Recurvataside, a new saponin from aerial parts of Mussaenda recurvata

Abstract A new glycoside, recurvataside (1) and six known compounds, quinovic acid (2), quinovic acid 28-O-β-D-glucopyranosyl ester (3), 3-O-β-D-glucopyranosylquinovic acid (4), 3-O-β-D-glucopyranosylquinovic acid 28-O-β-D-glucopyranosyl ester (5), pomolic acid (6), and ursolic acid (7) were isolated from aerial parts of Mussaenda recurvata. The structure of compound 1 was identified from its spectroscopic data and by comparison with the literature. Recurvataside represents the first occurrence of δ-oleanolic acid saponin bearing two D-glucose units at C-3 and C-28 in nature. This is the first time δ-oleanane-type saponin reported in the genus Mussaenda. Compounds 1-7 were evaluated the cytotoxicity against two cancer cell lines MCF-7 and HepG2. Among them, only compound 7 exhibited moderate activity against MCF-7 and HepG2 cell lines with IC50 value of 16.97 ± 1.55 and 20.28 ± 1.00 μM, respectively. Compounds 1–7 were also tested for their inhibitory NO production in LPS-stimulated RAW264.7 cells. Compounds 3, 5, and 7 showed significant reduction of nitrite accumulation in LPS-stimulated RAW 264.7 cells with the IC50 values of 8.81 ± 0.48, 13.42 ± 0.84, and 18.37 ± 0.67 μM, respectively. Graphical Abstract


Results and discussion
Compound 1, a white amorphous powder, exhibited a deprotonated molecular ion peak at [M-H] À 811.5207 on the HRESI mass spectrum, indicating its molecular formula as C 42 H 68 O 13 . 1 H NMR and HSQC analysis revealed seven tertiary methyls (d H 0.74, 0.78, 0.83, 0.87 Â 3, 0.95), two methines (d H 0.71 and 0.89) and eleven methylenes in the range 0.80-2.30 ppm. In the downfield region, the 1 H-NMR spectrum identified two anomeric protons [d H 4.13 (d,8.0 Hz) and 5.25 (d,8.0 Hz)] which were correlated with corresponding anomeric carbon signals at d C 105.3 and 94.0 in the HSQC spectrum, together with nine oxygenated methines, two oxymethylenes, and eight hydroxy groups in the range 2.93-5.25 ppm. These spectroscopic data indicated that 1 was a triterpenoid-type saponin having two monosaccharide units. The 13 C-NMR data in accordance with HRESI mass data showed 42 carbon signals, including 30 carbons of a terpenoid aglycone moiety and 12 carbons attributable to two pyranose-sugar units.
The aglycone component was found to share the planar structure of d-oleanolic acid (Caputo et al. 1974;Mahato and Kundo 1994;Yoshikawa et al. 1997;Kikuchi et al. 2011). This was confirmed by COSY and HMBC correlations. At first, HMBC cross peaks from both H 3 -23 (d H 0.95) and H 3 -24 (d H 0.74) to C-3 (d C 88.1) indicated the presence of a hydroxy group at C-3. HMBC cross peaks of both H 3 -23 and H 3 -24 to C-4 (d C 38.4) and C-5 (d C 55.0), of H 3 -25 (d H 0.78) to C-1 (d C 37.2), C-5, and C-10 (d C 37.8), and of H 3 -26 (d H 0.87) to C-7 (d C 37.1), C-8 (d C 40.6), and C-9 (d C 55.7) indicated the chemical structures of the rings A and B. This was supported by the 1 H-1 H COSY spectrum shown in Figure S1. The presence of two substituted olefinic carbons at d C 136.0 and 129.2 and HMBC cross peak of the methyl at d H 0.83 (H 3 -27) to carbon at d C 136.0 identified the position of the double bond at C-13 and C-18. Two remaining methyl groups were determined to locate at C-20 using HMBC and COSY correlations. Finally, a carboxylic acid group at d C 175.3 was assigned as C-17 by comparison with the NMR data of the related d-oleanolic acid derivatives (Kikuchi et al. 2011;Yoshikiwa et al. 1997;Caputo et al. 1974;Mahato and Kundo 1994). The absence of the CH-18 of oleanolic acid (d H 2.84, d C 41.6, see Tables S1 and S2) in 1 supported the d-oleanolic acid scaffold.
The nature of the sugar moieties was identified using J-coupling analysis, NOESY spectrum, and by comparison with the literature. The large J values of H-1 0 and H-1 00 were 8.0 Hz, indicating their axial position. NOESY correlations of H-1 0 with H-3 0 and H-5 0 and of H-1 00 with H-3 00 and H-5 00 hinted that each proton was at axial position. However, the coupling patterns of other protons of these sugars were complicated due to the presence of the hydroxy groups. Comparison of NMR data of 1 and coisolated saponins 3-5 (recorded in the same deuterated solvent DMSO-d 6 ), especially 3-O-b-D-glucopyranosylquinovic acid 28-O-b-D-glucopyranosyl ester (5) (Tables S1 and S2, Figures S10-S12) provided the NMR consistency of the sugar units of these compounds, even the chemical shifts of the hydroxy groups, indicating that two sugar units of 1 were b-D-glucopyranose.
Dozens of saponins were reported in the genus Mussaenda (Zhao et al. 1994(Zhao et al. , 1995(Zhao et al. , 1996a(Zhao et al. , 1996b(Zhao et al. , 1996c(Zhao et al. , 1997Mohamed et al. 2015Mohamed et al. , 2016Bach et al. 2019aBach et al. , 2019b. Most of them had the aglycone part derived from ursolic acid, quinolic acid, or oleanolic acid. Up to now, there is only d-oleanolic acid saponin reported previously in nature. Yoshikawa and co-workers isolated scoparionoside C, a saponin bearing two sugar units b-D-glucopyranosiduronic acid and b-D-xylopyranosyl isolated from Kochia scoparis (Yoshikawa et al. 1997). More recently, Liu and co-workers (2021) synthesized a series of d-oleanolic acid glycosides and evaluated their anti-inflammatory activity. The most similar product synthesized by Liu and co-workers was the saponin bearing one D-glucose unit at C-3 and one D-galactose unit at C-28. Recurvataside represents the first occurrence of d-oleanolic acid saponin bearing two D-glucose units at C-3 and C-28 in nature.
In this study, compounds 1-7 were evaluated the cytotoxicity against two cancer cell lines MCF-7 and HepG2 using SRB assay. Among them, only compound 7 exhibited moderate activity against MCF-7 and HepG2 cell lines with IC 50 value of 16.97 ± 1.55 and 20.28 ± 1.00 lM, respectively (ellipticine was used as a positive control) (Table S4). Other compounds failed to reveal any activity. A comprehensive review on cytotoxicity of saponins having oleanane-type, ursane-type, and lupane-type skeletons indicated that monodesmosides would enhance the higher activity than bidesmosides (Podolak et al. 2010). Among monodesmodides, saponins bearing an a-L-arabinose unit at C-3 would increase the cytotoxicity (Gauthier et al. 2009;Samarakoon et al. 2017). In 2017, Wang and co-workers synthesized a series of C-3-trisaccharide oleanolic acid saponins having strong cytotoxicity (Wang et al. 2017). All these compounds had the a-L-arabinose unit at C-3, indicating the important role of this sugar in cytotoxicity. In our case, recurvataside is a bidesmoside oleanolic acid-type that lacked the a-Larabinose unit, proposing its weak cytotoxicity against tested cancer cell lines.
Moreover, compounds 1-7 were also tested for their inhibitory NO production in LPS-stimulated RAW264.7 cells. The results indicated that compounds 3, 5, and 7 showed higher reduction of nitrite accumulation in LPS-stimulated RAW 264.7 cells with the IC 50 values of 8.81 ± 0.48, 13.42 ± 0.84, and 18.37 ± 0.67 lM, respectively, which are much lower than the positive control, L-NMMA (IC 50 value of 29.35 ± 0.30 lM) (Table S5). Other compounds are weaker or inactive in these assays (Table S5). Liu and co-workers evaluated the anti-inflammatory activity of the synthesized d-oleanolic acid and natural oleanolic acid saponins . Most d-oleanolic acid saponins inhibited LPS-induced secretion of pro-inflammatory factors TNF-a and IL-6 in THP1derived macrophages. Among eight tested bidesmosides, compounds having an a-Larabinose unit at C-3 exhibited much higher activity than D-glucose-bearing saponins. The weak activity of recurvataside (1) might come from the presence of two D-glucose units of 1. Comparison of the activities among 2-5 indicated the important role of a D-glucose unit at C-28 to enhance the NO inhibition. However, the presence of Dglucose moiety at C-3 does not affect the activity. Compounds 6 and 7 had much lower IC 50 values than that of 2 indicated that the 27-COOH group would decrease the activity but 19-OH group (in case of 6) would increase the activity.

General experimental procedures
NMR spectra (1 D and 2 D) were recorded on a Bruker Avance at 500 MHz for 1 H NMR and 125 MHz for 13 C NMR. HRESIMS were recorded on a Bruker MicrOTOF-Q II mass spectrometer. Thin layer chromatography was carried out on precoated Kieselgel 60 F 254 or silica gel 60 RP-18 F 254 S (Merck). Spots were visualized by spraying with 20% H 2 SO 4 solution, followed by heating.

Plant material
Aerial parts of Mussaenda recurvate were collected in Khanh Hoa province, Vietnam in November 2019. The scientific name of the plant was authenticated by the botanist Dang Van Son, Institute of Tropical Biology, Vietnam Academy of Science and Technology. A voucher specimen (VNM_Dang356) was deposited in the VNM Herbarium, Institute of Tropical Biology, Vietnam Academy of Science and Technology.

Cytotoxic assay
Cytotoxic activity was determined by Sulforhodamine B (SRB) assay following the previously described method (Skehan et al. 1990;Duong et al. 2015). Ellipticine was used as a positive control.

Measurement of NO production assay
Determination of NO production and cell viability assay was performed using the modified method (Le et al. 2019; Cheenpracha et al. 2010). L-NMMA was used as a positive control.

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