Investigation of the in Vitro Antioxidant Activity, in Vivo Antidiabetic Efficacy

Journal of Medicinal Plants Studies 2015; 3(1): 42-47 ISSN 2320-3862 Investigation of the in vitro antioxidant activity, JMPS 2015; 3(1): 42-47 in vivo antidiabetic efficacy and safety of Capparis © 2015 JMPS Received: 04-11-2014 tomentosa aqueous roots extracts in male Accepted: 26-11-2014 alloxanized mice Laura Nyawira Wangai Kirinyaga University College, School Health Sciences, Laura Nyawira Wangai, Brenda Wamae Waithera, Muriira Geoffrey P. O Box 143-10300, Kerugoya, Kenya Karau, Ndura Boniface Koimburi, Philip Karanja Ndura, Rebecca Karanja, Michael Kimani Gitau, Peter Kirira Brenda Wamae Waithera Jomo Kenyatta University of Agriculture and Technology, Abstract P. O Box 62000-00200, The current study investigated the in vitro antioxidant activity, in vivo antidiabetic efficacy and safety of Nairobi, Kenya Capparis tormentosa aqueous root extracts. Antioxidant activity was determined using 1, 1-diphenyl-2- picrylhydrazyl (DPPH), phosphomolybdate and reducing power assay with ascorbic and gallic acid as Muriira Geoffrey Karau references. Six groups of BALB/c mice each comprising of five were used in evaluating the antidiabetic Kenya Bureau of Standards activity. Diabetes mellitus was induced in five groups using 10% alloxan monohydrate at a dose of 186.9 Research and Development mg/kg body weight. Non-diabetic control mice was orally administered with 0.1 ml physiological saline; Department, P. O Box 54974-00200, diabetic mice with 0.075 mg of reference drug, glibenclamide at 3 mg/kg body weight; 1.25 mg, 2.5 mg, Nairobi, Kenya and 5 mg extracts in 0.1 ml physiological saline for 50, 100 and 200 mg/kg body weight, and the other group of diabetic mice was given 0.1 ml physiological saline. The blood glucose level was determined Ndura Boniface Koimburi after 0, 2, 4, 6 and 8 hours. Safety was evaluated by daily administration of a single dose of 1000 mg/kg Kitale Nature Conservancy body weight extract to BALB/c male mice of comparable age and weight over a period of one month, P. O Box 1556-3020, while recording body weights every 7 days and organs weights after the 28th day. The antioxidant activity Kitale, Kenya by DPPH was 35.50 ± 0.02%, by phosphomolybdate assay was 41.22 ± 0.17 mg/kg ascorbic acid Philip Karanja Ndura equivalent, and the reducing power increased with increase in concentration up to a maximum at 800 Kitale Nature Conservancy, µg/ml. The antidiabetic activity was dose dependent and significantly higher. There was no significant P. O Box 1556-3020, change in body weights for treated and untreated mice in safety studies (p = 0.69), and the weight gain Kitale, Kenya was normal for both experimental and control mice. Except kidneys, which changed significantly (p = 0.009), all the other organ weights were not affected. The study supports the claim that C. tormentosa is Rebecca Karanja effective and safe in the management of diabetes mellitus. Jomo Kenyatta University of Agriculture and Technology, Keywords: Antidiabetic, BALB/c mice, Capparis tormentosa, Safety, Efficacy P. O Box 62000-00200, Nairobi, Kenya 1. Introduction Diabetes mellitus is a chronic metabolic disorder caused by inherited and/or acquired Michael Kimani Gitau deficiency in production of insulin by the pancreas, or by the ineffectiveness of the insulin Kitale Nature Conservancy, produced. This deficiency in insulin production results to increase in the concentration of P. O Box 1556-3020, blood glucose, which in turn causes damage to many of the body’s systems particularly the Kitale, Kenya [1] blood vessels and nerves . Diabetes mellitus is a major health problem, affecting about 5% of [2] Peter Kirira the total population in the U.S and 3% of the world population. Epidemiological studies and Directorate of Research and clinical trials [3], strongly support the premise that most of the complications are caused by Development, Mount Kenya hyperglycemia. Effective blood glucose control is the critical intervention measure in the University, P. O Box 342-01000, management of diabetic complications and improving quality of life [4]. Thus, sustained Thika, Kenya reduction of hyperglycemia lowers the risk of developing microvascular complications and reduces the risk of macrovascular complications [5]. Capparis tomentosa belongs to the family Capparaceae commonly referred to as Woody Caper Bush in English [6]. It is an indigenous South African plant that grows naturally in the savanna Correspondence: forest of Western, Eastern and Southern Africa [7]. It is a scrambling shrub, sometimes Muriira Geoffrey Karau Kenya Bureau of Standards maturing into a tree that can grow as high as 10 meters tall and is covered with scattered Research and Development spines. It is well branched and the branches are normally covered with thick yellow hairs; even Department, the robust, recurved spines are often hairy. The twigs and leaves are yellow-green in colour P. O Box 54974-00200, and are covered in soft, velvety hairs. The oblong leaves are approximately 50 × 20 mm, with Nairobi, Kenya ~ 42 ~ Journal of Medicinal Plants Studies a pair of sharp, hook-like thorns at the junction of the stem and nm. The blank contained all reagents except the roots extract. leaf base. The white and pink flowers have multiple stamens. Ascorbic acid at a concentration of 1 mg/ml was used as The fruit is pink to orange in colour, round and stalked. The reference. The scavenging activity was calculated by using the seeds are surrounded by fleshy, grey fruit pulp [8, 6]. formula shown in equation 1. In herbal and traditional medicine, C. tomentosa is used to Percent scavenging activity = treat rheumatism, madness, snakebite, chest pain, jaundice, malaria, headache, coughs, pneumonia, constipation, infertility and to prevent abortions. It is used to treat leprosy, tuberculosis and gonorrhea [1]. The roots are boiled in water and half a cup of this infusion is drunk three times per day to manage cough and chest pain [7, 9]. Despite the continued use Equation 1 of C. tomentosa in management and treatment of many ailments among many communities, it has been reported to be 2.3 Total antioxidant activity by phosphomolybdate assay toxic to livestock [8, 10]. Data on its in vitro antioxidant activity, This was carried out according to the procedure described antidiabetic efficacy and safety in Kenya is scarce. Therefore, elsewhere [13]. The phosphomolybdate reagent was prepared by this study investigated the in vitro antioxidant activity, in vivo mixing equal volumes of 100 ml each of 0.6 M sulfuric acid, antidiabetic efficacy and safety of C. tomentosa in alloxan 28 mM sodium phosphate and 4 mM ammonium molybdate. induced diabetic male BALB/c mice, to validate the folkloric Test samples were prepared by dissolving 1 mg of plant claim among the herbalists and local communities. methanolic extract in 1 ml of methanol. Then, 0.1 ml of the test sample was dissolved in 1 ml of reagent solution in a test 2. Materials and Methods tube which was capped with silver foil and incubated in a 2.1 Preparation of the aqueous root extracts water bath for 90 min at 95 ºC. After cooling the sample to Fresh roots of C. tomentosa were collected from West Pokot room temperature, the absorbance was read at 765 nm against and Marakwet parts of the North Rift Valley of Kenya in the a blank. Ascorbic acid was used as a standard antioxidant with month of December 2012. The plant is likened to ‘the biblical a concentration ranging from 10 to 50 mg/L. The ascorbic acid hyssop’ by Mr. Ndura Boniface, who has been using it for absorbances were used in the construction of the standard over six years in treatment and management of diabetes curve. The results were expressed as μg of ascorbic acid mellitus, HIV/AIDS, reproductive problems and cancer among equivalent (AAE) per mg of the dried weight of the root many other diseases (Unpublished, personal communications). extracts of C. tomentosa. The AAE was determined according The fresh roots were washed with clean tap water, cut into to the expression in equation 2. small pieces and dried under shade at room temperature for 4 weeks. They were then milled into fine powder using electric mill, and the powder kept at room temperature away from direct sunlight in dry airtight plastic bags. 100 g of the root powder was extracted in 1 liter of distilled water at 60 ºC in a Equation 2 metabolic shaker for 6 hours. The extract was decanted into a 2.4 Reducing power assay clean, dry conical flask and then filtered through cotton gauze The reducing power assay was carried out by the method of [14] into another dry clean conical flask. The filtrate was freeze with some modifications. The root extract or gallic acid dried in 200 ml portions in a Modulyo Freeze Dryer (Edward, solution ranging from 25 to 800 µg/ml, each 2.5 ml was mixed England) for 48 hours. These extracts were weighed and stored with 2.5 ml of 0.2 M sodium phosphate buffer and 2.5 ml of in airtight, amber containers at the 4 ºC ready for use. 1% potassium ferricyanide. The mixture was incubated at 50 The extracts for in vivo antidiabetic studies were prepared ºC for 20 min and 2.5 ml of trichloroacetic acid solution (100 according to the protocol described elsewhere by Karau et al mg/L) was added. The mixture was centrifuged at 650 rpm for [12] , with slight modifications. Briefly, 125 mg to make an oral 10 min, and 5 ml of the supernatant was mixed with 5 ml of dose of 50; 250 mg to make 100, and 500 mg to make 200 distilled water and 1 ml of 0.1% ferric chloride solution.

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