Three new glycosides from the stems of Eurya chinensis R. Br

Abstract Two new phenolic glycosides (1 and 2), one known analogue (3), along with a new diterpene glucoside (4) were obtained from ethanolic extract of the stems of Eurya chinensis R. Br. The structures of these isolated compounds were identified by extensive analysis of HRESIMS and NMR spectroscopic data. The cytotoxicities of these compounds were evaluated on MCF-7, A549, HepG2, CaCo2 and 5-8 F cell lines by MTT method, but no obvious activities were observed. Graphical Abstract

Eurya chinensis R. Br. belonging to family Pentaphylacaceae, known as "Mi-Sui-Hua" in Chinese, is an evergreen shrub widely distributed in Jiangxi, Fujian, Hunan and Guangdong provinces of China.It is a Chinese medicine used to prevent influenza and treat nasopharyngeal cancer in the folk (Editorial Board of Chinese Materia Medica, State Administration of Traditional Chinese Medicine 1999).The previous phytochemical study on genus Eurya Thumb.revealed the presence of flavonoids, lignans glycosides, phenolic glucosides (Morita et al. 1974;Khan et al. 1992; Yang Kuo et al. 2013), while the study on E. chinensis discovered a series of new diterpenes, some displayed noticeable anti-inflammatory activity and cytotoxic activity (Song et al. 2016;Song et al. 2018aSong et al. , 2018b)).As part of our search for bioactive components with novel structures from natural resources, the stems of E. chinensis were thoroughly investigated, resulting in the isolation of two new phenolic glycosides, a known analogue, and a new diterpene glucoside (Figure 1).Herein, we describe the isolation and structure elucidation of these isolates, along with their cytotoxic activities against MCF-7, A549, HepG2, CaCo2 and 5-8 F cell lines.

Results and discussion
Compound 1 was obtained as a yellow amorphous powder, gave the molecular formula C 17 H 22 O 7 with seven degrees of unsaturation, as deduced by an HRESIMS ion at m/z 361.1254 [M þ Na] þ (calcd.361.1258).The IR spectrum suggested the presence of hydroxyl group (3392 cm À1 ), carbonyl group (1704 cm À1 ) and double-bond group (1636 cm À1 ).The 13 C NMR analysed with the HSQC spectrum showed 17 carbon signals, attributing to one methyl, two methylenes, 12 methines and two quaternary carbons (Figure S6).The presence of a trans-cinnamoyl moiety in  8 Hz), suggesting a b-configuration for the glucose.The D-configuration of 1 was determined according to the method of sequential derivatization with L-cysteine methyl ester and arylisothiocyanate (Tanaka et al. 2007).In addition to the signals of trans-cinnamoyl and glucose group, the remaining ones appearing at d C 66.3, 15.5 and d H 3.90 (1H, m), 3.63 (1H, m) and 1.23 (3H, t, J ¼ 7.1 Hz), indicated the existence of an oxygenated ethyl.The observed HMBC correlations from C-1 to H 2 -1 00 indicated the location of this oxoethyl group at C-1 (Figure S7).Similarly, the trans-cinnamoyl moiety was determined to be at C-6 by the correlations from C-1 0 to H 2 -6 in the HMBC experiment.The structure of 1 determined as 1-ethoxy-6-cinnamoyl-O-b-D-glucopyranose was further confirmed by its NMR spectroscopic data (Table S1-S2), and given the trivial name Euryacoside A.
To prove the natural occurrence of compound 1 (Venditti 2020), the following experiments were performed.Methanol extraction was freshly prepared instead of ethanol extraction, and compound 1 was clearly found by LCMS-IT-TOF (Figures S32-S33) in the methanol extraction.Notably, no methanol analogue of compound 1 was found in the methanol extraction, which further confirmed that compound 1 should not be an artifact.
Compound 2, isolated as a yellow amorphous powder, was found to possess a molecular formula of C 22 H 26 O 11 as deduced from its HRESIMS data, corresponding to an unsaturation of ten.The IR spectrum of 2 displayed the same absorption bands of hydroxyl (3542 cm À1 ) and double-bond group (1654 cm À1 ).The existence of the p- Additionally, the p-hydroxyphenethyl group was determined to be at C-1 by the downfield chemical shift of H-1 as well as the HMBC correlations from C-1 to H 2 -1 00 .Compound 2 identified as 1-O-(4-hydroxyphenyl)-6-(4-methoxy-galloyl)-b-D-glucopyranose was further confirmed by its NMR spectroscopic data (Tables S1-S2), and named Euryacoside B.
Compound 3 was identified as 1-cinnamoyl-6-galloyl-O-b-D-glucopyranose, firstly reported as a metabolite in rats after oral administration of rhubarb with absent NMR data (Xu et al. 2019).For this reason, the full NMR data of compound 3 was provided in our study (Tables S1-S2).To the best of our knowledge, compound 3 was isolated from natural resources for the first time in our study.
Compound 4 was gained as yellow amorphous powder.Its molecular formula was established as C 26 H 40 O 8 by its 13 C NMR and HRESIMS data, suggesting seven degrees of unsaturation.Two characteristic absorptions at 3379 and 1636 cm À1 in the IR spectrum indicated the presence of hydroxy groups and double-bond groups.The 13  ).Therefore, these four compounds were evaluated for their cytotoxic activities against human cancer cell lines, including MCF-7, A549, HepG2, CaCo2 and 5-8 F by MTT assay (Table S3).However, all these compounds displayed weak cytotoxic activities (IC 50 >50 lM).
To date, lots of natural phenolic glycosides have been extracted from a variety of plants and verified to possess extensive bioactivities.Among these bioactivities, the antitumor activity of phenolic glycosides is outstanding and thus massive effort has been made to search for the compounds with antitumor activity from the title plant, however, all the obtained compounds displayed weak cytotoxic activities in our study.From the structural point of view, it was found that the phenolic glycosides with high antitumor activity tended to have two sugar moieties or more in their structures (Shang et al. 2016;Wang et al. 2013;Zheng et al. 2012).These results permit assignment of some structure-activity relationships.And thus, it is likely that our obtained compounds showed weak antitumor activity due to the molecular features of only one sugar moiety in their structures.

General experimental procedures
The NMR (1 D and 2 D) spectra were measured on a Bruker AV-400 spectrometer (Bruker BioSpin AG, Fallanden, Switzerland, 400 MHz) at 298 K with TMS as internal reference. 1H-1 H COSY experiments were acquired with a spectral width of 13 ppm in both dimensions, 1 scan, a data matrix of 4k x 128 points, a recycle delay of 1.5 s. 1 H- 13 C HSQC experiments were acquired with a spectral width of 13 and 165 ppm for proton and carbon dimension respectively, 8 scans, a data matrix of 4k x 128 points, a recycle delay of 1.5 s and an average J 1 C-H coupling constant of 145 Hz. 1 H- 13 C HMBC experiments were acquired with a spectral of 13 and 220 ppm, 32 scans, a data matrix of 4k x 128 points, a recycle delay of 1.5 s and a long range J 3 C-H coupling constant of 8 Hz.HRESIMS data were measured on LCMS-IT-TOF (Shimadzu, Kyoto, Japan, Acquisition m/z 100-1000, Nebulizing Gas Flow 1.5 L/min, CDL Temperature 200.0 C, Heat Block Temperature 200.0 C, Detector Voltage 1.58 kV, MeCN-H 2 O, 5:95-95:5, v/v, 0.2 mL/min, 0-10 min).IR spectra were recorded on a Nicolet Magna FT-IR 750 spectrophotometer using KBr disks.Optical rotations were determined on a Perkin-Elmer 341 polarimeter (Perkin-Elmer, Boston, MA, USA).Preparative HPLC was performed on an Agilent 1100 series with a Waters Sunfire RP C18, 5 lm, 30 mm Â 150 mm column and a UV detector.HPLC was performed on Waters 2695 with a Waters Sunfire RP C18, 3.5 lm, 4.6 mm Â 100 mm column and a UV detector (MeCN-H 2 O, 5:95-95:5, v/v, 1.0 mL/min, 0-30 min, 254 nm).Column chromatography (CC) was performed on Silica gel (Qingdao Marine Chemical Industrials, Qingdao, China), Sephadex LH-20 (Pharmacia Fine Chemicals, Uppsala, Sweden) and MCI gel CHP20P (75-150 lm, Mitsubishi Chemical Industries, Tokyo, Japan), respectively.Thin layer chromatography (TLC) was carried out on precoated silica gel GF 254 and spots were visualized by spraying with 20% H 2 SO 4 solution, followed by heating.All solvents were analytical grade (Shanghai Chemical Plant, Shanghai, China).

Plant material
The stems of E. chinensis were collected from the Dasongling Sports Park (112.3E, 22.3 N, altitude, 113 meters) in Enping District of Guangdong Province, China, in January 2019.The voucher specimen (NO.2020032701) was identified by Dr. Yi Tong of Guangzhou University of Chinese Medicine and has been deposited in GZUCM (Guangzhou, China).

Cytotoxic evaluation
The compounds 1-4 were evaluated for cytotoxic activity against MCF-7 (human breast cancer cell), A549 (human lung epithelial carcinoma cells), HepG2 (human hepatic cancer cells), CaCo2 (human colon epithelial cancer cells), 5-8 F (human nasopharyngeal carcinoma cells).All the cell lines were purchased from Conservation Genetics CAS Kunming Cell Bank.Incubated under a humidified 5% CO 2 at 37 C, the cells suspension was adjusted to 1 Â 10 4 cell/well and seeded into 96-well plates.After preincubation for 12 h, the cells were treated with different concentrations (3.125, 6.25, 12.5, 25, 50, 100 lM) of compounds, respectively, while doxorubicin was used as a positive control.24 hours later, MTT reagent (5 mg/mL, 5% MTT) was added to each well and further incubated at 37 C for another 4 h.DMSO (150 lL) was added into each well after the removal of supernatants.The mixture was oscillated for 10 min and afterwards its absorbance was measured at 490 nm.The concentration resulting in 50% of cellgrowth inhibition (IC 50 ) was calculated by the Probit program in SPSS 25 for windows XP (SPSS Inc.Chicago).

The natural occurrence of 1 was confirmed by LCMS-IT-TOF
The dried-powdered stems of E. chinensis (the deposited specimen, 200 g) were extracted with methanol instead of ethanol and then processed in the same way mentioned above.Subsequently, the ethyl acetate-soluble fraction was analysed by LCMS-IT-TOF (see Figures S32 and S33).As a result, an ion at m/z 361.1261 could be observed from the methanol extract of E. chinensis, and this observed ion could be assigned to be the molecular ion since the calculated molecular weight of 1 was m/z 361.1258 ([M þ Na] þ ) as well as the MS peak of this ion appeared in the same retention time (% 17.20 min) with 1 (Figure S32).Additionally, the same molecular formula C 17 H 22 O 7 with 1 could be established with small diff value (0.83 ppm) by detailed MS analysis of the extracted ion (Figure S33).

Conclusions
Two new phenolic glucosides, one known compound, along with a new diterpene glucoside were isolated from the stems of E. chinensis.Cytotoxic activities against five human cancer cell lines of these compounds were examined by MTT method, but unfortunately no obvious activities were observed.

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

Funding
Financial support from the National Natural Science Foundation (No. 81903509) is gratefully acknowledged.