DOI:http://dx.doi.org/10.4314/njnpm.v20i1.2
BIOCHEMICAL AND HISTOLOGICAL CHANGES IN FEMALE WISTAR RATS FOLLOWING ORAL ADMINISTRATION OF AQUEOUS EXTRACT OF MANGIFERA INDICA L. LEAVES Yakubu, M.T.* and Salimon, S.S. Phytopharmacology, Toxicology and Reproductive Biochemistry Research Laboratory, Department of Biochemistry, University of Ilorin, Ilorin, Nigeria *Corresponding author’s e-mail:
[email protected]; Tel.:+234 803 754 4437 Keywords: Mangifera indica, Anacardiaceae, Structural toxicity, Functional toxicity, Histoarchitectural changes ABSTRACT This study was aimed at investigating the effect of aqueous extract of Mangifera indica leaves on some biochemical parameters and histology of liver, kidney and small intestine of rats. Twenty female rats (142.30 ± 7.56 g) were randomly assigned into four groups A, B, C and D. The rats were administered orally 1 mL of distilled water, 25, 50 and 100 mg/kg body weight of the extract once daily for 10 days. The biochemical parameters were determined using standard methods. All the doses of the extract significantly increased the activities of liver aspartate aminotransferase (AST), serum alanine aminotransferase (ALT) and chloride ion (Cl-) content whereas the liver ALT, serum urea, uric acid, potassium ion (K+) and small intestine body weight ratio decreased significantly in a dose dependent manner. The serum concentrations of sodium ion (Na +) and creatinine increased only at 50 and 100 mg/kg body weight of the extract while the levels of total protein, albumin, globulin, conjugated and total bilirubin significantly decreased in the serum. The liver and kidney body weight ratio significantly increased at 100 mg/kg body weight of the extract while the extract at all the doses did not significantly alter the activity of serum AST. Furthermore, the extract significantly decreased the activity of alkaline phosphatase (ALP) in the small intestine of the animals with corresponding increase in the serum. The extract at varying doses produced mild and moderate hepatocellular and submucosa layer degeneration in the liver and small intestine respectively while there was glomeruli shrinkage in the kidney. The extract caused structural and functional toxicity hence, should be consumed with caution.
INTRODUCTION Medicinal plants have recognized medical uses ranging from herbal medicine preparations to mainstream pharmaceutical products. Plants have been used in the treatment and management of human diseases from time immemorial to man [1]. They contain substances such as alkaloids, flavonoids, phenolics, saponins among others which are can be used for therapeutic purposes or serve as precursors for chemo-pharmaceutical semi-synthesis [2]. The stampeding use of medicinal plants for treatment of various ailments necessitates the evaluation of these plants for their safety. Mangifera indica Linn. (family: Anacardiaceae) commonly known as mango (English), Mangoro (Yoruba - Western Nigeria), Mangwaro (Hausa - Northern Nigeria), Mangolo (Igbo - Eastern Nigeria), Mano (Ghana), Manga (Portuguese) is native of tropical Asia [3], and now widely grown in different parts of Africa, especially the Southern part of Nigeria, where it is valued for its edible fruit[4]. M. indica is a large evergreen tree that can reach 15 to 30 m tall, anchored by a long unbranched taproot that can descend to a depth of 6-8 m. It bears green leaves (red or yellow at first) which are about 29-30 cm long with tiny red or yellowish– green flowers. The fruits vary in size, shape (oval, egg or kidney shaped) and colour (green, yellow or red). M. indica has been claimed to be used to treat ailments such as asthma, cough, diarrhoea, dysentery, leucorrhoea, jaundice, pains, malaria and diabetes [5-9]. Studies on biological activities of M. indica include antidiarrhoeal activity of aqueous extract of the leaves in female albino rats [10], phytochemical and antibacterial activity of the root extract [11], antihyperlipidemic activity of the leaf extract in rats fed with high cholesterol diet [12] and antiulcerogenic activity
4
of the leaf polyphenols [13]. Evaluation of the morphological and biochemical effect of crude aqueous extract of M. indica stem bark at 100 mg/kg body weight in Wistar rats indicated that the extract was hepatotoxic [14]. Despite the aforementioned studies on the uses of M. indica leaves, there is dearth of information on the effects of the aqueous extract of M. indica leaves on biochemical parameters and histoarchitecture of selected organs (liver, kidney and small intestine) of female rats. Only female animals were used in the present study because of the perceived advantages such as eliminating variations in results, difficulty in handling and caging of male animals due to aggressions [15], great sensitivity of females to some toxins [16] and ultimately to prevent pregnancy (in case of both sexes) that could cause unwanted hormonal changes which can adversely affect experimental outcome [17]. Therefore, this study was carried out to evaluate the toxicological implications of the aqueous extract of M. indica leaves at the doses of 25, 50 and 100 mg/kg body weight on some biochemical parameters and histology of the selected organs of female rats.
RESULTS AND DISCUSSION All the doses of the extract significantly decreased (p0.05) from the control (Table 1).
Table 1: Liver function indices of female rats treated with aqueous extract of M. indica leaves
Parameters
Extract (mg/kg body weight) Control
25
50
100
Total Protein (g/L)
6.07 ± 0.12c
5.63 ± 0.12b
5.76 ± 0.01b
4.40 ± 0.10a
Conj. Bilirubin (µmol/ml)
2.52 ± 0.12a
1.81 ± 0.02b
1.23 ± 0.41c
1.53 ± 0.23c
Total Bilirubin (µmol/ml)
4.97 ± 0.14a
4.30 ± 0.20b
3.89 ± 0.15c
3.22 ± 0.17d
Albumin (g/L)
4.91 ± 0.03a
3.03 ± 0.19b
2.62 ± 0.28b
2.47 ± 0.10c
Globulin (g/L)
1.16 ± 0.00a
1.00 ± 0.10b
0.84 ± 0.05c
0.63 ± 0.00d
Liver ALT (U/L)
1226.20 ± 77.40d 1025.10 ± 30.20c 835.50 ± 46.50b
667.10 ± 32.50a
Serum ALT (U/L)
39.60 ± 3.56a
45.95 ± 3.04b
58.50 ± 1.06c
83.68 ± 1.54d
Liver AST (U/L)
4946.00 ± 153a
5436.00 ± 350b
6079.00 ± 420b
5642.00 ± 300b
Serum AST (U/L)
80.70 ± 3.45a
87.50 ± 5.75a
88.75 ± 5.75a
82.00 ± 2.94a
Values are Mean ± SEM, n = 5. Values with different superscripts different from the control across the rows for each parameter are significantly (p < 0.05) different. All the doses of the extract significantly reduced the concentrations of urea, uric acid and K+ in the serum of the animals (Table 2). In contrast, the serum Cl- content increased significantly
at all the doses of the extract whereas the increases in creatinine and Na+ levels were only produced by the 50 and 100 mg/kg body weight of the extract (Table 2).
Table 2: Kidney function indices of female rats treated with aqueous extract of M. indica leaves
Parameters
Extract (mg/kg body weight) Control
25
50
100
Urea (mmol/L)
5.12 ± 0.04a
4.47 ± 0.05b
4.34 ± 0.10b
3.32 ± 0.07c
Uric acid (mg/dl)
0.72 ± 0.04a
0.65 ± 0.05b
0.51 ± 0.08c
0.40 ± 0.03d
Creatinine (µmol/ml)
45.99 ± 1.01a
46.00 ± 2.05a
48.90 ± 0.11b
55.54 ± 1.30c
Na+ (mmol/L)
134.00 ± 2.50a
136.00 ± 3.00a
142.00 ± 1.00b
156.00 ± 0.00c
K+ (mmol/L)
4.85 ± 0.15a
4.07 ± 0.13b
3.80 ± 0.05c
3.35 ± 0.10d
Cl- (mmol/L)
92.50 ± 2.70a
97.30 ± 0.50b
96.00 ± 2.00b
108.00 ± 2.00c
Values are Mean ± SEM, n = 5. Values with different superscripts different from the control across the rows for each parameter are significantly (p < 0.05) different. The dose dependent reduction in the activity of alkaline phosphatase in the small intestine of female rats was accompanied
Nig. J. Nat. Prod. and Med. Vol. 20 2016
by corresponding increase in the activity of the serum enzyme (Table 3).
5
Table 3: Alkaline phosphatase activity in small intestine and serum of female rats administered aqueous extract of M. Indica leaves
Group / Doses (mg/kg body weight)
Small intestine
Serum
Control
19.60 ± 0.13d
0.22 ± 0.01a
25
18.79 ± 0.31c
0.28 ± 0.01b
50
17.17 ± 0.15b
0.37 ± 0.03c
100
15.86 ± 0.54a
0.43 ± 0.02d
Values are Mean ± SEM, n = 5. Values with different superscripts different from the control down the column are significantly (p < 0.05) different. The liver- and kidney - body weight ratios increased only at the highest dose of 100 mg/kg body weight whereas all the doses of the extract significantly reduced small intestine – body weight ratio of the female rats (Table 4). Table 4: Organ-body weight ratio of female rats administered aqueous extract of M. indica leaves
Organ-body Weight Ratio (%) Groups/Doses Liver (mg/kg body weight)
Kidney
Small Intestine
Control
4.37 ± 0.11a 0.67 ± 0.02a 3.68 ± 0.09d
25
4.04 ± 0.21a 0.62 ± 0.03a 3.47 ± 0.02c
50
4.22 ± 0.01a 0.64 ± 0.01a 3.22 ± 0.01b
100
5.15 ± 0.07b 0.76 ± 0.05b 3.13 ± 0.01a
Values are Mean ± SEM, n = 5. Values with different superscripts different from the control down the column are significantly (p < 0.05) different.
The histoarchitecture of the liver were essentially within the normal histology following the administration of 25 mg/kg body weight of M. indica leaves just like the control (Plates 1a and 1b). In contrast, there was a mild and moderate hepatocellular degeneration in the 50 and 100 mg/kg body weight treated rats respectively (Plates 1c and 1d). There was no histoarchitectural changes in the kidney of rats administered 25 and 50 mg/kg body weight of the extract just like the control whereas there was shrinkage of glomeruli in the animals treated with the highest
6
dose (100 mg/kg body weight) of the extract (Plates 2a, 2b, 2c and 2d). Compared with the control (Plate 3a), the mucosal villi of the small intestine were intact in the animals that received 25 mg/kg body weight of the extract (Plate 3b), while there was mild degeneration of the submucosa layer (Plate 3c) and moderate degeneration of the submucosa layer with polymorph infiltration (Plate 3d) following the administration of 50 and 100 mg/kg body weight of the extract respectively.
Nig. J. Nat. Prod. and Med. Vol. 20 2016
Plate 1a: Cross section of the liver of control rat treated with distilled water (Mag. × 400; H & E)
Plate 1b: Cross section of the liver of rat treated with 25 mg/kg body weight of the extract (Mag. × 400; H & E)
Plate 1c: Cross section of the liver of rat treated with 50 mg/kg body weight of the extract (Mag. × 400; H & E)
Plate 1d: Cross section of the liver of rat treated with 100 mg/kg body weight of the extract (Mag. × 400; H & E)
Plate 2a: Cross section of the kidney of control rat treated with distilled water (Mag. × 400; H & E)
Plate 2b: Cross section of the kidney of rat treated with 25 mg/kg body weight of the extract (Mag. × 400; H & E)
Nig. J. Nat. Prod. and Med. Vol. 20 2016
7
Plate 2c: Cross section of the kidney of rat treated with 50 mg/kg body weight of the extract (Mag. × 400; H & E)
Plate 2d: Cross section of the kidney of rat treated with 100 mg/kg body weight of the extract (Mag. × 400; H & E)
Plate 3a: Cross section of the small intestine of control rat treated with distilled water (Mag. × 400; H & E)
Plate 3b: Cross section of the intestine of rat treated with 25 mg/kg body weight of the extract (Mag. × 400; H & E)
Plate 3c: Cross section of the intestine of rat treated with 50 mg/kg body weight of the extract (Mag. × 400; H & E)
Plate 3d: Cross section of the intestine of rat treated with 100 mg/ kg body weight of the extract (Mag. × 400; H & E)
8
Nig. J. Nat. Prod. and Med. Vol. 20 2016
Administration of herbal preparations without any standard dosage coupled with non-availability of adequate scientific studies on their safety has raised concerns on their toxicity [18]. The liver is prone to injuries induced by foreign substances including plant extract because of its role as the first line of defense in the body. The biochemical parameters investigated in this study are peculiar to the functioning of the liver and are related to their synthetic, secretory and excretory functions. The levels of albumin, globulin and protein in the liver could be used to ascertain the secretory and synthetic functions while conjugated and total bilirubin are used to monitor the excretory function of the organ [19]. Therefore, the reductions in the level of total protein, albumin, and globulin suggest impairment in secretory function of the liver arising probably from hepatocellular injury [20], abnormal secretion or increased catabolism [21]. The findings in this study were consistent with previous study by Yakubu et al. [21] on the toxicological evaluation of Senna alata on pregnant Wistar rats. Severe hemolysis causes the release of more bilirubin into the blood which manifests as elevated total bilirubin that the liver excretes [22]. The decrease in the level of both total and conjugated bilirubin could be as a result of the plant extract being able to influence the excretory function of the liver either by inhibiting its production or by increasing its uptake and elimination to bile duct by the liver cells. Enzyme pattern in the tissues are indicative of liver dysfunction while the level of such enzymes in the serum could corroborate the cellular integrity of the organs [23]. AST and ALT are two of the most important markers of hepatocellular injury or necrosis; their levels are elevated in various hepatic disorders or dysfunction. The dose dependent reduction in the activity of ALT in the liver accompanied by corresponding increase in the serum indicates hepatocellular leakage from the liver to the serum, a pattern that purports the hepatotoxic effect of the plant extract [24]. Elevation in activity of AST in the liver which did not manifest in the serum of the female rats suggests that the plant extract might have enhanced de novo synthesis of the enzyme [25]. Renal function indices are usually required to determine the normal glomerular and tubular functioning of the nephrons [26]. The decrease in the concentration of urea and uric acid at all the doses investigated might be attributed to impairment or obstruction of protein and nucleic acid catabolism respectively. This is an indication of the nephrotoxic activity of the extract as it adversely affected the tubular and glomerular function of the female rats. The findings of this study are similar to that earlier reported by Yakubu et al. on Fadogia agrestis stem [27].
Changes in serum concentrations of creatinine and electrolytes such as Na+, K+ and Cl are indicators of renal function at the tubular and glomerular levels [21]. Elevation in the concentration of serum creatinine, chloride and sodium ion as well as the reduction in concentration of serum potassium ion indicates that tubular dysfunction had developed following the administration of the extract. This finding contrasts previous report by Yakubu et al. [27] on effect of chronic administration of aqueous extract of
Nig. J. Nat. Prod. and Med. Vol. 20 2016
Fadogia agrestis stem in male rats kidney function indices which could be due to variation in sex or part of the plant used for the study. Intestinal alkaline phosphatase regulates the absorption of fats, hydrolysis of organic phosphate and maintains mucosal integrity of the gut [28]. Reduction in the activity of alkaline phosphatase in the small intestine of rats at all the doses that were accompanied by corresponding increase in the serum suggests alteration in the permeability of the plasma memebrane leading to leakage of the enzyme from the tissue to the serum [25]. This was corroborated by histoarchitectural alterations in the small intestine. Organ body weight ratio is a useful index for atropy and hypertropy [29]. Therefore, the increase in liver and kidney-body weight ratio by the aqueous extract of M. indica leaves at 100 mg/ kg body weight may suggest inflammation at hepatic and glomerular levels while the absence of significant changes at lower doses as revealed by histological examination in the present study indicates absence of structural toxicity. The dose dependent reduction in the small intestine-body weight ratio which corroborates the histoarchitectural changes could suggest that the integrity of the submucosa layer has been compromised as revealed by the loss of ALP.
CONCLUSION The aqueous extract of M. indica leaves at the doses of 50 and 100 mg/kg body weight caused structural and functional toxicity hence, should be consumed as oral remedy with caution while the dose of 25 mg/kg body weight appears to be relatively safe and could be explored for oral remedies.
MATERIALS AND METHODS Plant Material: Collection and Authentication The plant samples were obtained at the Government Reserved Area (GRA) Staff Quarters of University of Ilorin, Ilorin, Nigeria. It was identified and authenticated at the Herbarium Unit of the Department of Plant Biology, University of Ilorin. A voucher specimen (U.I.H. 454) was deposited at the Herbarium Unit of the Plant Biology Department. Diagnostic Kits and other Chemicals: The diagnostic kits for albumin, bilirubin, creatinine, urea, uric acid, alkaline phosphatase (ALP), aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were products of Randox Laboratory Ltd, Co. Antrim, United Kingdom while those of sodium, potassium and chloride were manufactured by Roche Diagnostic GmbH, Mannheim, Germany. Other reagents used were of analytical grade and were supplied by Sigma-Aldrich Inc., St. Louis, USA. Experimental Animals: Twenty female albino rats (Rattus norvegicus) weighing 142.30 ± 7.55 g were obtained from the Animal House of the Department of Biochemistry, University of Ilorin, Nigeria. All the animals were housed in clean aluminium cages placed in a well-ventilated house
9
conditions (temperature 23 ± 20C, photoperiod 12 hours natural light, 12 hours dark and humidity 45-55%). The animals were allowed free access to rat pellets (Premier Feeds, Ibadan, Nigeria) and clean tap water except when fasting was required during the study. The cages were cleaned of wastes on a daily basis. Preparation of Aqueous Extract of M. indica Leaves M. indica leaves were separated from the stem, washed under running tap and oven dried (Uniscope Laboratory Oven, SM9053, Surgifriend Medicals, England) at 400C for 72 hours until a constant weight was obtained. Thereafter, the dried leaves were pulverized using an electric blender (Super Master Co., Ltd, Osaka, Japan) and stored in an air-tight container prior to extraction. Forty gramme (40 g) of the powder was extracted in 1 Litre of distilled water for 48 hours. The resulting filtrate was concentrated on a water bath (Uniscope Laboratory Water Bath SM801A, Surgifriend Medicals, England) to give a yield of 4.85 g corresponding to a percentage yield of 12.13%. This was reconstituted separately in distilled water to give the required doses of 25, 50 and 100 mg/kg body weight used previously in the antidiarrhoeal study of the plant [10]. Animal Grouping and Treatment The animals were randomly assigned into four groups (A, B, C and D) consisting of five rats each such that rats in group A (control) were orally administered 1 mL of distilled water, once daily for 10 days while those in groups B, C, and D received equal volume of the aqueous leaf extract of M. indica at the doses of 25, 50 and 100 mg/kg body weight respectively which was also based on the study reported by Izunya et al. [14]. Preparation of Serum and Tissue Supernatants The animals were sacrificed by anesthetization using diethyl ether 10 days post administration of the extract. When the rats became unconscious, the neck area was quickly cleared of fur and the jugular veins were cut using a sterile scalpel blade. Blood samples were allowed to clot for 20 min. and then centrifuged at 685 x g for 10 minutes. The sera were used within 12 hr of preparation for various biochemical assays. The liver, kidney and small intestine were also removed, blotted with tissue paper, freed of fat and metabolic waste. They were weighed for the computation of organbody weight ratio. The organs were separately homogenized in ice-cold 0.25M sucrose solution (1:5 w/v) after which the homogenates were centrifuged at 894 x g for 15 min. The supernatants were stored frozen and used for biochemical analysis within 24 hr of preparation. Evaluation of Biochemical Parameters and Histological Examination The levels of total protein, albumin, bilirubin (total and conjugated) and globulin were determined in the serum of the animals using standard procedures [30-33]. The levels of urea and creatinine were determined by adopting the procedures described by Veniamin and Verkirtzi [34] and Bartels and Bohmer [35] respectively while the concentrations of uric acid, sodium, potassium and chloride ions were determined by adopting the procedure described by Tietz [33]. The activities of ALT, AST and
10
ALP were determined using standard procedures [36,37]. Histological studies were carried out following the procedures highlighted by Krause [38]. Data Analysis: Data were expressed as the means ± SEM of 5 determinations. Statistical analysis was performed using One-way Analysis of Variance (ANOVA) and Duncan Multiple Range Test (DMRT). The data were considered statistically significant at p < 0.05. REFERENCES 1. Saadabi, A.M. (2007). Evaluation of Lawsonia inermis Linn. (Sudanese henna) leaf extracts as an antimicrobial agent. Research Journal of Biological Sciences 2(4): 419-423. 2. Sre, P.R.R., Sheila, T. and Murugesan, K. (2012). Phytochemical screening and “in-vitro” anti-oxidant activity of methanolic root extract of Erythrina indica. Asian Pacific Journal of Tropical Biomedicine 2(3): S1696-S1700. 3. Ross, I.A. (1999). Medicinal Plants of the World. Human Press Inc. pp. 199-202. New Jersey, USA. 4. Nwinuka, N.M., Monanu, M.O. and Nwiloh, B.I. (2008). Effects of aqueous extract Mangifera indica L. (Mango) stem bark on haematological parameters of normal albino rats. Pakistan Journal of Nutrition 7(5): 663-666. 5. Madunagu, B.E., Ebana, R.U.B. and Ekpe, E.D. (1990). Antibacterial and antifungal activity of some medicinal plants of Akwa Ibom State. West African Journal of Biological and Applied Chemistry 35, 25-30. 6. Gill, L.S. (1992). Ethnomedicinal uses of plants in Nigeria. University of Benin Press. pp.155. Benin, Nigeria. 7. Perpetuo, J.M. and Salgado, J.M. (2003). Effect of mango (Mangifera indica L.) ingestion on blood glucose levels of normal and diabetic rats. Plant Foods and Human Nutrition 58: 1-12. 8. Muruganandan, K., Srinivasan, S., Gupta, P.K. and Gupta, J.L. (2005). Effects of mangiferin on hyperglycemia and atherogenicity in streptozotocin diabetic rats. Journal of Ethnopharmacology 97, 497-501. 9. Ojewole, J. (2005). Antiinflammatory, analgesic, and hypoglycemic effects of Mangifera indica Linn. (Anacardeacea) stem-bark aqueous extract. Methods & Findings in Experimental and Clinical Pharmacology 27, 547554. 10. Yakubu, M.T. and Salimon, S.S. (2015). Antidiarrhoeal activity of aqueous extract of Mangifera indica in female albino rats. Journal of Ethnopharmacology 163, 135-141. 11. Latha, M.S., Latha, K.P., Vagdevi, H.M., Virupaxappa, B.S. and Nagashree, A.S. (2011). Phytochemical investigation and antimicrobial activity of Mangifera indica L. Rasapuri root extract. International Journal Medicinal & Aromatic Plants 1(2): 45-47.
Nig. J. Nat. Prod. and Med. Vol. 20 2016
12. Shah, K.A., Patel, M.B., Shah, S.S., Chauhan, K.N., Parmar, P.K. and Patel, N.M. (2010). Antihyperlipidemic activity of Mangifera indica L. leaf extract on rats fed with high cholesterol diet. Der Pharmacia Sinica 1(2): 156-161. 13. Hiruma-Lima, C.K., Severi, J.A., Lima, Z.P., Kushima, H., Brito, M.S., Dos Santos, L.C. and Vilegas, W. (2009). Polyphenols with antiulcerogenic action from decoction of mango leaves. Molecules 14, 1098-1110. 14. Izunya, A.M., Nwaopara, A.O., Aigbiremolen, A., Odike, M.A.C., Oaikhena, G.A., Bankole, J.K. and Ogara, P.A. (2010). Morphological and biochemical effects of crude aqueous extract of Mangifera indica L. (Mango) stem bark on the liver in Wistar rats. Research Journal of Applied Science Engineering and Technology 2(5): 460-465. 15. Gatewood, J., Wills, A., Shetty, S., Xu, J., Arnold, A., Burgoyne, P. and Rissman, E. (2006). Sex chromosome complement and gonadal sex influence aggressive and parental behaviour in mice. Journal of Neuroscience 26(8): 2335-2342. 16. European Commission. Council Regulation EC-440-2008 (2008): Laying down test methods in pursuant to Regulation (EC) No. 1907/2008 of the European Parliament and of the Council on the Registration, Evaluation, Authorisation and Restriction of Chemicals (REACH). March, 2008, [LEX-FAOC114509]. 17. Becker, J., Arnold, A., Berkley, K., Blaustein, J., Eckel, L., Hampson, E., Herman, J., Marts, S., Sadee, W., Steiner, M., Taylor, J. and Young, E. (2005). Strategies and methods for research on sex differences in brain and behaviour. Endocrinology 146(4): 1650-1673.
722. 24. Akanji, M.A. and Yakubu, M.T. (2000). á-Tocopherol protects against metabisulphite- induced tissue damage in rats. Nigerian Journal of Biochemistry and Molecular Biology 15(2): 179183. 25. Akanji, M.A., Nafiu, M.O. and Yakubu, M.T. (2008). Enzyme activities and histopathology of selected tissues in rats treated with potassium bromate. African Journal of Biomedical Research 11, 87-95. 26. Panda, N.C. (1989). Kidney. In: Tawlar, G.P., Srivastava, L.M. and Moudgil, K.D. (Eds.) Textbook of Biochemistry and Human Biology. 2nd edition. Prentice-Hall of India Private Ltd. pp. 276-292. 27. Yakubu, M.T., Akanji, M.A. and Oladiji, A.T. (2008). Effect of chronic administration of aqueous extract of Fadogia agrestis stem in male rats kidney function indices. Recent Progress in Medicinal Plants 19, 359-367. 28. Milla´n, J.L. (2006). Mammalian Alkaline Phosphatases: From Biology to Applications in Medicine and Biotechnology. Wiley-VCH Verlag GmbH & Co. pp. 1-322. Weinheim, Germany. 29. Amresh, G.R., Singh, P.N. and Rao, C.V. (2008). Toxicological screening of traditional medicine Laghupatha (Cissampelos pareira) in experimental animals. Journal of Ethnopharmacology 116, 454–460. 30. Gornall, A.C., Bardawill, C.J. and David, M.M. (1949). Determination of serum protein by means of Biuret reaction. Journal of Biological Chemistry 177, 751-756.
18. Saad, B., Azaizeh, H., Abu-Hijleh, G. and Said, S. (2006). Safety of traditional Arab herbal medicine. Evidence Based Complementary and Alternative Medicine 3, 433-439.
31. Doumas, B.T., Watson, W.A. and Biggs, H.G. (1971). Albumin standards and measurement of serum albumin with bromocresol green. Clinica Chimica Acta 31, 87-92.
19. Yakubu, M.T., Bilbis, L.S., Lawal, M. and Akanji, M.A. (2003). Evaluation of selected parameters of rat liver and kidney function following repeated administration of yohimbine Biokemistri 15, 50-56.
32. Jendrassik, L. and Grof, P. (1938). Simplified photometric methods for the determination for direct and total bilirubin. Biochemical Zeitschrift 297(81): 2-23.
20. Lakmichi, H., Bakhtaoui, F.Z., Gadhi, C.A., Ezobeiri, A., El-Jahiri, Y., El-Mansouri, A., Zrara, B. and Loutfi, K. (2011). Toxicity profile of aqueous ethanol root extract of Corrigiola telephiifolia Pourr. (Caryophyllaceae) in rodents. Evidence Based Complementary and Alternative Medicine Volume 2011: 10 pages. Article ID 317090. 21. Yakubu, M.T., Adeshina, A.O. and Ibrahim, O.O.K. (2012). Toxicological evaluation of aqueous leaf extract of Senna alata in pregnant Wistar rats. International Journal of Current Research and Reviews 4(8): 89-109. 22. Naganna, B. (1989). Plasma proteins. In: Tawlar, G.P., Srivastava, L.M. and Moudgil, K.D. (Eds.). Textbook of Biochemistry and Human Biology. 2nd edition. Prentice-Hall of India Private Ltd. pp. 172. India. 23. Yakubu, M.T. and Omoniwa, B.P. (2012). Effect of aqueous stem extract of Massularia acuminata on some liver function indices of male rats. Iranian Journal of Toxicology 6(18): 716-
Nig. J. Nat. Prod. and Med. Vol. 20 2016
33. Tietz, N.W. (1995). Clinical Guide to Laboratory Tests. 3rd edition. W. B. Saunders Company. pp. 972. Philadelphia, USA. 34. Veniamin, M.P. and Varkirtzi, C. (1970). Chemical basis of the carbamidodi-acetyl micro-method for estimation of urea, citrulline and carbamyl derivatives. Clinical Chemistry 16: 3-6. 35. Bartels, H. and Bohmer, M. (1972). Kinetic determination of creatinine concentration. Clinical Chimica Acta 37, 193-197. 36. Reitman, S. and Frankel, S. (1957). Colourimetric method for determination of serum glutamate-oxaloacetate and pyruvate transaminase. American Journal of Clinical Pathology 28, 5659. 37. Wright, P.J., Leathwood, P.D. and Plummer, D.T. (1972). Enzymes in rat urine. Alkaline phosphatase. Enzymologia 42, 317-327. 38. Krause, W.J. (2001). The Art of Examining and Interpreting Histologic Preparations. A Student Handbook. Partheton Publishing Group. pp 9-10. United Kingdom.
11
