Two new oleanane triterpenoid saponins from Elsholtzia bodinieri

Abstract Two new oleanane triterpenoid saponins, bodiniosides Q (1) and R (2), along with five known saponins, niga-ichigoside F1 (3), 3-O-[β-D-glucopyranosyl]-28-O-[α-L-rhamnopyranosyl -(1→2)-β-D-glucopyranosyl] arjunolic acid (4), asiaticoside E (5), sericoside (6), and bodinioside E (7), were isolated from the aerial parts of Elsholtzia bodinieri. The structures of 1 and 2 were characterized by spectroscopic techniques and chemical evidence as 3-O-β-D- xylopyranosyl-2α, 23-dihydroxy-olean-12-en-28-oic acid 28-O-α-L-rhamnopyranosyl- (1→2)-β-D-glucopyranoside (1) and 3-O-β-D-xylopyranosyl-2α, 23-dihydroxy-olean-12-en-28-oic acid 28-O-[β-D-glucopyranosyl-(1→4)-α-L-rhamnopyranosyl -(1→2)]-β-D-glucopyranoside (2). Compounds 1, 3, and 5 exhibited weak anti-influenza activity against strain A/WSN/33/2009 (H1N1), with inhibition rate of 11.63%, 17.01%, and 16.98%, respectively. Graphical Abstract


Introduction
The genus Elsholtzia which grows throughout East Asia, Africa, North America, and European countries, belonging to the family Lamiaceae, has approximately 40 species. 33 species of the genus Elsholtzia were found in China. Among these, some are used as medicines, some are taken as food and some are source of honey manufacture (Guo et al. 2012). Elsholtzia bodinieri Van't is an annual herbaceous plant found in the northwest and southwest mountainous area of China (Chinese name "Dongzisu"and "yashuacao"). Mainly distributed in Yunnan and Guizhou Provinces. As a traditional Chinese medicine, E. bodinieri has been used as herbal tea or traditional folk medicine for the prophylaxis and treatment of cough, headache, pharyngitis, fever and hepatitis (Jiangsu New Medical College 1985). We previously has been isolated triterpenoid saponins (Zhu et al. 2002;Zhao et al. 2015;Xiang et al. 2019), flavonoid glycosides (Li et al. 2008;Zhong et al. 2016), sesquiterpene glycosides , clerodane diterpenoid glycosides (Hu et al. 2008), and phenolic constituents ) from the aerial parts of this plant. As a continuation of our work, we further systematically investigated the chemical components of the aerial parts of this plant. In our search for secondary metabolites with structural diversity and potential anti-influenza virus activity, two new oleanane triterpenoid saponins, bodinioside Q (1) and R (2), along with five known ones, were obtained from E. bodinieri. Among them, compounds 1, 3, and 5 exhibited weak inhibition of influenza virus activities with inhibition rate of 11.63%, 17.01%, and 16.98%. Here in, we report the isolation, structural elucidation and anti-influenza virus activities of isolated compounds.

Results and discussion
Compound 1 was obtained as white amorphous powder, its positive HR-ESI-MS spectrum indicated the molecular formula to be C 47 H 76 O 18 by the observation quasimolecular ion peak [M þ Na] þ at m/z 951.4916 and with the help of NMR spectroscopic date, indicating ten degrees of unsaturation. The IR spectrum exhibited the presence of hydroxyl (3441 cm À1 ), carbonyl (1722 cm À1 ), and olefinic (1635 cm À1 ) groups. The 1 H NMR spectrum (Table 1) of 1 revealed six methyl signals at d H 0.96 (3H, s), 1.03 (3H, s), 1.10 (3H, s), 1.15 (3H, s), 0.83 (3H, s), and 0.78 (3H, s), correlation with carbons at d C 14.6 (C-24), 17.4 (C-25), 17.4 (C-26), 30.6 (C-27), 33.0 (C-29), and 25.7 (C-30) in the HSQC spectrum, respectively. The signal at d H 5.42 (1H, br. s), corresponding to the carbon at d C 122.4 (C-12) which coupled with d C 144.1 (C-13) in the 13 C NMR spectrum, indicated the existence of a double bond. On the basis of the above spectroscopic data, compound 1 was suggested to possess an olean-12-ene skeleton. Comparison of its NMR spectroscopic data with those of compound 4 (Acebey-Castellon et al. 2011), suggested that they had same 2, 3, 23-trihydroxy-olean-12-en-28-oic acid as the aglycone. Acid hydrolysis of 1 with 1 M HCl produced L-rhamnose (Rha), D-glucose (Glc), and D-xylose (Xyl) as sugar residues by GC chromatography with the corresponding trimethylsilylated L-cysteine derivatives. The FAB-MS (negative) of 1 showed fragment ions at m/z 619 [M-Glc-Rha-H] À and 487 [M-Glc-Rha-Xyl-H] À . Since NMR signals of three sugar units have undesirable overlapped effects, the HMQC-TOCSY experiment was successfully used to distinguish and assign the 1 H and 13 C NMR signals of each sugar moiety. The correlations from the anomeric proton signal at d H 5.02 to three carbon signals at d C 75.6, 78.5, and 67.2, as well as from three proton signals at d H 4.04, 4.14, and 4.33 to the anomeric carbon, suggested the presence of D-xylopyranose. In a similar way, the 1 H and 13 C NMR signals for D-glucopyranosyl and L-rhamnopyranosyl were assigned. In addition, the J H 1, H2 coupling constants of two anomeric proton signals at d H 5.02 (d, J ¼ 7.3 Hz) and 6.19 (d, J ¼ 8.1 Hz) suggested that the b anomeric configuration for the xylopyranosyl and glucopyranosyl units. The inspection of the anomeric proton (6.11, br. s) deduced the a anomeric configuration for L-rhamnopyranosyl unit (Zhao et al. 2015). The NMR data of 1 was highly analogue to the 4, except for the replacement of signals for the Xyl in 1 by the Glc in 4. The observation suggests that 1 was glycosylated at C-3 by the Xyl residue instead of the Glc in 4, which was confirmed by HMBC correlation from H Xyl -1 (d H 5.02, d, J ¼ 7.3 Hz) to C-3 ( Figure 1). The other key HMBC correlations suggested that the remaining moiety of 1 was identical to that of 4. Based on the above evidence, the structure of 1 was elucidated as 3-O-b-D-xylopyranosyl-2a, 23-dihydroxy-olean-12-en-28-oic acid 28-O-a-L-rhamnopyranosyl-(1!2)-b-D-glucopyranoside, and named bodinioside Q.
The anti-influenza A virus activities of compounds 1-7 against strain A/WSN/33/ 2009 were evaluated in MDCK cells. The results showed that 1, 3, and 5 exhibited weak inhibition of influenza virus activities with inhibition rate of 11.63%, 17.01%, and 16.98%, while the inhibition rate of the positive control (oseltamivir) was 71.20%. A previous study revealed that pentacyclic triterpenoids including ursane, oleanane, and lupane type have anti-influenza virus activity (Wang et al. 2016). Our results further confirmed that the pentacyclic triterpenoids were active against influenza virus. Although the structure-activity relationship was not able to be established due to the limited samples in the present study, the pentacyclic system may be essential for the activity since the E ring break in the aglycone exampled by 7 significantly reduced the activity relative to that in 3 and 5.

Materials
The aerial parts of E. bodinieri were collected in Yuxi city, Yunnan Province, P. R. China, in May 2016, and identified by Dr Xuanqin Chen. A voucher specimen (KMUST 20160005) was deposited at the Laboratory of Phytochemistry, Faculty of Life Science and Technology, Kunming University of Science and Technology.

Extraction and isolation
Dried and finely powdered aerial parts of E. bodinieri (15 kg) was macerated in 75% aq. Me 2 CO (3 Â 35 L, 24 h, each) at room temperature, then concentrated in vacuo to yield an extract, which was suspended in H 2 O, and successively partitioned with CHCl 3 , AcOEt and n-BuOH.

Acid hydrolysis for sugar analysis
Each of 1 and 2 (1.0 mg for each compound) in 1 M HCl (0.4 mL) was heated at 90-100 C in a screw-capped vial for 5 h. The mixture was neutralized by addition of Amberlite IRA400 (OH À form) and then filtered. The filtrate was dried in vacuo, dissolved in 0.2 mL of pyridine containing L-cysteine methyl ester (10 mg/mL) and reacted at 60 C for 1 h. A solution (0.2 mL) of trimethylsilylimidazole in pyridine (10 mg/mL) was added to this mixture, and it was heated at 60 C for 1 h. The final mixture was directly analyzed by GC [30QC2/AC-5 quartz capillary column (30 m Â 0.32 mm) with the following conditions: column temperature: 180/280 C; programmed increase 3 C/ min; carrier gas: N 2 (1 mL/min); injection and detector temperature: 250 C; injection volume: 4 lL; split ratio: 1/50]. The authentic samples D-and L-glucose, D-and Lxylose, and L-rhamnose were treated in the same manner. Under these conditions, the retention times of authentic samples D-and L-glucose, D-and L-xylose, and L-rhamnose were 18.29, 18.87, 13.35, 14.01, and 14.97 min, respectively. During our studies, identical retention times observed between the different hydrolysates and authentic standards.

Anti-influenza virus activity
Influenza strain A/WSN/33/2009 (H1N1) was used in this study. Oseltamivir was used as a positive control and purchased from Tszchem and LKT laboratories. MDCK cells were seeded into 96-well plates, incubated overnight and infected with influenza virus (MOI 1 = 4 0.1). Cells were suspended in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 1% fetal bovine serum (FBS), containing test compound and 2 mg/mL TPCK-treated trypsin, and a final DMSO concentration of 1% was added in each well. After 40 h of incubation, Cell Titer-Glo reagent was added, and the plates were read using a plate reader (Wang et al. 2016). The inhibition rate was calculated by the following formula: inhibition rate (%) ¼ [1 À (luminescence with compounds À luminescence with compounds and virus)/(luminescence with DMSO À luminescence with DMSO and virus)] Â 100%. Assessment of anti-influenza virus activity was performed asdescribed previously (Song et al. 2014).

Conclusion
Our present work on the plants of E. bodinieri yielded two new oleanane triterpenoid saponins, along with five known saponins. Compound 1, 3, and 5 exhibited weak inhibition of influenza virus activities with inhibition rate of 11.63%, 17.01% and 16.98%, respectively. This investigation should provide valuable information for further understanding of E. bodinieri.

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