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Article

New Triterpenoids and Anti-Inflammatory Constituents from Glinus oppositifolius

1
Department of Pharmacy, School of Pharmaceutical Sciences, National Yang Ming Chiao Tung University, Taipei 112304, Taiwan
2
Department of Medical Research, China Medical University Hospital, China Medical University, Taichung 404327, Taiwan
3
Department of Chinese Pharmaceutical Sciences and Chinese Medicine Resources, China Medical University, Taichung 404327, Taiwan
4
Department of Food Nutrition and Health Biotechnology, Asia University, Taichung 41354, Taiwan
5
School of Pharmacy, College of Pharmacy, Kaohsiung Medical University, Kaohsiung 807377, Taiwan
6
Chinese Medicine Research Center, China Medical University, Taichung 404327, Taiwan
7
Department of Biotechnology, Asia University, Taichung 413305, Taiwan
*
Author to whom correspondence should be addressed.
These authors contributed equally to this work.
Molecules 2023, 28(7), 2903; https://doi.org/10.3390/molecules28072903
Submission received: 21 February 2023 / Revised: 20 March 2023 / Accepted: 21 March 2023 / Published: 23 March 2023

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 (420). 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.

Graphical Abstract

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 13 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) υmax: 3423 (OH), 2943, 1694 (C=O), 1458, 1385, 1155, 1113, 1061 cm−1 (Figure S1); 1H-NMR spectroscopic data, see Table 1 (Figure S2); 13C-NMR spectroscopic data, see Table 2 (Figure S3); ECD (c 0.25, MeOH) λmaε) 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) λmax nm (log λ): 229 (3.73); IR (KBr) υmax: 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) λmaxε) 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) υmax: 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) λmaxε) 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) υmax: 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) υmax: 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) υmax: 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) υmax: 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) υmax: 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) υmax: 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) υmax: 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) υmax: 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) υmax: 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) υmax: 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) υmax: 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) υmax: 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) υmax: 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) υmax: 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) υmax: 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) υmax: 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) υmax: 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 420 (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 [δH 2.23 (3H, s, H-30); δC 25.9 (C-30) and 217.2 (C-22)] and seven methyl signals [δH 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); δC 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 [δC 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 (δC 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 [δH 2.24 (3H, s, H-30); δC 25.9 (C-30) and 217.0 (C-22)] and seven methyl signals [δH 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); δC 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 [δH 5.83, 7.10 (each 1H, each d, J = 10.0 Hz, H-2 and H-1); δC 125.6 (C-2), 158.5 (C-1)] of 2 replaced C-1,2 single bond [δH 1.41, 1.94 (each 1H, m, H-1), 2.42, 2.48 (each 1H, m, H-2); δC 34.0 (C-2), 39.4 (C-1)] of 1. This was supported by the HMBC correlations between H-1 (δH 7.10) and C-3 (δC 205.3), C-4 (δC 39.2), C-5 (δC 53.3), and C-9 (δC 42.7); and between H-2 (δH 5.83) and C-4 (δC 39.2) and C-10 (δC 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 [δH 2.24 (3H, s, H-30); δC 26.1 (C-30); and 217.3 (C-22)] and seven methyl signals [δH 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); δC 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 [δC 210.9 (C-12)] of 3 replaced the 12β-OH group [δH 3.95 (each 1H, m, H-12); δC 69.5 (C-12)] of 1. This was supported by the HMBC correlations between H-11 (δH 2.22, 2.25) and C-9 (δC 49.6), C-12 (δC 210.9); and between H-9 (δH 1.70) and C-10 (δC 37.0) and C-12 (δC 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 120 on NO generation by macrophages are shown in Table 3 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 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].

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/molecules28072903/s1, Figure S1: The IR spectrum of 1; Figure S2: The 1H-NMR spectrum of 1 (CDCl3, 500 MHz); Figure S3: The 13C-NMR spectrum of 1 (CDCl3, 125 MHz); Figure S4: The 13C-DEPT spectrum of 1 (CDCl3, 125 MHz); Figure S5: The 1H–1H COSY spectrum of 1; Figure S6: The NOESY spectrum of 1; Figure S7: The HSQC spectrum of 1; Figure S8: The HMBC spectrum of 1; Figure S9: The IR spectrum of 2; Figure S10: The 1H-NMR spectrum of 2 (CDCl3, 500 MHz); Figure S11: The 13C-NMR spectrum of 2 (CDCl3, 125 MHz); Figure S12: The 13C-DEPT spectrum of 2 (CDCl3, 125 MHz); Figure S13: The 1H–1H COSY spectrum of 2; Figure S14: The NOESY spectrum of 2; Figure S15: The HSQC spectrum of 2; Figure S16: The HMBC spectrum of 2; Figure S17: The IR spectrum of 3; Figure S18: The 1H-NMR spectrum of 3 (CDCl3, 500 MHz); Figure S19: The 13C-NMR spectrum of 3 (CDCl3, 125 MHz); Figure S20: The 13C-DEPT spectrum of 3 (CDCl3, 125 MHz); Figure S21: The 1H–1H COSY spectrum of 3; Figure S22: The NOESY spectrum of 3; Figure S23: The HSQC spectrum of 3; Figure S24: The HMBC spectrum of 3; Table S1: Inhibitory effect of compounds 120 on production of nitric oxide in LPS-stimulated RAW 264.7 cells; Table S2: Inhibitory effect of compounds 120 on the production of pro-inflammatory, TNF-α in LPS-stimulated RAW 264.7 cells.

Author Contributions

J.-J.C. and C.-S.Y. performed the bioassay, analyzed the data, and wrote the manuscript. Y.-H.C., J.-J.C., C.-Y.C. and Y.-C.C. conducted the isolation and structure elucidation of the constituents. J.-J.C. analyzed bioassay data. J.-J.C. and Y.-H.K. planned, designed, and organized all of the research for this study and prepared the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by a grant from the Ministry of Science and Technology (MOST), Taiwan (No. MOST 109-2320-B-010-029-MY3), awarded to J.-J. Chen.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data are contained within the article.

Conflicts of Interest

The authors declare no conflict of interest.

Sample Availability

Samples of the compounds are available from the authors.

References

  1. Lu, S.U.; Yang, Y.P. Molluginaceae in Flora of Taiwan, 2nd ed.; Editorial Committee of the Flora of Taiwan: Taipei, Taiwan, 1996; pp. 325–328. [Google Scholar]
  2. Sheu, S.Y.; Yao, C.H.; Lei, Y.C.; Kuo, T.F. Recent progress in Glinus oppositifolius research. Phytochem. Pharm. Biol. 2014, 52, 1079–1084. [Google Scholar] [CrossRef] [PubMed]
  3. Yang, C.S.; Huang, H.C.; Wang, S.Y.; Sung, P.J.; Huang, G.J.; Chen, J.J.; Kuo, Y.H. New diphenol and isocoumarins from the aerial part of Lawsonia inermis and their inhibitory activities against NO production. Molecules 2016, 21, 1299. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  4. Ahmed, S.; Rahman, A.; Alam, A.; Saleem, M.; Athar, M.; Sultana, S. Evaluation of the efficacy of Lawsonia alba in the alleviation of carbon tetrachloride-induced oxidative stress. J. Ethnopharmacol. 2000, 69, 157–164. [Google Scholar] [CrossRef] [PubMed]
  5. Liou, J.R.; Mohamed, E.S.; Du, Y.C.; Tseng, C.N.; Hwang, T.L.; Chuang, Y.L.; Hsu, Y.M.; Hsieh, P.W.; Wu, C.C.; Chen, S.L.; et al. 1,5-Diphenylpent-3-en-1-ynes and methyl naphthalene carboxylates from Lawsonia inermis and their anti-inflammatory activity. Phytochemistry 2013, 88, 67–73. [Google Scholar] [CrossRef]
  6. Kitagwa, I.; Yamanaka, H.; Nakanishi, T.; Yosioka, I. Saponin and sapogenol. XXII. Structure of spergulatriol, a new bisnorhopane-type genuine sapogenol of isoanhydrospergulatriol, from the root of Mollugo spergula L. Chem. Pharm. Bull. 1977, 25, 2430–2436. [Google Scholar] [CrossRef] [Green Version]
  7. Karin, M.; Clevers, H. Reparative inflammation takes charge of tissue regeneration. Nature 2016, 529, 307–315. [Google Scholar] [CrossRef] [Green Version]
  8. Bradley, J.R. TNF-Mediated inflammatory disease. J. Pathol. 2008, 214, 149–160. [Google Scholar] [CrossRef]
  9. Chen, S.C.; Yang, C.S.; Chen, J.J. Main bioactive components and their biological activities from natural and processed rhizomes of Polygonum sibiricum. Antioxidants 2022, 11, 1383. [Google Scholar] [CrossRef]
  10. Hsu, J.H.; Yang, C.S.; Chen, J.J. Antioxidant, anti-α-glucosidase, antityrosinase, and anti-inflammatory activities of bioactive components from Morus alba. Antioxidants 2022, 11, 2222. [Google Scholar] [CrossRef]
  11. Zhang, D.; Fu, Y.; Yang, J.; Li, X.N.; San, M.M.; Oo, T.N.; Wang, Y.; Yang, X. Triterpenoids and their glycosides from Glinus oppositifolius with antifungal activities against Microsporum gypseum and Trichophyton rubrum. Molecules 2019, 24, 2206. [Google Scholar] [CrossRef] [Green Version]
  12. Lee, Y.J.; Wu, T.D. Total synthesis of kaempferol and methylated kampferol derivatives. J. Chin. Chem. Soc. 2001, 48, 201–206. [Google Scholar] [CrossRef]
  13. Youssef, T.A.; Ramadan, M.A.; Khalifa, A.A. Acetophenones, a chalcone, a chromone and flavonoids from Pancratium maritimum. Phytochemistry 1998, 8, 2579–2583. [Google Scholar] [CrossRef]
  14. Kuo, Y.H.; Chu, P.H. Studies on the constituents from the bark of Bauhinia purpurea. J. Chin. Chem. Soc. 2002, 49, 269–274. [Google Scholar] [CrossRef]
  15. Ralf, M. Flavonoids from Leptrospermum scoparium. Phytochemistry 1990, 29, 1340–1342. [Google Scholar]
  16. DellaGreca, M.; Monaco, p.; Pinto, G.; Pollio, A.; Previtera, L.; Temussi, F. Phytotoxicity of low-molecular-weight phenols from olive mill waters. Bull. Environ. Contam. Toxicol. 2001, 67, 352–359. [Google Scholar] [CrossRef]
  17. Yasuhara, A.; Kasano, A.; Sakamoto, T. An efficient method for the deallylation of allyl aryl ethers using electrochemically generated nickel. J. Org. Chem. 1999, 64, 4211–4213. [Google Scholar] [CrossRef]
  18. Huang, L.; Nardos, T.; Huang, X. A facile method for oxidation of primary alcohols to carboxylic acids and its application in glycosaminoglycan syntheses. Chem. Eur. J. 2006, 12, 5246–5252. [Google Scholar] [CrossRef] [Green Version]
  19. Sun, R.; Sacalis, J.N.; Chin, C.K.; Still, C.C. Bioactive aromatic compounds from leaves and stems of Vanilla fragrans. J. Agric. Food. Chem. 2001, 49, 5161–5164. [Google Scholar] [CrossRef]
  20. Syeda, F.A.; Habib-Ur, R.; Atta-Ur, R.M.; Iqbal, C. Phytochemical investigations on Iris germanica. Nat. Prod. Res. 2010, 24, 131–139. [Google Scholar]
  21. Song, Y.N.; Shibuya, M.; Ebizuka, Y.; Sankawa, U. Identification of plant factors inducing virulence gene expression in Agrobacterium tumefaciens. Chem. Pharm. Bull. 1991, 39, 2347–2350. [Google Scholar] [CrossRef] [Green Version]
  22. Lee, T.H.; Chiou, J.L.; Lee, C.K.; Kuo, Y.H. Separation and determination of chemical constituents in the root of Rhus javanica L. var. roxburghiana. J. Chin. Chem. Soc. 2005, 52, 833–841. [Google Scholar] [CrossRef]
  23. Choi, J.H.; Lee, D.U. A new citryl glycoside from Gastrodia elata and its inhibitory activity on GABA transaminase. Chem. Pharm. Bull. 2006, 54, 1720–1721. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  24. Pier, G.B.; Barbara, C.; Romeo, R.; Giampiero, S.; Angela, M.; Ennio, O.; Katia, V.; Pier, A.B. 7-Substituted 5-amino-2-(2-furyl)pyrazolo [4,3-e]-1,2,4-triazolo [1,5-c]pyrimidines as A2a adenosine receptor antagonists: A study on the importance of modifications at the side chain on the activity and solubility. J. Med. Chem. 2002, 45, 115–126. [Google Scholar]
  25. Miyazawa, M.; Okuno, Y.; Nakamura, S.; Kameoka, H. Suppression of SOS inducing activity of chemical mutagens by cinnamic acid derivatives from Scrophulia nangpoensis in the Salmonella typhimurium TA1536/pSK-1002 umu test. J. Agric. Food Chem. 1998, 46, 904–910. [Google Scholar] [CrossRef]
  26. Supaluk, P.; Saowapa, S.; Apilak, W.; Ratana, L.; Somsak, R.; Virapong, P. Bioactive metabolites from Spilanthes acmella Murr. Molecules 2009, 14, 850–867. [Google Scholar]
  27. Geller, D.A.; Billiar, T.R. Molecular biology of nitric oxide synthases. Cancer Metastasis Rev. 1998, 17, 7–23. [Google Scholar] [CrossRef]
  28. Moncada, S.; Palmer, R.M.; Higgs, E.A. Nitric oxide: Physiology, pathophysiology, and pharmacology. Pharmcol. Rev. 1991, 43, 109–142. [Google Scholar]
Figure 1. Plant material: Glinus oppositifolius (L.) Aug. DC.
Figure 1. Plant material: Glinus oppositifolius (L.) Aug. DC.
Molecules 28 02903 g001
Figure 2. The chemical structures of compounds 120.
Figure 2. The chemical structures of compounds 120.
Molecules 28 02903 g002aMolecules 28 02903 g002b
Figure 3. Key NOESY (Molecules 28 02903 i001) and HMBC (Molecules 28 02903 i002) correlations of 1.
Figure 3. Key NOESY (Molecules 28 02903 i001) and HMBC (Molecules 28 02903 i002) correlations of 1.
Molecules 28 02903 g003
Figure 4. Key NOESY (Molecules 28 02903 i001) and HMBC (Molecules 28 02903 i002) correlations of 1.
Figure 4. Key NOESY (Molecules 28 02903 i001) and HMBC (Molecules 28 02903 i002) correlations of 1.
Molecules 28 02903 g004
Figure 5. Key NOESY (Molecules 28 02903 i001) and HMBC (Molecules 28 02903 i002) correlations of 1.
Figure 5. Key NOESY (Molecules 28 02903 i001) and HMBC (Molecules 28 02903 i002) correlations of 1.
Molecules 28 02903 g005
Table 1. 1H-NMR data for Compounds 13 (δ in ppm, J in Hz).
Table 1. 1H-NMR data for Compounds 13 (δ in ppm, J in Hz).
Position1 a2 a3 a
11.94 (m). 1.41 (m)7.10 (d, J = 10.0 Hz)1.82 (m), 1.39 (m)
22.48 (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)
51.30 (m)1.54 (m)1.33 (m)
61.51 (m), 1.37 (m)1.56 (m), 1.43 (m)1.60 (m), 1.35 (m)
71.50 (m), 1.31 (m)1.50 (m), 1.44 (m)1.54 (m), 1.47 (m)
91.69 (m)1.59 (m)1.70 (dd, J = 9.6, 4.4Hz)
111.87 (m), 1.04 (m)1.57 (m), 1.46 (m)2.25 (m), 2.22 (m)
123.963.99 (m)
131.38 (d, J = 4.0 Hz)1.43 (m)2.23 (m)
151.72 (dd, J = 12.8, 4.0 Hz), 1.35 (m)1.72 (m), 1.32 (m)1.79 (m), 1.44 (m)
163.70 (m)3.71 (m)3.76 (m)
171.76 (d, J = 11.2 Hz)1.78 (m)1.64 (m)
192.02 (m), 1.27 (m)2.04 (m), 1.28 (m)2.17 (m), 1.02 (m)
202.05 (m), 1.84 (m)2.05 (m), 1.86 (m)2.10 (m), 1.93 (m)
231.03 (s)1.09 (s)1.06 (s)
241.08 (s)1.14 (s)1.10 (s)
250.96 (s)1.08 (s)1.00 (s)
261.07 (s)1.11 (s)1.21 (s)
271.01 (s)1.01 (s)0.99 (s)
281.04 (s)1.05 (s)1.14 (2)
291.43 (s)1.45 (s)1.43 (s)
302.23 (s)2.24 (s)2.24 (s)
a measured in CDCl3 at 500 MHz.
Table 2. 13C-NMR data for Compounds 13 (δ in ppm).
Table 2. 13C-NMR data for Compounds 13 (δ in ppm).
Position1 a2 a3 a
139.4158.538.9
234.0125.634.1
3217.3205.3216.9
448.139.247.6
554.953.355.1
619.719.119.9
732.532.632.0
845.545.547.2
947.342.749.6
1036.744.637.0
1132.932.539.6
1269.569.3210.9
1355.155.263.4
1441.442.341.6
1545.145.043.8
1665.865.765.8
1759.259.258.7
1846.346.344.9
1944.144.041.7
2035.935.835.8
2154.454.455.6
22217.2217.0217.3
2321.121.421.4
2426.627.826.6
2515.617.115.2
2616.617.216.7
2718.718.920.9
2817.218.817.7
2921.221.121.4
3025.925.926.1
a measured in CDCl3 at 125 MHz.
Table 3. Inhibitory effect of compounds 120 on production of nitric oxide in LPS-stimulated RAW 264.7 cells.
Table 3. Inhibitory effect of compounds 120 on production of nitric oxide in LPS-stimulated RAW 264.7 cells.
CompoundsNO 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 b16.74 ± 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.
Table 4. Inhibitory effect of compounds 120 on the production of pro-inflammatory cytokine, TNF-α in LPS-stimulated RAW 264.7 cells.
Table 4. Inhibitory effect of compounds 120 on the production of pro-inflammatory cytokine, TNF-α in LPS-stimulated RAW 264.7 cells.
CompoundsTNF-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 b5.08 ± 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.
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Chen, J.-J.; Yang, C.-S.; Chen, Y.-H.; Chao, C.-Y.; Chen, Y.-C.; Kuo, Y.-H. New Triterpenoids and Anti-Inflammatory Constituents from Glinus oppositifolius. Molecules 2023, 28, 2903. https://doi.org/10.3390/molecules28072903

AMA Style

Chen J-J, Yang C-S, Chen Y-H, Chao C-Y, Chen Y-C, Kuo Y-H. New Triterpenoids and Anti-Inflammatory Constituents from Glinus oppositifolius. Molecules. 2023; 28(7):2903. https://doi.org/10.3390/molecules28072903

Chicago/Turabian Style

Chen, Jih-Jung, Chang-Syun Yang, Yu-Hui Chen, Che-Yi Chao, Yu-Chang Chen, and Yeuh-Hsiung Kuo. 2023. "New Triterpenoids and Anti-Inflammatory Constituents from Glinus oppositifolius" Molecules 28, no. 7: 2903. https://doi.org/10.3390/molecules28072903

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