The chemical constituents from twigs of Hamamelis japonica and their antiviral activities

Abstract Three new monoterpenoid glycosides (1–3) and one new flavanol (4) along with 15 known compounds were isolated from the twig of Hamamelis japonica Sieb. et Zucc. The chemical constituent study of the twig of H. japonica has performed for the first time in the present investigation. Their structures were determined based on extensive spectroscopic methods including 1 D and 2 D NMR and CD spectra data. All isolated compounds were tested for their antiviral activities against HRV1B-, EV71-, PR8- and CVB3-infected Vero cells. Among the tested compounds, (–)-epigallocatechin 3-O-gallate exhibited the most consistent and effective antiviral activities against EV71 and PR8 infections. Graphical Abstract


Introduction
Hamamelis L., the witch hazel genus, of the Hamamelidaceae family is disjunctly distributed in eastern Asia mainly China and Japan and eastern North America (Wen and Shi 1999). Hamamelis genus comprises six major species of H. virginiana, H. ovalis, H. vernalis, H. mollis, H. mexicana and H. japonica. Among Hamamelis genus, H. virginiana, North American witch hazel, is the most well-known species due to its use as commercial ingredients for cosmetic products and treatment of the dermatological disorder (H€ ormann and Korting 1994;Wolff and Kieser 2007). However, compared to H. virginiana, only a few studies of chemical constituents have been done with H. japonica (Yim et al. 2015). From the ethyl acetate extract of H. japonica flowers, cytotoxic phenolic compounds were isolated (Yim et al. 2015). It is popularly cultivated as an ornamental plant in Korea and the flowers have been traditionally used as antipyretic and antihemorrhagic agents (Bae 2000). In a continuing project to identify plant natural products that exhibit antiviral effect, we performed extensive phytochemical study of H. japonica. This paper mainly deals with the isolation, structural characterization of four new (1-4) and fifteen known compounds from the plants. We also evaluated these isolated compounds as a natural antiviral source.
Compound 4 was isolated as yellow amorphous powder and its molecular formula was confirmed as C 16  þ , 321.0969). The 1 H NMR spectrum of 4 revealed a flavan-3-ol derivative. The 13 C NMR spectrum of 4 also showed the flavan-3-ol backbone: eight quaternary carbons, four methyl signals and two oxygenated methines, one methylene and one methoxy carbon. The analysis of 1 H and 13 C NMR data indicated the structure of 4 was similar to those of (þ)-gallocatechin (Davis et al. 1996) except for the addition of one methoxy group. The HMBC correlations between methoxy (d H 3.66) and C-7 (d C 160.8) suggested methoxy group is located at C-7 ( Figure S1). The flavan H-2 signal (d H 4.54) as a doublet with a large J-value (7.1 Hz) suggested the 2,3-trans configuration. The flavan-3-ols affect at longer wavelength band to chromophore A and shorter wavelength band to chromophore B in the CD spectra. Flavan-3-ols with a (2 R)-configuration show a negative Cotton effect at 280 nm (effect of chromophore B) that is independent of the configuration at C-3 (Slade et al. 2005). The CD spectrum of 4 showed two negative Cotton effects at 220 and 280 nm ( Figure S3), similar to those of (2 R,3S)-catechin-7-O-b-D-glucopyranoside (Friedrich and Galensa 2002) and confirmed the absolute stereochemistry at C-2 of compound 4 to be R. Based on the evidence above, compound 4 was elucidated as (-)-7-methoxygallocatechin.
In order to search for new and effective antiviral phytochemicals, all of the isolated compounds were evaluated for antiviral activities against HRV1B-, EV71-, PR8-and CVB3-infected Vero cells by employing the sulforhodamine B (SRB) method using cytopathic effect (CPE) reduction (Choi et al. 2009). H. japonica extract exhibited potent antiviral activities against PR8 infection with the antiviral activity index of 88.68%. Among the isolated compounds, the treatment of 10 mM of (-)-epigallocatechin 3-O-gallate (7) and 2-C-[[(3,4,5-trihydroxybenzoyl)oxy]methyl]-b-D-ribofuranose (13) showed effective antiviral activities with 75.07 and 53.53% survival rate of HRV1Binfected Vero cells, respectively (Table S3). Additionally, (-)-epigallocatechin 3-O-gallate (7) was the most consistent and effective antiviral component against both EV71-and PR8-infected Vero cell. Compound 7 is already known to possess antiviral activities against a wide range of DNA viruses and RNA viruses including herpes simplex virus, adenovirus, human papilloma virus, hepatitis B virus, hepatitis C virus, Zika virus, and dengue virus (Kaihatsu et al. 2018). Previous studies mentioned that it caused 5-fold increase in the viability of EV71-infected cells and suppressed replication of genomic RNA (Chaturvedula and Prakash 2011). To the best of our knowledge, there are no literature report about antiviral activity of compound 13.    Table S2.
3.4. Cell culture and antiviral assay: see supporting information

Conclusions
The detailed phytochemical study of twigs of H. japonica resulted in the isolation of three new monoterpenoid glycosides (1-3) and one new flavanol (4) along with fifteen previously reported compounds (5-19). These fifteen compounds were classified into flavanols, phenolics and stilbenes. To identify antiviral phytochemicals, isolated compounds were tested for their bioactivities using HRV1B-, EV71-, PR8-and CVB3infected Vero cells. 2-C- [[(3,4,5-trihydroxybenzoyl)oxy]methyl]-b-D-ribofuranose exhibited a significant antiviral effect against HRV1B. The antiviral assays in this study demonstrated that the compounds isolated from H. japonica twigs could be effective antiviral agents to treat viral infection caused by HRV1B, EV71, PR8 and CVB3.