| Summary: | Based on ethnobotanical information collected from Cuban diabetic patients in Cuba, medicinal plants such as Allophylus cominia (L.) Sw. (A. cominia) was identified as a possible source of new drugs that could be used for the treatment of type 2 diabetes mellitus. Type 2 diabetes mellitus or non-insulin dependent diabetes mellitus (T2-DM) is a chronic disease, and when associated with obesity (a condition known as diabesity) leads to an increase in the risk of a number of comorbidities, e.g. cardiovascular, kidney and liver diseases. A. cominia is a Cuban plant used traditionally by diabetic patients for the treatment of their diabetes symptoms. Preliminary studies of its leaves (Veliz et al., 2003; Veliz et al., 2005 Sanchez et al., 2014) have shown potential anti-diabetic activity and it is therefore being further investigated in the search for a novel, nontoxic, and efficacious anti-diabetic agent. The present project investigated the in vitro hypo-glycaemic activity of A. comin ia extracts. Chemical characterisation of the extracts was carried out using different phytochemical methods. Fatty acids, tannins, pheophytins (A and B), and a mixture of flavonoids were detected. The identified flavonoids (42.1 mg) were mearnsitrin, quercitrin, quercetin-3-alloside, and naringenin-7-glucoside. Some of these compounds have been reported in the literature as potent hypo-glycaemic agents. Separation of the mixture of quercitrin and mearnsitrin was carried out by high performance liquid chromatography using an amino column. Extracts from A. cominia were tested for their ability to inhibit the activity of four enzymes. DPPIV plays an essential role in glucose metabolism. PTP1B is important in inhibiting downstream signalling of the insulin and leptin receptors. Alpha-glucosidase is one of the enzymes responsible for the breakdown of carbohydrates into monosaccharides, and alpha-amylase breaks down large, insoluble starch molecules into soluble starches, producing successively smaller starches and ultimately, maltose. The flavonoids produced a concentration-dependent inhibition against DPPIV with a Ki value of 2.6 « 0.2 (So(Bg/ml. The flavonoids fraction from A. cominia revealed a competitive inhibition using DPPIV substrate comparable to the inhibition by the commercial (P32/98) inhibitor. In addition, PTP1B enzyme was 100 « 5% inhibited by the flavonoid mixture and 65 « 2% inhibited by pheophytin A and 61 « 1% inhibition by pheophytin B at 30 (So(Bg/ml respectively. The flavonoid mixture elicited a significant concentration-dependent inhibition against PTP1B with a Ki value of 3.2 « 0.09 (So(Bg/ml, as well as with pheophytin A with a Ki value of 0.64 « 0.05 (So(Bg/ml and pheophytin B with a Ki value of 0.88 « 0.03 (So(Bg/ml; both were lower than that of TFMS inhibitor, with a Ki value of 1.1 « 0.03 (So(Bg/ml. Both flavonoid and pheophytin A extracts from A. cominia revealed a competitive inhibition of PTP1B enzyme using DiFMUP as substrate . Competitive inhibition was also shown with TFMS inhibitor. On (Sa(B-glucosidase enzyme, a 79 « 1% inhibition was produced by the flavonoid mixture at 30 (So(Bg/ml. The flavonoid fraction from A. cominia showed a concentration-dependent pattern against (Sa(B-glucosidase, with a Ki value of 1.7 « 0.5 (So(Bg/ml that was lower than that of acarbose inhibitor (190 « 0.5 (So(Bg/ml). These extracts have shown a competitive inhibition using 4-nitrophenyl-glucopyranoside as substrate. Acarbose also produced a competitive inhibition against (Sa(B-glucosidase. No significant effect was found with any of the extracts from A. cominia at 30 (So(Bg/ml against (Sa(B-amylase enzyme. After separation of the flavonoids, mearnsitrin and quercitrin did not produce any effect (at 30 (So(Bg/ml) on any of the enzyme activities (DPPIV, PTP1B, (Sa(B-glucosidase and (Sa(B-amylase). Quercitrin and mearnsitrin were active only in synergy. On a glucose uptake assay using HepG2 cells, the crude methanolic extract from A. cominia enhanced insulin activity by increasing 2-NBDG uptake by two-fold (2-NBDG is a fluorescently-tagged glucose derivative). The 2-deoxy-D-glucose uptake by differentiated 3T3-L1 cell line showed an increase of the glucose uptake in the presence of 100 (So(Bg/ml of flavonoids by enhancing insulin activity (100 nM), whereas the uptake was increased in the presence of 100 (So(Bg/ml of pheophytin A without enhancing insulin activity. The effect of different compounds from A. cominia on 3T3-L1 cell differentiation was also confirmed by quantifying GLUT4 transporters in the pre-treated cells with flavonoids and pheophytin A. GLUT4 transporters in th e pre-treated cells were similar to those of the differentiated normal 3T3-L1 adipocytes. 2-NBDG glucose uptake assay was also performed using L6 myotubes. The uptake was significantly increased by two-fold in the presence of 100 nM insulin, and by four-fold in the presence of both 100 nM insulin and 100 (So(Bg/ml flavonoids. A significant increase was also shown in the presence of 100 (So(Bg/ml pheophytin A and 100 nM insulin with a 10-fold increase (P<0.05) of glucose uptake by L6 cells. An increase of 2-NBDG uptake by L6 cells was shown in the presence of flavonoids and pheophytin A in addition to 100 nM insulin. Both flavonoid and pheophytin extracts (100 (So(Bg/ml) blocked the differentiation of 3T3-L1 fibroblasts into adipocytes by decreasing the fat accumulation by two-fold (more than the TNF-(Sa (Binhibition at 10 ng/ml). A significant difference was shown (P<0.05) compared to the control. Troglitazone significantly enhanced 3T3-L1 differentiation by two-fold. Exposing 3T3-L1 cells to both extracts from the third day of the differentiation induction did not alter the adipogenesis. Exposing 3T3-L1 adipocytes to the extracts from A. cominia containing flavonoids and pheophytin A showed a significant decrease in the fat accumulation after five days of incubation with the extracts (P<0.05). However, no fat accumulation was observed after withdrawal of the extracts from the cell growth medium. These compounds may be responsible for the pharmacological effects observed in experimental diabetic models in Cuba. Therefore, all these results strongly suggest that this plant could be a new and promising candidate for treating diabesity with natural sources.
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