New Triterpenoids and Anti-Inflammatory Constituents from Glinus oppositifolius

Three new triterpenoids—spergulagenin B (1), spergulagenin C (2), and spergulagenin D (3)—were isolated from the aerial part of Glinus oppositifolius, along with 17 known compounds (4–20). The structures of these new compounds were identified by spectroscopic and MS analyses. Compounds 3, 5, 19, and 20 were evaluated for inhibition of nitric oxide production in LPS-stimulated RAW 264.7 cells with IC50 values of 17.03, 18.21, 16.30, and 12.64 μM, respectively. Compounds 3, 5, and 20 exhibited inhibitory effects on LPS-induced nitric oxide production in RAW 264.7 cells with IC50 values of 18.35 ± 1.34, 17.56 ± 1.41, and 14.27 ± 1.29 μM, respectively.


Introduction
Molluginaceae has about 13 genera and more than 120 kinds of plants in the world, mainly distributed in tropical and subtropical regions. Glinus oppositifolius (L.) Aug. DC. (Figure 1) is an annual herb mainly distributed at low altitudes in the southern part of Taiwan [1]. G. oppositifolius is a folk herb used in the treatment of dermatitis and chronic inflammatory diseases [2]. Flavonoids [3,4], triterpenoids [4], naphthalenes [4], and their derivatives are widely distributed in plants of the family Molluginaceae. Many of these compounds exhibit anti-inflammatory [3,5], antifungal, antiparasitic, and antibacterial activities [6]. Macrophages are one of the immune cells that can secrete nitric oxide (NO), a mediator of inflammatory responses that can participate in host defense [7]. Tumor necrosis factor alpha (TNF-α) is a cytokine with pleiotropic effects on a variety of cell types. It has been recognized as a master regulator of inflammatory responses and has a bearing on the pathogenesis of certain inflammatory diseases [8]. Inhibition of abnormal activation of macrophages by medicines has been proposed as a way to improve inflammatory diseases. G. oppositifolius was one of many species that we screened for the anti-inflammatory constituents of Formosan plants. Current phytochemical studies of G. oppositifoliu have led to the isolation of three new triterpenoids-spergulagenin B (1), spergulagenin C (2), C (2), and spergulagenin D (3)-together with 17 known compounds. This article describes the structural elucidation of 1-3 and the anti-inflammatory activity of the isolated compounds.

Plant Material
Glinus oppositifolius was collected from Neipu Township, Pingtung County, Taiwan, in February 2010 and identified by J.-J. Chen. A voucher specimen (GO-100514) was deposited in the Department of Pharmacy, National Yang Ming Chiao Tung University, Taipei, Taiwan.

MTT assay
The MTT assay was performed by the reference method with slight modifications [9].

Nitric Oxide Inhibitory Assay
The NO inhibition assay was followed with a slight modification of the reference method [10].
2.8. Enzyme-Linked Immunosorbent Assay RAW264.7 cells (4 × 10 5 cells in 96 well plates) were pre-treated with isolated compounds or vehicle (0.05% DMSO) for 1 h and then stimulated with LPS (100 ng/mL) for 20 h. Supernatants were collected and analyzed for production of TNF-α by using appropriate ELISA kits (R&D, Minneapolis, MN, USA) in accordance to the manufacturer's instructions.

Conclusions
Twenty compounds, including three new triterpenoids-spergulagenin B (1), spergulagenin C (2), and spergulagenin D (3)-were isolated from aerial part of G. oppositifolius. The structures of these new compounds were elucidated on the basis of spectral data. The effects on macrophage pro-inflammatory responses of isolated compounds were evaluated by suppressing LPS-induced NO generation by macrophage RAW264.7 cells. The results of anti-inflammatory assays show that compounds 3, 5, 19, and 20 can obviously inhibit LPS-induced NO generation. Trans-ferulic acid (20) is the most effective among the isolated compounds, with IC 50 value of 12.64 ± 1.14 µM, against LPS-induced NO generation. Furthermore, compounds 3, 5, and 20 exhibited inhibitory effects on LPS-induced TNF-α production in RAW 264.7 cells with IC 50 values of 18.35 ± 1.34, 17.56 ± 1.41, and 14.27 ± 1.29 µM, respectively. Our research indicates G. oppositifolius and its isolates (especially 3, 5, 19, and 20) are worth further research and may be expectantly developed as candidates for the treatment or prevention of various inflammatory diseases (such as dermatitis and arthritis). This study also provides anti-inflammatory scientific evidence for the use of traditional herbal medicine (G. oppositifolius) in the treatment of dermatitis and chronic inflammatory diseases [2].

Conflicts of Interest:
The authors declare no conflict of interest.
Sample Availability: Samples of the compounds are available from the authors.