The liver is vulnerable to a wide variety of metabolic, toxic, microbial, circulatory and neoplastic insults. The level of impairment and the state of injury to the hepatocyte can be ascertained by various markers. The most commonly used markers of hepatocyte injury are L-aspartate aminotransferase and L-alannine aminotransferase. Although, if hepatic disease is primarily of an obstructive nature (cholestatic), alkaline phosphatase will be significant enzyme marker. It has been shown that hepatic injury or disease results in the leakage of these enzyme markers into the circulation thereby altering their concentration (Ulièná et al., 2003). However, the pathogenesis of induced hepatic injury is not quite clear, but there is no doubt that Reactive Oxygen Species (ROS) and free radicals play important roles in biochemical changes taking place in the liver. Biochemical membranes are particularly prone to the effect of ROS. The peroxidation of unsaturated fatty acids in biological membranes lead to a decrease of membrane fluidity and a disruption of membrane integrity and function which may cause serious biochemical changes.
Carbon tetrachloride (CCl4) is a hepatotoxin. Trichloromethyl radicals are generated from it in vivo. The radicals stimulate a sequence of reactions that culminate in the initiation of the peroxidation of membrane lipids (Reinke et al., 1988) and hence liver damage. Trichloromethyl radical is believed to be the immediate product of the reductive dechlorination of CCl4, catalysed by certain cytochrome P450 isoenzymes (Sipes et al., 1977) particularly the ethanol inducible isoform of the cytochrome (Reinke et al., 1988). Several endogenous protective mechanisms have been evolved to limit ROS and the damage caused by them (Ulièná et al., 2003), but this may not provide complete protection from oxidative stress. Hence, there is increased need of natural and artificial agents possessing antioxidant properties.
Nauclea pobeguini, a family of Rubiaceae, consist of indole alkaloids having such significant biological activities as antiproliferative. This plant can also be thought to possess other components, yet to be identified, which has antioxidant activity. The aqueous extract of the plant is used by the local community in Abraka, Nigeria as a remedy for jaundice. It is usually prepared by boiling the leaves in water. CCl4-induced lipid peroxidation process provides a model with which N. pobeguinii can be assessed for antioxidant property. The ability of extracts of N. pobeguinii to exhibit antioxidant action was therefore investigated by administering it to rats prior to CCl4 treatment. The effect was compared with that in extract-free, CCl4 -treated rats.
MATERIALS AND METHODS
Forty male albino rats, Wistar strain (150-170 g) bred in the animal house
of the College of Medicine, University of Lagos, Nigeria were used for the study
after permission from appropriate authorities. They were divided into four experimental
groups with ten animals per group. The animals were left to acclimatize to laboratory
conditions for two weeks, before the commencement of the study.
L-alanine/L-aspartate aminotransferase kits were products of Quimica Clinica Applicada (QCA), Spain. Para-nitrophenol phosphate and para-nitrophenol were purchased from BDH chemicals (Poole, England). Tartaric and citric acids were obtained from Merck chemicals (Darmstadt). Growers mash was obtained from Livestock Feeds Plc., Nigeria. The fresh leaves of N. pobeguinii were obtained from a farm in Abraka, Nigeria and authenticated by the department of Botany, Delta State University, Abraka, Nigeria.
Preparation of Aqueous Extract of Nauclea pobeguinii
The aqueous extract was prepared by boiling 100 g of the leaves in 1000
mL of water for 15 min with subsequent standing to allow for cooling down to
room temperature. After separation of undissolved residue, the solution was
used for the study.
Treatment of Animals
Rats in groups 2 and 4 were given 5 mL of extract kg-1 body weight
orally by incubations. At the same time animals in groups 1 and 3 received an
equal volume of de-ionized water kg-1 body weight by the same route.
This treatment was carried out three times daily for ten days during which the
rats were allowed free access to food and water. Following the last treatment,
rats in group 3 and 4 received a mixture of CCl4 in olive oil (1:1)
subcutaneously at a dose of 6 mL kg-1. The CCl4-free control
rats (group 1 and 2) were given 6 mL corn oil kg-1 body weight subcutaneously.
Treatment of the animals was in accordance with the principles of laboratory
animal care (NIH, 1985).
Preparation of Serum
Eighteen hours after CCl4 treatment, each rat was anaesthetized in a chloroform
saturated chamber. The thoracic and abdominal regions were opened to expose
the heart. Blood was obtained through heart puncture by means of a 5 mL hypodermic
syringe and needle and placed in ice-cold 10 mL centrifuge tube. It was allowed
to clot and then centrifuged at 3000 g for 5 min. The serum sample was collected
and left standing on ice until required.
Serum aspartate and alanine aminotransferases activities were assessed using
Quimmica Applicada kits based on the methods of Reitman and Frankel (1957).
The activities of L-ALT and L-AST are expressed as units mL-1. Alkaline
phosphatase activity was determined by the method of Annino and Giese (1976).
The enzyme activity is expressed in units L-1 in which one unit represent
one micromole p-nitrophenol produced per minute.
The results are expressed as mean±SEM and the mean values of the
groups were compared using ANOVA and least square difference. The significance
level was set at p<0.05.
It was observed that the size of the liver was increased in CCl4-intoxicated rats, but it was not significantly different from the control in rats both treated with extract alone and those pretreated with extract before CCl4 administration. The body weight gain of rats did not differ significantly between the groups. Thus the study indicates that aqueous extracts of N. pobeguinii restored to normal the CCl4-induced increase in liver/body weight ratio of rats (Table 1).
Table 2 present the effect of N. pobeguinii extract
on liver function indices of CCl4 treated rats. The activities of
serum aminotransferases (L-ALT and L-AST) of rats administered CCl4
(group 3) and extract alone (group 2) were significantly increased relative
to control. Prior administration of the extract to CCl4 treated rats
(group 4) decreased the activities of the serum aminotransferases to levels
that were similar to the control. Similarly the serum alkaline phosphatase activity
was significantly increased in CCl4 treated extract-free rats as
compared to control and extract treated rats. However pretreatment of CCl4
administered rats with the extract significantly decreased the activity of alkaline
phosphatase relative to CCl4 treated extract-free rats.
|| Effect of N. pobeguinii extract on liver/body
weight ratio and body weight gain of CCl4 treated rats
|Values are mean±SEM, Values on the same row with different
superscripts differ significantly (p<0.05) from each other
||Effect of N. pobeguinii extract on serum L-aspartate
aminotransferase (L-AST), L-alanine aminotransferases (L-ALT) and alkaline
phosphatase (ALP) activities of CCl4 treated rats
|Values are mean±SEM, Values on the same row with different
superscripts differ significantly (p<0.05) from each other
Therefore the study indicates that the extract protects rats from CCl4-
induced liver damage.
Direct change in organ weight or organ/body weight ratio has been used as an index of CCl4 toxicity (Uemitsu et al., 1986; Uemitsu and Nakayoshi, 1984). The latter method was used in this study since it has been shown to be a more sensitive indicator of CCl4 toxicity than absolute liver weight (Uemitsu et al., 1986). Therefore the observed increase in liver/body weight ratio of rats administered CCl4 (Table 1) is consistent with these reports and that of others (Vilstrup, 1983; Okazaki et al., 1985). Increase in organ weight after exposure to a toxicant may be due to a tumor, fluid or triglyceride accumulation. Triglyceride accumulation in liver (fatty liver) is a common response to CCl4 toxicity (Timbrell, 1991; Junnila et al., 2000) and this may account for the observed increase in the liver/body weight ratio of the CCl4-treated rats. In this study, the hepatoprotective activity of N. pobeguinii extract in CCl4-induced toxicity was evaluated using changes in liver/bodyweight ratio and body weight gain of rats since they are well known indices of CCl4 toxicity. The reversal of the CCl4-induced increase in liver/body weight ratio of rats by the extract is an indication of its protective effect.
Serum aminotransferases (ALT and AST) and serum alkaline phosphatase activities were also used to measure both CCl4-induced hepatotoxicity and protection of N. pobeguinii extract against the same effect of CCl4 in rats. In agreement with previous reports (Reinke et al., 1988), our results show that CCl4 caused an elevation in the serum levels of ALT, AST and ALP which is indicative of damage to the liver. Treatment of rats with aqueous extract of N. pobeguinii caused less hepatotoxicity than with CCl4 alone (Table 2) as evidenced by the decreased serum content of the above enzymes relative to the CCl4-treated extract-free group. Various mechanisms have been proposed for CCl4-induced liver damage (Brattin et al., 1985). One view is that a trichloromethyl radical (CCl) is produced from CCl4 by reductive dechlorination. The trichloromethyl radical in turn abstracts a hydrogen atom from a fatty acid to form chloroform and a lipid radical. The lipid radical may then react with molecular oxygen to initiate lipid peroxidation which is thought to ultimately cause the cytotoxic response (Sipes et al., 1977; Recknagel, 1983; Brattin et al., 1985; Reinke et al., 1988). In the present study, we did not attempt to address any mechanistic concept but merely adopted CCl4-induced liver injury as a model for assessing N. pobeguinii for antioxidant activity. Since the mechanism of action of CCl4 involves oxidation, we are of the view that if N. pobeguinii possesses antioxidant action it would prevent lipid peroxidation and therefore membrane damage. Present results clearly demonstrate that N. pobeguinii is an excellent preventive agent for CCl4-induced liver damage in rats and this finding appears to suggest that the aqueous extract of N. pobeguinii possesses antioxidant activity.
Many natural and artificial agents possessing anti-oxidative properties have been proposed to prevent and treat hepatopathies induced by oxidative stress (Lieber, 1997; Èervincová and Drahota, 1998). There is increasing evidence for the hepatoprotective role of hydroxyl- and polyhydroxy-organic compounds particularly from vegetables, fruits and some herbs (Bass, 1999). Although the biochemical mechanism of the antioxidant effect of N. pobeguinii extract against CCl4-induced hepatotoxicity was not examined in the present study, it is possible that the bioactive principles in the extract may have acted directly or indirectly in protecting the liver against damage. Directly it may be breaking the sequence of events between the reductive dechlorination of CCl4 and the subsequent abstraction of hydrogen from unsaturated fatty acids in the membrane and peroxide formation. Indirectly, it may inhibit the activities of cytochrome P450 isoenzymes (Sipes et al., 1977; Reinke et al., 1988) required for trichloromethyl radical formation or it may be an effective scavenger of the reactive metabolite, the trichloromethyl radical. Besides the increase in serum L-ALT and L-AST of rats exposed to the extract alone (Table 2) may arise following increased synthesis of these enzymes in the liver. Activation of liver aminotransferases may promote regeneration of damaged hepatocytes and this could contribute to the restoration of the CCl4-induced liver damage by prior treatment of rats with the extract.
In conclusion the present study indicates that aqueous extract of N. pobeguinii has anti-oxidant activity since it improves recovery or reduces the toxic effects of CCl4 in liver cells of male rats.