Isolation and characterisation of anti-diabetic pharmacological activities of phytoestrogens and components of Moringa peregrina (Forssk) Fiori

Diabetes is an endocrine disease characterised by a chronic increase in blood sugar levels caused by a deficiency of insulin production, which leads to type 1 diabetes, or by a loss of tissue response to insulin, which leads to type 2 diabetes. The disease leads to disruption of metabolism, vascular damage and damage to the nervous system, as well as damage to other organs and systems. Type 2 diabetes is becoming more common throughout the world, due to poor nutrition and lifestyle, and genetic background. Efforts have accordingly been increased towards developing and refining treatments as well as to addressing the underlying causes of the disease. Many parts of the world have a documented history of the use of plants to treat diabetes, and these can be an attractive, local, alternative to expensive pharmaceutical medicines. Accordingly there is an increasing interest in identifying new phytochemicals with proven pharmacological effects on diabetes. The tree Moringa peregrina is commonly found throughout the Middle East and the oil from its seeds has been used for thousands of years. Other members of the Moringa family have recorded anti-diabetic family and Moringa peregrina was chosen for investigation in this work with the aim of characterising anti-diabetic activity from its leaves. Six extracts were prepared using solvents water, methanol, butanol, ethyl acetate, chloroform and hexane, based on standard extraction techniques. The study was designed to assess the effect of these six extracts on the uptake of glucose in a human hepatoma cell line (HepG2) using a well-studied fluorescent derivative of glucose, 2-2-[N-(7-nitrobenz-2-oxa-1, 3-diazol-4-yl) amino glucose (2-NBDG). The cells can take up 2-NBDG instead of glucose via glucose transporters and its uptake is indicative of the capacity of cells to take up glucose, i.e. of the number of active glucose transporters on the cell surface. The results revealed that the extracts prepared with ethyl acetate and chloroform increased glucose uptake significantly more than the other extracts. Moreover the effects of the extracts were rapid, with a one hour treatment producing a similar stimulation to a 24 hour treatment. These data were confirmed in a second method of investigation of the hypoglycaemic effect of the extracts, by measuring consumption of glucose from cell culture medium. A preliminary assessment of the effect of active extracts on expression of the main glucose transporter of HepG2 cells, GLUT1, by western blotting indicated no large changes in expression. Dietary phytoestrogens have been shown to play a beneficial role in obesity and diabetes, so the second part of this study investigated the effect of phytoestrogens on glucose uptake. Three phytoestrogens (daidzein, formonontein and genestin, which are naturally occurring isoflavones) were chosen. HepG2 cells showed a significant increase in glucose uptake after treatment with phytoestrogens compared to the control. In an attempt to identify the active phytochemicals that could account for the observed effects, extensive purification and characterisation of components from the ethyl acetate fraction was undertaken. Seven components were identified: (1) OEthyl 4-[(α-L-rhamnosyloxy)benzyl] thiocarbamate (E), (2) O-Butyl 4-[(α-Lrhamnosyloxy)benzyl] thiocarbamate (E), (3) 4-(α-L-Rhamnosyloxy)benzyl isothiocyanate, (4) ȕ-Sitosterol, (5) Daucosterol, (6) γ’-methyl-quercetin-3-Orutinoside (also known as isorhamnetin-3-O-rutinoside), (7) Rutin. In a final analysis an attempt was made to assess the effect of a representative set of three out of the seven components on basic metabolic activity of HepG2 cells using a Seahorse XF-24 analyser. The three components chosen were O-ethyl 4-[(α-L-rhamnosyloxy) benzyl] thiocarbamate (E), ȕ-sitosterol from the phytosterols family and Rutin. Real-time monitoring of cell metabolism by a Seahorse XF-24 auto analyser after two hours incubation with the three chosen compounds revealed that maximal respiration, non-mitochondrial respiration and spare respiratory capacity have trended towards an increase with ȕ-sitosterol and3-O-ethyl 4-[(α-Lrhamnosyloxy) benzyl] thiocarbamate (E) treatments compared to control. As no similar trends were observed in ATP production, the increased maximal respiration could increase metabolic activity at higher concentrations of glucose and account, in part for the effects observed on glucose consumption