Iridoid, phenylethanoid and flavonoid glycosides from Forsythia suspensa

Abstract As part of our continuing efforts to explore bioactive compounds from natural resources, a new iridoid glycoside, adoxosidic acid-6′-oleuroperic ester (1), together with one known phenylethanoid glycoside (2) and two known flavonoid glycosides (3–4) were isolated from the fruit of Forsythia suspensa. The structure of the new compound (1) was elucidated through 1D and 2D NMR spectroscopic data and HR-ESIMS. Interestingly, compound 1 was a monoterpene ester of one iridoid glycoside. Compounds 2–4 were identified as calceolarioside A (2), kaempferol-3-O-rutinoside (3), kampferol-3-O-robinobioside (4) on the basis of NMR spectroscopic data analyses and comparison with the data reported in the literature. The antiviral activity aganisist influenza A (H5N1) virus of compound 1 was studied as well. Graphical Abstract


Introduction
The dried fruit of Forsythia suspensa (Thunb.) Vahl, which is a widely used herbal medicine in China, is distributed in China, Korea, Japan and many European countries. It is used for treating infections (Wang et al. 2018). Previous phytochemical inestigations on the fruits of F. suspensa have led to the isolation of a variety of compounds including phenylethanoid glycosides , lignan glycosides (Piao et al. 2008), quinoid glycosides (Li et al. 2014), flavonoids and diterpenoids (Kuo et al. 2017;Zhang et al. 2017). Some of them possess different bioactivities, including antiviral (Law et al. 2017), antibacterial (Qu et al. 2017), antioxidant activities (Lu et al. 2010;Yang and Kang 2012) and neuroprotective effect (Chen et al. 2017;. Our ongoing search for new bioactive compounds from this plant led to the isolation of one new iridoid glycoside (1), one known phenylethanoid glycoside (2) and two known flavonoid glycosides (3-4). The structure of the new compound (1) was elucidated through 1D and 2D NMR spectroscopic data, and HR-ESIMS. Compound 1 was tested for its antiviral activity against influenza A (H5N1) virus, but it showed no obvious activity at the concentration of 83.0 mmol/L.

Results and discussion
The study reports the isolation and identification of one iridoid glycoside (1), one phenylethanoid glycoside (2), and two flavonoid glycosides (3-4) (Figure 1) from the fruit of F. suspensa. Compound 1 was a new monoterpene ester of one iridoid glycoside.
The linkage of two moieties was established on the basis of the HMBC correlation between H-6 0 and 7 00 -CO, which showed the structure of 1. The relative configuration of the iridoid moiety was confirmed by analysisof the NOESY spectrum, the NOESY correlations between H-9 and H-5 indicated that H-5 and H-9 were both b-oriented, and NOESY correlations between H-8 and H-1, H-9 and H-10 implied that H-1and H-8 were a-oriented. The configuration of C-8 00 was assumed to be b-oriented on the basis of compounds forsythenside G and H, which were isolated from F. suspensa previously containing the same oleuropeic acid unit.
Acid hydrolysis and HPLC analysis were performed according to the method of Tanaka et al (Tu et al. 2011), which showed that the monosaccharide was D-glucose. Moreover, the b-glucopyranosyl configuration of the sugar moiety was evident from the 13 C NMR spectrum. Therefore, compound 1 was elucidated as adoxosidic acid-6 0oleuroperic ester.

Plant material
The fruit of F. suspensa was purchased from Shangluo, ShanXi province, and identified as the fruit of F. suspensa by Professor Li Bo Wang (College of Pharmacy, HarBin Medical University). A voucher specimen (FS20161210) was deposited at room 610, the college of pharmacy, HarBin Medical University.

Acid hydrolysis and HPLC analysis of compound 1
The absolute configuration of the sugar unit was determined by the method of Tanaka et al. (Tu et al. 2011). Compound 1 afforded D-glucose (t R = 21.27 min; L-glucose t R = 19.47 min) 3.5. Indirect immunofluorescence assay (IFA) Indirect immunofluorescence assay was used to perform a rapid evaluation of antiviral activities of compounds against influenza A virus (IAV) infection (Huang et al. 2012;Reina et al. 1998). For immunostaining, the IAV-infected or control cells were fixed with 4% paraformaldehyde for 10 min, then permeabilized with 0.25% Triton X-100 for 10 min at room temperature (RT). Cells were blocked with 1% bovine serum albumin (BSA) for 60 min at RT and then incubated with a mouse monoclonal antibody against influenza nucleoprotein (1:500 dilution, Sino Biological, Beijing, China) at 4 C overnight. After three washes with PBS, the cells were incubated for 1 h at RT with an antimouse IgG antibody conjugated with Alexa FluorV R 488 (green) (CST, USA) at 1:1000 dilution. Nuclei were counterstained using 50 ll of 4, 6-diamidino-2-phenylindole (DAPI, 300 nM; Sigma Chemical Co., USA). Immunofluorescence was captured using the Leica DMI 4000B fluorescence microscope (Leica, Wetzlar, Germany).

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