A comparative study of the beneficial effects of Osbeckia octandra and Osbeckia aspera in liver dysfunction in rats

A study was conducted to compare the protec­ tive effects of aqueous extracts of Osbeckia octandra and Osbeckia aspera against carbon tetrachloride (CC1 4)mediated liver damage in Sprague Dawley rats by assessing their ability to protect livers against the toxin-mediated alterations in the liver histopathology and the serum levels of alanine aminotransferase (ALT), aspartate aminotrans­ ferase (AST) and alkaline phosphatase.


Introduction
Osbeckia octandra and Osbeckia aspera are two very closely related plant species belonging to the family Melastomaceae, that are found in Sri Lanka (1).They are both referred to as "Heenbovitiya" by the local population and are both used in tra ditional medicine for the alleviation of jaundice related to liver disease (2,3), the species used de pending on its local availability.Although many investigations have been carried out to assess the hepatoprotective ability of O.octandra (4-7), no information is available on the relative potencies of O.octandra and O.aspera as hepatoprotective agents.Since both plant species are being used by the local population for the therapy of liver disease, it was decided to conduct an investiga tion to determine if both plant species possess

Experiment animals
In all experiments, male Sprague-Dawley rats of 150 -200 g body weight were used.The animals were maintained on a standard laboratory diet purchased from the Oils and Fats Corporation of Sri Lanka.All animals had access to food and water ad libitum.

Preparation of plant extracts
Fresh leaves of O.octandra and O.aspera were col lected from areas in the Gampaha district and the Galle district of Sri Lanka, respectively.The bo tanical identities of the plants were confirmed by the Curator of the Royal Botanical Gardens, Peradeniya, in comparison with authentic samples in the herbarium.
The leaves of each plant species were cut into small pieces and 200g of each type was homog enized in 1.5 L distilled water and refluxed for 4h.The mixtures were then strained through muslin and the final volume of each extract re duced to 100 ml in vacuo.The extracts were cen trifuged at 3000 rpm to remove any plant debris and the supernatants then freeze dried and stored at -20°C until required.For each experiment, the appropriate weight required was reconstituted in distilled water.

Treatment of animals
The rats were randomly divided into 9 groups of 10 animals each.Group 1 served as the controls and were dosed orally by gavage with distilled water (10 mL,/kg).Groups

Results
The effects of CO. and leaf extracts of O.octandra and 0.aspera on the activities of serum ALT, AST and alkaline phosphatase in rats is summarized in Tables 1 and 2. As expected from results of pre vious investigations (4,9), a significant increase in the activities of serum ALT, AST and alkaline phosphatase occurred within 24 h of exposure of rats to sub-lethal doses (0.2mL/100g) of CC1 4 .
Results in Tables 1 and 2 2).In these post-treatment experi ments also, both plant extracts appear to be equally effective in modulating the toxin-medi ated alterations in serum enzyme activities.Thus, on the 4 ,h day after CC1 4 treatment, the serum ALT, AST and alkaline phosphatase activities were still 162.0%,76.5% and 90.1% higher than the corre sponding values in control animals.In the O.octandra and O.aspera post-treated groups re spectively, activities of ALT were 53.8% and 35.5% higher; AST were 39.2 % and 41.6 % higher while alkaline phosphatase were 29% and 18.6% higher than the corresponding values in control animals.
Histopathological evidence also provided sup portive evidence for the enzyme results (Table 3).

1 .
Associate Professer, Department of Biochemistry and Clinical Biochemistry, Faculty of Medicine, University ofKelaniya, Talagolla Road, Ragama.similar hepatoprotective abilities or whether one species is more effective than the other.Carbon tetrachloride (CC1 4 ) is an established hepatotoxin (8).A single sub-lethal dose of the toxin has been shown to cause marked alterations in liver function as observed by elevations in the serum levels of several enzymes such as alanine aminotransferase (ALT), aspartate aminotrans ferase (AST) and alkaline phosphatase.In the present investigation therefore, due to conve nience of obtaining the toxin, hepatic injury was induced in rats by administration of CC1 4 .The hepatoprotective potentials of O.octandra and O.aspera were assessed by comparison of the ex tent to which aqueous leaf extracts of the two plants could prevent the CCl 4 -mediated alter ations in (a) serum levels of ALT, AST and alka line phosphatase and (b) liver histopathology.

2 and 3 were similarly dosed with 10
mL (equivalent to 20 g wet weight of leaves)/kg/day respectively of either O.octandra or O.aspera leaf extract, for 7 days.Groups 4 and 5 were treated with O.octandra and O.aspera extracts respectively, in a similar man ner to Groups 2 and 3, but after 7 days pre-treatment, these animals were administered a single sub-lethal dose (intraperitoneally) of CC1 4 (CC1 4 : Olive oil (1:1) and 0.2 ml CC1 4 /100g/day), and killed after 24 h.Groups 6 and 7 were adminis tered only the sub-lethal dose of CC1 4 and killed after 24 h and 4 days respectively.Groups 8 and 9 were administered the sub-lethal dose of CC1 4 and after 24 h, dosed for a futher 3 days with 10 mL/ kg/day of the O.octandra and O.aspera extracts respectively.All animals were sacrificed by cervical decapitation at the end of the prescribed regime of treatment.Livers of all animals were excised and fixed in buffered formalin for histo pathological assessment of hepatic damage.The animals were dosed for only 3 days in the posttreatment experiments and for 7 days in the pre-treatinent experiments because in a previous investigation with O. octandra (4) it was found that these post-treatment and pre-treatment time pe riods were sufficient for the plant extract to offer maximum protection against CC1 4 mediated liver damage in rats.Assessment of liver functionLiver function was assessed by a) the estimation of serum levels of alanine aminotransferase (ALT) aspartate aminotransferase (AST) and al kaline phosphatase and b) histopathological examination of liver slices.Blood collection for enzyme assaysBlood from rats subjected to cervical dislocation were collected into clean, dry, centrifuge tubes.The serum was allowed to separate and used in the enzyme assays.The Ceylon Journal of Medical ScienceEnzyme assays Serum ALT and AST levels were determined us ing a combination of the methods of Mohun and Cook (10) and Reitman and Frankel (11) as de scribed by BDH Chemicals Ltd., (Poole, UK) in their assay kits for the respective enzymes.The method of King et al. (12) was used for the esti mation of serum alkaline phosphatase.Statistics Results are expressed as the mean ± S.E.M.The level of significance was determined by the Student's t-test and analyses of significant differ ences between groups was carried out by one-way analysis of variance (ANOVA).
Units; K.A. Units = King Armstrong Units Results statistically different from controls are indicated by asterisks -** p<0.001;NS -Not significantly different; a -results significantly different from group treated with only CCl 4 at p<0.001.
from controls are indicated by asterisks -* p<0.001;** p<0.01;NS -Not significantly different from corresponding CC1 4 control group at p<0.001.Administration of O.octandra or 0.aspera extract 24 h after exposure to CC1 4 also resulted in a faster recovery of the liver as can be judged by compar ing the serum enzyme activities in these animals with those treated with the toxin and left to re cover for 4 days on their own, without the plant extracts (Table the portal tract, fatty depo sition and loss of cell boundaries were observed in livers of rats challenged with CC1 4 alone.The toxin-mediated changes in livers of rats pre-or post-treated with either O.octandra or O.aspera extracts were of much less intensity than those observed in livers of CC1 4 controls untreated with the plant extracts.The extent of protection against the toxin-mediated liver injury in the groups of rats pre-or post-treated with either O.octandra or 0.aspera extract appeared to be very similar.