Toxicity of Mucuna Pruriens Seed Extract on the Kidney of Adult Sprague-Dawley Rats
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Gbotolorun et al, Afr. J. Pharmacol. Ther. 2018. 7(1): 27-33 African Journal of Pharmacology and Therapeutics Vol. 7 No. 1 Pages 27-33, 2018 Open Access to full text available at http://www.uonbi.ac.ke/journals/kesobap/ Research Article Toxicity of Mucuna pruriens seed extract on the kidney of adult Sprague-Dawley rats Stella C. Gbotolorun a,*, Perpetual K. Isah a, and Oluwaseye A. Adebajo a a Department of Anatomy, Faculty of Basic Medical Sciences, College of Medicine, University of Lagos, Nigeria _____________ * Corresponding author: Department of Anatomy, Faculty of Basic Medical Sciences, College of Medicine, University of Lagos, P.M.B. 12003, Lagos, Nigeria; Tel: +234-803-8098631; E-mail: [email protected] Background: The commonly acceptable knowledge that herbal medications have little or no toxicity and are absolutely safe makes people consume them indiscriminately. All parts of Mucuna pruriens have been reported to possess valuable medicinal properties, but its potential toxicity on vital organs remains unexplored. Objective: To determine the deleterious effect of Mucuna pruriens on the Kidney of Adult Sprague-Dawley Rats. Methodology: Twenty Sprague-Dawley rats were used and divided into four groups of five rats per group. Group I served as control and received distilled water and groups II-IV received 50, 100 and 200 mg/kg of the extract respectively for 2 weeks. The animals were sacrificed, blood was collected for kidney function test and the kidneys were excised via ventral laparatomy. The right kidney was fixed for histological studies while the left kidney was analysed for biochemical markers of oxidative stress Results: Lipid peroxidation increased significantly while superoxide dismutase and glutathione recorded a significant decrease in activities when the treated groups were compared to control. Creatinine decreased significantly and urea increased significantly when treated groups were compared to control. Histological sections showed degenerative changes and tubular necrosis in the kidney at higher doses. Conclusion: Mucuna pruriens causes degenerative changes in glomerular epithelia and reduced urea clearance possibly by an oxidative stress mechanism. Keywords: Kidney, Mucuna pruriens, superoxide dismutase (SOD), creatinine, urea. Received: March, 2018 Published: May, 2018 1. Introduction (Sathiyanarayanan and Arulmozhi 2007). In vitro and in vivo studies on Mucuna pruriens extracts revealed the Medicinal plants are used as one of the available presence of substances that exhibit a wide variety of medicines especially in developing countries (Hashim et pharmacological effects, including anti-diabetic, anti- al, 2010). Plants used as traditional medicine contain a inflammatory, neuroprotective and anti-oxidant wide range of substances that are effective in the properties, probably due to the presence of L-dopa, a treatment of sicknesses and diseases. Clinical precursor of the neurotransmitter dopamine (Misra and microbiologists also have great interest in screening of Wagner, 2007). medicinal plants for new therapeutics (Penyasamy et al, 2010). Among the various under-utilized wild legumes, It is known that the main phenolic compound of Mucuna is the velvet bean called Mucuna pruriens that is found pruriens seeds is L-dopa (approximately 5%) (Vadivel in tropical and sub-tropical regions of the world. It is and Pugalenthi 2008). Phytochemical screening of the considered a viable source of dietary proteins plant revealed that it contains alkaloids, flavonoids, (Janardhanan et al, 2003; Pugalenthi et al, 2005) due to tannins, saponins, cardiac glycosides, anthraquinones its high protein concentration of 23–35%. All parts of and carbohydrates (Minari et al, 2016). Mucuna pruriens possess valuable medicinal properties A KeSoBAP Publication ©2018. All rights reserved. 27 Gbotolorun et al, Afr. J. Pharmacol. Ther. 2018. 7(1): 27-33 Available data on the toxicity of herbal drugs is limited. chloroform as anaesthesia, a ventral laparotomy was The commonly acceptable knowledge that herbal performed and the kidneys were dissected. The right medications have little or no toxicity and thus are kidney was fixed in 10% formal saline for histological absolutely safe makes people consume herbal studies while the left kidney was stored in -20 °C for medicines indiscriminately. In lieu of this fact, there is a biochemical analysis of oxidant status and blood was growing concern about the safety of most herbal drugs. collected by cardiac puncture for kidney function test This study was carried out to investigate the effect of and analysed immediately. Mucuna pruriens on the kidney in Sprague-Dawley rats. 2.5 Histological Procedures 2. Methods Following fixation, the harvested tissues were 2.1 Plant Materials dehydrated in graded ethanol, embedded in wax and sectioned to 5 µm thickness. The sections were stained Mucuna pruriens plants with mature seeds were with routine haematoxylin and eosin stains and procured from Mushin market, a local market in Lagos photomicrographs were made at a magnification of 100 State, Nigeria. They were identified and authenticated in and 400 using Olympus and Leica microscopes (Haneia the Department of Botany, Faculty of Science, University et al, 2013). of Lagos, Nigeria where the voucher specimens of the fruits are kept in the herbarium with a voucher number 2.6 Biochemical Analysis of Oxidant status of LUH 4922. The kidneys were washed in ice cold 1.15% KCl 2.2 Preparation of Extract solution, blotted and weighed. They were then homogenized with 0.1 M phosphate buffer (pH 7.2). The The extraction was carried out in the Department of tissues were introduced into mortar and laboratory Pharmacognosy, Faculty of Pharmacy, University of sand was then added. This was crushed using a pestle. Lagos. The seeds of the plants were collected in the The resulting homogenate was centrifuged at 2500 rmp month of August and September. Briefly, seeds were speed for 15 mins. Thereafter, it was removed from the obtained from the pods, air-dried and pulverized into centrifuge and the supernatant was decanted and fine powder using a mortar and pestle. 1.5 kg of fine stored at -20 °C until analysis. powder was mixed with absolute methanol and placed Superoxide Dismutase (SOD) was assayed by its in the soxhlet apparatus for duration of 6 hrs at 60 °C. The solvent was removed by distillation and semisolid ability to inhibit the auto-oxidation of epinephrine, determined by the increase in absorbance at 480 nm as mass was dried by using a hot water bath at 40-50 °C described by Sun and Zigma (1978). The enzyme and the % yield of the methanolic extract of Mucuna pruriens seed was calculated to be 147.8 g and stored at activity was calculated by measuring the change in absorbance at 480 nm for 5 min. room temperature of 25 °C before use. All dilutions of the extract were made in distilled water. Glutathione (GSH) activity was estimated according to the method described by Sedlak and Lindsay, 1968. To 2.3 Experimental Animals the tissue homogenate, 10% TCA was added, centrifuged. 1.0 ml of supernatant was treated with 0.5 A total of twenty healthy Sprague-Dawley rats with ml of Ellman’s reagent (19.8 mg of 5, 5-dithiobisnitro weights ranging between 120 and 140 g were used for benzoic acid (DTNB) in 100 ml of 0.1% sodium nitrate) this study. They were housed in well standard and 3.0 ml of phosphate buffer (0.2 M, pH 8.0). The ventilated wire mesh plastic cages in the Animal House absorbance was read at 412 nm. of the Department of Anatomy, College of Medicine of the University of Lagos, Nigeria under standard room Malondialdehyde (MDA) an index of lipid temperature (26-28 °C) and relative humidity (50- peroxidation was determined using the method of 55%). The rats were exposed to twelve hours light and Buege and Aust (1978). The supernatant was removed twelve hours dark cycle. They were allowed and the absorbance was read at 532 nm. MDA was unrestricted access to water and commercial rat chow calculated using the molar extinction coefficient for 5 -1 -1 ad libitum. They were left to acclimatize for a period of MDATBA- complex of 1.56 × 10 M cm . two weeks before the commencement of the experiment. The animals were identified by different 2.7 Determination of Creatinine concentration ear tags and the weights of the animals were taken weekly. All experimental procedures and techniques The creatinine concentration in serum was measured were in compliance with the guiding principles for using Creatinine K commercial kits (LabtestDiagnostica research involving animals (Helsinki 2002). SA, Lagoa Santa, Brazil), which uses a two-point optimized kinetic procedure based on the modified-Jaffe 2.4 Experimental Design reaction. For disposing purposes, 50 µL of the serum sample was added to 50 µL of alkaline picrate, mixed The animals were divided into four groups of five and aspirated into the automatic analyser bucket set to animals each. Group I received distilled water and zero at 510 nm, and then measured the absorbance at served as control. Groups II to IV orally received varying 30 seconds. The results were expressed in µmol/l. doses of methanolic seed extract of Mucuna pruriens dissolved in distilled water at 50, 100 and 200 mg/kg 2.8 Determination of Urea concentration respectively for 2 weeks. The rats were monitored for toxicity and their body weights were measured. At the The urea concentration in serum was measured using end of the experiment, the rats were sacrificed using Liquiform Urea UV test (LabtestDiagnostica SA, Lagoa Santa, Brazil) which uses an enzymatic system by UV A KeSoBAP Publication ©2018. All rights reserved. 28 Gbotolorun et al, Afr. J. Pharmacol. Ther. 2018. 7(1): 27-33 photometry and two-point Kinetics. For disposing weights were lower in groups II and III when compared purposes, 10 µL of the serum were aspirated into the to IV in the treatment groups (Table 1).