The phytochemical content and anti-diabetic properties of Aloe ferox and Aloe greatheadii var. davyana

Abstract

Motivation: Diabetes mellitus is a non-communicable disease considered to be one of the five leading causes of death worldwide, characterized by hyperglycemia and hyperlipidemia as a result of altered glucose and lipid metabolism. Recently the search for suitable ant diabetic agents has focused on plants used in traditional medicine. Various Aloe species have been used for centuries in the management of various diseases, including diabetes. The majority of the scientifically 1Jased research on this topic was done on Aloe Vera (or Aloe barbadensis) and Aloe arborescence. However, in the rural communities, the type of Aloe which is chosen as a traditional medicine would depend on its immediate availability to the specific community. Hence, various communities in different parts of the world would use the species of Aloe indigenous to their immediate surroundings. Aloe ferox (indigenous to the Western provinces of South Africa) and Aloe greatheadii var. davyana (indigenous to the Northern provinces of South Africa) are the most frequently used among the rural communities of South Africa to treat diabetes, even though very little scientific evidence, if any, exists to substantiate its use in diabetes. Different Aloe species would have varying phytochemical contents, health benefits and possible toxicities. Hence, it is of relevance for scientists, industry, and rural communities to not only investigate the relevant medicinal uses of their indigenous Aloe species, but also to determine the active components and their individual or combined mechanisms of biological function. Objectives: The main objective of this study was to determine and compare the anti-diabetic effects of A. ferox and A. greatheadii ethanol leaf gel extracts using a streptozotocin (STZ)-induced diabetic rat model. In order to provide a foundational body of evidence for the aforementioned, a secondary objective included the characterization and comparison of the phytochemical content of A. ferox and A. greatheadii leaf gel extract (LGE) and 95% ethanol

leaf gel extract (ELGE) using gas chromatography mass spectrometry (GCMS) and spectrophotometry prior to this, in order to confirm the presence of phytochemicals with health related benefits and to determine the most optimal extraction conditions for these. Methods: The phytochemical content of both A. ferox and A. greatheadii var davyana LGE and ELGE were analyzed and compared via standard extraction methods and analysis on GC-MS (Agilent, USA) and spectrophotometric ally (Shimadzu UV-160l spectrophotometer). The extract obtained from the extraction method providing the most phytochemicals with previously proposed ant diabetic action, was chosen for the intervention study that followed. The intervention study was done using a STZ diabetic rat model in order to confirm the predicted ant diabetic effects based on the phytochemical characterization. In order to accomplish this, fifty male Wistar rats were divided into five groups: Group 1 consisted of normal control rats (NC), group 2 of diabetic control rats (DC), group 3 of diabetic rats receiving 300 mg/kg A.. greatheadii (DAG), group 4 of diabetic rats receiving 300 mg/kg A. ferox (DAF), and group 5 of diabetic rats receiving glibenclamide (DGL). After a 16 hour fast, the rats in the DC, DAG, DAF and DGL groups were injected (intraperitoneally) with 40mg/kg STZ dissolved in 0.1M cold sodium citrate buffer (PH 4.5) and left for one week, in order for diabetes to develop. Diabetes was confirmed after a 12 hour fast (blood glucose> 13.875mmol/L or 250mg/dL) by measuring blood glucose from a cut to the tail. The A.forox ELGE, A greatheadii ELGE and glibenclarnide were• given with an intragastric tube once daily for 5 weeks during which the rats had unlimited access to food and water. At the end of the intervention period, the rats were sacrificed and tissue and blood samples were collected. The effects of these interventions on the STZ induced diabetic state was monitored by measurement of various biochemical diabetes markers which included: serum

glucose, insulin, insulin resistance, fructosamine, triacylglycerol (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), very low-density lipoprotein cholesterol . (VLDL-C), alanine transaminase (ALT), alkaline phophatase (ALP), ferric reducing antioxidant power (FRAP), and diacron reactive metabolites (dROMs). Results: GC-MS and spectrophotometric analyses revealed a wide range of compounds with potential health benefits in both A. ferox and A. greatheadii LGE and ELGB. GC-MS analysis revealed that separate ethyl acetate/diethyl ether and hexane extractions of the LGE, is better suited to general . phytochemical characterization purposes, whereas 95% aqueous ethanol extraction effectively concentrated selective groups of health related compounds, hence justifying its application to biological in vivo efficacy studies. Apart from these health related phytochemicals, sugar determinations revealed that A. ferox ELGE consisted of 96.9% sugar and A. greatheadii ELGE consisted of 83.75% sugar. In the animal study, diabetes was confirmed one week after the injection of 40 mg/kg STZ by measuring fasting glucose concentrations via a cut to the tail. Compared to the NC group, STZ resulted in increased relative liver and kidney mass, end-point plasma glucose, fructosamine, oxidative stress, liver enzymes, total cholesterol, triglycerides, VLDL-C, and TC:HDL-C values, and reduced serum insulin levels. The majority of these diabetes markers, including fasting end-point glucose concentrations, fasting serum insulin levels, insulin resistance, and lipid levels, returned to near normal levels with glibenclamide supplementation, confirming that STZ injections resulted in an insulin independent diabetes that closely resembles type 2 diabetes biochemical abnormalities in human subjects.

Treatment with A. greatheadii moderately increased serum insulin accompanied by a modest decreased end-point plasma glucose and decreased liver enzyme ALP, in addition to moderately increased HDL-C and decreased• TC:HDL-C values. A. ferox supplementation resulted in moderately increased serum insulin, accompanied by slight corrections in ALP and HDLC, however, without a decrease in end-point plasma glucose. Little effect was seen on other diabetes markers. Conclusion: Oral administration of the Aloe extracts, A. greatheadii in particular, resulted in moderate improvements in the STZ induced diabetic state, especially when considering the changes observed in the end-point. plasma glucose and serum insulin levels, hence, justifying further investigations into the use of these traditional remedies for the treatment of diabetes. However, considering the phytochemical contents and previous literature using other Aloe species, more significant results were expected. Consequently, it is proposed that these effects should be studied using higher dosages, longer intervention periods, alternative extracts and perhaps larger sample groups for future ant diabetic investigations using these indigenous plants.