ORIGINAL ARTICLE Rec. Nat. Prod. 16:1 (2022) 84-91 A New 2,3-Dioxygenated Flavanone and Other Constituents from Dysosma difformis Bui Van Thanh 1, Nguyen Thi Van Anh 1, Chu Thi Thu Ha 1, Do Hoang Giang 2, Truong Thi Lien 2, Ninh Khac Thanh Tung 2, and Nguyen Tien Dat 2 1Institute of Ecology and Biological Resources, Vietnam Academy of Science and Technology, 18- Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam 2Center for Research and Technology Transfer, Vietnam Academy of Science and Technology, 18- Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam (Received March 16, 2021; Revised May 19, 2021; Accepted May 26, 2021) Abstract: A novel 2,3-dioxygenated flavanone, dysosmaflavanone (2), along with five known phenolic compounds including podophyllotoxin (1), podoverin A (3), kaempferol (4), 8,2′-diprenyl quercetin 3-methyl ether (5), and ethyl β-D-glucoside (6) were isolated from the roots of the plant Dysosma difformis. Their structures were elucidated via spectroscopic analysis. Besides podophyllotoxin and kaempferol, the rest of the compounds were isolated from the genus Dysosma for the first time. Dysosmaflavanone, which possesses a rare 2,3-dioxygenated skeleton, could be regarded as an important chemotaxonomic marker. The antioxidant and antidiabetic activities of the isolated compounds were evaluated. Keywords: Dysosma difformis; Berberidaceae; 2,3-dioxygenated flavanone; dysosmaflavanone. © 2021 ACG Publications. All rights reserved. 1. Introduction Dysosma difformis (Hemsl. & E.H.Wilson) T.H.Wang (syn: Podophyllum tonkinense Gagnep) (Berberidaceae) is widely used as a traditional medicine to treat sore throats, pimples, and snakebites [1]. Aglycone, aryltetralin lignan glycosides, and flavonols [2–5], which exhibit antiproliferative [6], anticancer [7], and angiogenesis-inhibiting [8] activities, are produced by species of the Dysosma genus. Podophyllotoxin (1), the major aryltetralin lignan in Dysosma plants, exhibits anticancer properties [9]. However, few investigations into the phytochemical constituents of D. difformis have been conducted [10, 11]. In the present study, we describe the isolation and structure of a new 2,3- dioxygenated flavanone (2) and five other known compounds isolated from the roots of D. difformis (Figure 1). Corresponding author: E-Mail: [email protected]; Phone: 84-24-37568422 The article was published by ACG Publications http://www.acgpubs.org/journal/records-of-natural-products January-February 2022 EISSN:1307-6167 DOI: http://doi.org/10.25135/rnp.256.21.03.2017 Available online June 03, 2021 85 Thanh et.al., Rec. Nat. Prod. (2022) 16:1 84-91 Figure 1. Structures of the isolated compounds 1-6 2. Materials and Methods 2.1. Plant Material The underground parts of Dysosma difformis were collected from Ha Giang province, Vietnam, in December 2018. The sample was identified by one of the authors (Bui Van Thanh) and a voucher specimen (No. Berb_HG_10) was deposited at the herbarium of the Institute of Ecology and Biological Resources. 2.2. General Procedures NMR experiments were performed on a Bruker AM500 FT-NMR spectrometer with tetramethylsilane (TMS) as an internal standard. Optical rotations were read on a JASCO P-2000 digital polarimeter. High-resolution mass spectra (ESI positive mode) were obtained with a Thermo LTQ Orbitrap XL mass spectrometer. Thin-layer chromatography (TLC) was performed on precoated silica gel 60 F254 plates (Merck, Germany), and spots were detected under UV illumination 254 nm and spraying with H2SO4 10% reagents followed by heating. Column chromatography (CC) was carried out using D101 resin (0.3-1.5 mm, Extrepure, China), silica gel 60 (70-230 mesh, Merck, Germany), or YMC RP-C18 resin (150 µm, YMC, Japan). Preparative HPLC was conducted on a Thermo Dionex Ultimate 3000 system, using a YMC-Pack ODS-A (5 μm, 250 x 20 mm i.d., YMC Co., Ltd., Kyoto, Japan) column, with a 5 mL/min flow rate. 2.3. Extraction and Isolation The air-dried powdered materials (1.3 kg) were extracted with ethanol (EtOH) (2 L × 4 times) in an ultrasonic bath for 30 min. The combined extracts were concentrated to obtain an ethanol crude residue (160.5 g), which was then loaded on a column (300 × 100 mm i.d.) filled with 1 kg of D101 resin. After the sample was completely adsorbed, the column was eluted by 10 L of deionized water, 86 New 2,3-dioxygenated flavanone from Dysosma difformis following by increasing concentrations of MeOH (25%, 50%, and 100%) in water, to obtain four fractions, F1-F4. Fraction F3 was subjected to a silica gel CC with gradient mixtures of n-hexane– acetone (10/1-1/1, v/v) to afford eleven subfractions (F3.1 – F3.11). Fraction F3.7 was separated using silica gel eluted with CH2Cl2–MeOH (70/1, v/v) to yield podophyllotoxin (1) (416.6 mg) and ten subfractions (F3.7.2-F3.7.11). Fraction F3.7.6 was purified by preparative HPLC (60 min, 40-75% MeOH in H2O) to afford the new flavanonol, dysosmaflavanone (2) (9.1 mg), and podoverin A (3) (40.1 mg). Kaempferol (4) (53.9 mg) was purified by recrystallizing the fraction F3.7.11 in MeOH- H2O (1/1, v/v). Fraction F3.6 was isolated using a silica gel column eluted with CH2Cl2–MeOH (60/1, v/v), following by a YMC RP-C18 column eluted with MeOH–water (3:1, v/v) to yield 8,2'-diprenyl quercetin 3-methyl ether (5) (7.5 mg). Fraction F2 was chromatographed on a silica gel column and eluted with CH2Cl2–MeOH–H2O (5/1/0.1, v/v/v) to yield ethyl β-D-glucoside (6) (75.3 mg). The purity of all isolated compounds was > 97% via HPLC (data not shown). 25 Dysosmaflavanone (2): Pale yellow powder; [α]D +0.15 (c 0.2, MeOH); HRMS (ESI positive) m/z + + 389.1218 ([M+H] , calcd for C20H21O8, 389.1236), 411.1070 ([M+H] , calcd for C20H20O8Na, + + + 411.1056), 799.2259 ([2M+Na] ), 373.0957 ([M-CH3] ), 343.0864 ([M-OC2H5] ). NMR data of the compound in CD3OD and DMSO-d6 were shown in Table 1. 2.4. DPPH Radical Scavenging Assay 2,2-Diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging activity was conducted by modified a previous method [12]. Briefly, 10 μL of each sample was mixed with 190 μL of DPPH (Sigma-Aldrich) in methanol before incubated at 37oC for 20 minutes. The absorbance was measured at 517 nm. Ascorbic acid was used as a positive control. 2.5. Superoxide Radical Scavenging Assay Superoxide radical scavenging activity was measured by a reported method with some modification [13]. In brief, 100 μL of the sample dissolved in DMSO was mixed with 300 μL of the phosphate buffer 50 mM pH 7.8, 200 μL xanthine 0.5 mM, 100 μL nitroblue tetrazolium 0.2 mM, and 100 μL of xanthine oxidase. The mixture was incubated at 37oC for 60 minutes then measured at the wavelength of 550 nm. (+)-catechin was used as a positive control. 2.6. Hydroxyl Radical Scavenging Assay Hydroxyl radical inhibition was evaluated by a modified method of the previously reported assay [13]. The mixture containing 50 μL of the test sample, 100 μL of the phosphate buffer 50 mM pH 7.8, 100 μL of deoxyribose 2.8 mM, and 100 μL of Fe(NH4)2(SO4)2 500 μM was incubated for 1 h at 37 oC. After adding 250 μL of trichloroacetic acid (10%, w/v) and 250 μL of thiobarbituric acid (1%, w/v), the reaction mixture was boiled for 15 min in a water bath. The color development was measured at 532 nm. (+)-catechin was used as a positive control. 2.7. α-Glucosidase Inhibition Assay The α-glucosidase enzyme inhibition activity was assessed by modifying a previous method [14]. 50 μL of the sample solution in methanol was mixed with 100 μL of α-glucosidase (G0660- 750UN, Sigma-Aldrich) 0.5 U/mL and 100 μL of phosphate buffer 100 mM (pH 6.8-7.0). After 10 min of pre-incubation at room temperature, 50 μL of 5 mM p-nitrophenyl-α-D-glucopyranoside solution was added, and the solution was incubated at 37oC for 30 min. The absorbance of released 4- nitrophenol was measured at 405 nm by using a microplate reader. Acarbose was used as a positive control. 2.8. α-Amylase Inhibition Assay 87 Thanh et.al., Rec. Nat. Prod. (2022) 16:1 84-91 The α-amylase enzyme inhibitory activity was evaluated by the previously reported method [14] with some modifications. The substrate was prepared by boiling 80 mg of potato starch in 4 mL phosphate buffer (pH 7.0) for 5 min, then it was left at room temperature to cool down. Next, 100 μL of the sample solution was mixed with 50 μL of the substrate and 30 mL of 100 mM phosphate buffer (pH 7.0). After 5 min of pre-incubation, 50 µg/mL α-amylase (A8220, Sigma-Aldrich) solution was added, and the solution was incubated at 37oC for 15 min. The reaction was stopped by adding 50 μL of glacial acetic acid, then 50 mL iodine solution was added. The absorbances were measured at 650 nm by using a microplate reader. Acarbose was used as a positive control. 3. Results and Discussion 3.1. Structure Elucidation Compound 2 was obtained as a pale-yellow powder, which generated [M+H]+ and [M+Na]+ ions with mass-to-charge ratio (m/z) values of 389.1218 and 411.1070, respectively, when analysed by high resolution electrospray ionization mass spectrometry; this suggested that the compound had a + molecular formula of C20H20O8. Fragment ions with m/z 373.0957 [M-CH3] and 343.0864 [M- + 1 OC2H5] revealed the presence of an ethoxy group in the compound 2 structure.