α-Glucosidase inhibitors from the bark of Mangifera mekongensis

Background Mangifera mekongensis (Anacardiaceae) is cultivated for its edible fruit and has been used in traditional Vietnamese medicine for its anti-aging properties and for treating diabetes, vermifuge, and dysentery. As part of a search for biologically active compounds with reduction of the rate of glucose absorption, a screening has been initiated to evaluate natural product extracts for the inhibition of enzyme α-glucosidase. A n-hexane extract of the bark of M. mekongensis showed strong α-glucosidase inhibitory activity with IC50 value of 1.71 µg/mL. Thus, the constituents of this plant were examined. Results Two new steroids named mekongsterol A (1) and mekongsterol B (2), were isolated from the n-hexane extract of the bark of M. mekongensis (Anacardiaceae), together with seven known compounds (3–9). Their chemical structures were elucidated on the basis of spectroscopic data. All compounds possessed significant α-glucosidase inhibitory activity in a concentration-dependent manner, except for 3 and 4. Compounds 1, 2, 5–9 showed more potent inhibitory activity, with IC50 values ranging from 1.2 to 112.0 µM, than that of a positive control acarbose (IC50, 214.5 µM). Conclusions These results suggested that the traditional use of the bark of M. mekongensis for the treatment of diabetes diseases in Vietnam may be attributable to the α-glucosidase inhibitory activity of its steroid and cycloartane constituents. Electronic supplementary material The online version of this article (doi:10.1186/s13065-016-0193-9) contains supplementary material, which is available to authorized users.


Background
Mangifera mekongensis (Anacardiaceae), commonly known as mango, is widely distributed in tropical and subtropical regions of Asia. In Vietnam, M. mekongensis is called as "Xoai Thanh Ca", and this plant is cultivated for its edible fruit and has been used in traditional Vietnamese medicine for treating anti-aging, diabetes, vermifuge, dysentery [1,2]. A research for biologically active compounds with reduction of the rate of glucose absorption, a screening has been initiated to evaluate natural product extracts for the inhibition of enzyme α-glucosidase. It is effective in controlling postprandial hyperglycaemia and prevents complications associated with type-II diabetes, such as microvascular (i.e., retinal, renal, and possibly neuropathic), macrovascular (i.e., coronary and peripheral vascular), and neuropathic (i.e., autonomic and peripheral) complications [3,4]. Previously, we reported that the methanolic extracts of Embelia ribes, Oroxylum indicum, and Artocarpus altilis exhibited significant inhibitory activity on α-glucosidase [5][6][7][8]. In a part of our continued research on the screening of medicinal plants of different origins, we also found that the n-hexane extract of the bark of M. mekongensis showed strong α-glucosidase inhibitory activity with IC 50 value of 1.71 µg/mL. Thus, we carried out the bioactivity-guided fractionation of n-hexane extract of this plant and isolated two new steroids, mekongsterols A (1) and B (2), together with seven known compounds (3-9) (Fig. 1). In this paper, we describe the isolation and structural elucidation of these compounds by spectroscopic methods as well as their α-glucosidase inhibitory activity.

Biological assay
Among three fractions extracted from the bark of M. Mekongensis, n-hexane fraction showed α-glucosidase inhibitory activity with IC 50 value of 17.1 µg/mL. This fraction was subjected to silica gel column chromatography to yield twelve fractions. All these fractions possessed inhibitory activity, with IC 50 values ranging from 1.9 to 69.3 μg/mL ( Table 2).
The isolated compounds were tested for their α-glucosidase inhibitory activity ( Table 3). The assay was carried out at various concentrations ranging from 1 to 250 µM. Compounds 1, 2, 5-9 possessed significant α-glucosidase inhibitory activity in a concentrationdependent manner, and showed more potent inhibitory activity, with IC 50 values ranging from 1.2 to 112.0 μM, than that of a positive control acarbose (IC 50 , 214.5 μM), which is currently used clinically in combination with either diet or anti-diabetic agents to control blood glucose level of patients [14]. Among isolated compounds, the sterol compounds (1-5) with saturated fatty ester chain or sugar group at C-3 (1, 2, and 5) showed potent α-glucosidase inhibitory activity, while the compounds with hydroxyl or ketone grop at C-3 (3 and 4) were inactive. On the other hand, all isolated cycloartane triterpenes (6-9) showed strong α-glucosidase inhibitory activity, however, their structure-activity relationships have not been discussed yet due to the limited number of compounds. These results indicated that the strong active compounds such as mekongsterol B (2; IC 50 , 2.5 μM) and magiferonic acid (8; IC 50 , 1.2 μM) can potentially be developed as a novel natural nutraceutical to decrease the blood glucose level because of their strong α-glucosidase inhibitory activity.

General experimental procedures
The

α-Glucosidase inhibitory assay
The inhibitory activity of α-glucosidase was determined according to the modified method of Kim et al. [15]. 3 mM p-nitrophenyl-α-d-glucopyranoside (25 μL) and 0.2 U/mL α-glucosidase (25 μL) in 0.01 M phosphate buffer (pH = 7.0) were added to the sample solution (625 μL) to start the reaction. Each reaction was carried out at 37 °C for 30 min and stopped by adding 0.1 M Na 2 CO 3 (375 μL). Enzymatic activity was quantified by measuring absorbance at 401 nm. One unit of α-glucosidase activity was defined as amount of enzyme liberating p-nitrophenol (1.0 μM) per min. The IC 50 value was defined as the concentration of α-glucosidase inhibitor that inhibited 50 % of α-glucosidase activity. Acarbose, a known α-glucosidase inhibitor, was used as positive control.

Conclusions
In this paper, we have reported two new compounds, mekongsterol A (1) and mekongsterol B (2), together with seven known compounds isolated from the bark of M. mekongensis. Seven compounds possessed α-glucosidase inhibitory activity. This is the first report on α-glucosidase inhibitory activity of the bark of this plant. These results suggested that the traditional use of the bark of M. mekongensis for the treatment of diabetes diseases in Vietnam may be attributable to the α-glucosidase inhibitory activity of its steroid and cycloartane constituents.