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A New 2,3-Dioxygenated Flavanone and Other Constituents from Dysosma difformis

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.


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.

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 37 o C for 20 minutes. The absorbance was measured at 517 nm. Ascorbic acid was used as a positive control.

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 37 o C for 60 minutes then measured at the wavelength of 550 nm. (+)-catechin was used as a positive control.

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 o C. 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.

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 37 o C for 30 min. The absorbance of released 4nitrophenol was measured at 405 nm by using a microplate reader. Acarbose was used as a positive control.

Structure Elucidation
The near-zero optical rotation and lack of significant Cotton effect on the compound 2 circular dichroism spectrum (data not shown) suggested that the mixture was racemic. To observe the -OH groups of compound 2 in more detail, one-dimensional and two-dimensional NMR experiments were conducted using deuterated dimethyl sulfoxide. Nuclear Overhauser effect spectroscopy revealed correlations between the 3-OH proton and H-1'', and between H-1''' and H-3', while there was no correlation between H-1'' and H-1''' (Figures 2 and S12); this suggested an erythro-orientation between the ethoxy group at C-2 and the hydroxyl group at C-3 of compound 2. Thus, we determined compound 2 to be (2RS,3SR)-2-ethoxy-3,5,7-trihydroxy-2-(4-hydroxyphenyl)-3-(2-oxopropyl)chroman-4-one and named the compound "dysosmaflavanone". Next, we performed high-performance liquid chromatography (HPLC) to further examine compound 2. Extractions were performed on the roots of D. difformis with methanol and acetone, then analysed with an HPLC-diode array detector system. Compound 2 was not detected in the methanol or acetone extracts (data not shown), suggesting that the compound was an artefact of the ethanol extraction process. Recently, similar 2,3-dioxygenated flavanones were discovered to be artificial products generated during flavonoid extraction from plant materials [16]. Furthermore, flavonoids that are oxygenated at both the C-2 and C-3 positions are rarely found in nature [15,17]; thus, the origin of compound 2 must be investigated further.

Biological Activities
In this study, the isolated compounds were evaluated for their antioxidant capacity via scavenging 2,2-diphenyl-1-picrylhydrazyl (DPPH), hydroxyl, and superoxide radicals. Their antidiabetic activity was also tested on digestive enzymes α-glucosidase and α-amylase. As shown in Table 2, the five phenolic compounds exhibited higher DPPH scavenging activity than ascorbic acid, while showing weaker hydroxyl radical and superoxide radical inhibition compared to catechin. The inhibitory activities of all isolated compounds against α-glucosidase and α-amylase were evaluated in comparison with acarbose, an antidiabetic agent. Dysosmaflavanone inhibited α-glucosidase and α-amylase activities at lower doses than acarbose. Among the six compounds, kaempferol showed the highest antioxidant and while 8,2'-diprenyl quercetin 3-methyl ether exhibited strongest inhibition against digestive enzymes, while ethyl β-D-glucoside did not exhibit any antioxidant or antidiabetic activity. The new dysosmaflavanone showed similar effects with kaempferol in inhibiting αglucosidase and α-amylase.

Chemotaxonomy
In the present investigation, we isolated and identified six metabolites from the roots of D. difformis (Berberidaceae), including one aryltetralin lignan, three flavonols, a novel 2,3-dioxygenated flavanone, and an alkyl glycoside. Our findings highlight the need for further chemotaxonomic studies of the Dysosma genus and the Berberidaceae family. Podophyllotoxin (1) has previously been isolated from the Dysosma [3], Sinopodophyllum [24], Podophyllum [25], and Diphylleia [18] genera of the Berberidaceae family. However, this compound is not present in other well-known genera in the same family, such as Epimedium, Vancouveria, Mahonia, and Berberis. Thus, podophyllotoxin might be a valuable marker for distinguishing between plants within the Berberidaceae family. Moreover, we isolated podoverin A (3), 8,2'-diprenyl quercetin 3-methyl ether (5), and ethyl β-D-glucoside (6) from a Dysosma species for the first time. The discovery of these compounds has extended our phytochemical knowledge of the genus Dysosma. While podoverin A (3) is present Sinopodophyllum [26] and Podophyllum [19] species, 8,2'-diprenyl quercetin 3-methyl ether (5) has only been found in Sinopodophyllum [21]; this suggests a close relationship between the Dysosma and Sinopodophyllum genera. A previous phylogenetic study of Berberidaceae found that the Dysosma genus was more closely related to Sinopodophyllum than to Podophyllum, despite their similar morphology [27]. Therefore, compound 5 could be an important chemotaxonomic marker, enabling the identification of these genera.