A New Cycloartane-Type Triterpenoid Saponin Xanthine Oxidase Inhibitor from Homonoia riparia Lour

A new cycloartane-type triterpenoid saponin named riparsaponin (1) was isolated from the stem of Homonoia riparia Lour together with six known compounds. The structure of riparsaponin was determined by using NMR and mass spectroscopy and X-ray crystallography techniques. Additionally, riparsaponin has a significant inhibitory effect on xanthine oxidase in vitro, and the IC50 was 11.16 nmol/mL.


Introduction
Homonoia riparia Lour, (family Euphorbiaceae), is widely distributed in the south part of China [1]. The roots of H. riparia are commonly used as an effective traditional Chinese herbal medicine for treating hepatitis and joint gall, stomach ache, and ambustion based on its antipyretic choleretic, anti-inflammatory, detoxification, and diuretic activities [1,2]. However, phytochemical and pharmacological investigations of this plant are currently lacking. Previous chemical research reported that it contains triterpenes, steroids, and phenolics [3][4][5]. In our present investigation, a new cycloartane-type triterpenoid saponin (compound 1, named riparsaponin) was isolated from the stems of H. riparia together with six known compounds ( Figure 1). In addition, riparsaponin showed OPEN ACCESS significant inhibitory activity on xanthine oxidase. Here, we report the isolation, identification and activity of the new compound, which could be helpful for treating gouty arthritis.

Identification of the Riparsaponin
The new compound riparsaponin (1) was identified by using NMR and mass spectroscopy, and X-ray crystallography techniques. Compound 1 was obtained as colorless prismatic crystal (MeOH); mp: 291-292 °C. Its Liebermann-Burchard response was positive. FAB-MS (m/z) (%): 620 (99), 712 (29), 487 (55). The 13 C-NMR, 1 H-NMR data are listed in Table 1. The 13 C-NMR spectrum (100 MHz, DMSO-d 6 ) showed 36 carbon signals, and lots of carbon signals were between 60-80 (δ C ), which indicated the existence of a saccharide group. The carbon signals of 105.9, 73.82, 105.9, 73.82, and 65.62 (δ C ) could further demonstrate the existence of pentose, and the carbon signal at 105.9 ppm is the terminal group carbon of the pentose (β-type) [6]. The two high-field doublets observed at δ H 0.99 (1H, m) and 0.3 (1H, s) are characteristic of the two germinal protons of a cyclopropane moiety. What's more, two olefinic protons at δ H 4.65 (2H, brs) indicated the presence of an olefinic methylene moiety. There are two carbon signals at 106.13 and 156.22 (δ C ), suggesting a double bond. The 1 H-NMR spectrum (400 MHZ, DMSO-d 6 ) indicated seven methyl groups (δ H 0.87, 0.88, 0.96, 0.98, 1.05, 1.08, 1.12). These spectroscopic data suggested this compound to be a cycloartane-type triterpene with an exocyclic methylene group. Combined the reference data [3], we found the 1 H-NMR and 13 C-NMR spectra of compound 1 were analogous to that reported for a cycloartane-type triterpene which was isolated from H. riparia previously. Furthermore, the structure of compound 1 was confirmed by HMBC, ROESY ( Figure 2) and X-ray diffraction (CCDC deposition number is 1021164) (Figure 3, Table 2).

Inhibitory Effect of Riparsaponin on Xanthine Oxidase Activity in Vitro
Gout is one of the common human metabolic diseases and caused by hyperuricemia, which can result in depositions of urate crystals in joints, leading to gouty arthritis [7]. Xanthine oxidase plays an important role during the formation of uric acid, and the accumulation of uric acid can result in hyperuricaemia, leading to gout [8]. Previous investigations revealed that inhibitors of xanthine oxidase could be potentially beneficial for treating gouty arthritis [9]. Herbal remedies have been used in China for more than millennium, and lots of investigations have reported that the herbal medicines and its derived compounds can safely and effectively in treatment of various diseases [10,11]. In our present study, the inhibitory activities of the six known compounds on xanthine oxidase were weak, but riparsaponin could significantly inhibit xanthine oxidase activity in vitro at the doses during 9.68 to 161.29 nmol/mL compared with the DMSO group (p < 0.01), in a dose-dependent manner (Table 3). In our present study, the IC 50 of riparsaponin was 11.16 nmol/mL, which is a better value compared to allopurinol used as positive control drug (IC 50 11.84 nmol/mL). The results above indicated that riparsaponin is a potential powerful xanthine oxidase inhibitor.

Plant Material
The stems of H. riparia were collected in Jinping County, Yunnan Province, China, in October 2010. The plant was identified by Jingxiu Li (Kunming Institute of Botany Chinese Academy of Science, Kunming, Yunnan, China). A voucher specimen was deposited in our laboratory.

Extraction and Isolation
Dried stems of H. riparia (58.8 kg) were powered and extracted three times with 60% ethanol (total 300 L) at reflux. Then the extracts were spray-dried to obtain a dry fine powder (4.8 kg). The powder was extracted three times by maceration with petroleum ether, CH 2 Cl 2 , EtOAc, and 95% ethanol, respectively (total 40 L, each extraction lasted 2 days).

Xanthine Oxidase Inhibitory Assay
The inhibitory effect of compound 1 against xanthine oxidase activity was carried out according to the previous investigations [15]. The experiment was performed by using 96 holes plate, and the allopurinol and DMSO were used as the positive and negative control. Firstly, xanthine (50 μmol/L) was added into each reaction system, and then the tested samples were added. After that, the xanthine oxidase (0.1 U/mL), quinoline solution (5 μmol/L), (NH 4 ) 2 Fe (SO 4 ) solution (1 μmol/L), H 2 SO 4 solution (50 μmol/L) were added subsequently. The reaction system incubated at room temperature for 18 min, then added NaOH solution (1 mol/L) into total reaction system to 150 μL. Finally, adding 150 μL ethanol to terminate the reaction, and the reaction production was determined by measuring the absorbance at 380 nm by using fluorescent chemiluminescence detector on a microplate spectrophotometer. The inhibitory percentage against xanthine oxidase (%) was calculated using the following equation: inhibition (%) = [1 − (A sample/A control) × 100 In addition, the IC 50 values were calculated

Conclusions
In the present study, a new cycloartane-type triterpenoid saponin named riparsaponin was isolated from the stem of H. riparia, and it has a significant inhibitory effect on xanthine oxidase.