Flavonoids from the fruits of Phyllanthus acidus (L.) Skeels with anti-α-glucosidase activity

Abstract Eleven flavonoids including one new flavonol glycoside, quercetin-3-O-(2-α-L-rhamnopyranosyl)-β-D-glucuronopyranosyl methyl ester (1), were isolated for the first time from the fruits of Phyllanthus acidus (L.) Skeels (Phyllanthaceae). Their structures were determined by extensive spectroscopic data. The known flavonoids, quercetin-3-O-β-D-glucuronide methyl ester (3), quercetin-3-O-(2''-α-L-rhamnopyranosyl-6''-O-α-L-rhamno pyranosyl)-β-D-glucopyranoside (5), myricetin (9), and 6-methoxy-naringenin (11) were isolated for the first time from the genus Phyllanthus. Flavonoids 4, 6 and 9 (IC50 = 6.01, 6.32, and 7.84 μM, respectively) showed stronger α-glucosidase inhibitory activities than the positive control, acarbose (IC50 = 306.45 μM). The fruits of P. acidus might be further developed as an anti-diabetic food supplement. Graphical Abstract


Introduction
Phyllanthus acidus (L.) Skeels (Phyllanthaceae), an evergreen tree growing mainly in tropical countries of the world, e.g. Thailand, Vietnam, Myanmar, Laos, and Malaysia, has been introduced and cultivated in Xishuangbanna and Yuanjiang areas of Yunnan Province, China, for a few decades. It has been used widely to treat various diseases, such as hypertension, asthma, diabetes, dermatitis, fever, and smallpox, by the local people of its distributing area (Thyagarajan et al. 1988;Jain and Singhai 2011;Tram et al. 2017). So far, studies on P. acidus have focused mainly on its roots, stems, barks and leaves, resulting in the identification of a series of sesquiterpenes, diterpenes, triterpenes and flavonoids. The norbisabolane sesquiterpenes, e.g. phyllanthacidoids A-D, F-I and M from the roots, displayed potential anti-HBV activities, with IC 50 values of 0.8-36 lmol/L against HBV surface antigen (HBsAg) and HBV excreted antigen (HBeAg) (Lv et al. 2014), and the rare dichapetalins (pacidusins A-D) isolated from the leaves exhibited cytotoxicities against five human cancer cell lines (HL-60, SMMC-7721, A-549, MCF-7, and SW-480) with IC 50 values ranging from 3.38 to 22.38 lM (Geng et al. 2021). The fruits are edible, and displayed antioxidant, antibacterial, and blood pressure reduction activities (Melendez and Capriles 2006;Das and De 2013). Moreover, the fruit juice was reported to have the highest inhibitory rate (95.37%) on a-glucosidase among 40 different fruit juices (Sulaiman and Ooi 2014). Only one compound, sitosterol-3-O-b-D-glucoside, was reported to be isolated from the fruits of P. acidus (Khatun et al. 2012). Our detailed phytochemical study led to the isolation and identification of 11 flavonoids (1-11) from the fruits of P. acidus for the first time. Most of the isolates 2-11 were evaluated for their inhibitory effects on a-glucosidase.
Compound 1 was isolated as a yellow amorphous powder and possessed a molecular formula C 28 Hz, 2.0 Hz, H-6 0 ), 7.54 (d, J ¼ 2.0 Hz, H-2 0 ) and 6.86 (d, J ¼ 8.3 Hz, H-5 0 ) arising from the A and B rings of a quercetin aglycone, and two anomeric protons at d H 5.75 (d, J ¼ 5.8 Hz, H-1 0 ') and 5.21 (brs, H-1 0 ''). The 13 C-NMR and DEPT spectra of 1 displayed 28 carbon signals including 11 quarternary carbons, 15 methines, and two methyls, assignable to a quercetin aglycone, one rhamnopyranosyl and one glucuronopyranosyl moieties. The presence of an a-L-rhamnopyranosyl and a b-D-glucuronopyranosyl unit were confirmed by the detailed analysis of 1 D and 2 D NMR (HSQC, HMBC) spectral data. In which, HMBC correlations of the glucuronosyl H-2 0 ' (d H 3.69) with rhamnosyl C-1 0 '' (d c 102.7), and glucuronosyl H-1 0 ' (d H 5.75) with aglycone C-3 (d c 134.2) were observed, indicated the sugar linkage and position as depicted. The above 1 H and 13 C-NMR data of 1 were quite similar to those of 2, except for an additional methoxy group d H 3.64 (3H, s) appeared in 1. The location of the methoxy group was further confirmed by the HMBC correlation of methoxy protons at d H 3.64 (6 0 '-OCH 3 ) with glucuronopyranosyl carboxyl carbon at d c 170.8 (C-6 0 ') ( Figure S1). Therefore, compound 1 was determined to be quercetin-3-O-(2-a-L-rhamnopyranosyl)-b-D-glucuronopyranosyl methyl ester. Although artificial products with methyl esters are easy to appear (Venditti 2020), all experiments were carried out carefully at room temperature. Since similar methyl ester of flavonoid glycoside was also reported from the leaves of P. acidus (Tram et al. 2017), it suggested that compound 1 should be a natural product.
Compounds 2-11 were evaluated for their inhibitory effects on a-glucosidase. As shown in Table S1, compounds 4 (IC 50 ¼ 6.01 lM), 6 (IC 50 ¼ 6.32 lM) and 9 (IC 50 ¼ 7.84 lM) showed stronger a-glucosidase inhibitory activities than the positive control, acarbose (IC 50 ¼ 306.45 lM). While, 3, 8, 10 and 11 showed only weak activities with inhibition rates of 14.2%, 24.3%, 20. 9% and 9.4%, respectively, at a concentration of 50 mM. On the basis of the above results, quercetin and myricetin derivatives, 4, 6, and 9, with ortho di-or tri-hydroxy B-ring possessed stronger a-glucosidase inhibitory activity than kaempferol derivatives (7 and 8) with only one hydroxy group at B-ring. Moreover, it suggested that O-glycosylation at flavonoid C-3 had negative effect on a-glucosidase inhibitory activity, and the presence of a bulky substituents (sugar) at C-3 position will reduce the activity probably for the steric hindrance. The results of this study were in good agreement with previously published research on structure-activity relationship of flavonoids against a-glucosidase activity (Kokanova-Nedialkova et al. 2021).

Plant material
The fruits of P. acidus were collected from Yuanjiang country (geographical coordinates: 102 E, 23.

a-Glucosidase inhibition activities assay
The a-glucosidase inhibition activities assay was referred to the method of literature (Ooi et al. 2011) with some modifications. In briefly, the sample (gradient dilution at the final concentration of 50 lM), enzyme solution (final concentration of 0.025 U/mL), buffer and substrate (final concentration of 1 mM) were added into 96-well enzyme standard plate, fully mixed, and set three well repeat. At the same time, a blank control without tested samples, and a positive control (acarbose) were set. After the reaction mixture was incubated at 37 C for 50 min, the OD value at 405 nm was measured by microplate reader. The a-glucosidase inhibition rate was calculated as the following equation.
The inhibition rate ð%Þ ¼ OD Blank hole À OD Sample hole ð Þ =OD Blank hole Â 100% The IC 50 value of each compound was calculated with Reed and Muench's method (Reed and Muench 1938).

Conclusions
In summary, 11 flavonoids including one new flavonol glycoside, quercetin-3-O-(2-a-Lrhamnopyranosyl)-b-D-glucuronopyranosyl methyl ester (1), were isolated for the first time from the fruits of P. acidus. The known compounds included five quercetin (2-6), two kaempferol (7-8) and two myricetin (9-10) derivatives, as well as one dihydroflavone, 11. Four of them, 3, 5, 9 and 11 were isolated for the first time from the genus Phyllanthus. Compounds 4, 6 and 9 (IC 50 ¼ 6.01, 6.32, 7.84 lM, respectively) displayed stronger a-glucosidase inhibitory activities than the positive control, acarbose (IC 50 ¼ 306.45 lM). These flavonoids may play an important role for the anti-diabetic activity of the plant. The results suggested that the fruits of P. acidus might be further developed as an anti-diabetic food supplement.

Disclosure statement
No potential conflict of interest was reported by the authors.

Supporting information available
Funding This work was supported by the National Natural Science Foundation of China (82074124) and the Key Project of Basic Research Plan of Yunnan Province, China (202001AS070017). We are grateful to the staffs of the analytical and bioactivity screening group at the State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, for measuring the spectroscopic data and antidiabetic activity.