Four new phenolic glycosides from Dobinea delavayi

Abstract Three new benzophenone glycosides, 2-O-β-D-glucopyranosyl-6,3',4'-trihydroxy-4-methoxybenzophenone (1), 4'-O-β-D-glucopyranosyl-2,4,6,3'-tetramethoxybenzophenone (2) and 2-O-β-D-glucopyranosyl-4'-hydroxy-6-methylbenzophenone (3), and a new flavonoid glycoside 5'-hydroxyombuoside (6), along with three known phenolic glycosides 4-O-β-D-glucopyranosyl-2,6,4'-trihydroxybenzophenone (4), mangiferin (5), and ombuoside (7), were isolated from the 80% ethanol extract of roots of Dobinea delavayi. Their structures were determined by the comprehensive analyses of the physicochemical properties and spectroscopic data (1D NMR, 2D NMR, and HR-ESI-MS). Cytotoxicity and in vitro antimalarial activity of compounds 1, 3, 4, 6, and 7 were evaluated by MTT colorimetric assay and β-hematin formation inhibition assay, respectively. Graphical Abstract


Introduction
Dobinea delavayi which belongs to the Anacardiaceae family, is a perennial herb with purple-brown, cylindrical, and bulky roots, and mainly distributed in Yunnan and Sichuan provinces of China at altitudes ranging from 1100 to 2300 m above sea level (ECFCCAS 1980). Owing to the root shaped like a sheep's horn after processing, this plant was called "Yang-Jiao-Tian-Ma" in China. In addition, D. delavayi was used to treat cough caused by lung-heat, mastitis, traumatic injury, and so on, in the folk (EBCHSATCM 1999). Previous phytochemical investigation of this plant led to the isolation of several benzophenones, eudesmane sesquiterpenoids and their dimers (Liu et al. 1995(Liu et al. , 2001Cheng, Yang, Ma, Dai, et al. 2012;Cheng et al. 2013;Wu et al. 2020). In this paper, four new phenolic glycosides (1-3, and 6), together with three known phenolic glycosides (4, 5, and 7), were isolated from the roots of D. delavayi. Their structures were elucidated by extensive spectroscopic data analyses. Herein, we report the isolation and structural elucidation of those new compounds, as well as the results of cytotoxicity and in vitro antimalarial activity of compounds 1, 3, 4, 6, and 7.

Results and discussion
Seven compounds of the ethyl acetate and n-butanol soluble fraction derived from the 80% ethanol extract of the roots of D. delavayi were isolated and purified by the repeated column chromatography (normal and reverse phase silica gel, sephadex LH-20, macroporous adsorption resin, and MCI gel stationary phases, respectively).
Compound 1 was isolated as a yellow solid, and assigned a molecular formula C 20 H 22 O 11 (10 of unsaturation) from its HR-ESI-MS (m/z 437.1092 [M À H] À ) and NMR spectra (Table S1). According to 1 H NMR spectrum (Table S1), 1 contained a methoxy at d H 3.78 (s), three ABX coupling system aromatic protons at d H 6.78 (1H, d, J ¼ 8.3 Hz), 7.23 (1H, dd, J ¼ 8.3, 2.1 Hz), and 7.31 (1H, d, J ¼ 2.1 Hz), and two aromatic protons at d H 6.18 (1H, d, J ¼ 2.1 Hz) and 6.39 (1H, d, J ¼ 2.1 Hz). Another seven proton resonances from either oxymethine or oxymethylene groups were found to resonate in a region between d H 3.17 and 4.90. Analyses of the 13 C NMR spectrum with the aid of DEPT experiments (Table S1) revealed the existence of 20 carbon resonances including a methoxy (d C 54.6), a sugar moiety (d C 100.9, 76.8, 76.4, 73.4, 69.7, and 61.1), 12 aromatic carbons, and a carbonyl (d C 195.9). These spectroscopic data obviously indicated a benzophenone glycoside for 1, which suggested a highly structural similarity of dobiniside A (Cheng et al. 2013). The obvious difference between the two compounds was the lack of a methoxy group in 1. The HMBC correlation ( Figure S1) from the methoxy (d H 3.78, s) to C-4 (d C 162.7) suggested that the methoxy was linked to C-4. The ROESY correlations ( Figure S1) of H-1" (d H 4.90, d, J ¼ 7.7 Hz) to H-3" (d H 3.40, overlap) and H-5" (d H 3.40, overlap) suggested that the sugar moiety in 1 was a glucose. In addition, a HMBC correlation between H-1 00 and C-2 (d C 156.9) indicated that the glucose moiety was bound to C-2. For identification of stereochemistry of the glucose, acid hydrolysis of 1 was performed. The stereochemistry of the glucose was determined as D-configuration by comparing its specific rotation data with that of the authentic D-glucose. Then the b-configuration of the glucose was determined by coupling constant of H-1 00 (d, J ¼ 7.7 Hz). Based on the further analyses of 1 H-1 H COSY, ROESY, and HMBC spectra ( Figure S1), compound 1 was finally assigned as shown in Figure  1, and named 2-O-b-D-glucopyranosyl-6,3 0 ,4 0 -trihydroxy-4methoxybenzophenone.
Compound 6 was obtained as a yellow amorphous powder, and assigned a molecular formula C 29 H 34 O 17 (thirteen degrees of unsaturation) from its HR-ESI-MS (m/z 653.1724 [M À H] À ) and NMR spectra (Table S2). According to 1 H NMR spectrum (Table S2), 6 contained a hydroxy proton with intramolecular hydrogen bond at d H 13.00 (s, 5-OH), a methyl at d H 1.52 (3H, d, J ¼ 5.8 Hz), two methoxy groups at d H 3.72 (s) and 4.03 (s), and two 1,3,4,5-tetrasubstituted benzene rings with protons at d H 6.42 (1H, d, J ¼ 2.2 Hz), 6.55 (1H, d, J ¼ 2.2 Hz), and 8.00 (2H, s), respectively. In addition, twelve protons from oxymethines or oxymethylenes at the range between d H 3.94 and 6.14 were also observed. Analyses of the 13 C NMR spectrum with the aid of DEPT experiments (Table S2) 69.1, and 19.0) and a flavonol moiety (one carbonyl and 14 aromatic carbons) were recognized readily. The absolute configuration of rhamnose in rutinose moiety was arbitrarily assigned as L configuration, due to the natural rhamnose presence as L configuration. Therefore, compound 6 was considered as a flavonol glycoside.
The HMBC correlation ( Figure S3) from H-1 00 (d H 6.14, d, J ¼ 7.4 Hz) to C-3 (d C 136.7) indicated that the rutinose moiety was bound to C-3. In addition, HMBC correlations of a methoxy (d H 3.37) to C-7 (d C 166.4), and of the other methoxy (d H 4.03) to C-4 0 (d C 140.4) indicated that the two methoxyls were linked to C-7, and C-4 0 , respectively. Based on the further analyses of 1 H-1 H COSY, ROESY, and HMBC spectra ( Figure S3), compound 6 was finally assigned as shown in Figure 1, and named 5 0hydroxyombuoside.

Plant materials
Roots of D. delavayi (Baill.) Baill. were purchased from the herbal medicine market of Eryuan county, Dali, Yunnan Province, P. R. China in October 2017. The plant material was identified by Prof. Dr. Bei Jiang from Dali University, P. R. China. A voucher specimen (NO. 20171008-1) has been deposited at the Yunnan Key Laboratory of Screening and Research on Anti-pathogenic Plant Resources from Western Yunnan, Dali University.

Acid hydrolysis for identification of sugars
The hydrolysis experiments of compounds 1 (18 mg) and 3 (15 mg) were performed as we described previously .

Cytotoxicity assay
The procedure for cytotoxic assay was measured by MTT colorimetric assay that we previously described (Huang et al. 2014;Qiu et al. 2020). Cells were seeded into 96well plates at the concentration of 7 Â 10 3 cells/well. Docetaxel used as positive control. The final concentrations of docetaxel were 5, 1, 0.5, 0.25, and 0.125 lM. The final concentrations of the isolated compounds were 200, 100, and 50 lM.

b-Hematin formation inhibition assay
The in vitro antimalarial activity was tested by b-hematin formation inhibition assay as we described previously (Xiang et al. 2019;Li et al. 2021). The final concentrations of the isolated compounds were 277.8, 55.6, 11.1, 2.2, and 0.4 lg/mL.

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