An in vitro investigation into the glucose regulating activity of Gnidia canoargentea and Osyris lanceolata

Abstract

Diabetes mellitus, is an endocrine disorder characterized by insulin resistance and glucose tolerance impairment with mainly two classifications (type 1 diabetes and type 2 diabetes mellitus). Type 2 diabetes mellitus has been reported to be the third leading natural cause of death in South Africa. While mainstream Euro-Western medical treatment and management of type 2 diabetes tend to be effective, there remain strong societal and cultural beliefs in using traditional medicine. It is believed that traditional medicines offer beneficial properties with regards to managing health conditions and have less side effects compared to pharmaceutical drugs. Gnidia canoargentea and Osyris lanceolata are herbal remedies used traditionally to treat and manage diabetes in North West, South Africa. The aim of this study was to evaluate the in vitro glucose regulating activity of the plant extracts. Extracts were prepared using hot water, methanol dichloromethane (DCM:MeOH) and chloroform partition which involved brewing and maceration techniques, respectively. The phytochemical constituents of the plant extracts were determined using thin layer chromatography and ultra-performance liquid chromatography. Furthermore, carbohydrate hydrolyzing enzyme (α-amylase and α-glucosidase) inhibition activity was monitored using a colorimetric method. Cytotoxicity against C2C12 mouse myoblast and Ea.hy926 cell lines were determined using the sulforhodamine B staining assay. The effect of the extracts on oxidative stress (reactive oxygen species) was assessed in Ea.hy926 cells using permeable fluorogenic dichlorodihydrofluorescein diacetate assay. Glucose uptake was assessed fluorometrically in C2C12 cells using 2-[N-(7-nitrobenz-2-oxa-1,3-diaxol-4-yl) amino]-2-deoxyglucose assay. Phytochemical analyses revealed the presence of kaempferol-3-rutinoside, rutin (flavonoid) and Pelargonidin-3-O-rutinoside and pelargonidin-3-O-glucoside (phenolics) compounds within the plant extracts. The aqueous extracts of O. lanceolata (IC50 = 13.79 μg/mL) possessed more potent scavenging activity than the DCM:MeOH extract (IC50 = 23,51 µg/mL), whereas for G. canoargentea, the DCM:MeOH (IC50 < 3.125 μg/mL) extract possessed more potent scavenging activity than the aqueous extract (IC50 = 3.55 µg/mL) . Moreover, the chloroform partition of O. lanceolata exhibited a more cytotoxic reaction (~35% cell cytotoxicity) in C2C12 cells, while the combination (O. lanceolate & G. canoargentea) plant extract had concentration dependent cytotoxicity in C2C12 cells; thus, indicating potential susceptibility of muscle tissue to the herbal remedy. Only the DCM:MeOH (IC50=35.48 µg/mL) and the chloroform (IC50=17.48 µg/mL) extract of G. canoargentea displayed inhibitory activity against α-amylase. The aqueous and organic plant extract of the selected plant species did exhibit in vitro oxidative stress regulation in Ea.Hy926 cells, however the activity was insignificant compared to the positive control Trolox. In the glucose uptake study the organic extract of G. canoargentea improved the glucose uptake by two-folds in C2C12 cells, however was not significant in comparison to the positive control insulin. The combination plant extract was unsuccessful in upregulating glucose uptake in C2C12 cells. Based on the results, it is evident that O. lanceolata and G. canoargentea extracts play part in reducing free-radical damage by scavenging radicals which play part in the disease progression and also inhibit the carbohydrate hydrolyzing enzyme, α-amylase. Furthermore, O. lanceolata and G. canoargentea offer minimal to no glucose regulation when extracted with water, neither does the combination of the plants offer improved activity and thus the herbal remedies studied do not offer beneficial attributes to glucose regulation when evaluated with the assays employed in this study.