Megastigmane sesquiterpenoids from Saussurea medusa and their anti-inflammatory activities

Abstract Objectives: An ethanol extract of the whole plants of Saussurea medusa had been investigated to find novel anti-inflammatory sesquiterpenoids. Methods: Extensive spectroscopic data and chemical methods were applied to elucidate the structures of the compounds. Results: One new megastigmane sesquiterpenoid (1), along with 11 known analogues (2–12), were obtained from S. medusa. All isolates, except compounds 3 and 6, were mentioned from the studied plant for the first time. Compounds 1, 2, 4, 5, 7, 8 and 12 were firstly isolated from the genus Saussurea. Compounds 2, 9 and 10 were found to inhibit the lipopolysaccharide (LPS)-induced release of NO by RAW264.7 cells with IC50 values ranging from 21.1 ± 1.7 to 46.7 ± 1.9 μM. Furthermore, iNOS expression experiment was performed to examine the interactions between the active compounds and the iNOS enzyme. Graphical Abstract


Introduction
Saussurea medusa is a rare subnival plant known as 'snow lotus' that belongs to the genus Saussurea. Saussurea DC is a large genus of about 400 species in the family Asteraceae with high diversity in alpine habitats, the Himalayas and Central Asia (Xu et al. 2009). Among them, approximately 289 species have been found in China, most of which are distributed in Qinghai-Tibet Plateau (Shih and Raab-Straube 2011). S. medusa is a very important traditional Chinese medicinal herb used for the treatment of rheumatic arthritis, menoxenia, gynopathy, traumatic bleeding, anthrax, febrile tingling and headache (Committee of Pharmacopoeia of the People's Repubulic of China 1995; Fan and Yue 2003;Xie et al. 2005).
A literature search found that previous researches mainly focused on the extraction and activity study of lignans and flavonoids from S. medusa (Takasaki et al. 2000;Duan et al. 2002;Fan et al. 2015), no comprehensive study had been conducted to explore terpenoids of S. medusa. As a result of our ongoing phytochemical investigation on S. medusa, one new megastigmane sesquiterpenoid (1), along with 11 known analogues (2-12) were isolated from the whole plants of S. medusa. The structure of compound 1 was elucidated by extensive spectroscopic data and chemical methods. The absolute configuration of the 3,4-diol moiety in compound 1 was determined by observing the circular dichroism induced after addition of dimolybdenum tetracetate [Mo 2 (OAc) 4 ] in DMSO solution. The anti-inflammatory activities of the compounds were evaluated by determining their inhibitory activities on the production of NO by LPS-stimulated RAW264.7 cells. Furthermore, iNOS expression experiment was performed to preliminarily explore the possible mechanism of inhibiting NO production. The planar structure of 1 was determined by two dimensional (2 D) NMR spectra. The HMBC correlations ( Figure S1) of H 3 -11/C-1, C-2, C-6, C-10; H 3 -9/C-4, C-5, C-6; and H-4/C-6, together with 1 H-1 H COSY correlations ( Figure S1) of H 2 -2/H-3/H-4, indicated the existence of an hexatomic ring in compound 1, with the existence of the gemdimethyl groups (C-10/C-11) located at C-1 and the methyl group (C-9) at C-5. The 1 HÀ 1 H COSY correlations of H 2 -7/H 2 -8 and H 2 -1 0 /H 3 -2 0 revealed the presence of two spin coupling units on the side chain. These two partial structures were connected through an ether bond on the basis of the HMBC correlations of H 2 -8 to C-1 0 and the dramatically downfield chemical shifts of C-8 (d C 62.1) and C-1 0 (d C 70.1) compared to typical hydroxylated carbons. The side chain (-CH 2 CH 2 OCH 2 CH 3 ) was located at C-6 via the HMBC correlations of H 2 -7/C-6. Thus, the planar structure of 1 was unambiguously determined as depicted.

Results and discussion
The orientations of the two hydroxy substituents at C-3 and C-4 were determined to be equatorial and axial, respectively, which were deduced from the coupling constants of H-3 (d H 3.80, dt, J H2ax-H3 ¼ 12.9 Hz and J H3-H4 and H2eq ¼ 3.6 Hz) and H-4 (d H 3.52, d, J H3-H4 ¼ 3.6 Hz) (Yu et al. 2002). The absolute configurations of the C-3 and C-4 in 1 were determined by observing the circular dichroism induced after in-situ complexation with dimolybdenum tetracetate in DMSO solution (Snatzke's method, one of the most reliable methods for assigning the absolute configuration of vic-diols) (Bari et al. 2001). The sign of the diagnostic band at 305 nm is correlated to the absolute configurations of the chiral centers in the 3,4-diol moiety. According to the rule proposed by Snatzke, a positive Cotton effect at 301 nm in the Mo 2 (OAc) 4 -induced CD spectrum ( Figure S2) indicated the 3S,4R configuration for 1 (G orecki et al. 2006). Thus, compound 1 was elucidated and named Medulignin A ( Figure 1).
Compound 2   methyls (d C 11.8, 25.8, 25.8), two sp 3 methylenes (d C 35.2, one oxygenated at d C 61.0), two sp 2 methines (d C 126.0, 160.2), four quaternary carbons (two sp 2 quaternary carbons at d C 133.7, 160.9, one aliphatic quaternary carbons at d C 41.9, and one ketone at d C 188.3). The aforementioned functional groups accounted for three IHDs, implying the presence of a ring in compound 2.
The above-mentioned evidence of 2 showed many similarities to those of 1, suggesting that they had the same megastigmane sesquiterpenoid skeleton, which was corroborated by the 2 D NMR data. The main difference was the existence of an a, b-unsaturated ketone moiety in 2, and this was evident from the 13 C NMR spectroscopic data (d C 160.2/C-2, 126.0/C-3 and 188.3/C-4) of 2. The double bond was unsubstituted, which was deduced from the proton signals at d H 6.96 (1H, d, J ¼ 9.9 Hz, H-2) and 6.15 (1H, d, J ¼ 9.9 Hz, H-3), as verified by the 1 HÀ 1 H COSY correlation ( Figure S1) of H-2/H-3. The other difference was the replacement of an ethoxy unit by a hydroxy group at C-8 in 2, which was established based on the 1 HÀ 1 H COSY correlation of H 2 -7/H 2 -8 and the HMBC correlation of H 2 -7/C-6, suggesting a hydroxyethyl group was located at C-6. Therefore, compound 2 was structurally demonstrated and featured a rare megastigmane sesquiterpenoid, which had only been reported from Chimonanthus salicifolius recently and named 3-(2-hydroxyethyl)-2,4,4-trimethylcyclohexa-2,5-dienone (Xu and Shen 2021).
Inflammation represents the basis of severe acute and chronic syndromes like rheumatic arthritis (Selmi et al. 2014), inflammatory bowel disease (Wang et al. 2014), Parkinson's disease (Su and Federoff 2014) and cancer (Hagerling et al. 2015). Nitric oxide (NO), a gaseous free radical, has been identified as an important molecule involved in immune responses and inflammation (Guzik et al. 2003). The anti-inflammatory activities of the compounds were evaluated by determining their effect on the production of NO by LPS-stimulated RAW264.7 cells. Cell viability assay in response to the purified constituent treatment was simultaneously determined to verify that the decrease of the NO level was not attributed to cell death. Quercetin was used as the positive control (IC 50 ¼ 15.9 ± 1.2 lM).
As a result, all the tested compounds (up to 50 lM) did not show any significant cytotoxicity (Table S2). Compounds 2, 9 and 10 showed moderate inhibitory effects with IC 50 values of 46.7 ± 1.9, 21.1 ± 1.7 and 31.5 ± 2.3 lM, respectively (Table S3). As far as I know, this was the first time that the anti-inflammatory effects of compounds 1 À 4 and 12 had been reported. In addition, compounds 9 and 10 showed the considerable moderate inhibitory effects as reported in the previous literature, which also further confirmed their anti-inflammatory activities (Cuong et al. 2021;Jin et al. 2014;Yang et al. 2017).
The large amount of NO production is mainly catalyzed by the upstream protein iNOS in the inflammatory signaling pathway (Lee et al. 2017;Lee et al. 2019). Inhibition the overproduction of NO means reducing the expression of iNOS. Compound 9, which had relatively strong activity and enough content, had been selected to detect the iNOS expression by western blot to explore primary anti-inflammation mechanism. The experimental methods were referred to the previous literature (Sheeba and Asha 2009). As demonstrated in Figure S3, the iNOS expression stimulated with LPS was significantly increased, and compound 9 showed a certain dose dependent reduction in the expression of iNOS in LPS treated RAW264.7 cells. The results revealed that compound 9 inhibited the production of NO by reducing the iNOS expression.

General experimental procedures
Optical rotation (Na lamp, 589 nm) was determined on an Autopol VI automatic polarimeter at room temperature. ECD spectra were recorded on a JASCO J-815 spectrometer using a 0.1 cm path length sample cell. IR spectra were carried out on a Thermo IS5 spectrometer with KBr panels. UV spectra were measured on a Shimadzu UV-2550 UV-visible spectrophotometer. NMR spectra were obtained on a Bruker Avance III 600 MHz spectrometer (Bruker Biospin AG, Switzerland) with TMS as an internal standard. (±)-ESIMS and (±)-HRESIMS spectra were carried out on a Bruker Daltonics Esquire 3000 Plus LC-MS instrument and a Waters Q-TOF Ultima mass spectrometer, respectively. MCI gel (CHP20P, 75 À 150 lm, Mitsubishi Chemical Industries, Ltd.), Sephadex LH-20 (GE Healthcare, Uppsala, Sweden) and silica gel (200 À 300 and 300 À 400 mesh, Qingdao Haiyang Chemical Co. Ltd.) were used for column chromatography (CC). Precoated silica gel GF254 plates (Qingdao Haiyang Chemical Co. Ltd.) were used for TLC detection. Semipreparative HPLC was performed on a Waters 2695 binary pump system equipped with a Waters 2489 detector (210 and 254 nm) using a YMC-Pack ODS-A column (250 Â 10 mm, S-5 lm) or a Waters X-Bridge Prep C18 column (250 Â 10 mm, S-5 lm). All solvents were of analytical grade (Shanghai Chemical Reagents Co. Ltd., China) and solvents used for HPLC were of HPLC grade (J & K Scientific Ltd., China).

Plant material
The whole plants of S. medusa were collected from Yeniu Ditch (altitude 4100 m), Qilian County, Xining City, Qinghai Province in August 2018, and were identified by Professor Lijuan Mei. A voucher specimen (No. 0341202) was deposited in the Key Laboratory of Tibetan Medicine of the Chinese Academy of Sciences.

Determination of NO production and cell viability assay
The level of NO production was determined by measuring the amount of nitrite present in cell culture supernatants as described previously (Cuong et al. 2012). Briefly, the RAW264.7 cells (1 Â 10 5 cells/well) were cultured in 96-well plates with DMEM high-glucose medium supplemented with 10% fetal bovine serum (FBS), 1 mM pyruvate, 2.0 mM glutamine, 10.0 lg/mL of streptomycin and 100.0 U/mL of penicillin at 37 C in a humidified atmosphere with 5% CO 2 . The cells were treated with 1.0 lg/mL of LPS and with the test compounds for 24 h. The absorbance at 540 nm was measured after incubating culture supernatant (100 lL/each well) with Griess reagent (100 lL) (Sigma-Aldrich) at room temperature. The concentration of NO was calculated using a NaNO 2 solution standard. Cell viability was measured using the MTT-based colorimetric assay.

Conclusion
In summary, one new megastigmane sesquiterpenoid (1), along with 11 known analogues (2-12), were obtained from S. medusa. Their structures were established by spectroscopic data and chemical methods. All isolates, except compounds 3 and 6, were mentioned from the studied plant for the first time. Compounds 1, 2, 4, 5, 7, 8 and 12 were firstly isolated from the genus Saussurea. All compounds were screened for their anti-inflammatory activities on NO production in LPS-induced RAW264.7 macrophages. Compounds 2, 9 and 10 showed moderate inhibitory effects. Furthermore, compound 9 showed a certain dose dependent reduction in the expression of iNOS in LPS treated RAW264.7 cells. Overall, these findings suggested that except for lignans and flavonoids reported previously, megastigmane sesquiterpenoids might also be the main and the important anti-inflammatory constituents of S. medusa.