α-Glucosidase inhibitory and α-amylase inhibitory activities of compounds isolated from Uvaria rufa Blume

Abstract A new C-benzylated flavone, uvariaruflavone (1), along with 13 known compounds (2–14) were isolated from the twig and leaf extracts of Uvaria rufa Blume. Their structures were established by extensive spectroscopic methods. Flavones (5–8) and cyclohexene (10) were isolated from U. rufa for the first time. Most of the isolated compounds were evaluated for their α-glucosidase and α-amylase inhibitory activities. Of these, uvariaruflavone (1) showed the highest α-glucosidase inhibitory activity with an IC50 value of 44.3 µM, while ferrudiol (12) displayed the highest α-amylase inhibitory activity with an IC50 value of 73.5 µM. Graphical Abstract


Introduction
Uvaria rufa Blume (Annonaceae) is a woody climber tree, which is widely distributed over the tropical zones of Asia, Africa, and Australia (Tip-Pyang et al. 2011). The roots of this plant have been used in traditional medicine purposes as a stimulant for childbirth and to treat fever and kidney failure (Buncharoen et al. 2016). Previous phytochemical investigations of U. rufa resulted in the isolation and identification of several types of secondary metabolites, including flavonoids (Deepralard et al. 2009;Tip-Pyang et al. 2011), polyoxygenated cyclohexenes (Macabeo et al. 2012), alkaloids (Tip-Pyang et al. 2011, lignans (Nguyen et al. 2015), and terpenoids (Thang et al. 2014). Some of these compounds exhibited interesting biological activities, including antimicrobial (Macabeo et al. 2012), cytotoxicity (Nguyen et al. 2015), advanced glycation end-products (AGEs) (Deepralard et al. 2009), and a-glucosidase inhibitory (Hamid et al. 2015). As part of an ongoing investigation for anti-diabetes compounds from Thai medicinal plants (Meesakul et al. 2019;Suthiphasilp et al. 2019;Raksat et al. 2020;Phukhatmuen et al. 2021), the twig and leaf extracts of U. rufa were examined and these showed good a-glucosidase and a-amylase inhibitory activities with IC 50 values in the range of 62.6-87.1 lg/mL. These findings prompted us to further investigate their phytochemicals and anti-diabetes activities, including a-glucosidase and a-amylase inhibitory activities. This report describes the isolation and structure elucidation of a new C-benzylated flavone, uvariaruflavone (1), along with 13 known compounds (2-14) (Figure 1). The a-glucosidase and a-amylase inhibitory activities of some of the isolated compounds are also reported.
Uvariaruflavone (1)  . These NMR data suggested that compound 1 had a flavone skeleton (Meesakul et al. 2019). The HMBC correlations between the hydrogen-bonded hydroxy proton (d H 13.83) and C-4a (d C 105.6), C-5 (d C 157.1) and C-6 (d C 112.1) confirmed that the hydrogen-bonded hydroxy proton was located at C-5. The location of methoxy group at C-7 was confirmed by the HMBC correlations between OMe-7 (d H 4.01) and H-8 (d C 6.57) with C-7 (d C 163.2). The linkage of the hydroxybenzyl group at C-6 of flavone moiety was deduced from the 3 J HMBC of H-1 00 (d H 3.96) to C-5 (d C 157.1), C-6 (d C 112.1), C-7 (d C 163.2), C-2 00 (d C 125.6), C-3 00 (d C 154.7), and C-7 00 (d C 131.7) (Table S1). Furthermore, HMBC cross-peaks of H-8 (d H 6.57) to C-6 (d C 112.1) supported the position of the hydroxybenzyl moiety at C-6. Therefore, compound 1 has been identified as a new C-benzylated flavone and named uvariaruflavone. Compounds 1, 3-8, and 10-14 were evaluated for their a-glucosidase inhibitory activities. All tested compounds displayed a-glucosidase inhibitory activity with IC 50 values in the range of 44.3-646.7 mM. Of these, uvariaruflavone (1) showed the highest a-glucosidase inhibitory activity with IC 50 value of 44.3 mM, which is comparable to that of the positive control (acarbose, IC 50 ¼ 77.2 mM). The a-glucosidase inhibition of compounds 1, 5, 6, and 10-12 are reported for the first time in this study.
In the case of a-amylase inhibitory activity, compounds 1-4, 9, and 11-14 showed a-amylase inhibitory activity with an IC 50 values ranging from 73.5-265.5 mM and compounds 1 (uvariaruflavone) and 12 (ferrudiol) displayed the activity better than that of positive control (acarbose, IC 50 ¼ 103.4 mM) with the IC 50 values of 92.5 and 73.5 mM, respectively (Table S2). The a-amylase inhibition of compounds 1-4, 9, 11 and 12 are reported for the first time in this study.

Experimental
For the details of all experimental parts see the Supplementary material.

Conclusion
The phytochemical investigation of the twig and leaf of U. rufa afforded one new Cbenzylated flavone, uvariaruflavone (1), together with 13 known compounds (2-14). Flavones (5-8) and a cyclohexene (10) were isolated for the first time from this plant. The biological activities of tested compounds were evaluated using a-glucosidase and a-amylase inhibitory activities. C-benzylated flavone (1) exhibited the significant a-glucosidase and a-amylase inhibitory activities. An analysis of the structure-activity relationship suggested that C-hydroxybenzyl moiety at C-6 may enhances the a-glucosidase and a-amylase inhibitory activities of this family of flavonoids.