New Triterpenoids and Anti-Inflammatory Constituents from Glinus oppositifolius

Abstract

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.

1. 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), 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.

2. Materials and Methods

2.1. General

Infrared (IR) spectra (KBr or neat) were measured using a Shimadzu IR prestige-21 Fourier transform infrared spectrophotometer (Shimadzu, Kyoto, Japan). Optical rotations were recorded on a Jasco P-1020 polarimeter (Jasco, Kyoto, Japan) in MeOH and CHCl₃. Electronic circular dichroism (ECD) spectra were recorded on a Chirascan CD spectrometer (Applied Photophysics Ltd., Leatherhead, UK). High resolution electron ionization mass spectrometry (HR-EI-MS) was measured at Chung Hsing University (Taichung, Taiwan). Ultraviolet (UV) spectra were measured using a Shimadzu Pharmaspec-1700 UV-Visible spectrophotometer (Shimadzu, Kyoto, Japan). Nuclear magnetic resonance (NMR) spectra—including heteronuclear single-quantum coherence (HSQC), correlation spectroscopy (COSY), heteronuclear multiple-bond correlation (HMBC), and nuclear Overhauser effect spectrometry (NOESY) experiments—were measured using a Bruker DRX-500 FT-NMR (Bruker, Bremen, Germany) operating at 125 MHz (¹³C) and 500 MHz (¹H), respectively. Chemical shifts are given in ppm (δ) using tetramethylsilane (TMS) as internal standard. HPLC separations were carried out utilizing a P230 HPLC system (NATIONAL ANALYTICAL CORPORATION, Maharashtra, India) equipped with P230 HPLC Pump and an IOTA 2 detector, utilizing ChromNav software (version 2.0, Jasco). TLC analysis was performed utilizing aluminum pre-coated Si plates (Merck, Darmstadt, Germany). Column chromatography was carried out utilizing LiChroCART Si gel (5 μM) (Merck, Darmstadt, Germany).

2.2. Chemicals

ACS grade solvents (methanol, ethyl acetate, n-hexane, acetone, and chloroform), HPLC grade solvents (ethyl acetate, acetone, and n-hexane) and deuterated solvents (CDCl₃, acetone-d₆, or CD₃OD) for NMR measurements were procured from Merck, Taipei, Taiwan. LPS (endotoxin from Escherichia coli, serotype 0127:B8), Carr (type IV), and quercetin were purchased from MedChemExpress (Monmouth Junction, NJ, USA). MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) was acquired from Sigma Chemical Co. (St. Louis, MO, USA).

2.3. 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.

2.4. Extraction and Isolation

The dried whole plant (10.58 kg) of Glinus oppositifolius was extracted 3 times with methanol (80 L each) for 7 days. The extract was concentrated under reduced pressure at 38 °C, and the residue (1.48 kg) was partitioned between H₂O and EtOAc (1:1) to provide the EtOAc-soluble fraction (fraction A, 285 g). Fraction A (285 g) was separated by column chromatography (CC) (10.0 kg of SiO₂, 70–230 mesh; n-hexane/EtOAc/methanol gradient) to afford 20 fractions: A1–A20.

Fraction A13 (7.69 g) was purified by Sephadex LH 20 CC (chloroform:methanol = 3:7), silica gel CC (n-hexane:acetone = 8:3), and then HPLC (chloroform:acetone = 6:1) to obtain 4 (12.8 mg), 5 (27.4 mg), 6 (12.2 mg), 7 (8.4 mg), and 5 (7.3 mg). Fraction A14 (16.7 g) was purified by silica gel CC (ethyl acetate: n-hexane = 1:6) and HPLC (acetone:n-hexane = 1:8) to obtain 6 (12.6 mg), 7 (6.4 mg), and 8 (13.4 mg). Fraction A16 (15.5 g) was purified by silica gel CC (n-hexane:ethyl acetate = 4:1) and HPLC (n-hexane:acetone = 3:1) to obtain 9 (8.2 mg), 10 (27.5 mg), 11 (25.0 mg), 12 (8.4 mg), 13 (13.4 mg), 14 (24.5 mg), 15 (7.8 mg), 16 (6.2 mg), 17 (4.3 mg), 18 (32.4 mg), 19 (4.5 mg), and 20 (32.4 mg). Fraction A18 (13.3 g) was purified by Sephadex LH 20 CC (chloroform:methanol = 3:7), silica gel CC (n-hexane:acetone = 5:1), and then semi-preparative HPLC (chloroform: ethyl acetate = 3:2) to obtain 1 (6.6 mg), 2 (4.2 mg), and 3 (3.6 mg).

Spergulagenin B (1): colorless needle; mp 306.2–307.6 °C; IR (KBr) υ_max: 3423 (OH), 2943, 1694 (C=O), 1458, 1385, 1155, 1113, 1061 cm⁻¹ (Figure S1); ¹H-NMR spectroscopic data, see

Table 1. ¹H-NMR data for Compounds 1–3 (δ in ppm, J in Hz).

Position	1 a	2 a	3 a
1	1.94 (m). 1.41 (m)	7.10 (d, J = 10.0 Hz)	1.82 (m), 1.39 (m)
2	2.48 (m), 2.42 (m)	5.83 (d, J = 10.0 Hz)	2.52 (m), 2.38 (ddd, J = 16.0, 5.6, 3.2 Hz)
5	1.30 (m)	1.54 (m)	1.33 (m)
6	1.51 (m), 1.37 (m)	1.56 (m), 1.43 (m)	1.60 (m), 1.35 (m)
7	1.50 (m), 1.31 (m)	1.50 (m), 1.44 (m)	1.54 (m), 1.47 (m)
9	1.69 (m)	1.59 (m)	1.70 (dd, J = 9.6, 4.4Hz)
11	1.87 (m), 1.04 (m)	1.57 (m), 1.46 (m)	2.25 (m), 2.22 (m)
12	3.96	3.99 (m)
13	1.38 (d, J = 4.0 Hz)	1.43 (m)	2.23 (m)
15	1.72 (dd, J = 12.8, 4.0 Hz), 1.35 (m)	1.72 (m), 1.32 (m)	1.79 (m), 1.44 (m)
16	3.70 (m)	3.71 (m)	3.76 (m)
17	1.76 (d, J = 11.2 Hz)	1.78 (m)	1.64 (m)
19	2.02 (m), 1.27 (m)	2.04 (m), 1.28 (m)	2.17 (m), 1.02 (m)
20	2.05 (m), 1.84 (m)	2.05 (m), 1.86 (m)	2.10 (m), 1.93 (m)
23	1.03 (s)	1.09 (s)	1.06 (s)
24	1.08 (s)	1.14 (s)	1.10 (s)
25	0.96 (s)	1.08 (s)	1.00 (s)
26	1.07 (s)	1.11 (s)	1.21 (s)
27	1.01 (s)	1.01 (s)	0.99 (s)
28	1.04 (s)	1.05 (s)	1.14 (2)
29	1.43 (s)	1.45 (s)	1.43 (s)
30	2.23 (s)	2.24 (s)	2.24 (s)

^a measured in CDCl₃ at 500 MHz.

(Figure S2); ¹³C-NMR spectroscopic data, see

Table 2. ¹³C-NMR data for Compounds 1–3 (δ in ppm).

Position	1 a	2 a	3 a
1	39.4	158.5	38.9
2	34.0	125.6	34.1
3	217.3	205.3	216.9
4	48.1	39.2	47.6
5	54.9	53.3	55.1
6	19.7	19.1	19.9
7	32.5	32.6	32.0
8	45.5	45.5	47.2
9	47.3	42.7	49.6
10	36.7	44.6	37.0
11	32.9	32.5	39.6
12	69.5	69.3	210.9
13	55.1	55.2	63.4
14	41.4	42.3	41.6
15	45.1	45.0	43.8
16	65.8	65.7	65.8
17	59.2	59.2	58.7
18	46.3	46.3	44.9
19	44.1	44.0	41.7
20	35.9	35.8	35.8
21	54.4	54.4	55.6
22	217.2	217.0	217.3
23	21.1	21.4	21.4
24	26.6	27.8	26.6
25	15.6	17.1	15.2
26	16.6	17.2	16.7
27	18.7	18.9	20.9
28	17.2	18.8	17.7
29	21.2	21.1	21.4
30	25.9	25.9	26.1

^a measured in CDCl₃ at 125 MHz.

(Figure S3); ECD (c 0.25, MeOH) λ_ma (Δε) 284 (+0.88), 250 (–0.12), 217 (+0.98), 198 (–1.34) nm; HI-EI-MS: 472.3549 [M]⁺ (calcd. for C₃₀H₄₈O₄, 472.3547).

Spergulagenin C (2): colorless needle; mp 305.4–306.8 °C; UV (MeOH) λ_max nm (log λ): 229 (3.73); IR (KBr) υ_max: 3493 (OH), 3416 (OH), 2972, 1690 (C=O), 1458, 1385, 1355, 1254, 1076 cm⁻¹ (Figure S9); ¹H-NMR spectroscopic data, see Table 1 (Figure S10); ¹³C-NMR spectroscopic data, see Table 2 (Figure S11); ECD (c 0.18, MeOH) λ_max (Δε) 283 (+0.96), 249 (–0.14), 219 (+1.05), 198 (–1.09) nm; HI-EI-MS: 470.3409 [M]⁺ (calcd. for C₃₀H₄₆O₄, 470.3406).

Spergulagenin D (3): colorless needle; mp 286.4–287.0 °C; IR (KBr) υ_max: 3447 (OH), 2938, 1697 (C=O), 1558, 1420, 1387, 1354, 1327 cm⁻¹ (Figure S17); ¹H-NMR spectroscopic data, see Table 1 (Figure S18); ¹³C-NMR spectroscopic data, see Table 2 (Figure S19); ECD (c 0.21, MeOH) λ_max (Δε) 284 (+1.02), 249 (–0.20), 218 (+1.02), 197 (–0.91) nm; HI-EI-MS: 470.3407 [M]⁺ (calcd. for C₃₀H₄₆O₄, 472.3403).

Kaempferol (4): yellow powder; mp 274~276 °C; IR (KBr) υ_max: 3348, 3278~2509, 1661, 1616, 1570, 1089, 1010 cm⁻¹; ¹H -NMR (500 MHz, acetone-d₆) δ (ppm): 6.26 (1H, d, J = 1.9 Hz, H-6), 6.52 (1H, d, J = 1.9 Hz, H-8), 7.01 (2H, d, J = 8.9 Hz, H-3′ and H-5′), 8.14 (2H, d, J = 8.9 Hz, H-2′ and H-6′), 12.15 (1H, s, OH-5).

6,8-Dimethyl-5,7,4′-trihydroxyflavone (5): yellow powder; mp 220~225 °C; IR (KBr) υ_max: 3427, 3704~2509, 1654, 1611, 1576, 1555 cm⁻¹; ¹H-NMR (500 MHz, acetone-d₆) δ (ppm): 2.13 (3H, s, Me-6), 2.36 (3H, s, Me-8), 6.64 (1H, s, H-3), 7.05 (2H, d, J = 8.8 Hz, H-3′, H-5′), 7.98 (2H, d, J = 8.8 Hz, H-2′ and H -6′), 13.24 (1H, s, OH-5).

5,7-Dihydroxy-6,8-dimethylflavone (6): yellow powder; mp 289~290 °C; IR (KBr) υ_max: 3400, 3587~2403, 1650, 1602, 1486 cm⁻¹; ¹H-NMR (500 MHz, acetone-d₆) δ (ppm): 2.19 (3H, s, Me-6), 2.37 (3H, s, Me-8), 6.68 (1H, s, H-3), 7.54 (3H, m, H-3′, H-4′ and H-5′), 7.91 (2H, d, J = 7.2 Hz, H-2′ and H-6′), 12.95 (1H, s, OH-5).

5,4′-Dihydroxy-7-methoxy-6,8-dimethylflavone (7): yellow powder; mp 286~287 °C; IR (KBr) υ_max: 3502~2423, 3430, 3072, 2920, 1650, 1612, 1585, 1466 cm⁻¹; ¹H-NMR (500 MHz, CDCl₃) δ (ppm): 2.18 (3H, s, Me-6), 2.36 (3H, s, Me-8), 3.90 (3H, s, OMe-7), 5.40 (1H, s, OH -4′), 6.89 (1 H, s, H-3), 7.03 (2H, d, J = 8.8 Hz, H-3′ and H-5′), 7.87 (2H, d, J = 8.8 Hz, H-2′ and H-6′), 13.03 (1H, s, OH-5).

4-Hydroxybenzoic acid (8): white solid; mp 210~212 °C; IR (KBr) υ_max: 3300~2500, 1696 cm⁻¹; ¹H-NMR (500 MHz, CDCl₃) δ (ppm): 6.33 (1H, br s, Ar-OH), 6.81 (2H, d, J = 8.8 Hz, H-3 and H -5), 7.87 (2H, d, J = 8.8 Hz, H-2 and H-6).

4-Hydroxybenzaldehyde (9): white solid; mp 110~112 °C; IR (KBr) υ_max: 3170, 1676, 1600, 1519, 1454 cm⁻¹; ¹H-NMR (500 MHz, CDCl₃) δ (ppm): 5.81 (1H, s, Ar-OH), 6.95 (2H, d, J = 8.4 Hz, H-3 and H-5), 7.81 (2H, d, J = 8.4 Hz, H-2 and H-6), 9.87 (1H, s, CHO).

4-Hydroxyacetophenone (10): white solid; mp 106~107 °C; IR (KBr) υ_max: 3312, 1664, 1602, 1578 cm⁻¹; ¹H-NMR (500 MHz, CDCl₃) δ (ppm): 2.56 (3H, s, COMe), 6.09 (1H, s, Ar-OH), 6.89 (2H, d, J = 8.8 Hz, H-3 and H-5), 7.91 (2H, d, J = 8.8 Hz, H-2 and H-6).

Methyl 4-Hydroxybenzoate (11): white solid; mp 124~125 °C; IR (KBr) υ_max: 3358, 1689, 1608, 1585, 1514 cm⁻¹; ¹H-NMR (500 MHz, CDCl₃) δ (ppm): 3.89 (3H, s, COOMe), 5.37 (1H, s, Ar-OH), 6.95 (2H, d, J = 8.0 Hz, H-3 and H -5), 7.95 (2H, d, J = 8.0 Hz, H-2 and H-6).

p-Anisic acid (12): white solid; mp 182~184°C; IR (KBr) υ_max: 3307~2503, 2926, 1686, 1605, 1578, 1516 cm⁻¹; ¹H-NMR (500 MHz, CDCl₃) δ (ppm): 3.85 (3H, s, OMe-4), 6.97 (2H, d, J = 8.8 Hz, H-3 and H-5), 7.96 (2H, d, J = 8.8 Hz, H-2 and H-6).

Vanillin (13): white solid; mp 210~212 °C; IR (KBr) υ_max: 3213, 2724, 2858, 1667, 1589, 1510 cm⁻¹; ¹H-NMR (500 MHz, CDCl₃) δ (ppm): 3.97 (3H, s, OMe-3), 6.21 (1H, s, Ar-OH), 7.04 (1H, d, J = 8.0 Hz, H-5), 7.42 (1H, d, J = 2.0 Hz, H-2), 7.43 (1H, dd, J = 8.0, 2.0 and H-6), 9.83 (1H, s, CHO).

4-Hydroxy-3-methoxyacetophenone (14): white solid; mp 182~184 °C; IR (KBr) υ_max: 3323, 2912, 1658, 1575, 1518 cm⁻¹; ¹H-NMR (500 MHz, CDCl₃) δ (ppm): 2.56 (3H, s, COMe), 3.96 (3H, s, OMe-3), 6.05 (1H, s, Ar-OH), 6.95 (1H, d, J = 8.0 Hz), 7.54 (2H, br s, H-2 and H-6).

Acetosyringone (15): white solid; mp 105~107 °C; IR (KBr) υ_max: 3307, 1672, 1608 cm⁻¹; ¹H-NMR (500 MHz, CDCl₃) δ (ppm): 2.57 (3H, s, COMe), 3.96 (6H, s, OMe-3, OMe-5), 6.05 (1H, s, Ar-OH), 7.25 (2H, s, H-2 and H-6).

4-Hydroxy-3, 5-dimethoxybenzaldehyde (16): white solid; mp 112~114 °C; IR (KBr) υ_max: 3410, 2727, 1685, 1605, 1514 cm⁻¹; ¹H -NMR (500 MHz, CDCl₃) δ (ppm): 3.98 (6H, s, OMe-3, OMe-5), 5.91 (1H, s, Ar-OH), 7.15 (2H, s, H-2 and H-6), 9.82 (1H, s, CHO).

4-Hydroxybenzyl alcohol (17): white solid; mp 116–117 °C; IR (KBr) υ_max: 3370, 1585, 1512 cm⁻¹; ¹H-NMR (500 MHz, CDCl₃) δ (ppm): 4.62 (2H, s, H-7), 4.79 (1H, s, Ar-OH), 6.82 (2H, d, J = 8.4 Hz, H-3 and H-5), 7.25 (2H, d, J = 8.4 Hz, H-2 and H-6).

2-(4-Hydroxyphenyl)ethanol (18): white solid; mp 92~93 °C; IR (KBr) υ_max: 3392, 1599, 1514 cm⁻¹; ¹H-NMR (500 MHz, CDCl₃) δ (ppm): 2.80 (2H, t, J = 8.0 Hz, H-7), 3.83 (2H, br. t, J = 8.0 Hz, H-8), 4.75 (1H, s, Ar-OH), 6.79 (2H, d, J = 8.0 Hz, H-3 and H-5), 7.10 (2H, d, J = 8.0 Hz, H-2 and H-6).

Cinnamic acid (19): white solid; mp 133~135 °C; IR (KBr) υ_max: 3267~2582, 2962, 1684, 1629 cm⁻¹; ¹H-NMR (500 MHz, CDCl₃) δ (ppm): 6.49 (1H, d, J = 16.0 Hz, H-8), 7.41 (3H, m, H-3, H-4 and H-5), 7.60 (2H, dd, J = 7.6, 2.0 Hz, H-2 and H-6), 7.68 (1H, d, J = 16.0 Hz, H-7).

trans-Ferulic acid (20): white solid, mp 168~169 °C; IR (KBr) υ_max: 3435, 3481~2750, 1690, 1662, 1515 cm⁻¹; ¹H-NMR (500 MHz, acetone-d₆) δ (ppm): 3.89 (3H, s, OMe-3), 6.30 (1H, d, J = 15.0 Hz, H-8), 6.80 (1H, d, J = 8.0 Hz, H-5), 7.05 (1H, d, J = 8.0 Hz, H-6), 7.17 (1H, s, H-2), 7.59 (1H, d, J = 15.0 Hz, H-7), 8.17 (1H, br s, Ar-OH).

2.5. Cell Culture

Murine RAW264.7 macrophages were cultured in DMEM medium containing 10% fetal bovine serum (FBS) and 1% penicillin at 37 °C, 5% CO₂ [9].

2.6. MTT assay

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

2.7. 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 × 10⁵ 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.

2.9. Statistical Analysis

All the data are expressed as mean ± SEM. Statistical analysis was carried out using the Student’s t-test. A probability of 0.05 or less was considered statistically significant. All the experiments were performed at least 3 times.

3. Results and Discussion

3.1. Isolation and Structural Elucidation

Chromatographic isolation of the EtOAc-soluble fraction of MeOH extract of aerial part of G. oppositifolius on column chromatography and high-performance liquid chromatography (HPLC) afforded three new triterpenoids—spergulagenin B (1), spergulagenin C (2), and spergulagenin D (3)—and 17 known compounds 4–20 (Figure 2).

Spergulagenin B (1) was isolated as colorless needle with molecular formula C₃₀H₄₈O₄ as confirmed by HR-EI-MS, showing an [M]⁺ ion at m/z 472.3549 (calcd. 472.3547) and supported by the ¹H- and ¹³C-NMR data. The IR absorption bands implied the presence of OH (3423 cm⁻¹) and acetyl group (1694 cm⁻¹). The ¹H- and ¹³C-NMR data of 1 showed the acetyl group [δ_H 2.23 (3H, s, H-30); δ_C 25.9 (C-30) and 217.2 (C-22)] and seven methyl signals [δ_H 0.96 (3H, s, H-25), 1.01 (3H, s, H-27), 1.03 (3H, s, H-23), 1.04 (3H, s, H-28), 1.07 (3H, s, H-26), 1.08 (3H, s, H-24) and 1.43 (3H, s, H-29); δ_C 15.6 (C-25), 16.6 (C-26), 17.2 (C-28), 18.7 (C-27), 21.1 (C-23), 21.2 (C-29) and 26.6 (C-24)]. Comparison of the ¹H- and ¹³C-NMR data of 1 with those of spergulagenin A (1a) [6] suggested that their structures were closely related, except that the carbonyl group [δ_C 217.3 (C-3)] at C-3 of 1 replaced the 3β-hydoxyl group of spergulagenin A (1a) [6]. This was supported by both HMBC correlations between H-1, H-2, H-23 and C-3 (δ_C 217.3). The relative stereochemistry of 1 was elucidated on the basis of NOESY experiments (Figure 2). The NOESY cross-peaks between H-5/H-9, H-9/H-12, H-12/H-27, H-13/H-17, H-13/H-26, H-16/H-29, H-23/H-25, H-25/H-26, H-27/H-28, and, H-28/H-29 suggested that H-13, H-17, Me (23), Me (25) and Me (26) on the β-side and H-5, H-9, H-12, H-16, Me (27), Me (28) and Me (29) are on the α-side of 1. The full assignment of ¹³C- and ¹H-NMR resonances was determined by ¹³C-DEPT (Figure S4), ¹H–¹H COSY (Figure S5), NOESY (Figure 3 and Figure S6), HSQC (Figure S7), and HMBC (Figure 3 and Figure S8) techniques. The absolute configuration of 1 was evidenced by the ECD Cotton effects at 284 (Δε +0.88), 250 (Δε −0.12), 217 (Δε +0.98), and 198 (Δε −1.34) nm, in analogy with those of glinusopposide D [11]. According to the evidence above, the structure of 1 was elucidated as (3R,4S,5aR,5bR,11aR,13R,13bR)-3-acetyl-4,13-dihydroxy-3,5a,5b,8,8,11a,13b-heptamethylicosahydro-9H-cyclopenta[a]chrysen-9-one, named spergulagenin B.

Spergulagenin C (2) was obtained as colorless needle crystal. Its molecular formula, C₃₀H₄₆O₄, was confirmed by the positive HR-ESI-MS at m/z 470.3409 [M]⁺ (calculated for C₃₀H₄₆O₄, 470.3406) and supported by the ¹³C, ¹H, and DEPT NMR data. IR absorptions for OH (3493 and 3416 cm⁻¹) functions were observed. The presence of the acetyl group was supported by a band at 1690 cm⁻¹ in the IR spectrum and was affirmed by signal at δ 25.9, and δ 217.0 in the ¹³C-NMR spectrum. The ¹³C- and ¹H-NMR data of 2 revealed the acetyl group [δ_H 2.24 (3H, s, H-30); δ_C 25.9 (C-30) and 217.0 (C-22)] and seven methyl signals [δ_H 1.01 (3H, s, H-27), 1.04 (3H, s, H-28), 1.08 (3H, s, H-25), 1.09 (3H, s, H-23), 1.11 (3H, s, H-26), 1.14 (3H, s, H-24), and 1.45 (3H, s, H-29); δ_C 17.1 (C-25), 17.2 (C-26), 18.8 (C-28), 18.9 (C-27), 21.4 (C-23), 21.1 (C-29) and 27.8 (C-24)]. The ¹H- and ¹³C-NMR data of 2 were similar to those of 1, except that the double bond at C-1,2 [δ_H 5.83, 7.10 (each 1H, each d, J = 10.0 Hz, H-2 and H-1); δ_C 125.6 (C-2), 158.5 (C-1)] of 2 replaced C-1,2 single bond [δ_H 1.41, 1.94 (each 1H, m, H-1), 2.42, 2.48 (each 1H, m, H-2); δ_C 34.0 (C-2), 39.4 (C-1)] of 1. This was supported by the HMBC correlations between H-1 (δ_H 7.10) and C-3 (δ_C 205.3), C-4 (δ_C 39.2), C-5 (δ_C 53.3), and C-9 (δ_C 42.7); and between H-2 (δ_H 5.83) and C-4 (δ_C 39.2) and C-10 (δ_C 44.6). The NOESY cross-peaks between H-5/H-9, H-9/H-12, H-12/H-27, H-13/H-17, H-13/H-26, H-16/H-29, H-23/H-25, H-25/H-26, H-27/H-28, and, H-28/H-29 suggested that H-13, H-17, Me (23), Me (25) and Me (26) are on the β-side and H-5, H-9, H-12, H-16, Me (27), Me (28) and Me (29) are on the α-side of 1. The full assignment of ¹³C- and ¹H-NMR resonances was confirmed by ¹³C-DEPT (Figure S12), ¹H–¹H COSY (Figure S13), NOESY (Figure 4 and Figure S14), HSQC (Figure S15), and HMBC (Figure 4 and Figure S16) techniques. The absolute configuration of 2 was evidenced by the ECD Cotton effects at 283 (Δε +0.96), 249 (Δε −0.14), 219 (Δε +1.05), and 198 (Δε −1.09) nm, in analogy with those of 1 and glinusopposide D [11]. On the basis of the evidence above, the structure of 2 was elucidated as (3R,4S,5aR,5bR,11aR,13R,13bR)-3-acetyl-4,13-dihydroxy-3,5a,5b,8,8,11a,13b-heptamethyl-1,2,3,3a,4,5,5a,5b,6,7,7a,8,11a,11b,12,13,13a,13b-octadecahydro-9H-cyclopenta[a] chrysen-9-one, named spergulagenin C.

Spergulagenin D (3) was obtained as colorless needle. Its molecular formula, C₃₀H₄₆O₄, was determined on the basis of the positive HR-EI-MS at m/z 470.3407 [M]⁺ (calcd. 470.3403) and supported by the ¹H, ¹³C, and DEPT NMR data. IR absorptions for OH (3447 cm⁻¹) and carbonyl (1697 cm⁻¹) functions were observed. The ¹H- and ¹³C-NMR data of 3 showed the acetyl group [δ_H 2.24 (3H, s, H-30); δ_C 26.1 (C-30); and 217.3 (C-22)] and seven methyl signals [δ_H 0.99 (3H, s, H-27), 1.00 (3H, s, H-25), 1.06 (3H, s, H-23), 1.10 (3H, s, H-24), 1.14 (3H, s, H-28), 1.21 (3H, s, H-26), and 1.43 (3H, s, H-29); δ_C 15.2 (C-25), 16.7 (C-26), 17.7 (C-28), 20.9 (C-27), 21.4 (C-23), 21.4 (C-29), and 26.6 (C-24)]. The ¹H- and ¹³C-NMR data of 3 were similar to those of 1, except that the carbonyl group at C-12 [δ_C 210.9 (C-12)] of 3 replaced the 12β-OH group [δ_H 3.95 (each 1H, m, H-12); δ_C 69.5 (C-12)] of 1. This was supported by the HMBC correlations between H-11 (δ_H 2.22, 2.25) and C-9 (δ_C 49.6), C-12 (δ_C 210.9); and between H-9 (δ_H 1.70) and C-10 (δ_C 37.0) and C-12 (δ_C 210.9). The relative stereochemistry of 3 was elucidated on the basis of NOESY experiments (Figure 4). The NOESY cross-peaks between H-5/H-9, H-13/H-17, H-13/H-26, H-16/H-29, H-23/H-25, H-25/H-26, H-27/H-28, and H-28/H-29 suggested that H-13, H-17, Me (23), Me (25) and Me (26) were on the β-side and H-5, H-9, H-16, Me (27), Me (28), and Me (29) were on the α-side of 3. The full assignment of ¹³C- and ¹H-NMR resonances was determined by ¹³C-DEPT (Figure S20), ¹H–¹H COSY (Figure S21), NOESY (Figure 5 and Figure S22), HSQC (Figure S23), and HMBC (Figure 5 and Figure S24) experiments. The absolute configuration of 3 was evidenced by the ECD Cotton effects at 284 (Δε +0.76), 249 (Δε −0.09), 218 (Δε +1.00), and 197 (Δε −0.91) nm, in analogy with those of 1 and glinusopposide D [11]. On the basis of the evidence above, the structure of 3 was elucidated as (3R,4S,5aR,5bR,11aR,13bS)-3-acetyl-4-hydroxy-3,5a,5b,8,8,11a,13b-heptamethyloctadecahydro-9H-cyclopenta[a]chrysene-9,13(8H)-dione, named spergulagenin D.

3.2. Structure Identification of Known Isolated Compounds

The known isolated compounds were readily determined by a comparison of physical and spectroscopic data (¹H-NMR, ¹³C-NMR, MS, UV, and IR) with the literature values or corresponding authentic samples, and this included four flavonoids, kaempferol (4) [12], 6, 8-dimethyl-5, 7, 4′-trihydroxyflavone (5) [13], 5,7-dihydroxy-6,8-dimethylflavone (6) [14], and 5,4′-dihydroxy-7-methoxy-6,8-dimethylflavone (7) [15], and thirteen aromatics, 4-hydroxybenzoic acid (8) [16], 4-hydroxybenzaldehyde (9) [17], 4-hydroxyacetophenone (10) [17], methyl 4-Hydroxybenzoate (11) [17], p-anisic acid (12) [18], vanillin (13) [19], 4-hydroxy-3-methoxyacetophenone (14) [20], acetosyringone (15) [21], 4-hydroxy-3,5-dimethoxybenzaldehyde (16) [22], 4-hydroxybenzyl alcohol (17) [23], 2-(4-hydroxyphenyl)ethanol (18) [24], cinnamic acid (19) [25], and trans-ferulic acid (20) [26].

3.3. Biological Studies

Nitric oxide (NO) is derived from the oxidation of L-arginine by NO synthase (NOS) and is a mediator in the inflammatory response involved in host defense [27]. In inflammation and carcinogenesis conditions, there is an increased production of NO by inducible NO synthase (iNOS) [28]. The anti-inflammatory effects of the compounds isolated from the aerial part of G. oppositifolius were also evaluated by suppressing lipopolysaccharide (LPS)-induced NO generation in macrophage cell line RAW264.7. The inhibitory activity data of the isolates 1–20 on NO generation by macrophages are shown in

Table 3. Inhibitory effect of compounds 1–20 on production of nitric oxide in LPS-stimulated RAW 264.7 cells.

Compounds	NO Inhibition IC50 (μM) a
Spergulagenin B (1)	24.76 ± 1.41 ***
Spergulagenin C (2)	28.26 ± 2.78 **
Spergulagenin D (3)	17.03 ± 1.28
Kaempferol (4)	38.87 ± 1.68 ***
6,8-Dimethyl-5,7,4′-trihydroxyflavone (5)	18.21 ± 1.15
5,7-Dihydroxy-6,8-dimethylflavone (6)	43.61 ± 2.96 ***
5,4′-Dihydroxy-7-methoxy-6,8-dimethylflavone (7)	32.08 ± 2.75 **
4-Hydroxybenzoic acid (8)	75.83 ± 6.63 **
4-Hydroxybenzaldehyde (9)	88.20 ± 7.78 **
4-Hydroxyacetophenone (10)	76.24 ± 6.55 **
Methyl 4-Hydroxybenzoate (11)	78.50 ± 8.00 **
p-Anisic acid (12)	115.58 ± 10.35 **
Vanillin (13)	94.95 ± 10.99 **
4-Hydroxy-3-methoxyacetophenone (14)	111.29 ± 12.91 **
Acetosyringone (15)	75.43 ± 6.63 **
4-Hydroxy-3, 5-dimethoxybenzaldehyde (16)	86.62 ± 7.74 **
4-Hydroxybenzyl alcohol (17)	78.64 ± 7.23 **
2-(4-Hydroxyphenyl)ethanol (18)	28.47 ± 1.94 ***
Cinnamic acid (19)	16.30 ± 1.41
trans-Ferulic acid (20)	12.64 ± 1.14 **
Quercetin b	16.74 ± 1.26

^a The IC₅₀ value was defined as half-maximal inhibitory concentration and was expressed as mean ± SD (n = 3); ^b Quercetin was used as positive control; ** p < 0.01, and *** p < 0.001 compared with the control.

and Table S1. Quercetin was used as the positive control. From the results of our anti-inflammatory tests, the following conclusions can be drawn: (a) Compounds 3, 5, 19, and 20 exhibited inhibitory effects on lipopolysaccharides (LPS)-induced nitric oxide production in RAW 264.7 cells with IC₅₀ values of 17.03 ± 1.28, 18.21 ± 1.15, 16.30 ± 1.41, and 12.64 ± 1.14 μM, respectively (Table 1); (b) Among new triterpenoids, spergulagenin D (3) (with 3,12-dioxo groups) exhibited more effective inhibition than its analogues, spergulagenin B (1) (with 3-oxo-12β-hydroxy groups) and spergulagenin C (2) (with 1,2-dehydro-3-oxo-12β-hydroxy groups) against LPS-induced NO generation. (c) Among the flavonoids, 6,8-dimethyl-5,7,4′-trihydroxyflavone (5) (with 6,8-dimethyl-5,7,4′-trihydroxy groups) exhibited more effective inhibition than its analogues, kaempferol (4) (with 5,7,4′-trihydroxy groups), 5,7-dihydroxy-6,8-dimethylflavone (6) (with 5,7-dihydroxy-6,8-dimethyl groups), and 5,4′-dihydroxy-7-methoxy-6,8-dimethylflavone (7) (with 5,4′-dihydroxy-7-methoxy-6,8-dimethyl groups) against LPS-induced NO generation. (d) trans-ferulic acid (20) is the most effective among the isolated compounds against LPS-induced NO generation. In addition, compounds 3, 5, and 20 exhibited inhibitory effects on LPS-induced TNF-α 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 (

Table 4. Inhibitory effect of compounds 1–20 on the production of pro-inflammatory cytokine, TNF-α in LPS-stimulated RAW 264.7 cells.

Compounds	TNF-α Inhibition IC50 (μM) a
Spergulagenin B (1)	30.49 ± 2.20 **
Spergulagenin C (2)	31.36 ± 2.59 **
Spergulagenin D (3)	18.35 ± 1.34 **
Kaempferol (4)	35.71 ± 4.74 *
6,8-Dimethyl-5,7,4′-trihydroxyflavone (5)	17.56 ± 1.41 **
5,7-Dihydroxy-6,8-dimethylflavone (6)	39.48 ± 3.06 **
5,4′-Dihydroxy-7-methoxy-6,8-dimethylflavone (7)	34.17 ± 2.49 **
4-Hydroxybenzoic acid (8)	80.02 ± 7.10 **
4-Hydroxybenzaldehyde (9)	86.38 ± 6.28 ***
4-Hydroxyacetophenone (10)	79.03 ± 5.26 ***
Methyl 4-Hydroxybenzoate (11)	82.33 ± 7.25 **
p-Anisic acid (12)	125.84 ± 11.47 **
Vanillin (13)	102.35 ± 9.36 **
4-Hydroxy-3-methoxyacetophenone (14)	123.07 ± 11.37 **
Acetosyringone (15)	68.38 ± 5.48 **
4-Hydroxy-3, 5-dimethoxybenzaldehyde (16)	77.39 ± 6.73 **
4-Hydroxybenzyl alcohol (17)	69.38 ± 6.24 **
2-(4-Hydroxyphenyl)ethanol (18)	26.44 ± 2.35 *
Cinnamic acid (19)	22.00 ± 1.51 **
trans-Ferulic acid (20)	14.27 ± 1.29 **
Quercetin b	5.08 ± 0.23

^a The IC₅₀ value was defined as half-maximal inhibitory concentration and was expressed as mean ± SD (n = 3); ^b Quercetin was used as positive control; * p < 0.05, ** p < 0.01, and *** p < 0.001 compared with the control.

and Table S2).

The above findings indicated that the promising inhibitory activity against LPS-induced NO and TNF-α generation of G. oppositifolius and its isolates could stimulate future development of new anti-inflammatory agents.

4. 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 IC₅₀ 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 IC₅₀ 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].