Triterpenoids From Kadsura coccinea With Their Anti-inflammatory and Inhibited Proliferation of Rheumatoid Arthritis-Fibroblastoid Synovial Cells Activities

One new 3,4-seco-17,13-friedo-lanostane triterpenoid heilaohuacid A (1), one new 3,4-seco-17,14-friedo-lanostane triterpenoid heilaohuacid B (2), five new 3,4-seco-lanostane triterpenoids heilaohuacids C-D (3–4) and heilaohumethylesters A-C (7–9), one new 3,4-seco-cycloartane triterpenoid heilaohuacid E (5), and one new intact-lanostane triterpenoid heilaohuacid F (6), together with twenty-two known analogues (10–31), were isolated from heilaohu. Their structures were determined using HR-ESI-MS data, 1D and 2D NMR spectra, 13C NMR calculations, and electronic circular dichroism (ECD) calculations. Heilaohuacids A and B (1 and 2) contain a 3,4-seco ring A and unprecedented migration of Me-18 from C-13 to C-17 or C-14 to C-18. This type of lanostane triterpenoid derivatives was rarely reported so far. More importantly, all compounds against inflammatory cytokines IL-6 and TNF-α levels on LPS-induced RAW 264.7 macrophages were evaluated, and compounds 4 and 31 significantly inhibited the release level of IL-6 with IC50 values of 8.15 and 9.86 μM, respectively. Meanwhile, compounds 17, 18, and 31 significantly inhibited proliferation of rheumatoid arthritis-fibroblastoid synovial (RA-FLS) cells in vitro with IC50 values of 7.52, 8.85, and 7.97 μM, respectively.

The relative configuration of 2 was determined following the NOE effects, which showed correlations (Figure 3) of H 3 -19 with H-9 and H-12b, H 3 -18 with H-15b and H-20, and H 3 -30 with H-11a and H 3 -21, suggesting that H-9 and H 3 -18 were β-oriented, and H 3 -21 and H 3 -30 were a-oriented. Moreover, the no NOE correlations of H 3 -27 with H-24 were found, suggesting that the double bond between C-24 and C-25 has a trans-configuration. Additionally, the relative configuration of C-20 was investigated by the TDDFT to calculate the 13 C NMR data for 2-1 and 2-2. As shown in Figure 6, the 13 C NMR chemical shifts of isomers were calculated at the mPW1PW91/6-31+G** level. The calculation result of 2-1 (R 2 0.9961) matched the experimental data better than 2-2 (R 2 0.9952), which indicated that H 3 -21 has an a-orientation. The absolute configuration of 2 was determined by ECD data. The experimental ECD spectrum of 2 exhibited a negative cotton effect around 250 nm, which was consistent with the calculated ECD data of the (5S,9S,10R,13S,17S,20R) model ( Figure 7). Thus, heilaohuacid B (2) was elucidated as a 5S, 9S, 10R, 13S, 17S, and 20R absolute configuration.
Compound 3  Comprehensive analysis of the 1D and 2D NMR data revealed it to be the derivative of seco-coccinic acid K (Wang et al., 2012). The differences were that a methylene group at C-12 was replaced by a conjugated ketone group (δ C 205.3, C-12) and the absence of a methoxy group at C-31 in 3, which were confirmed by the HMBC correlations of H-11 with C-12, C-9, and C-13, H 3 -18 with C-12, and H 2 -1 and H 2 -2 with C-3. The relative configuration of H-8 was determined to be β-oriented, through the NOE correlations of H 3 -18 with H-8. The 5, 8, 10S, 13R, 14S, 17R, and 20R absolute configuration of 3 was determined by comparing the experimental and calculated ECD spectra ( Figure 8). Accordingly, the structure of compound 3 was deduced as shown and given the trivial name heilaohuacid C.
Compound 4   Comprehensive analyses of its NMR data ( Tables 1, 3) suggested 4 to be a structural analogue of 3 as both shared the same 3,4-secolanostane triterpenoid skeleton. However, the obvious differences were that an α,β-conjugated ketone group (δ C 200.2, C-6; δ C 123.7, C-7; δ C 175.3, C-8) shifted from ring C to ring B, a double bond at C-24/C-25 was present, but a ketone group was absent at C-23 in 4, as supported using the HMBC spectral analyses. The relative configuration was confirmed by ROESY spectral analyses. Based on the NOE correlation of H 3 -19 with H-9, the H-9 was classified as β-oriented. Thus, the structure of 4 was assigned as shown in Figure 1, and named heilaohuacid D. ) and δ C 179.4 (C-3), 149.4 (C-4), 111.6 (C-28), and a pair of methylene doublets at δ H 0.42 (J 3.8 Hz) and 0.74 (J 3.8 Hz), characteristics of the C-19 protons and carbon of the cyclopropane ring, suggesting that six was a 3,4-secocycloartane triterpenoid (Yang et al., 2015). Analysis of the 1D NMR data (Tables 2, 3) revealed that the structure of 5 was very similar to nigranoic acid (25) (Sun et al., 1996). The obvious differences were the presence of a ketone group at C-23, and the absence of double bond at C-24/C-25 and carboxyl groups at C-27 in 5.  -30), and two olefinic protons at δ H 5.31 (d, J 6.0 Hz, H-11) and 6.91 (t, J 7.0 Hz, H-24). Analyses of the 13 C NMR and DEPT data ( Table 3) showed that compound 6 contained seven methyls, nine methylenes, six methines (two olefinics), and eight quaternary carbons (two carboxyl group). These evidences indicated that compound 6 was an intact lanostane-type triterpenoid, whose 1 H and 13 C NMR spectroscopic data were very similar to those of coccinic acid (Li and Xue, 1986). The only difference was in the geometry of the double bond between C-24 and C-25. Because the carbon chemical shift of C-27 was shifted upfield by 8.0 ppm, compared with coccinic acid, this showed the presence of a double bond between C-24 and C-25 in 6, which was different with that of coccinic acid. Additionally, the configuration of 6 was determined using the ROESY spectrum (Supplementary Figure S42), in which H-27 showed correlation with H-23 but no NOE correlation was observed between H-27 and H-24, demonstrating that the geometry of the double bond must be E configuration ( Figure 3). Therefore, the structure of 6 was elucidated as shown and assigned name heilaohuacid F. Heilaohumethylester A (7) was assigned a molecular formula of C 31 H 50 O 4 based on the HR-ESI-MS spectra and NMR data analysis (Tables 2, 3), suggesting that 7 was a methylated analogue of seco-coccinic acid C (16) . The presence of a methoxy group (δ C 51.6, δ H 3.67) was located at C-31, confirmed by the HMBC correlations of H 3 -31 with the carbonyl (δ C 175.1) at C-3. The similar chemical shifts, coupling constants, and NOE correlations with 16 determined the relative configurations of 7. Therefore, the structure of 7 was elucidated as shown.
Compound 8 was deduced to have the molecular formula of C 31 H 52 O 4 from the molecular ion at m/z 511.3734 [M + Na] + (calcd. for C 31 H 52 O 4 Na, 511.3758) in the HR-ESI-MS data. The NMR data of 8 were highly similar to those of 7; HMBC spectral analysis showed that the obvious differences were absence of a ketone group at C-23 and a hydroxyl group was present at C-24 in 8. Comparison of the NMR data of 8 with those of a pair of 24-epimers (Hong et al., 2013) with the OH at C-24 possessed different orientations, 24(S)-24,25-dihydroxytirucall-7-en-3-one (δ C 78.6, δ H 3.32) and (24R)-24,25-dihydroxytirucall-7-en-3-one (δ C 79.5, δ H 3.29). This indicated that the NMR data of 8 (δ C 78.8, δ H 3.34) were almost similar to the corresponding 24(S)-24,25dihydroxytirucall-7-en-3-one, suggesting that the C-24 stereochemistry should be assigned as 24S in 8. According to analysis of the NOE effect, and the ROESY cross-peaks of H 3 -19 with H-9, the H-9 was classified as β-oriented. Finally, the structure of 8 was identified, and named heilaohumethylester B accordingly.
The inhibited proliferation activity in RA-FLS cells of the isolated compounds (1-31) were evaluated using the MTT method, and methotrexate was used as the positive control (IC 50 4.10 µM). The results (Table 4) indicated that compounds 17, 18, and 31 exhibited good inhibition activities against RA-FLS cells with IC 50 values of 7.52, 8.85, and 7.97 µM, respectively. Furthermore, all isolated compounds were evaluated for their inflammatory activity on inflammatory cytokines (IL-6 and TNF-α) released by LPS-induced RAW 264.7 cells. The inflammatory activity of the isolated compounds was determined using ELISA kits, with methotrexate as positive control. The results (

General Experimental Procedures
Optical rotations of compounds were determined by a Rudolph Research Analytical Autopol Ⅲ automatic polarimeter. UV analysis of compounds was performed on a Shimadzu 2450 UV-vis spectrometer. An Applied Photophysics Chirascan plus CD spectrometer was used to determine ECD spectrum. An Agilent Technologies Cary 630 FTIR spectrometer

ECD Calculations
Methods of quantum chemical ECD calculations for compounds 1-3 are described in the Supporting Information (Supplementary Figure S1).

NMR Calculations
Methods of 13 C NMR calculations for compounds 1 and 2 are described in the Supporting Information (Supplementary Figure S1).

Cell Culture
Human

Anti-Inflammatory Bioassay
Inhibition effects of all compounds (1-31) on release of inflammatory cytokines (IL-6 and TNF-α) in the supernatants on LPS-induced RAW264.7 cells were determined using ELISA kits (BOSTER Biological Technology Co. Ltd., Wuhan, China) following the manufacturer's instructions. Methotrexate was used as a positive control.

Inhibited Proliferation Activity Against RA-FLS Bioassay
Inhibited proliferation activity against RA-FLS cells was determined by the standard MTT assay methods as described previously. RA-FLS cells were seeded into 96-well plates and treated with different concentrations of all compounds for 48 h. Ten microliters of MTT (5 mg/ml) was then added to each well and incubated for 4 h. The supernatants were retrieved, and 100 μl of DMSO was added to each well and mixed by shaking for 5 min. Optical density values at 490 nm were measured using a microplate reader.

CONCLUSION
The roots of K. coccinea, as a Tujia ethnomedicine, have been used to the treat rheumatoid arthritis for a long time in China. The present study has reported that nine new triterpenoids (1-9), along with 22 known analogues (10-31), were isolated from the roots of K. coccinea. Heilaohuacids A and B (1 and 2) contain a 3,4-seco ring A and unprecedented migration of Me-18 from C-13 to C-17 or C-14 to C-18; their relative and absolute configurations were determined by 13 C NMR calculations and ECD data analysis. To the best of our knowledge, this type of lanostane triterpenoid derivative was rarely reported so far, which enriched the structural types of lanostane triterpenoids in K. coccinea. Additionally, compounds 4, 17, 18, 29, and 31 showed good anti-RA and/or anti-inflammatory activities. These findings suggest that lanostane triterpenoids from K. coccinea might serve as therapeutic agents for RA treatment.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material. Further inquiries can be directed to the corresponding authors.