Further isolation and identification of anti-diabetic principles from root bark of Myrianthus arboreus P. Beauv.: The ethyl acetate fraction contains bioactive phenolic compounds that improve liver cell glucose homeostasis.

ETHNOPHARMACOLOGICAL RELEVANCE: We recently reported that ethanol extract of Myrianthus arboreus P. Beauv. root bark demonstrated antidiabetic activity by modulating hepatocyte glucose homeostasis. This activity was associated significantly to the ethyl acetate (EAc) fraction. The current study sought to identify the active compounds responsible of the antidiabetic effect of M. arboreus in the EAc fraction using bioassay-directed sub-fractionation. MATERIALS AND METHODS: EAc fraction was sub-fractionated using Flash chromatography. Preparative HPLC was used to isolate the pure compounds. The structures of the isolated compounds were confirmed by analysis of NMR spectroscopic and mass spectrometric data. Hepatic (H4IIE, HepG2) cells were treated with maximum non-toxic concentrations of ethanol extract, its EAc fraction and isolated compounds thereof. Glucose-6-phosphatase (G6Pase) activity was measured using the glucose oxidase method. To measure glycogen synthase (GS) activity, radioactive assays were used. Phosphorylation of AMP-activated protein kinase (AMPK) and Glycogen Synthase Kinase-3 (GSK-3) were probed by Western blot. RESULTS: Six sub-fractions were obtained, and the antidiabetic activity was found in two sub-fractions (SFE1 and SFE2). For the first time, two known C-glycosylflavone regio-isomers, isoorientin (1) and orientin (2) were detected and isolated from M. arboreus plant, especially from SFE2 as well as protocatechuic acid, 3,4-dihydroxybenzaldehyde (4), and chlorogenic acid isolated from SFE1. The compounds 1, 2, 4 were determined to decrease the activity of G6Pase by increasing AMPK phosphorylation and to stimulate GS through GSK-3 phosphorylation. Isoorientin which is one of the main compounds of EAc fraction, expressed the strongest effect in all bioassays, similar to that of the EAc fraction. A significant and linear correlation was found between the phosphorylation of AMPK and the activity of G6Pase modulated by all samples (R2 = 0.54; p < 0.05). Similar to G6Pase assay, a correlation was determined between the capacity of M. arboreus extracts/fractions/compounds to stimulate GS activity and to phosphorylate GSK-3 (R2 = 0.57, p < 0.01). CONCLUSION: Results demonstrate that 1, 2, 4 are responsible, at least in part, for the antidiabetic activity of M. arboreus. These compounds can be used to ensure the quality and efficacy of M. arboreus antidiabetic preparations and standardize such preparations.

Root bark extracts of Myrianthus arboreus P. Beauv. (Cecropiaceae) exhibit anti-diabetic potential by modulating hepatocyte glucose homeostasis.

ETHNOPHARMACOLOGICAL RELEVANCE: Myrianthus arboreus P. Beauv. is a tropical tree used in African folk medicine, including for diabetes. However, little research has yet been conducted to support this ethnopharmacological use of this plant. The present study sought to determine the antidiabetic potential of root bark extracts through cell-based bioassays of liver and muscle glucose homeostasis. MATERIALS AND METHODS: Four extracts were obtained from crude root bark powder: 1 aqueous (AQ), 2 ethanol (EtOH), 3 alkaloid enriched (Alk) (obtained from methanol extract) and 4 dichloromethane (Dic) extracts. Moreover, extract 2 was further separated into two fractions: 2.1 ethyl acetate (EAc) and 2.2 hexane (Hex). To assess the antidiabetic activity of the plant extracts, inhibition of glucose-6-phosphatase (G6Pase), stimulation of glycogen synthase (GS) and modulation of glucose uptake were determined in cultured H4IIE and HepG2 hepatocytes as well as C2C12 myocytes, respectively. Phosphorylation of three kinases, AMP-activated protein kinase (AMPK), Akt and Glycogen Synthase Kinase-3 (GSK-3) were probed by Western blot. RESULTS: M. arboreus extracts/fractions did not stimulate glucose uptake in C2C12 cells albeit 2.2 (Hex) fraction showed a mild positive tendency. In contrast, extract 2 and its fractions as well as extract 3 were able to decrease hepatocyte G6Pase activity. Their effect on G6Pase activity involved both Akt and AMPK phosphorylation. No significant correlation was observed between activation of Akt and inhibition of G6Pase (R2 = 0.50 p < 0.14), whereas that between stimulation of AMPK and inhibition of G6Pase was statistically significant (R2 = 0.75 p < 0.05). On the other hand, extract 2, its fraction 2.2 and extract 3 were able to stimulate GS through GSK-3 phosphorylation. A high correlation was observed between the ability of M. arboreus extracts and fractions to phosphorylate GSK-3 and modulate GS activity (R2=0.81 p < 0.01). Extract 2 and its fraction 2.2 together with extract 3 were the only plant products to simultaneously and potently regulate G6Pase and GS, the key players of hepatic glucose homeostasis. CONCLUSION: Overall, these data support the traditional antidiabetic uses of the root bark of M. arboreus.