Three new compounds from Anoectochilus roxburghii (Wall.) Lindl

Abstract In this study, three new compounds, roxburic acid A (1) and two flavone glycosides isorhamnetin-3-O-α-L-rhamnosyl-(1→6)-β-D-glucopyranose-(1→3)-β-D-glucopyranoside (2), and kaempferol-7-O-β-D-glucopyranosyl-(1→3)-β-D-glucopyranoside (3) were isolated from an ethanol extract of the fresh Anoectochilus roxburghii (Wall.) Lindl., together with 10 known compounds (4-13). The structures of these compounds were comprehensively characterized by HR-ESI-MS, 1H NMR, 13C NMR, and 2 D-NMR. The DPPH free radical scavenging activity of the isolated compounds was evaluated, and the results showed that kaempferol-7-O-β-D-glucopyranosyl- (1→3) -β-D-glucopyranoside (3) and rutin (11) has the potential antioxidant activity with IC50 values of 139 μg/mL and 22.5 μg/mL respectively. Graphical Abstract


Introduction
Anoectochilus roxburghii (Wall.) Lindl. (A. roxburghii) is a perennial herb in the Orchidaceae family. A. roxburghii is mainly distributed in China, including Fujian, Zhejiang, Jiangxi, Guizhou, Taiwan provinces, and Japan, Sri Lanka, India, and Nepal (Shao et al. 2016;Chen et al. 2021). As a traditional Chinese herbal medicine, A. roxburghii has been used to prevent and treat diabetes, hypertension, hepatitis, bronchitis and tumours (Cui et al. 2013;Xu et al. 2017). A. roxburghii has attracted considerable attention as a rich source of glycosides , flavonoids and flavonoid glycosides , organic acids and volatile compounds (Yang et al. 2007;Wang et al. 2011), triterpenes (Cai et al. 2008;Han et al. 2008) and other components. Recent research has reported the biological scavenge free radical activity for flavonoids, while flavonoids and glycosides components are also meaningful for liver and tumour protection and treatment of diabetes (Wu et al. 2007;Liu et al. 2013).
UV spectrum were determined on a Shimadzu UV-260 spectrometer (Shimadzu Corporation, Tokyo, Japan). Bruker ALPHA FT-IR infrared spectrometer (Bruker Corporation, Karlsruhe, Germany) were used in IR spectrum detection with KBr pellets. The 1 D ( 1 H, 13 C and DEPT) and 2 D ( 1 H-1 H COSY, HSQC, HMBC and NOESY) NMR spectrum were confirmed by a Brucker Avance III 600 FT NMR spectrometer (Bruker Corporation, Billerica, Massachusetts, USA) operating at 600 MHz (151 MHz for 13 C and DEPT) in DMSO, with TMS as internal standard. HR-ESI-MS analysis was recorded on a Thermo Scientific Q-Exactive (Thermo Fisher Scientific Corporation, Waltham, Massachusetts, USA) in positive or negative mode, and the instrument were settled with following ion source settings: drying gas flow, 5 L/min of nitrogen; capillary temperature, 350 C; heater temperature, 300 C; spray voltage, 2.5 kV; sheath gas pressure, 40 psi.

Anoectochilus roxburghii sample
The sample (XMU170311029) was purchased from Luyan Pharmaceuticals (Xiamen, China). It is a wrinkled, curved herbal and its slender roots are below 1.5 mm in diameter with brown pubescence growing. The leaf shape presents oval or ovoid, while the leaf length range is 1.5 À 4 cm and width range is 1 À 3 cm. Besides, the sample smells faint and tastes slightly sweet. (Wu and Li 1994) According to the above characteristics, the sample was identified as Anoectochilus roxburghii (Wall.) Lindl. by Professor Ying-Kun Qiu (Xiamen University, Xiamen, China). The HPLC chromatogram of A. roxburghii extract showed as Figure S3.

Extraction and isolation
Fresh A. roxburghii herbal (10 kg) was refluxed with 60% ethanol by thermal reflux condenser for three times, two hours each time. The extract obtained was evaporated to dryness under vacuum to give 169 g crude extract. Then dissolved the crude extract with water, and added it to the microporous resin column for adsorption. Using water and 95% ethanol for the column adsorption and elution, 156 g water component and 12.6 g alcohol component of A. roxburghii was obtained. The samples were stored at À20 Cbefore use.

Natural products or artifacts
There is a suspicion that the artifact may have been produced during the decoction extraction and probably came out at a high temperature and the long infusion time (Venditti 2020). Considering this, we took the same batch of sample (XMU170311029) for extraction at room temperature and performed a high-performance liquid chromatography (HPLC) analysis. The result showed that the chromatographic peaks of compounds 1-3 could still be detected by HPLC, excluding the possibility of artifacts.
3.5. DPPH free radical scavenging activity assay DPPH (1,1-diphenyl-2-picrylhydrazyl) method was determined by colorimetric procedure. In brief, 2.0 mg of compound 1-13 and 0.5 mg of vitamin C were accurately weighted and dissolved in DMSO, respectively. These compound sample solutions were diluted to different concentrations until experiment. Then 180 lL DPPH solution (3.2 mg DPPH in 40 mL of ethanol) was mixed with 20 lL for each individual compound in various concentrations. The resulting solution was thoroughly mixed and placed in the dark at room temperature for 30 min, and absorbance was measured at 517 nm. Vitamin C was used as a positive control. The antioxidant activity and 50% inhibition (IC 50 ) for each pure compound was calculated based on graph plot-inhibition percentage against concentration. -, 381.1189, supporting information Figure S4). 1 H NMR (600 MHz, DMSOd 6 ) and 13 C NMR (151 MHz, DMSO-d 6 ) was in Table S1 in the supporting information.  Figure S11). 1 H NMR (600 MHz, DMSO-d 6 ) and 13 C NMR (151 MHz, DMSO-d 6 ) was in Table S2 in the supporting information.  Figure S18). 1 H NMR (600 MHz, DMSO-d 6 ) and 13 C NMR (151 MHz, DMSO-d 6 ) was in Table S2 in the supporting information.

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

Funding
The project was supported by Fujian Provincial Health Commission Project (2017FJZYZY105).