mean + SD and statistical analysis was performed using students t-test and P < 0.05 being considered as statistically significant.

RESULTS

The results of the effect of aqueous leaf extract of Vernonia amygdalina (V.A.) on CCl4-treated rats are shown in Table 1. In the CCl4-treated group, serum ALT, AST and ALP levels were quite high. In contrast, the CCl4 group treated with leaf extracts of V.A. had significantly (P < 0.05) lower levels of ALT, AST and ALT when compared with the CCl4 control group. The reduction of the ALT, AST and ALP activities by V.A. extract though not statistically significant (P < 0.05) was dose dependent. The effect of time of administration of V.A. extracts on ALT, AST and ALP activities was statistically significant (P < 0.05). (Table 2)

Table 1: The effect of aqueous leaf extract of Veronica amygdalina (V.A.) on activities of ALT, AST and ALP on rats treated with CCl4.

|Groups |ALT (U/L) |AST (U/L) |ALP (U/L) |

|1 normal |35.10 + 3.77b |120.17+ 2.91b |247.30+ 3.56b |

|n = 3 | | | |

|2 veg oil |35.30 +5.42b |122.58 + 3.25b |214.24 + 6.43ab |

|n = 3 | | | |

|3 ccl4 |68.95 + 21.38a |165.65 +17.75a |364.65 + 37.75a |

|n = 4 | | | |

|4 CCl4 + |40.24 +4.03b |123.40 + 9.38ab |213.44 + 17.52ab |

|5% V.A n = 3 | | | |

|5 CCl4 + |37.38 +4.63b |115.77 + 18.17ab |201.14 + 20.23ab |

|10% V.A n = 3 | | | |

|6 CCl4 + |39.31 + 3.30b |116.90 + 1.75ab |207.35 + 5.51ab |

|15% V.A n = 3 | | | |

Data are mean + SD

a – significantly different from normal control group

b – Significantly different from CCl4 treated group.

Table 2: The effect of time (duration) of administration of aqueous leaf extract on ALT, AST and ALP activities on rats treated with CCl4

|Group |Period |ALT (U/L) |AST (U/L0 |ALT (U/L) |

|3 CCl4 |7 days |80.60 + 0.59a |177.50 + 0.54a |375.36 + 0.94a |

|N |14 days |57.70 + 0.29b |157.00 + 1.30b |381.92 + 0.26b |

| |21 days |48.80 + 0.40c |145.00 + 0.10c |309.00 + 0.90c |

|4 CCl4 + 5% |7 days |44.30 + 1.20d |133.40 + 0.70d |222.76 + 0.88d |

|V.A. |14 days |36.26 + 0.94e |114.80 + 0.26e |194.92 + 0.44e |

| |21 days |40.17 + 0.18f |122.00 + 2.25f |215.64 + 0.86f |

|5 CCl4 + 10% |7 days |36.00 + 0.53e |122.00 + 1.93f |210.00 + 0.61g |

|V.A. |14 days |42.55 + 0.67g |130.00 + 0.21g |215.43 + 0.40f |

| |21 days |33.60 + 0.50h | 95.30 + 2.95h |178.00 + 1.47g |

|6 CCl4 + 15% |7 days |42.60 + 0.37g |124.90 + 0.38f |201.20 + 1.20h |

|V.A. |14 days |36.00 + 0.52e |116.80 + 0.40g |211.84 + 0.83g |

| |21 days |39.33 + 0.77f |109.00 + 0.46j |209.00 + 1.05g |

Mean with different superscripts are significantly different from each other at (P < 0.05).

DISCUSSION

Carbon tetrachloride (CCl4) is one common hepatotoxin used in the experimental study of liver diseases (Obi et al, 1998, Ulicina et al., 2003, Yan-Jun luo et al., 2004). CCl4 treatment generates free radicals that trigger a cascade of events resulting in hepatic fibrosis. In this study, when treated with CCl4, the liver exhibited drastic alterations, extensive fatty change, distended hepatocytes vacuolated cytoplasm, compressed sinusoids, fatty degeneration, area of necrosis and infiltration by inflammatory cells. In addition, serum levels of ALT, AST and ALP were elevated.

This is in agreement with the report by previous workers (Rienke et al, 1988, Obi et al, 1998, Ulicna et al, 2003, Yan Jun luo et al, 2004).

A primary consideration in the assessment of the efficacy of a potential therapeutic agent for hepatic injury (damage) is its effect on liver histology. Those livers that were treated with CCl4 (Group 3) had a high degree of fatty change, compression of sinusoid and distention of hepatocytes. V.A. extract in the dosage range administered to liver damage rats apparently accelerated the reversion of the liver damage and lowered the high levels of serum ALT, AST and ALP activities when compared to rats treated with CCl4 alone. The effect was time and dose dependent.

Akah and Okafor (1992), reported that the leaves of Vernonia amygdalina contain saponins, sesquiterpenes, lactone, steroid glycosides, alkaloids, tannins and flavonoids. Flavonoids are reported to exhibit antioxidant activity (Ramanthan, et al, 1989) and are effective scavengers of superoxide anions (Robak and Gygkwski, 1988). The aqueous leaf extract of Vernonia amygdalina may have exhibited hepatoprotective activity due to its antioxidant property attributable to flavonoids.

In conclusion, our results indicate that treatment with V.A. extracts after establishment of CCl4 – induced liver damage significantly reduced and even reversed the damage in rats. Hence the leaf extracts of Vernonia amygdalina might be effective hepatoprotectors in the diets of patients with hepatopathies since the model of ccl4 – induced liver damage in rats simulates many of the features of human liver fibrosis (damage).

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( Corresponding author (c/o uwadik@).

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