Effect of Alkaloidal and Aqueous Ethanol Extracts of Roots of Boscia angustifolia (Capparidaceae) on Hepatorenal Functions in Albino Rats

MATERIALS AND METHODS

Chemicals
All chemicals used were of analytical grade.

Collection of Plant and Authentication
The roots of Boscia angustifolia were obtained from Kara market, Sokoto, Nigeria. It is preferred than other parts of the plant by traditional healers in northern Nigeria for treatment of bacterial diseases. The plant was botanically authenticated at the Herbarium, Botany unit, Usmanu Danfodiyo University, Sokoto, Nigeria. Voucher specimen was deposited in the Herbarium of the same institution. The root obtained was open-air-dried under the shade, pulverized in to a moderately coarse powder (using a wooden pestle and mortar) and stored until required for use (Onoruvwe and Olorunfemi, 1998).

Animals
Male albino rats (Wister strains) weighing 223-263 g were purchased from animal house, Department of Biological science, Usmanu Danfodiyo University, Sokoto, Nigeria. The animals were kept at the animal house of the same department in wire mesh cages and were fed with pellet diet, seasonal vegetables and tap water Ad libitum for one week to acclimatize them before starting the experiment.

Preparation of Plant Extract
The dried powdered (40 g) root was extracted with 50% ethanol at room temperature over night and filtered through Whatman No. 1 filter paper. The filtrate was concentrated to dryness in an oven at 40°C and the yield was 9.0% (w/w). The extract was stored in the refrigerator until required for reconstitution in distilled water (for oral administration). This procedure enabled us obliterate the possible contributory effect of the organic solvents.

Extraction of Alkaloids
Fifty gram powdered plant sample (root) was extracted with a liter of methanol: water (1:1 V: V) mixture. It was filtered through Whatman No. 1 filter paper and the filtrate evaporated. The resultant residue was mixed with 200 mL of 0.0025M H₂SO₄ and partitioned with ether to remove unwanted materials. The aqueous fraction was basified with strong NH₃ solution and then extracted with excess chloroform to obtain the alkaloidal fraction or separated by filtration. The chloroform extraction was repeated several times and the bulk extract was concentrated to dryness. The final alkaloid was weighed and the percentage was calculated with reference to the initial weight of the powder (Trease and Evans, 1978; Nuhu et al., 2000).

Administration of Extracts
The crude alkaloidal and aqueous ethanol extracts of Boscia angustifolia were used for the toxicity test. The rats were divided into two sets of 4 groups of 4 each. In first set, Groups 2, 3 and 4 were orally administered with 1 mL of alkaloidal extract (281.43, 703.60 and 1125.70 mg kg^-1 body weight, respectively) and the second set, with aqueous-ethanol residue (335.71, 839.30 and 1342.80 mg kg^-1 body weight, respectively) daily for a period of 28 days. Group 1 in each set served as controls and received only drinking water by the same route. The weight of the animals was taken prior to the administration of the plant extract and after 28 days of the experiment.

Body Weight
The body weights of all the animals before and after 28 days of the extracts administration were recorded.

Blood Samples and Clinical Chemistry
Animals were sacrificed and blood samples were collected, allowed to clot and centrifuged to obtain sera. The biochemical parameters, serum alanine amino transferase (ALT) and aspartate amino transferase (AST) were determined using Randox assay kit by standard methods of Reitman and Frankel (1957). Alkaline phosphatase (ALP) was estimated by the Randox (colorimetric) method of Rec (1972). The 5^I-nucleotidase (5^I-NLT) was determined by the methods of Campbell (1962); Harold et al. (1980). Total bilirubin (TBL), conjugated bilirubin (CBL) and unconjugated bilirubin (UCBL) were analyzed (Randox kit) using the methods of Jendrassik and Grof (1938); Sherlock (1951). Albumin (Bromocresol green) and urea (Diacetylmonoxime) were done by the methods of Cheesbrough (1991) and Wybenga et al. (1971), respectively. Electrolytes, creatinine (colorimetric with deproteinization) were estimated by the methods of Uriyo and Singh (1974) and Henry (1974), respectively. Uric acid was by the method of Collins and Diehl (1959) and Morin and Prox (1973).

Histopathological Assessment
Histopathological examinations were carried out on the liver and kidney of the rats. These were fixed in 10% formalin, dehydrated in gradual ethanol concentrations (50-100%), cleared in xylene and embedded in paraffin. Sections (4-6 μM thick) were prepared and then stained with hematoxylin and eosin (H-E) dye for photomicroscopic observation under light microscope at high (x 400 objective) power magnifications (Wasfi et al., 1994; Guntupalli et al., 2006) .

Statistics
The data collected in the study was subjected to one way analysis of variance (ANOVA), Benferoni compare all columns using Graph Pad Prism. Values were expressed as mean±standard error.

RESULTS

Alkaloidal Composition
The amount of alkaloids detected in 50 g powdered extract was 11.44 g% (w/v).

Body Weight
There was noticeable significant (p<0.05) changes in the body weight of the animals that received higher doses of the alkaloidal and aqueous ethanol extracts of Boscia angustifolia compared with control group (Table 1).

Table 1:	Mean body weight changes in rats administered alkaloidal and aqueous ethanol root extracts of Boscia angustifolia
WBT = Weight of rats before administration of extract, WAT = Weight of rats after administration of extract. ALK = Alkaloidal, AQE = Aqueous ethanol extract. Values are mean±standard deviation, *significantly different (p<0.05) compared with weight of rats after (28 days) administration of extract by using the student t-test (n = 4)

Subacute Toxicity (Hepatorenal function)
Some hepatorenal Indices (Table 2 and 3) are significantly different at concentrations of 703.60 and 1125.70 (alkaloidal extract) and 839.30 and 1342.80 mg kg^-1 body weight (aqueous ethanol extract).

Histopathological Studies
Aqueous ethanol and alkaloidal root extracts produced histopathological lesions of the kidney and liver (Table 4). The lesions are perivascular cuffs and protein cast, infiltration (kidney) and slight infiltration with perivascular cuffs (liver).

Table 2:	Serum liver function indices in rats administered alkaloidal and aqueous ethanol root extracts of Boscia angustifolia
Values are mean±standard error of the mean, *Significantly different from the control (p<0.05) by using the analysis of variance (ANOVA) (n = 4), ALT = Alanine transaminase, AST = aspartate transaminase ALP = alkaline phosphatase, ALB = albumin, 51-NLT= 51-nucleotidase, TB = total bilirubin, CB = conjugated bilirubin, UCB = unconjugated bilirubin, ALK = Alkaloidal and AQE = Aqueous Ethanol Extracts

Table 3:	Serum kidney function indices in rats administered alkaloidal and aqueous ethanol root extracts of Boscia angustifolia
Values are mean±standard error of the mean. *Significantly different from the control (p<0.05) by using the analysis of variance (ANOVA) (n = 4), ALK = Alkaloidal and AQE = Aqueous Ethanol Extracts

Table 4:	Histopathological features of liver and kidney of rats administered alkaloidal and aqueous ethanol root extracts of Boscia angustifolia
ALK = Alkaloidal and AQE = Aqueous Ethanol Extracts

DISCUSSION

Many medicinal plants have been used to treat bacterial diseases without risk assessments. Sub-acute toxicity studies of the alkaloidal and crude aqueous ethanol root extracts of B. angustifolia were elucidated in small laboratory animals. The alkaloidal composition in the study presents an easy method that can be used for analyzing the composition of alkaloids. The detection of some amount of alkaloids in the plant extract could possibly contribute to the antibacterial activity of Boscia angustifolia, since alkaloids and other phenolic compounds have been reported to have antimicrobial activity (Hostettman and Nakanishi, 1979; Okwute and Mann, 1999). However, in the present investigation the doses of the extracts were calculated according to the body weight of the animals, which shows appreciable results in the rats studied in dose dependent manner.

In this study, the alkaloidal and aqueous ethanol root extracts of Boscia angustifolia caused significant changes in hepatorenal indices at only concentrations of 703 to 1343 mg kg^-1 body weight (Table 2 and 3). Thus, this finding did not provide evidence of clinical safety of the plant at higher concentrations as indicated by the results. The significant (p<0.05) increases of ALT and AST resulted from possible necrotic injury of the liver and cholestasis (Speech and Liehr 1983; Panteghini et al., 1984; Lott and Wolf, 1986). Elevations of ALP and 5'-NLT are indicative of cholestasis (Birkett et al., 1986; Van Hoof and De Broe, 1994). The significant (p<0.05) increase of serum TBL and UCBL with decrease of albumin indicate defective liver excretory function (Weiss et al., 1983; Cheesbrough, 1991) and impaired synthetic function of the liver (Harold et al., 1980; Cheesbrough, 1991). The significant (p<0.05) marked increase in serum urea, creatinine and uric acid at higher doses of the extracts are indication of damaged renal function (Harold et al., 1980; Cheesbrough, 1991) and the significant (p<0.05) decrease of sodium and increase of potassium are also signs of renal failure (Cheesbrough, 1991).

Histopathological examinations of the tissue (Fig. 1 to 10) indicated presence of lesions in the kidney and liver of rats treated with higher doses of Boscia angustifolia and have justified the significant alterations of the hepatorenal indices. This may not be a surprise since these organs are sites for biotransformation and excretion, respectively. Thus, the 2 organs may have been exposed to the toxic principle present in the extracts. This finding in the present study is in agreement with the observation of Muyibi et al. (2000) who noted tubular necrosis and cast in animals fed with Cassia occidentalis. On the basis of this and at higher doses of the extracts, it is clear that the plant has no clinical safety. Alkaloids and saponins present in the root extracts may highly be responsible for the altered hepatorenal indices. Some alkaloids have cytotoxic effect on organs; they damage the cells of the liver, lungs, heart and kidney (Harbone, 1972) and saponins cause haemolysis of the red cells (Clark and Myra, 1975). The significant (p<0.05) decreases in weight loss after 28 days of extracts administration compared with control could be due to reduced feed and water intake observed from the animals.

Fig. 1:	Kidney photomicrograph section of normal rat (control). Hematoxylin and Eosin (H and E) X 400

Fig. 2:	Kidney photomicrograph section of rat administered alkaloidal (281.43 mg kg-1) and aqueous ethanol (335.71 mg kg-1) root extracts of Boscia angustifolia: Showing no visible lesion. Hematoxylin and Eosin (H and E) X 400

Fig. 3:	Kidney photomicrograph section of rat administered (839.29 mg kg-1) aqueous ethanol root extract of Boscia angustifolia: Showing perivascular cuffs. Hematoxylin and Eosin (H and E) X 400

Fig. 4:	Kidney photomicrograph section of rat administered (1342.86 mg kg-1) aqueous ethanol root extract of Boscia angustifolia: Showing protein cast. Hematoxylin and Eosin (H and E) X 400

Fig. 5:	Kidney photomicrograph section of rat administered (703.57 mg kg-1) alkaloidal root extract of Boscia angustifolia: Showing protein cast. Hematoxylin and Eosin (H and E) X 400

Fig. 6:	Kidney photomicrograph section of rat administered (1125.71 mg kg-1) alkaloidal root extract of Boscia angustifolia: Showing infiltration. Hematoxylin and Eosin (H and E) X 400

Fig. 7:	Liver Photomicrograph section of normal rat (control). Hematoxylin and Eosin (H and E) X 400

Fig. 8:	Liver photomicrograph of section of rat administered alkaloidal (281.43 mg kg-1 ) and aqueous ethanol (335.71 mg kg-1) root extracts of Boscia angustifolia: Showing no visible lesion. Hematoxylin and Eosin (H and E) X400

Fig. 9:	Liver photomicrograph section of rat administered alkaloidal (703.57 mg kg-1) and aqueous ethanol (839.29 mg kg-1) root extracts of Boscia angustifolia: Showing slight infiltration and perivascular cuffs. Hematoxylin and Eosin (H and E) X 400

Fig. 10:	Liver photomicrograph section of rat administered alkaloidal (1125.71 mg kg-1) and aqueous ethanol (1342.86 mg kg-1) root extracts of Boscia angustifolia: Showing slight infiltration and perivascular cuffs. Hematoxylin and Eosin (H and E) X 400

The presence of alkaloids and saponins may have caused loss of appetite resulting in decreased weight gain as do extracts of Cassia occidentalis containing these compounds (Muyibi et al., 2000).

The present research has shown the effect of roots extracts of Boscia angustifolia on kidney and liver tissues. The plant must therefore, be used cautiously and in small therapeutic doses, since herbs that have higher toxicity can be used safely and effectively if taken in small doses (Klein, 1996).

ACKNOWLEDGMENTS

Authors appreciate the assistance of Mr. Bassey Jonah, histopathology laboratory, Usmanu Danfodiyo University Teaching Hospital, Sokoto, Nigeria.