Changes in some liver lipids of non-diabetic and diabetic rats following administration of combined extracts of Vernonia amygdalina

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1 AGRICULTURE AND BIOLOGY JOURNAL OF NORTH AMERICA ISSN Print: , ISSN Online: , doi: /abjna , ScienceHuβ, Changes in some liver lipids of non-diabetic and diabetic rats following administration of combined extracts of Vernonia amygdalina and Azadirachta indica Item J. Atangwho 1*, Ime F. Ani 2, Godwin E. Egbung 1 and Mary A. Iyam 2 Departments of Biochemistry 1 and Public Health 2, College of Medical Sciences, University of Calabar, P.M.B. 1115, Calabar Nigeria. ABSTRACT Some lipid fractions of whole liver homogenate (WLH) of normal and streptozotocin diabetic rats administered combined extracts of Vernonia amygdalina (VA) and Azadirachta indica (AI) for 28 days were evaluated. There was significant decrease (p<0.05) in HDL-cholesterol concentration in hepatocytes of diabetic control rats compared to the normal control. Whereas administration of single extracts of VA or AI further reduced the HDL-cholesterol non-significantly (p>0.05) and significantly (p<0.05), respectively, co-administration of the two extracts significantly increased (p<0.05) the HDL-cholesterol to levels comparable to the insulin treated group. Total cholesterol (TC) concentration of diabetic rats was not significantly altered by the extracts. In the non diabetic counterparts, AI extract alone, caused a significant elevation (p<0.05) of TC concentration. However, this effect was modulated by VA extract when given as combined extract. Triacylglycerol (TG) and very low density lipoprotein (VLDL) cholesterol levels in hepatocytes of diabetic control rats which reduced significantly (p<0.05) following diabetes induction, became significantly increased (p<0.05) upon administration of both single and combined extracts. The effect of extracts on these latter indices was better than insulin treated group. Combined extract of VA and AI suppresses hyperlipidemia of diabetes via hepatic lipid modulation and may be better in this respect than insulin. Keywords: Polyherbal therapy; Vernonia amygdalina; Azadirachta indica; diabetes liver lipids. INTRODUCTION Polyherbal therapy, sometimes called polyherbalisma combination of herbs or phytochemicals from more than one source, a concept originally peculiar to Ayurveda (Singh, 2005), has today become accepted as an effective therapeutic approach in sourcing medicament for degenerative ailments. The herbs are selected according to the disease and have several advantages over monotherapies namely, reduced toxicity/side effects and maximum/synergistic therapeutic efficacy (Tiwari and Rao, 2002; Singh, 2005). This approach has proven more useful and beneficial in the management of various ailments especially those that seem to defile conventional medication. The World Health Organisation (WHO) collaborations are taking place in several countries including Burkina Faso, the Democratic Republic of Congo, Ghana, Mali, Nigeria, Kenya, Uganda and Zimbabwe in the search and evaluation of herbal treatments for such ailments including HIV / AIDS, malaria, sickle cell anaemia and diabetes mellitus (WHO, 2003). mellitus; Recent report from our laboratory has shown enhanced efficacy in glycemic control with combined extracts from known antidiabetic plants, Vernonia amydalina and Azadirachta indica over their respective single extracts, in alloxan diabetic rat models (Ebong et al., 2008). However, detailed scientific studies have not been carried out with this combined extracts option from these plants. Meanwhile a sound basic and rigorous clinical investigation to confirm and advocate the excellence over the existing therapies of traditional medicinal plants, preparation(s), mechanism(s) of action and therapeutic effect(s) is absolutely required (Tiwari and Rao, 2002). The cardinal lesion leading to the high morbidity and mortality in diabetes and perhaps its frequency and severity is atherosclerosis (Tomkin and Owens, 1991). This is made more visible in the increased incidence of gangrene of the foot, myocardial infarction and strokes in diabetics than in non diabetics. It is well known also that the direct and independent link and/or relationship between atherosclerosis and diabetes is a faulty lipoprotein-

2 cholesterol metabolism. Hyperglycemia the primary clinical diagnostic feature of diabetes contributes to diabetes complication by altering vascular cellular metabolism and hence circulating lipoprotein (Chattopadhyay and Bandyopadhyay, 2005) This underscores the imperative of testing the effect of a potential therapy for diabetes such as this, on lipid metabolism particularly in the liver, the centre for all biotransformation reactions and metabolic coordination. The liver is the principal organ occupied with xenobiotics metabolism, such as medicinal plant extracts and is also a target tissue where possible toxicity effect of same is first expressed. The present study therefore investigated the effect of the combined extracts of Vernonia amydalina (African bitter leaf) and Azadirachta indica (neem) on lipid profile of liver hole homogenate of diabetic and non diabetic rats with the aim of ascertaining the influence of our potential medicament on lipoprotein and choleterol processing ability of the liver. MATERIALS AND METHODS Plant material: Matured leaves of Vernonia amygdalina Del. and Azadirachta indica A. Juss were respectively collected from the Endocrine Research Farm, and the staff village, University of Calabar, after authentication. The leaves were rinsed severally with clean tap water to remove dust particles and debris and thereafter allowed to completely drain. The plant materials were separately cut and chopped with a knife after which one kilogram (1kg) each of A. indica and V. amygdalina was homogenized in 1.95 and 2.25 liters of 80% (v/v) ethanol respectively. The mixtures were allowed for 48hrs in the refrigerator at 4 0 C for thorough extraction of the plants active components. These were firstly filtered with cheesecloth and later with Whatman No. 1 filter paper and the filtrates concentrated in vacuo at low temperature ( C) to about one tenth the original volume using a rotary evaporator. The concentrates were allowed open in a water bath (40 0 C) for complete dryness yielding 40.54g (4.054%) and 34.71g (3.471%) of greenish brown and brown oily substances for V. amygdalina and A. indica respectively. The extracts were then refrigerated at C until use. Experimental: Sixty male albino rats of Wistar strain weighing about g obtained from the animal house of the Department of Zoology and Environmental Biology, University of Calabar, Calabar, were allowed to acclimatize for three weeks in the animal house of the Department of Biochemistry. The animals were housed in well ventilated cages (wooden bottom and wire mesh top) and kept under controlled environmental conditions of temperature (25 ± 5 0 C), relative humidity (50 ± 5%) and 12 hour light / dark cycle. The animals were maintained on palletised Growers Feed (Vital Feeds, Jos, Plateau State, Nigeria) and tap water ad libitum. Diabetes was induced by intraperitoneal injection of 65mg/kg b.w. of streptozotocin (STZ) (Sigma St. Louis, MO, U.S.A) reconstituted in normal saline after a 12 hour fast. Control animals received saline only. Seven days after STZ treatment, diabetes was confirmed in STZ treated rats with a fasting blood glucose concentration 200mg/dl using One Touch Glucometer (Lifescan, Inc Milpas, California, U.S.A) with blood obtained from the tail vein of the rats. The 60 rats were divided into 5 parallel groups consisting of a diabetic and non-diabetic pair of 6 animals each (table 1). The diabetic and non-diabetic animals were accordingly, treated with extracts and insulin as shown in table 1. The dosages of the plant extracts were as determined from preliminary work in our laboratory whereas insulin dose, NPH (5U/kg b.w. s.c.) was as previously used by Sonia and Srinivasan (1999). The plant extracts were administered via gastric intubation, twice per day (6.00am: 6.00pm) and insulin once per day post prandial (6.00pm) for 28 days. Tissue collection and preparation of sample for assays: At the end of the 28 days, food was withdrawn from the rats and they were fasted overnight but had free access to water. They were then euthanized under chloroform vapour and sacrificed. Immediately the liver tissues were surgically removed then perfused in heparinized saline (0.9% NaCl) to remove blood cells. Thereafter the livers were blotted with blotting paper, and the whole weight measured with an analytical balance. Exactly 1g of the tissue was weighed and thoroughly homogenized in 10ml of freshly prepared phosphate buffer (20mM; ph 7.4). The homogenates were then centrifuged at 3,000g for 10 minutes using table centrifuge (B. Bran Scientific and Instrument Company, England) and the supernatant (whole liver homogenate, WLH) decanted into clean tubes and used for the lipid assays. Biochemical assays: Assay kits used in the biochemical assays were obtained from Randox Laboratories Ltd., Admore Diamond Road, Crumlin, Co., Antrim, United Kingdom, Bt294QY. Serum lipids including total cholesterol, triacylglycerol and HDLcholesterol were estimated according to the method 1097

3 of Tietz (1995), whereas VLDL- cholesterol calculated from relationship established by Friedewald et al (1972). Statistical analysis: The results were analysed for statistical significance by one way ANOVA using the SPSS statistical program and Post Hoc Test (LSD) between groups using MS excel program. All data were expressed as Mean ± SEM. P values < 0.05 were considered significant. Table 1: Experimental design Non-Diabetic Batch Group No. of animals Treatment 1. NC 6 Placebo (non diabetic control ) 2. NVA 6 V. amygdalina extract (200mg/kg b.w.) 3. NAI 6 A. indica extract (200mg/kg b.w.) 4. NVA/AI 6 V. amygdalina and A. indica combined extracts (100mg/kg b.w. each) 5. NHU 6 Insulin (5 unit/kg b.w.) Diabetic Batch Group No. of animals Treatment 1. DC 6 Placebo ( diabetic control ) 2. DVA 6 V. amygdalina extract (200mg/kg b.w.) 3. DAI 6 A. indica extract (200mg/kg b.w.) 4. DVA/AI 6 V. amygdalina and A. indica combined extracts (100mg/kg b.w. each) 5. DHU 6 Insulin (5unit/kg b.w.) RESULTS Result of effects of a 28-day administration of combined extracts of VA and AI on WLH lipid indices of normal and diabetic rats is respectively shown on tables 2.1 and 2.2. There was significant decrease (p<0.05) in HDL-cholesterol concentration in livers of diabetic control rats compared to the normal control. Whereas administration of single extracts of VA and AI further reduced the HDL-cholesterol nonsignificantly (p>0.05) and significantly (p<0.05), respectively, co-administration of the two extracts significantly increased (p<0.05) the HDL-cholesterol to levels comparable to the insulin treated rats. Total cholesterol (TC) concentration of diabetic rats was not significantly altered by the extracts. In the non diabetic counterparts, AI extracts alone, caused a significant elevation (p<0.05) of TC concentration. However, this effect was modulated by VA extract when given as combined extracts from these plants. Changes in HDL/TC following extracts administration were analogous to those of TC. Triacylglycerol (TG) and very low density lipoprotein (VLDL) cholesterol levels in hepatocytes of diabetic control rats which reduced significantly (p<0.05) following diabetes induction, became significantly increased (p<0.05) upon administration of both single and combined extracts. The effect of extracts on these latter indices was also non-significantly higher (p>0.05) than insulin. Combined administration of extracts of VA and AI may reverse altered lipid metabolism in the hepatocytes, hence possible amelioration of risk of atherosclerosis of diabetes. DISCUSSION Although products of lipid digestion are emptied directly into blood via the thoracic duct (lymph) as chylomicrons, chylomicrons travel through the blood stream to supply fatty acids to needed tissues, and their remnants are removed from blood by the liver for recycling. The liver also recovers cholesterol from bile, synthesize more cholesterol and triacylglycerol from excess acetyl units of diets and process them into transport forms lipoproteins (Gorman, 1992). Moreover, many genetic and acquired disorders may lead to deposits of lipids in vital organs such as liver and kidney, resulting in their impaired function (Crook, 2006). We therefore assayed the most common lipids and lipoproteins usually implicated in complications of diabetes in the hepatocytes of diabetic and non-diabetic rats which received our treatments. The result of this study showed significant decrease in HDL- cholesterol, TG and VLDL- cholesterol levels and non significant decrease in total cholesterol in hepatic tissue of untreated diabetic rats compared to non-diabetic 1098

4 control. Gorman (1992) had noted that abnormal lipid results, often have an underlying hormonal defect; and went ahead to list them to include hormones of the thyroid, pancreas and gonad. The first two including T 3, T 4 and insulin are well known or studied in diabetes, particularly type 1 such as is our model in this study. In physiological states, insulin stimulates hepatocytes to synthesise triacylglycerol and their subsequent export for storage in the adipose tissue and also inhibits lipolysis in these tissues apparently clearing off or impeding the formation of VLDLcholesterol and LDL-cholesterol in blood stream (Granner, 2000). The consequence is increased levels of these lipids in liver of non-diabetic rats (as observed in this study) where insulin response is potent and effective, and a correlated decreased level in serum. The reverse is therefore expected in untreated diabetic rats with defective insulin secretion or synthesis and reported increased T 3 and T 4 concentration (Atangwho, 2010). Compared to our earlier report it appears therefore that the relationship between concentration of lipid fractions in tissue and serum is alternate or inverse. Table 2.1. Effect of treatments on some lipid levels in liver whole homogenate of non diabetic rats. Group/ HDL-Ch. T-Chol HDL/TC TG Treatment VLDL-Ch. NC ± ± ± ± ±4.11 NVA ± ± ± ±25.32 c 81.88±11.51 c,d NAI ± ±13.15*,b,c,d 0.92±0.11*,b,c,d ±10.11 c 67.39±3.53 c NVA/AI 92.22± ± ± ±13.54 b,d 39.62±6.15 b,d NHU 91.90± ± ± ± ±5.88 Mean ± SE, n = 6, N = non diabetic, HU = insulin, b = p < 0.05 vs NC, c = p < 0.05 vs NVA/AI, d = p < 0.05 vs NHU, * = p < 0.05 vs NVA. Table 2.2. Effect of treatments on some lipid levels in liver whole homogenate diabetic rats. Group / VLDL-Ch. HDL-Ch. T.chol HDL/TC TG Treatment DC 54.75± ± ± ± ±4.50 NC ±4.53 a 70.97± ± ±9.04 a 78.95±4.11 a DVA 41.05±3.89 c 60.04± ±0.06 c ±19.04 a,d 86.27±8.66 a,d DAI 38.22±2.73 a,c 43.54± ± ±7.03 a,d 95.93±3.19 a,d DVA/AI 62.76± ± ± ±16.77 a,d 88.72±7.62 a,d DHU 50.30± ± ± ± ±8.26 Mean ± SE, n = 6, D = diabetic, HU = insulin, a = p < 0.05 vs DC, c = p < 0.05 vs DVA / AI, d = p < 0.05 vs DHU, * = p < 0.05 vs DVA. The four treatments in our study did not have severe effect on HDL- cholesterol and total cholesterol, but triglycerides and VLDL-cholesterol improvements except extracts of AI which respectively decrease HDL- cholesterol in diabetic rats and increase TC levels in hepatocytes of non diabetic rats. Some phytochemicals more abundant in AI than VA leaves including tannins, alkaloids and hydrocyanic acids reported ealier (Atangwho et al., 2009) may have endowed it this selective action over VA. Tannins have been implicated in hypolipidemic effects 1099

5 (Nimenibo-Uadia, 2003) and alkaloids from cocoa were also reported to ameliorate dietarily induced obesity in rats via their hypolipidemic action (Eteng et al., 2006). However when administered in combination, this solo effect of AI extract was effectively modulated by VA extract and the indices brought to levels similar to normal control. Furthermore, the extracts singly and in combination increased hepatocyte levels of TG and VLDLcholesterol of diabetic rats following 28 days treatment, to levels comparable with non-diabetic control and even to a better extent compared with insulin treatment. The converse of this observation is decreased serum levels of TG and VLDL cholesterol, hence effective antihyperlipidemic action particularly when used in combination. VA extracts alone (Atangwho et al., 2007a; Nwanjo, 2005) and AI extracts alone (Chattopadhyay and Bandopadhyay, 2005) have seperately been reported to possess potent antihyperlipidemic action in experimental diabetes using serum as the analytic sample. From this work, it is clear therefore that the antihyperlipidemic effects are exerted via hepatocyte processing and in combination the effects are conserved, but may be better than insulin in ameliorating atherosclerosis of diabetes at least in rats. REFERENCES Atangwho, I. J., Ebong, P. E., Eyong, E. U. and Egbung, G. E. (2010). Combined extracts of Vernonia amygdalina and Azadirachta indica may substitute insulin requirement in the management of type I diabetes. Res. J. Med. Med. Sci. 5(1): Atangwho I. J., Ebong P. E., Eyong E. U., Eteng M. U. and Uboh, F. E. (2007a): Vernonia amygdalina Del.: A potential prophylactic antidiabetic agent in lipids complication. Glob. J. Pure Appl. Sci. 13 (1), Atangwho I. J., Ebong, P. E., Eyong, E.U., Williams, I. O., Eteng, M. U. and Egbung, G. E. (2009): Comparative chemical composition of leaves of some antidiabetic medicinal plants: Azadirachta indica, Vernonia amygdalina and Gongronema latifolium. Afri. J. Biotechnol. 8(18): Chattopadhyay R. R. and Bandyopadhyay, M. (2005): Effect of Azadirachta indica leaf extract on serum lipid profile changes in normal and streptozotocin induced diabetic rats. Afri. J. Biomed. Res. 8, Crook M. A. (2006): Clinical Chemistry and Metabolic Medicine. Edward Arnold Publishers Ltd. Ebong P. E., Atangwho I. J., Eyong E. U. and Egbung G. E. (2008): The antidiabetic efficacy of combined extracts from two continental plants: Azadirachta indica (A. Juss) (Neem) and Vernonia amygdalina (Del.) (African bitter leaf). Am. J. Biochem. Biotechnol. 4(3), Eteng M. U., H. A. Ibekwe, U. I. Umoh, P. E. Ebong, I. B. Umoh and E. U. Eyong. (2006): Theobromine Rich cocoa powder induces weight loss and changes in lipid profile of obese Wistar rats. Discov. Innovat. 18(3), Friedewald W. T., Levy R. T. and Fredickson D. S. (1972): Estimation of the concentration of LDL cholesterol in plasma without use of ultracentrifugation. Clin. Chem. 185, Gorman L. S. (1992): Lipids and Lipoproteins. In: Clinical chemistry. Principles, Procedures, Correlations (2 nd ed.) (eds. Bishop ML, Duben-Engelkirk JL and Fody EP.) J. B Lippincott Company, Philadelphia, pp Granner D. K. (2000): Hormones of the pancreas and gastrointestinal tracts. In Harper s Biochemistry (25th ed.), McGraw Hill, New York, pp Nimenibo-Uadia R. (2003): Effect of Vernonia amygdalina in alloxan-induced diabetic albino rats. J. Med. Lab. Sci Nwanjo H. U. (2005): Efficacy of aqueous leaf extract of Vernonia amygdalina on plasma lipoprotein and oxidative status in diabetic rat models. Nig. J. Physiol. Sci Singh, A. P. (2005). Relevance of polyherbal formulations. Retrieved June from Sonia B. and Scrinivasan B. P. (1999): Investigations into the anti-diabetic activity of Azadirachta indica. Ind. J. Pharm. 31, Tietz, N. W. (1995): Clinical Guide to Laboratory Tests (3rd ed.). WB Saunders Company, PA., Philadelphia, pp Tiwari A. K. and Rao J. M. (2002): Diabetic mellitus and multiple therapeutic approaches of phytochemicals: Present status and future prospects. Curr. Sci. 83 (1), Tomkin G. H. and Owens D. (1991): Abnormalities of cholesterol metabolism in diabetes. Proceed. Nutrit. Societ. 50, WHO (World Health Organisation) (2003). Traditional Medicine. Retrieved June 09, 2008 from centre/fact sheet/fs134/en/ 1100

The Antidiabetic Efficacy of Combined Extracts from Two Continental Plants: Azadirachta indica (A. Juss) (Neem) and Vernonia amygdalina

American Journal of Biochemistry and Biotechnology 4 (3): 239-244, 2008 ISSN 1553-3468 2008 Science Publications The Antidiabetic Efficacy of Combined Extracts from Two Continental Plants: Azadirachta