Characterisation of four new triterpenoid saponins with nitric oxide inhibitory activity from aerial parts of Gouania leptostachya

Abstract Four new triterpenoid saponins (gouaniasides VII–IX [1–3] and joazeiroside C [4]) and one known triterpenoid (5) were isolated from the aerial parts of Gouania leptostachya DC. (Rhamnaceae). Their structures were elucidated via one-dimensional and two-dimensional nuclear magnetic resonance spectroscopy, high-resolution electrospray ionisation–mass spectrometry, and analyses of hydrolytic cleavage results. The anti-inflammatory potential of compounds 1–3 was evaluated according to their ability to inhibit the production of nitric oxide (NO) by RAW 264.7 macrophages. All compounds at noncytotoxic concentrations significantly inhibited NO production by macrophages in a concentration-dependent manner. Graphical Abstract


Results and discussion
Crude ethanol extract from the aerial parts of G. leptostachya DC. (Rhamnaceae) were fractionated, isolated, and purified through a combination of chromatographic methods to obtain four new triterpenoid saponins (1-4) and one known compound, epigouanic acid A (5) (Leal et al. 2010;Nganso et al. 2020) (Figure 1). The structures of the compounds were characterised via one-dimensional and two-dimensional nuclear magnetic resonance (NMR) spectroscopy, high-resolution electrospray ionisation-mass spectrometry (HR-ESI-MS), and analyses of the derivatives of chemical reactions.
Compound 3 was isolated as a white powder. Its molecular formula (C 55 H 90 O 23 ) was determined via HR-ESI-MS. The spectrum exhibited a quasi-molecular ion peak at m/z 1153.5464 [M þ Cl] -(calculated for C 55 H 90 O 23 Cl -, 1153.5567). The 1 H-and 13 C-NMR spectra of 3 agreed well with those of 1, except for the presence of an additional b-ᴅ -glucopyranosyl moiety (see Table S1) at C-6 000 Glc . The presence of the moiety was indicated by the HMBC between H-1 0000 Glc (d H 4.34) and C-6 000 Glc (d C 70.2) and the downfield-shifted signal of C-6 000 Glc at d C 70.2 ( Figures S35 and S59). Therefore, the structure of 3 was established as 16b,22a:16a,18-diepoxy-24-methylidenedammarane-3b,20-diol- Compound 4 was obtained as a white powder. Its molecular formula (C 49 H 82 O 19 ) was determined via HR-ESI-MS (m/z 1009.5068 [M þ Cl] -). The 1 H and 13 C NMR spectra 4 resembled those of 1, except for the presence of D ring signals. The absence of signals at d C 66.8 and 38.9, corresponding to C-18 and C-15 in the structure of 1, respectively, and the presence of one methyl group (d H 0.99, d C 10.0) and one hydroxymethine group (d H 3.96, d C 77.6), suggested that the D ring was arranged differently from that in 1. The presence of upfield-shifted signals at C-14 (-3.5 ppm), C-16 (-9.2 ppm), and C-22 (-6.2 ppm) indicated that 4 had a comparable structure with the aglycone of joazeiroside B (Renault et al. 1997). The relative configuration of 4 was proposed based on the comparison of their NMR data and the agreement of the biosynthetic pathway of 1-3. Furthermore, the NMR data suggested that 4 possessed the same trisaccharide as 1 (Table S1). From these results, the structure of 4 was determined as 16,22-epoxy-24-methylidenedammarane-3b,15a,16a,20b-tetrol- To determine the cytotoxicity of compounds 1-3, we treated RAW 264.7 cells with serially diluted single-molecule concentrations of the compounds for 3 days and verified the number of cells in the culture via MTT analyses (Vinh et al. 2020). The results (data not shown) revealed that compounds 1-2 exhibited no cytotoxicity at a concentration of 5 mM, whereas compound 3 showed weak cytotoxicity at 25 mM.
NO is a key mediator of inflammatory response and pathogenesis (Nguyen et al. 2019). We examined the inhibitory activity of isolated compounds 1-3 against the activation of RAW 264.7 macrophages for NO production. RAW 264.7 macrophages were treated with any of compounds 1-3 for 24 h and then incubated with lipopolysaccharide. The level of NO produced by the cells decreased in a concentration-dependent manner. All tested compounds (1-3) showed strong inhibitory activity against NO production in a dose-dependent manner ( Figure S61).
In conclusion, we identified four new triterpenoid glycosides from 96% EtOH extracts of G. leptostachya. Their structures were characterised via one-dimensional and two-dimensional NMR spectroscopy and HR-ESI-MS. G. leptostachya is rich in saponin. The cytotoxicity and NO inhibition result also provide a scientific rationale for further investigations of anti-inflammatory activity for this valuable medicinal plant.

Extraction and isolation
Powdered aerial parts of G. leptostachya (1.5 kg) were mixed with 96% EtOH (10 L Â3), and the mixture was left at room temperature for 4 days. The extract was filtered and concentrated under reduced pressure using a rotary evaporator at 40-50 C, and a greenish-brown residue (148.0 g) was obtained. The residue was suspended in distilled water and successively fractionated with solvents of increasing polarity to obtain n-hexane (17.0 g), ethyl acetate (38.0 g), and n-butanol (21.0 g) fractions and a water layer.