Bodiniosides S–Y, Seven New Triterpenoid Saponins from Elsholtzia bodinieri and Their Anti-Influenza Activities

Investigation of the n-BuOH extract of the aerial parts of Elsholtzia bodinieri led to the isolation of seven new triterpenoid saponins, Bodiniosides S–Y (1–7, resp.). Their strictures were elucidated on the basis of spectroscopic techniques, including HSQC, HSBC, and HSQC–TOCSY experiments, together with acid hydrolysis and GC analysis. The anti-influenza activities of compounds 1–7 were evaluated against A/WSN/33/2009 (H1N1) virus in MDCK cells. The results showed that compounds 2 and 5 exhibited moderate anti-influenza activities against A/WSN/33/2009 (H1N1), with inhibition rates of 35.33% and 24.08%, respectively.


Introduction
Elsholtzia bodinieri Vaniot (Chinese name "Dongzisu"), belonging to the taxonomically diverse group of the family Labiatae, is a medicinal plant that grows in Yunnan and Guizhou Provinces in China. It is commonly known as "yashuacao" and is used as a traditional Chinese medicine for the treatment of cough, headache, pharyngitis, fever and hepatitis [1]. Previous studies on E. bodinieri led to the isolation of triterpenoid saponins [2][3][4][5][6], flavonoid glycosides [7,8], sesquiterpene glycosides [9], clerodane diterpenoid glycosides [10], and phenolic constituents [11] 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, seven new triterpenoid saponins, Bodiniosides S-Y (1-7, resp.), were obtained from E. bodinieri. Among them, compounds 2 and 5 exhibited moderate inhibition of influenza virus activities with inhibition rates of 35.33% and 24.08%, respectively. Herein, we report the isolation, structural elucidation and anti-influenza virus activities of the isolated compounds.

Results
The n-BuOH soluble fraction of the 75% aqueous acetone extract of the aerial parts of E. bodinieri was subjected to repeated column chromatography over silica gel, Sephadex LH-20, RP-18, and semipreparative reversed-phase HPLC, eluting with various solvent systems, to afford seven new triterpenoid saponins ( Figure 1). The spectrums can be found in supplementary materials. The IR spectrum showed the presence of hydroxyl (3441 cm −1 ), carbonyl (1722 cm −1 ), and olefinic (1635 cm −1 ) groups. NMR data analysis indicated that 1 was a saponin containing a triterpene sapogenin and five monosaccharides.
In the 1 H and 13 (Tables 1 and 2). This information suggested that the aglycone moiety of compound 1 was 3β, 23-dihydroxyolean-12-en-28-oic acid [12]. Except for the signals for the aglycone, the remaining 31 signals were assigned as five sugar moieties and an acetoxy group, due to signals of δC 172.7 and 20.9. Moreover, comparison of its 1 H and 13 C NMR spectroscopic data with those of Bodinioside H [4], suggested that they had same 3-hydroxy-23-acetoxy-olean-12-en-28-oic acid as the aglycone, but differed in the sugar moiety. ; δ C 123.2 (d, C-12)], as well as 8 quaternary carbons (including a carbonyl carbon (δ C 176.4, C-28) and a unsaturated one (δ C 144.0, C-13)) were observed (Tables 1 and 2). This information suggested that the aglycone moiety of compound 1 was 3β, 23-dihydroxyolean-12-en-28-oic acid [12]. Except for the signals for the aglycone, the remaining 31 signals were assigned as five sugar moieties and an acetoxy group, due to signals of δ C 172.7 and 20.9. Moreover, comparison of its 1 H and 13 C NMR spectroscopic data with those of Bodinioside H [4], suggested that they had same 3-hydroxy-23-acetoxy-olean-12-en-28-oic acid as the aglycone, but differed in the sugar moiety.   The 1 H NMR spectrum showed five signals for anomeric protons at δ H 6.45 (1H, br. s), 6.09 (1H, d, J = 7. The inspection of the anomeric proton (6.45, br. s) deduced the α anomeric configuration for the L-rhamnopyranosyl unit [4].
The anti-influenza A virus activity of compounds 1-7 against strain A/WSN/33/2009 was evaluated in MDCK cells, and their cytotoxicity was measured in parallel with the determination of antiviral activity, using oseltamivir as a positive control. It was found that compounds 1-7 displayed no significant cytotoxicity at 50 µM concentration. Then, under this concentration, the in vitro potential anti-influenza A virus effects of all isolates were investigated. The results showed that compounds 2 and 5 exhibited moderate inhibition of influenza virus activities with inhibition rates of 35.33% and 24.08%, while the inhibition rate of the positive control (oseltamivir) was 71.20%.

Discussion
In summary, seven new triterpenoid saponins, including six oleanane triterpenoid saponins and a ursane one, named Bodiniosides S-Y, were isolated from the aerial parts of Elsholtzia bodinieri. Elucidation of their structures was performed based on extensive spectroscopic analyses. The anti-influenza activities of the isolates against A/WSN/33/2009 (H1N1) virus were investigated. The results demonstrated that compounds 2 and 5 exhibited potent inhibition of influenza virus activities, with inhibition rates of 35.33% and 24.08%; meanwhile, compounds 1, 3, 4, 6 and 7 were found to be inactive, with an inhibition rate lower than 10%. A previous study revealed that pentacyclic triterpenoids, including ursane, oleanane, and lupane types, have anti-influenza virus activity [15]. Our results further confirmed that the pentacyclic triterpenoids were active against influenza virus. This investigation should provide valuable information for further understanding of E. bodinieri.

Materials
The aerial parts of E. bodinieri were collected in Yuxi city, Yunnan Province, P. R. China, in May 2016, and identified by Dr. Jindong Zhong. 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
The powered air-dried aerial parts of E. bodinieri (15 kg) were extracted with 75% aq. Me 2 CO (3 × 35 L, 24 h, each) at room temperature and filtered. The filtrate was concentrated in vacuo, and the resulting residue was extracted successively with CHCl 3 , AcOEt and n-BuOH, respectively.