Anti-osteoclastogenic cycloartane saponins from Mussaenda pubescens

Abstract The methanolic extract of Mussaenda pubescens Dryand leaves exhibited significant anti-osteoclastogenic activity. Chemical investigation of M. pubescens led to the isolation of one new cycloartane saponin, mussaendoside X (1) along with eight known compounds: heinsiagenin A 3-O-[α-L-rhamnopyranosyl-(1→2)-O-β-D-glucopyranosyl-(1→2)]-O-β-D-glucopyranoside (2), mussaendoside O (3), heinsiagenin A 3-O-[α-L-rhamnopyranosyl-(1→2)-O-β-D-glucopyranosyl-(1→2)]-O-β-D-glucopyranosyl-(1→4)-O-β-D-glucopyranoside (4), mussaendoside G (5), mussaendoside U (6), shanzhiside methyl ester (7), barlerin (8) and musaenoside (9). Their structures were elucidated by extensive spectroscopic methods including 1D- and 2D-NMR as well as MS analysis and comparison with the literature. Cycloartane saponins 1–6 positively suppressed osteoclast formation in an anti-osteoclastogenic screening assay. Consequently, treatment of RANKL-stimulated RAW 264.7 cells with compounds 1–4 significantly decreased the number of osteoclasts in a concentration-dependent manner. Six compounds from M. pubescens, with the new cycloartane, mussaendoside X, were shown for the first time as potential effective inhibitors of osteoclastogenesis. Graphical Abstract


Introduction
Osteoblasts, osteoclasts, osteocytes and lining cells are the four types of cells that make up bone. The damaged bone is eliminated by osteoclasts and replaced by new bone created by osteoblasts in the process of bone remodelling (Feng and McDonald 2011). An imbalance between bone formation and resorption can result in too much bone (osteopetrosis) or too little bone (osteoporosis) (Lazner et al. 1999). Bisphosphonates and estrogen therapy are popular and effective in treating osteoporosis, but they have a number of adverse effects (Kennel and Drake 2009;Baron et al. 2011). As a result, new, safer anti-osteoporosis medications are desperately needed. Previous studies indicated that receptor activator of nuclear factor kappa B ligand (RANKL) is produced by osteoclasts and interacts with a receptor activator of nuclear factor kappa B (RANK) on osteoclasts and promotes their change into completed osteoclasts. In addition, the RANK/RANKL interaction promotes bone resorption (Kostenuik et al. 2009). This opens up many options for future research in terms of evaluating anti-osteoporotic activity by affecting osteoclast formation in vitro.
For thousands of years, traditional herbs have been used in folklore with few side effects. As a result, finding chemicals from herbal sources that contain anti-osteoporotic properties have raised concerns (Sun et al. 2017;Wang et al. 2020;Wei et al. 2021). Mussaenda is a genus of flowering plants in the family Rubiaceae and includes 188 accepted species. Mussaenda species native to the African and Asian tropics and subtropics are important sources of oriental medicine. Mussaenda pubescens Dryand has been used in traditional medicine for the treatment of some inflammatory diseases, such as laryngopharyngitis and acute gastroenteritis (Chi 1999). Phytochemical studies of M. pubescens have revealed the presence of N-triterpene cylcoartane saponins (Zhao et al. 1994;Weimin et al. 1995Weimin et al. , 1996Zhao et al. 1996bZhao et al. , 1997, phenolics, and iridoids (Hien et al. 2017).
In the search for anti-osteoclastogenic activity in Vietnamese traditional plants, the methanol extract of M. pubescens leaves was found to inhibit strongly the progression of osteoclastogenesis by 98.51% at a concentration of 25 mg/mL (Huong et al. 2021). The plant was selected for further chemical investigation. This paper reports the isolation, structural elucidation and anti-osteoclastogenic activity of one new saponin and eight known compounds from M. pubescens.

Results and discussion
The methanolic extract of M. pubescens leaves was suspended in water and then partitioned with n-hexane and ethyl acetate (EtOAc) to obtain layers. Using various chromatography methods, nine compounds were isolated. The known compounds were identified as heinsiagenin (Zhao et al. 1994 (Weimin et al. 1996), mussaendoside U (6) (Zhao et al. 1997), shanzhiside methyl ester (7), barlerin (8) and musaenoside (9) (Hien et al. 2017) (Supplementary material, Figure S1). Their structures were elucidated by extensive spectroscopic methods including 1D-and 2D-NMR as well as MS analysis and by comparison with the literature.
Compound 1 was obtained as an amorphous powder and its molecular formula determined to be C 78  3.16, dd, J ¼ 4.0, 12.0 Hz) and the NOESY correlation between H-3 (d H 3.16) and H-5 (d H 1.33). Acid hydrolysis of 1 gave L-rhamnose and D-glucose (identified as trimethylsilyl derivatives by gas chromatography). In addition, the monosaccharides were determined to be b-D-glucopyranosyl and a-L-rhamnopyranosyl based on the multiplicity of H-1 of glc I: To identify the anti-osteoclastogenic effect of the isolated compounds, we examined them on RANKL-induced RAW 264.7 cells at the screening concentration of 10 mM. Cycloartane saponins 1-6 exhibited strong inhibitory effects against RANKLinduced osteoclast formation (Supplementary material, Figure S13). The cell viability assay revealed that all compounds had no impact on cell growth (Supplementary material, Figure S14). This result ruled out the possibility that the test compounds induced cell toxicity. Therefore, the concentration-dependent effects of the saponins were examined. As expected, treatment of RAW 264.7 cells with compounds 1-4 decreased significantly the number of osteoclasts in a concentration-dependent manner (Supplementary material, Figures S15 and S16), suggesting that the saponins 1-4 suppress the RANKL-induced formation of mature osteoclasts. Compounds 5-6 moderately inhibited osteoclastogenesis at a concentration of 10 mM; therefore, these compounds were selected to evaluate the concentration-dependent effects at 3, 10 and 30 mM. As shown in Figures S14 and S15 (Supplementary material), compounds 5-6 exhibited significant effects in reducing the formation of osteoclasts only at the highest concentration (30 mM). Bone remodelling is modulated by the essential roles of bone-resorbing osteoclasts and bone-formation osteoblasts. In particular, the hyperactivity of osteoclasts results in several bone-related diseases, such as osteoporosis, rheumatoid arthritis or bone metastasis. In recent years, natural products have been used widely in the treatment of bone-loss-related diseases (Sethi and Aggarwal 2007;An et al. 2016). M. pubescens is a rich source of triterpenes and saponins (Weimin et al. 1996;Zhao et al. 1996aZhao et al. , 1996bZhao et al. , 1997. Saponins have been reported to exhibit several biological effects, including anti-inflammatory and anti-osteoclastogenic activities. For example, triterpenoid saponin W3 from Anemone flaccida inhibits osteoclast differentiation through suppressing RANKL-induced MAPKs and NF-jB pathways (Kong et al. 2015). The present study found for the first time that the cycloartane saponins from M. pubescens inhibited RANKL-induced osteoclast differentiation in macrophages. Further studies should be performed to identify the molecular mechanism underlying the anti-osteoclastogenic activity of these cycloartane saponins.

Plant materials
The leaves of Mussaenda pubescens Dryand were collected in Vinhphuc province, Vietnam in May, 2019, and identified by Dr. Nguyen The Cuong, Institute of Ecology and Biological Resources, VAST. A voucher specimen (ML-29) was deposited at the University of Science and Technology Hanoi, VAST.

Acid hydrolysis
Compound (1, 2.0 mg) was separately dissolved in 1.0 N HCl (dioxane À H 2 O, 1:1, v/v, 1.0 mL) and heated to 80 C in a water bath for 3 h. Acidic solution was neutralized with Ag 2 CO 3 with the solvent thoroughly removed under a N 2 stream overnight. After extraction with CHCl 3 , the aqueous layer was concentrated to dryness using N 2 . The residue was dissolved in dry pyridine (0.1 mL), followed by addition of L-cysteine methyl ester hydrochloride in pyridine (0.06 M, 0.1 mL). The reaction mixture was heated at 60 C for 2 h. Trimethylsilylimidazole solution (0.1 mL) was then added, followed by heating at 60 C for 1.5 h. The dried product was partitioned with n-hexane and H 2 O (0.1 mL each), and the organic layer was analyzed by gas chromatography: column DB-5 (0.32 mm ID Â 30 m length), detector FID, column temp 210 C, injector temp 270 C, detector temp 300 C, carrier gas He (2 mL/min). Under these conditions, the standard sugars gave peaks at t R (min) 14.11 and 14.26 for D-and L-glucose and 4.50 for L-rhamnose, respectively. Peaks at t R (min) 14.11 and 4.50 of D-glucose and Lrhamnose were observed.