A new methylene bisflavan-3-ol from the branches and leaves of Potentilla fruticosa

Abstract Dasiphora fruticosa L. (Rosaceae), also known as Potentilla fruticosa L. (syn.), is a hardy deciduous shrub widely distributed in the north temperate regions of the world. Three methylene bisflavan-3-ols (1-3), together with a procyanidin dimer, (-)-afzelechin-(4α→8)-(-)-afzelechin (4) were isolated for the first time from the branches and leaves of the titled plant, in addition to 11 known compounds (5-15). Their structures were elucidated by means of extensive spectroscopic analysis and by comparison with data reported in the literatures. Methylene 6,8-bis(7-O-glucosyl) catechin (1) was determined to be a new dimeric flavan-3-ol glycoside through a methylene linkage between C-8 and C-8 of two units. At a concentration of 128 μg/mL, the known compounds 9 – 13 exhibited antibacterial activities on Escherichia coli, Staphylococcus aureus subsp. aureus, Salmonella enterica subsp. enterica, and Pseudomonas aeruginosa. Compound 12 also showed certain glucose uptake stimulating activity. Graphical Abstract


Introduction
The genus Potentilla L. (Rosaceae), comprising about 500 species is widely distributed in the temperate regions of the northern hemisphere of Europe, Asia and America, as well as Chile, New Zealand and Australia, with about 90 species growing in China (The flora of China's editorial board 1985; Sun et al. 2010). Many Potentilla species have been used commonly as traditional medicine for the treatment of inflammations, wounds, cancer, diarrhoea, diabetes, and infections caused by bacteria, fungi and virus. More than 100 compounds including flavonoids, terpenoids, and tannins were isolated from some Potentilla species (Tomczyk & Latt e 2009).
Dasiphora fruticosa (L.) Rydb, also known as Potentilla fruticosa L. (syn.), Dasiphora riparia Raf. (syn.), and Pentaphylloides fruticosa (L.) O. Schwarz (syn.), is a hardy deciduous shrub widely distributed in the north temperate regions of the world, often growing in the grassland, meadow, alpine bushes and edge of the forest with high altitudes of 1000 -4200 m (The flora of China's editorial board 1985). Because of the hardiness and low maintenance, it is a popular ornamental plant with dozens of small, flat, and round flowers on each bush. The whole plants of D. fruticosa displayed various pharmacological activities, particularly the antimicrobial and anti-viral activities, as well as the repairment potential for immune system (Tomczyk et al. 2008;Tomczyk & Latt e 2009). The branches and leaves with potential effects to reduce cholesterol and sugar levels in blood have also been used as food additives (Havsteen 1983). The leaf extract of D. fruticosa was reported to contain higher amounts of hyperoside, ellagic acid and (þ)-catechin, which displayed significant antioxidant activity in vitro and protective effects on Escherichia coli under peroxide stress (Luo et al. 2016). Moreover, the combination of D. fruticosa leaf extracts (PFE) with green tea polyphenols (GTP) at a ratio of 3:1 exhibited stronger synergistic effect, due to the promotion of CAT and SOD genes expression and enhancing the activities of CAT and SOD enzyme (Liu et al. 2018). In order to explore the bioactive components of the titled plant, the branches and leaves of D. fruticosa were studied chemically, which led to the isolation and identification of 15 compounds. Their structures were determined on the basis of detailed spectroscopic analyses and compared with literature values. Considering the reported bioactivity for the whole plants and the leaves, all the isolates were tested for their anti-bacterial and the glucose uptake stimulating activities.

General
Shown in supporting information.

Plant Material
The branches and leaves of Dasiphora fruticosa were collected in Qinghai Province, China, in July 10-12, 2015 and identified by Professor Zhen-Ning Chen of Qinghai Normal University, China. A voucher specimen (QNU-SLC-2015009) has been deposited in School of Life Science, Qinghai Normal University.

Anti-bacterial assay
The assay was performed according to the Performance standards (Clinical Lab Standards Institute 2009). Briefly, a mixture of tested sample (100 lL) and bacteria (1 Â 10 6 CFU/mL, 100 lL) was added to the 96-well plates. After culturing at 37 C for 24 hrs, the OD value of each well was recorded by ELIASA under 625 nm. Blank control (culture medium), bacterial control, and positive control (ceftazidime and penicillin G sodium) were set up at the same time. The inhibition values (%) were obtained according to the following equation.

Glucose uptake assay
The assay for glucose uptake was conducted with minor modifications (Zhou et al. 2007;Hu et al. 2015), and 3T3-L1 fibroblasts (ATCC, USA) were cultured and differentiated into adipocytes as reported (Xiong et al. 2010). Differentiated adipocytes were plated into 96-well plates and pre-incubated with FBS-DMEM containing BSA (0.2%) for 12 hours, and then further incubated for 24 hrs with either 25 mM compounds (1-15) or insulin (0.1 lM) or berberine (10 lM) as positive controls, or DMSO as negative control. The medium (10 lL) was collected to determine its glucose concentrations by the glucose oxidase method using a Glucose Kit. The amount of glucose uptake was calculated by the glucose concentrations of blank wells subtracting the remaining glucose in the cell-plated wells. At the same time, the remaining medium was added with 20 lL of 3-(4,5-dimethylthiazol-2-yl)-5(3-carboxymethoxyphenyl)-2-(4-sulfopheny)-2H-tetrazolium (MTS). The absorption at 492 nm was measured after incubation at 37 C for 2 hrs to determine the toxicity of the tested sample to adipocytes.
The new compound 1 was determined to be methylene 6,8-bis(7-O-glucosyl)catechin. All the isolates 1-15 were reported from the titled plant for the first time. The known ones 9-13 displayed obvious antibacterial activities on E. coli, S. aureus subsp. aureus, S. enterica subsp. enterica, and P. aeruginosa. Moreover, compound 12 showed weak glucose uptake stimulating activity, which may be related to the folk uses of this plant.

Conflicts of interest
The authors declare that there are no conflicts of interest.