A new epicatechin glucopyranoside derivative from Styrax suberifolius

Abstract A new derivative of epicatechin glucopyranoside, (2R,3R)-3,7,4'-trihydroxy-5,3'-dimethoxyflavan 7-O-β-d-glucopyranoside (1), together with three mononuclear phenolic acid esters, methyl orsellinate (2), ethyl orsellinate (3) and methyl β-orcinolcarboxylate (4) were isolated from the bark of Styrax suberifolius. The structures of 1–4 were determined on the basis of extensive analysis of NMR and MS spectra combined with chemical hydrolysis. The antifungal activities of the isolated compounds against three plant pathogenic fungi, Alternaria solani, Fusarium oxysporum and Phomopsis cytospore were evaluated using radial growth inhibition assay. Compounds 2, 3 and 4 exerted selective inhibitory activities against the tested fungi. Among of them, methyl β-orcinolcarboxylate (4) exhibited obvious inhibitory effect against P. cytospore, with an inhibition rate of 86.72% at 100 μg/ml. Graphical Abstract


Introduction
Styrax Linn. is a genus of plants belonging to the family Styracaceae. The genus consists of more than 130 species mainly occurring in eastern Asia and southeastern America. Styrax suberifolius Hook, a tall tree, is distributed in south of the Yangtze River in China and Vietnam that displays remarkable morphological variation throughout its range (Editorial Committee of Chinese flora 1987). Many species of Styrax have been used as traditional folk medicine worldwide (Chinese Pharmacopoeia Commission 2010) and about fifteen Styrax species have been phytochemically investigated (Liu et al. 2018). Previous phytochemical studies on this genus revealed various biologically active secondary metabolites including lignan derivatives, nor-neolignans, phenylpropanoids, phenolic acids, lipids, saponins and triterpenes (Pauletti et al. 2006;Bertanha et al. 2012;Timmers et al. 2015;Pazar and Akg€ ul 2015;Scheuba et al. 2017). However, there have been very few studies about the chemical constituents of S. suberifolius in the literatures. Very recently, we have successfully isolated and identified seventeen phenylpropanoids from the bark of S. suberifolius, which represent the first phytochemical investigation of the species (Liu et al. 2018). In continuation of our phytochemical studies on this plant, herein, we report the isolation and structure identification of a new (-)-epicatechin glucopyranoside derivative (1) and three known mononuclear phenolic acid esters (2-4), as well as the antifungal activity of these compounds.
In the course of this research, it was noticed that CH 3 OH-soluble extracts of S. suberifolius are resistant to plant pathogenic fungi. The antifungal activities of 1À4 against A. solani, F. oxysporum and P. cytospore were evaluated using radial growth inhibition assay at 100 lg/ml. As shown in Table 1, compound 1 has no inhibitory effect against the three fungi, but compounds 2, 3 and 4 exerted selective inhibitory activities. Among which, compounds 2 and 3 showed moderate activity against A. solani and F. oxysporum, while 4 against F. oxysporum and P. cytospore. Methyl b-orcinolcarboxylate (4) exhibited obvious inhibitory effect against P. cytospore with an inhibition rate of 86.72%. It is noteworthy that the compounds 2À4 are a class of antimicrobial mononuclear phenolic acid esters, which are frequently isolated from lichen species (Moln ar and Farkas 2010). Previous reports have demonstrated that methyl orsellinate (2) and methyl-b-orcinolcarboxylate (4) showed significant antifungal activity against Trichophyton longifusis, Microsporum canis, Aspergillus flavus and Fusarium solani at a concentration of 200 lg/ml (Thadhani et al. 2012). The present study extends the antifungal activity of metabolites 2À4 to plant pathogenic fungi A. solani, F. oxysporum and P. cytospore.

Plant material
Bark from trunks of S. suberifolius were collected in western Hunan province, China, in December 2012, which identified by Prof. Dai-gui Zhang (Key laboratory of Plant Resources Conservation and Utilization, Jishou University). A voucher specimen (zdg20121214) was deposited in Hunan Agricultural University.

Acid hydrolysis of 1 and identification of its aglycone and D-Glucose
Compound 1 (11.0 mg) in 50 ml of 1 M HCl (H 2 O/1,4-dioxane, 1:1) was heated under reflux for 6 h. After removal of the solvent, the residue was partitioned between CHCl 3 and H 2 O. The CHCl 3 -soluble portion was evaporated and subjected to ODS column chromatography using 90% CH 3 OH to obtain the aglycone 1a (4.5 mg). Aglycone (1a) gave as a yellow powder, its optical rotation [a] 25 D À69.9 (c 0.2, CH 3 OH) as well as 1 H and 13 C NMR (CD 3 OD, 600 MHz) data are in accordance with those reported in the previous papers (Joo et al. 2014).
The water layer was neutralized with 5% NaOH and desalted using a Sephadex LH-20 column (CH 3 OH) to afford a sugar residue (3.5 mg). The sugar was confirmed to be

Antifungal assay
Alternaria solani, Fusarium oxysporum and Phomopsis cytospore were obtained from Hunan Provincial Key Laboratory for Biology and Control of Plant Diseases and Insect Pests. The three fungi were incubated on a slant that had 10 ml of a potato dextrose agar (PDA) medium for 7 days at 28 C in dark. The effects of compounds 1 2 4 on colony growth of the three plant pathogenic fungi were evaluated according to the procedure described by Vargas-Arispuro et al. (2005). Pure compounds were dissolved in 500 ll of acetone and 10 ll of Twain-80 was added as emulsifier. Then diluted with sterile water to 5 ml. These solutions were incorporated into 5 ml of PDA media at final concentrations of 100 lg/ml. Three plates of solid PDA media containing the compounds at the specified concentrations were centrally point-inoculated with mushroom cakes from 7-day-old cultures of A. solani, F. oxysporum and P. cytospore. The control plates were included as blanks that contained solvent without the compounds. Cultures were incubated in the dark at 28 C. The percentage of growth inhibition was obtained by comparing the experimental with the control, which was considered to be 100% growth. Experimental set was run in triplicate, and the mean colony diameter and standard deviation (S.D.) of the mean values were calculated.

Conclusions
In this study, we identified a new derivative of epicatechin glucopyranoside and three known mononuclear phenolic acid esters in the bark of S. suberifolius. Methyl orsellinate (2), ethyl orsellinate (3) and methyl b-orcinolcarboxylate (4) have selective inhibitory activities against plant pathogenic fungi. Compound 4 exhibited obvious inhibitory effect against P. cytospore at 100 lg/ml, with an inhibition rate of 86.72%. Therefore, these compounds may have a biopesticidal potential as control agents for plant pathogenic fungi.

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

Funding
This work was financially supported by the Hunan Provincial Natural Science Foundation (grant number 2017JJ3114) and the Hunan Province science and technology plan project (grant number 2018NK2094).