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Article

Triterpenoids and Their Glycosides from Glinus Oppositifolius with Antifungal Activities against Microsporum Gypseum and Trichophyton Rubrum

1
Southeast Asia Biodiversity Research Institute, Chinese Academy of Sciences, Yezin, Nay Pyi Taw 05282, Myanmar
2
Key Laboratory of Economic Plants and Biotechnology and Yunnan Key Laboratory for Wild Plant Resources, State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650201, China
3
Forest Research Institute, Yezin, Nay Pyi Taw 05282, Myanmar
*
Authors to whom correspondence should be addressed.
Molecules 2019, 24(12), 2206; https://doi.org/10.3390/molecules24122206
Submission received: 5 May 2019 / Revised: 6 June 2019 / Accepted: 7 June 2019 / Published: 12 June 2019

Abstract

:
Four new triterpenoids, 3β,12β,16β,21β,22-pentahydroxyhopane (1), 12β,16β,21β,22-tetrahydroxyhopan-3-one (2), 3-oxo-olean-12-ene-28,30-dioic acid (3), and 3β-hydroxyoleana-11,13(18)-diene-28,30-dioic acid 30-methyl ester (4); 21 new triterpenoid saponins, glinusopposides A–U (525); and 12 known compounds (2637) were isolated from the whole plants of Glinus oppositifolius. The structures of the new compounds were elucidated based on the analysis of one-dimensional (1D) and two-dimensional (2D) nuclear magnetic resonance (NMR) and mass spectrometry (MS) data. All compounds from the plants were measured for antifungal activities against Microsporum gypseum and Trichophyton rubrum. Glinusopposide B (6), glinusopposide Q (21), glinusopposide T (24), and glinusopposide U (25) showed strong inhibitory activities against M. gypseum (MIC50 7.1, 6.7, 6.8, and 11.1 μM, respectively) and T. rubrum (MIC50 14.3, 13.4, 11.9, and 13.0 μM, respectively). For those active compounds with an oleanane skeleton, glycosylation (2126) or oxidation (3) of 3-OH was helpful in increasing the activity; replacement of the 30-methyl group (29) by a carboxymethyl group (26) enhanced the activity; the presence of 11,13(18) double bonds (20) decreased the activity.

Graphical Abstract

1. Introduction

Dermatophytosis is one of the most common skin diseases in animals and humans, which is mainly caused by Epidermophyton, Microsporum and Trichophyton [1,2]. As a chronic disease, dermatophytosis is difficult to treat due to the drug resistance developed by the related fungus [2]. Therefore, it is important to search for novel agents to treat dermatophytosis.
Glinus oppositifolius (L.) Aug. DC. (Syn: Mollugo spergula L. and Mollugo oppositifolia L; family: Molluginaceae) is a small herb widely distributed in tropical Asia, tropical Africa, and Australia [3]. Traditionally it has been used for treating skin and various infectious diseases in Bangladesh, China, India, Mali and Myanmar [4,5,6]. As a Chinese folk medicine, the whole plants of G. oppositifolius are used to treat diarrhea, coughs, hyperthermia, heat rashes, pinkeye, furuncles, snakebites, and burns [6]. The plant is reputed in Indian medicine due to its antiseptic and antidermatitic properties [7]. It is used to treat leprosy, leukoderma, heart, and skin diseases in the traditional medicine of Myanmar [5]. The major secondary metabolites from G. oppositifolius are triterpenoids and their glycosides, which exhibit α-glucosidase inhibitory [8], cytotoxic [9], and antiprotozoal activities [10]. There is little research reported the anti-fungal activities of G. oppositifolius. In this study, we isolated 25 new triterpenoids and triterpenoid saponins (Figure 1), along with 12 known compounds in the whole plants of G. oppositifolius. Their antifungal properties against Microsporum gypseum and Trichophyton rubrum were analyzed.

2. Results and Discussion

2.1. Structure Elucidation of the Compounds

Compound 1 had the molecular formula C30H52O5 based on 13C-NMR data (Table 1) and the positive ion at m/z 515.3718 [M + Na]+ (calcd. for C30H52NaO5, 515.3712) in the high resolution electrospray ionization mass spectroscopy (HRESIMS). The 1H-NMR spectrum showed resonances for eight methyl groups at δH 1.61 (s), 1.56 (s), 1.21 (s), 1.19 (s), 1.13 (s), 1.01 (s), 0.97 (s), and 0.82 (s), as well as three oxymethines at δH 4.48 (m), 4.23 (m), and 3.44 (br t, J = 8.3 Hz) (Table 1). The 13C-NMR spectrum showed resonances for thirty carbon atoms as expected from high resolution mass spectrum, which were sorted by DEPT into eight methyls, eight methylenes, seven methines (three oxymethines, δC 78.0, 69.1, and 66.8), and seven quaternary carbons group, including two oxygenated quaternary carbons. These NMR data were very similar to those of a known hopane triterpenoid saponin from this plant, glinoside C, except for the lack of signals for glucopyranose [8]. The full NMR assignments and connections were determined by 1H-detected heteronuclear single quantum coherence (HSQC), 1H-detected heteronuclear multiple bond correlation (HMBC), and 1H-1H correlation spectroscopy (COSY) analyses.
According to the 1H–1H COSY correlations in the 2D spectra of 1 (Figure 2), five connections, C-1-C-2-C-3, C-5-C-6-C-7, C-9-C-11-C-12-C-13, C-15-C-16-C-17, and C-19-C-20, were confirmed. The planar structure of 1 was further deduced as 3,12,16,21,22-pentahydroxyhopane by the HMBC correlations from H3-23 and H3-24 to C-3, C-4, and C-5; from H3-25 to C-1, C-5, C-9, and C-10; from H3-26 to C-7, C-9, and C-14; from H3-27 to C-8, C-13, and C-15; from H3-28 to C-13, C-17, C-18, and C-19; from H3-29 and H3-30 to C-21 and C-22; and from H-17 to C-19 and C-22. The configurations of 3-OH, 12-OH, 16-OH, and 21-OH were deduced as 3β,12β,16β,21β by the key nuclear overhauser effect spectroscopy (ROESY) correlations of H-3/H-5, H-5/H-9, H-9/H-12, H-16/H3-27, H-16/H3-28, H-16/H3-30, and H3-30/H3-28. Thus, the structure of 1 was determined to be 3β,12β,16β,21β,22-pentahydroxyhopane. The absolute configuration was assigned by Cu Kα X-ray crystallographic analysis (Figure 3).
Compound 2 showed a molecular formula of C30H50O5 based on 13C-NMR data (Table 1) and the [M + Na]+ ion at m/z 513.3551 (calcd. for C30H50NaO5, 513.3556) in the HRESIMS. The NMR data (Table 1) of 2 were analogous to those of 1 except that the signal (δC 78.0) for an oxygenated methine in the 13C-NMR spectrum of 1 was replaced by the signal (δC 216.4) for a carbonyl group in the 13C-NMR spectrum of 2. The structure of 2 was easily established as 12β, 16β, 21β, 22-tetrahydroxyhopan-3-one by the COSY, HMBC, and ROESY spectra of 2 (Supplementary Materials).
Compound 3 was assigned the molecular formula C30H44O5 based on 13C-NMR data (Table 2) and positive ion mode HRESIMS, which showed a pseudomolecular ion peak at m/z 507.3084 [M + Na]+ (calcd. for C30H44NaO5, 507.3086). The 1H-NMR data of 3 (Table 2) indicated the presence of six methyl groups at δH 1.43 (s), 1.30 (s), 1.14 (s), 1.00 (s), 0.99 (s), and 0.86 (s) and one olefinic group at δH 5.72. The 13C-NMR data of 3 (Table 2) indicated the presence of six methyl groups, two carboxylic carbons at δC 180.1 and 179.5, one carbonyl carbon at δC 216.3, and two olefinic carbons (one quaternary at δC 144.8 and one methane at δC 122.8, suggesting the presence of a double bond), 10 sp3 methylenes, three sp3 methines, and seven sp3 quaternary carbon atoms. The NMR data of 3 were very similar to those of 3-oxo-olean-12-en-28,29-dioic acid [11], implying that 3 was also an oleanane triterpenoid.
Six fragments, C-1-C-2, C-5-C-6-C-7, C-9-C-11-C-12, C-15-C-16, C-18-C-19, and C-21-C-22, were deduced from the 1H–1H COSY correlations in the 2D-NMR spectra of 3 (Figure 2). The structure of 3 was deduced as 3-oxo-olean-12-ene-28,30-dioic acid by the HMBC correlations from H3-23 and H3-24 to C-3, C-4, and C-5; from H2-1 and H2-2 to C-3; from H3-25 to C-1, C-5, C-9, and C-10; from H3-26 to C-7, C-9, and C-14; from H3-27 to C-8, C-13, and C-15; from H-18 to C-12, from H2-19 to C-17; from H2-16 and H2-22 to C-28; and from H3-29 to C-19, C-20, C-21 and C-30; as well as the key ROESY correlations of H-19α/H3-27 and H-19α/H3-29 (Figure 2).
The molecular formula of compound 4, C31H46O5, with nine degrees of unsaturation, was determined by the 13C-NMR data in methanol-d4 (Table 2) and positive ion mode HRESIMS, which showed a pseudomolecular ion peak at m/z 521.3234 [M + Na]+ (calcd. for C31H46NaO5, 521.3237). The 1H-NMR data in methanol-d4 (Table 2) showed signals for six methyl groups at δH 1.11 (s), 1.00 (s), 0.98 (s), 0.94 (s), 0.81 (s), and 0.78 (s); a methoxy group at δH 3.67 (s); and a disubstituted double bond at δH 6.33 (dd, J = 11.2, 2.8 Hz) and 5.72 (br d, J = 11.2 Hz). The NMR data (Table 2) were very similar to those of 30-O-methyl spergulagenate (27) [12]. However, compound 4 had one more degree of unsaturation than 30-O-methyl spergulagenate, which was supported by four olefinic carbons at δC 139.8, 130.8, 129.0, and 126.2 for two double bonds in the 13C-NMR spectrum of 4 measured in methanol-d4. Finally, the structure of 4 was elucidated to be 3β-hydroxyoleana-11,13(18)-diene-28,30-dioic acid 30-methyl ester by the key HMBC correlations from H-11 to C-10, from H-12 to C-8, and from H3-27 to C-13, as well as the key ROESY correlations of H-3/H-5, H-5/H-9, H-9/H3-27, H3-29/H-19α, H3-29/H-16α, and H3-29/H3-27 (Figure 2).
The HRESIMS of glinusopposide A (5) indicated a molecular formula of C35H58O7, with the positive ion at m/z 613.4068 [M + Na]+ (calcd. for C35H58NaO7, 613.4080). By comparing its NMR data (Table 3) with those of spergulagenin A 3-O-β-d-xylopyranoside (31) [13], compound 5 might be combined by a modified hopane and a β-xylopyranose [δH 4.87 d (J = 7.6 Hz)]. The configuration of xylopyranose in the plant was determined as the d-configuration by acidic hydrolysis of 31 followed by acetylation to yield 1,2,3,4-tetra-O-acetyl-d-xylopyranose. The genin was deduced as 16-deoxyspergulagenin A by 1H–1H COSY, HMBC, and ROESY experiments. The ROESY correlations (Figure 2) of H-3/H-5, H-5/H-9, H-9/H-12, H-12/H3-28, and H3-28/H3-29 indicated that 3-OH, 12-OH, and Me-29 were β-, β-, and α-oriented, respectively. The xylose was located at 3-OH based on the HMBC correlations from H-3 to C-1′ and from H-1′ to C-3 (Figure 2). Finally, the structure of 5 was elucidated to be 16-deoxyspergulagenin A 3-O-β-d-xylopyranoside (glinusopposide A).
Both glinusopposides B (6) and C (7) have the same molecular formula, C41H68O11, based on 13C-NMR data (Table 3) and HRESIMS. The NMR data of 6 and 7 (Table 3) indicated the presence of the same genin in the two saponins as in compound 5, with differences in the sugars. There are two sugars, β-d-xylopyranose and α-l-rhamnopyranose, in the structures of 6 and 7. Base on the key HMBC correlations from H-1′′ to C-2′ and from H-1′ to C-3 in 6, along with the correlations from H-1′′ to C-3′ and from H-1′ to C-3 in 7 (Supplementary Materials), the linkages between the two sugars were easily established to be Rha-(1→2)-Xyl-O-C-3 and Rha-(1→3)-Xyl-O-C-3 for saponins 6 and 7, respectively. Therefore, the structures of 6 and 7 were determined to be 16-deoxyspergulagenin A 3-O-[α-l-rhamnopyranosyl-(1→2)]-β-d-xylopyranoside (glinusopposide B) and 16-deoxyspergulagenin A 3-O-[α-l-rhamnopyranosyl-(1→3)]-β-d-xylopyranoside (glinusopposide C), respectively.
Based on 13C-NMR data (Table 4) and HRESIMS, the molecular formulae of glinusopposides D–G (811) were deduced to be C43H70O13, C45H72O13, C47H74O14, and C44H70O13, respectively. By comparing their NMR data (Table 4) with those of spergulagenin A 3-O-β-d-xylopyranoside (31) [13], saponins 8–11 were deduced to be disaccharide glycosides of spergulagenin A. The presence of trans-2-butenoyl (crotonyl) group in 9 was confirmed by the 1H-NMR signals at δH 7.06 (m), 6.02 (dq, J = 15.6, 1.6 Hz), and 1.66 (3H, d, J = 6.8 Hz), along with COSY correlations of H-2′′′/H-3′′′ and H-3′′′/H-4′′′ (Supplementary Materials). The closely similar data and correlations can also be found in 10 and 11, herein the trans-2-butenoyl group was assigned in 10 and 11 as same way. The trans-2-butenoyl moiety of 911 was located at C-4′ by the key HMBC correlation from H-4’ to C-1"’. According to the correlations in the 1H–1H COSY, HMBC, and ROESY spectra (Supplementary Materials), the structures of 8–11 were easily elucidated to be 3-O-[α-l-rhamnopyranosyl-(1→3)-4-O-acetyl-β-d-xylopyranosyl] spergulagenin A (glinusopposide D), 3-O-[α-l-rhamnopyranosyl-(1→3)-4-O-trans-2-butenoyl-β-d-xylopyranosyl] spergulagenin A (glinusopposide E), 3-O-[α-l-rhamnopyranosyl-(1→3)-4-O-trans-2-butenoyl-β-d-xylopyranosyl] 12-O-acetylspergulagenin A (glinusopposide F), and 3-O-[β-d-xylopyranosyl-(1→3)-4-O-trans-2-butenoyl-β-d-xylopyranosyl] spergulagenin A (glinusopposide G), respectively.
Glinusopposide H (12) was assigned the molecular formula C36H58O8, with eight degrees of unsaturation as determined by 13C-NMR data (Table 5) and the positive ion at m/z 641.4021 [M + Na]+ (calcd. for C36H58NaO8, 641.4024) in the HRESIMS. The 1H and 13C-NMR data indicated the compound might be a hopane triterpenoid saponin with eight methyl groups [δH 1.67 (s), 1.20 (s), 1.44 (s), 1.44 (s), 0.99 (s), 0.94 (s), 0.88 (s), and 0.79 (s)], one tetrasubstituted double bond (δC 152.5 and 146.8), and one β-glucopyranose [δH 5.19 (d, J = 7.6 Hz); δC 100.1, 78.8, 78.4, 75.9, 72.4, and 63.4]. In addition to the signals for the sugar, signals (δC 84.2, 78.1, 76.0, and 74.5) for four oxygenated carbon atoms were observed. The sugar was attached to C-12 based on the HMBC correlations from H-12 to C-1′ and from H-1′ to C-12, and the 17(21)-double bond was confirmed by the correlations from H3-28 to C-17 and from H3-29 and H3-30 to C-21 (Figure 2). The other three oxygenated carbon atoms were C-3, C-16, and C-22 based on the correlations from H3-23 and H3-24 to C-3, from H-16 to C-21, and from H3-29 and H3-30 to C-22. According to the deduced molecular formula and the degrees of unsaturation, a dihydrofuran ring containing the C-16-C-17-C-21-C-22-O fragment must be formed in the structure of 12, which was further confirmed because of the shift in the 13C-NMR signals for C-16 (δC 76.0) and C-22 (δC 84.2) to downfield compared with the analogues 1, 2, and 22,24,28-trihydroxy-hop-17(21)-ene [14]. The 3β,12β,16β configurations were determined by the key ROESY correlations of H-3/H3-23, H-3/H-5, H-5/H-9, H-9/H-12, H-12/H3-27, H-12/H3-28, H-16/H3-27, and H-16/H3-28 (Figure 2). Thus, the structure of 12 was elucidated to be 3β,12β-dihydroxy-16β,22-epoxyhop-17(21)-ene 12-O-β-d-glucopyranoside (glinusopposide H).
Based on 13C-NMR data (Table 5 and Table 6) and HRESIMS, the molecular formulae of glinusopposides I–K (1315) were deduced to be C35H56O7, C41H66O11, and C41H66O11, respectively. Comparison of the NMR data of 1315 (Table 5 and Table 6) with those of 12 (Table 5) which are closely similar that were suggested these compounds with the same genin, 3β,12β-dihydroxy-16β,22-epoxyhop-17(21)-ene. The position of connectivity of sugars to sapogenin were established according to the correlations in the 2D-NMR spectra (Supplementary Materials). Therefore, the structures of saponins 1315 were determined to be 3β,12β-dihydroxy-16β,22-epoxyhop-17(21)-ene 3-O-β-d-xylopyranoside (glinusopposide I), 3β,12β-dihydroxy-16β,22-epoxyhop-17(21)-ene 3-O-[α-l-rhamnopyranosyl-(1→2)]-β-d-xylopyranoside (glinusopposide J), and 3β,12β-dihydroxy-16β,22-epoxyhop-17(21)-ene 3-O-[α-l-rhamnopyranosyl-(1→3)]-β-d-xylopyranoside (glinusopposide K), respectively.
The HRESIMS of glinusopposide L (16) exhibited an ion peak at m/z 731.4353 [M + Na]+ (calcd. for C39H64NaO11, 731.4346), implying a molecular formula of C39H64O11. The NMR data of 16 (Table 6) were highly similar to those of spergulin B (35) [13], indicating that the compound might also be a bisnor hopane saponin with the same genin, spergulatriol, and the same sugars, xylose and rhamnose. The difference between the two saponins was the linkage mode of the two sugars. The rhamnose was linked to 3-OH of the inner sugar, xylose, based on the HMBC correlations from H-1″ to C-3′ and from H-3′ to C-1″ (Supplementary Materials). Finally, the structure of 16 was elucidated to be spergulatriol 3-O-[α-l-rhamnopyranosyl-(1→3)]-β-d-xylopyranoside (glinusopposide L).
According to 13C-NMR data (Table 6) and the positive ion HRESIMS at m/z 731.4347 [M + Na]+ (calcd. for C39H64NaO11, 731.4346), glinusopposide M (17) had the same molecular formula, C39H64O11, as saponin 16. The 1D and 2D-NMR spectra (Supplementary Materials) indicated that 17 had a tetrasubstituted double bond rather than the terminal double bond of 16. The 17(21) double bond was identified based on the HMBC correlations from H3-28 to C-17 and from H3-22 to C-17 and C-21. Therefore, the structure of 17 was determined to be 29,30-bisnor-3β,12β,16β-trihydroxyhop-17(21)-ene 3-O-[α-l-rhamnopyranosyl-(1 → 3)]-β-d-xylopyranoside (glinusopposide M).
According to 13C-NMR data (Table 7) and HRESIMS, the molecular formulae of glinusopposides N (18) and O (19) were deduced to be C33H52O6 and C39H62O10, respectively. Comparison of their NMR data (Table 7) with those of 17 indicated that saponins 18 and 19 were 29,30-bisnor hopane saponins with two double bonds and two hydroxy substitutions in the structure of the genin. 3β-OH and 12β-OH were determined based on the key HMBC correlations from H3-23 and H3-24 to C-3 and from H-9 to C-12, as well as the key ROESY correlations of H-3/H-5, H-5/H-9, H-9/H-12, H-12/H3-27, and H-12/H3-28 (Supplementary Materials). The 15,17(21) double bonds were identified by the HMBC correlations from H3-22 to C-17 and C-21, from H3-27 to C-15, from H3-28 to C-17, and from H-16 to C-14 and C-18. Finally, based on other correlations in the 2D-NMR spectra (Supplementary Materials), 18 and 19 were elucidated to be 29,30-bisnor-3β,12β-dihydroxyhopa-15,17(21)-diene 3-O-β-d-xylopyranoside (glinusopposide N) and 29,30-bisnor-3β,12β-dihydroxyhopa-15,17(21)-diene 3-O-[α-l-rhamnopyranosyl-(1→3)]-β-d-xylopyranoside (glinusopposide O), respectively.
The molecular formula of glinusopposide P (20) was determined to be C44H66O15 based on 13C-NMR data (Table 7) and the positive ion at m/z 857.4300 [M + Na]+ (calcd. for C44H66NaO15, 857.4299) in the HRESIMS. The NMR data (Table 7) indicated a moiety of 3β-hydroxyoleana-11,13(18)-diene-28,30-dioic acid 30-methyl ester (4), an α-rhamnopyranosyl group [δH 6.33 (br s), 5.08 (m), 4.76 (dd, J = 3.3, 1.4 Hz), 4.57 (dd, J = 9.3, 3.3 Hz), 4.35 (dd, J = 9.3, 9.3 Hz), and 1.71 (d, J = 6.1 Hz); δC 103.4, 74.6, 73.2, 73.0, 70.3, and 19.1], and a 6-O-methyl-β-glucuronopyranosyl group [δH 4.92 (d, J = 7.9 Hz), 4.58 (d, J = 9.3 Hz), 4.45 (dd, J = 9.3, 8.7 Hz), 4.41 (dd J = 9.3, 9.3 Hz), 4.07 (dd, J = 8.7, 7.9 Hz), and 3.79 (s); δC 171.3, 107.6, 82.3, 77.6, 76.2, 71.9, and 52.7]. The linkage of the sugar chain was determined to be Rha-(1→3)-[6-O-methyl-GlcA]-O-C-3 based on the key HMBC correlations from H-1″ to C-3′, from H-3′ to C-1″, from H-1′ to C-3, and from H-3 to C-1′ (Supplementary Materials). Thus, the structure of 20 was elucidated to be 3-O-[α-l-rhamnopyranosyl-(1→3)-6-O-methyl-β-d-glucuronopyranosyl]-3β-hydroxyoleana-11,13(18)-diene-28,30-dioic acid 30-methyl ester (glinusopposide P).
Based on 13C-NMR data (Table 8 and Table 9) and HRESIMS, the molecular formulae of glinusopposides Q–U (2125) were deduced to be C39H61NO10, C44H68O15, C45H70O15, C36H56O9, and C42H66O13, respectively. By comparing their NMR data with those of 30-O-methyl spergulagenate (27) [12], these saponins were determined to have the same genin, 30-O-methyl spergulagenate. The NMR signals of 21 at δH 8.94 (d, J = 9.0 Hz) and 2.15 (s), along with δC 170.3 and 23.8 manifested the presence of an acetylamino unit which was further confirmed by the HMBC correlations from δH 2.15 (H-2″) to δC 170.3 (C-1″) and from δH 8.94 (NH) to δC 170.3 (C-1″). The position of the acetylamino moiety of 21 was determined by the HMBC correlation from H-2’ to C-1″. The location of the sugar in 21 was also confirmed by the HMBC correlations from H-3 to C-1’ and H-1’ to C-3. Two anomeric carbons at δC 107.1 and 102.9 of 22 suggested that the presence of two sugars, of which the positions were assigned by the key HMBC from H-3’ to C-1″, from H-1″ to C-3’, from H-1′ to C-3, and from H-3 to C-1’. The NMR date of 23 were almost identical to those of 22 except for the replacement of the methoxy group in 22 by ethoxy group (δC 61.4 and 14.3). Comparison of NMR data of 30-O-methyl spergulagenate (27), signals for an additional sugar in compound 24 and for two additional sugars in compound 25 were observed. According to these correlations in the 2D-NMR spectra (Supplementary Materials), saponins 2125 were determined as 3-O-(2-acetylamino-2-deoxy-β-d-glucopyranosyl)-30-O-methyl spergulagenate (glinusopposide Q), 3-O-[α-l-rhamnopyranosyl-(1→3)-6-O-methyl-β-d-glucuronopyranosyl]-30-O-methyl spergulagenate (glinusopposide R), 3-O-[α-l-rhamnopyranosyl-(1→3)-6-O-ethyl-β-d-glucuronopyranosyl]-30-O-methyl spergulagenate (glinusopposide S), 30-O-methyl spergulagenate 3-O-β-d-xylopyranoside (glinusopposide T), and 30-O-methyl spergulagenate 3-O-[α-l-rhamnopyranosyl-(1→3)]-β-d-xylopyranoside (glinusopposide U), respectively.
The NMR data of compound 26 in methanol-d4 (Supplementary Materials) were the same as those of coryternic acid 3-O-β-d-glucuronopyranoside-6′-O-methyl ester [3β-O-(6-O-methyl-β-d-glucuronopyranosyl)-olean-12-ene-28,29-dioic acid 29-methyl ester] [15]. Based on the 1D and 2D-NMR spectra of 26 both in methanol-d4 and pyridine-d5 (Table 9, Figure 2, and Supplementary Materials), especially on the ROSEY correlations of H3-29/H-19α and H-19α/H3-27, the structure of 26 was determined to be 3β-O-(6-O-methyl-β-d-glucuronopyranosyl)-olean-12-ene-28,30-dioic acid 30-methyl ester. Therefore, the structure of coryternic acid 3-O-β-d-glucuronopyranoside-6′-O-methyl ester reported in the literature is suggested to be revised to 3β-O-(6-O-methyl-β-d-glucuronopyranosyl)-olean-12-ene-28,30-dioic acid 30-methyl ester.
Other known compounds, 30-O-methyl spergulagenate (27) [12], 28-β-d-glucopyranosyl-30-methyl 3β-hydroxyolean-12-en-28,30-dioate (28) [16], oleanolic acid 3-O-6′-O-methyl-β-d-glucuronopyranoside (29) [17], oppositifolone (30) [18], spergulagenin A 3-O-β-d-xylopyranoside (31) [13], spergulin A (32) [13], spergulacin A (33) [13], spergulacin (34) [13], spergulin B (35) [13], grasshopper ketone (36) [19], and β-carboline (37) [20], were determined by comparing their NMR data (for all compounds) and optical rotation values (for all compounds except 37) with those reported in the literature.

2.2. Biological Evaluation

We proposed antifungal activities of G. oppositifolius according to traditional healthcare use. The 70% ethanol extract of the whole plants of G. oppositifolius showed inhibitory activity against M. gypseum with an inhibition of 23.0 ± 1.9% at a concentration of 128 μg/mL. All the isolated compounds (137) were measured for antifungal activities against M. gypseum and T. rubrum, and the results are presented in Table 10. Glinusopposide B (6), glinusopposide Q (21), glinusopposide T (24), and glinusopposide U (25) showed the most notable inhibitory activities against M. gypseum (MIC50 7.1, 6.7, 6.8, and 11.1 μM, respectively) and T. rubrum (MIC50 14.3, 13.4, 11.9, and 13.0 μM, respectively) compared with the positive control terbinafine hydrochloride (MIC50 0.008 μM against M. gypseum and 1.647 μM against T. rubrum). Glinusopposide K (15), glinusopposide N (18), glinusopposide O (19), glinusopposide R (22), glinusopposide S (23), glinusopposide U (25), and 3β-O-(6-O-methyl-β-d-glucuronopyranosyl)-olean-12-ene-28,30-dioic acid 30-methyl ester (26) showed moderate inhibitory activities against M. gypseum, with MIC50 values ranging from 22.0 to 46.8 μM. Additionally, 3β,12β,16β,21β,22-pentahydroxyhopane (1), 3-oxo-olean-12-ene-28,30-dioic acid (3), glinusopposide H (12), and glinusopposide L (16) showed weak activities against M. gypseum, with MIC50 values ranging from 105.0 to 260.1 μM. Other compounds did not display activity against M. gypseum or T. rubrum (MIC50 > 300 μM).
The active compounds of G. oppositifolius against M. gypseum and T. rubrum have two types of carbon skeletons, hopane and oleanane. For those oleanane-type compounds, glycosylation (2126) or oxidation (3) of 3-OH was helpful in increasing the activity based on a comparison of the MIC50 values of 3 and 2126 with those of 27 and 28. Replacement of the 30-methyl group (29) with a carboxymethyl group (26) enhanced the activity. The presence of 11,13(18) double bonds (20) decreased the activity. The structure-activity relationships (SARs) of the hopane-type compounds against the two fungi were not clear.

3. Experimental Section

3.1. General Experimental Procedures

This part can be found in the Supplementary Materials.

3.2. Plant Material

Whole plants of G. oppositifolius were bought from Zay cho market of Mandalay in Myanmar, in December 2015. The plants were identified by author, Jun Yang. A voucher specimen (No. MD1612078) was deposited at the Yunnan Key Laboratory for Wild Plant Resources, Kunming Institute of Botany, Chinese Academy of Sciences.

3.3. Extraction and Isolation

Powdered whole plants of G. oppositifolius (3.0 kg) were extracted with 70% EtOH at 60 °C for six times (each for 4 h) to obtain a crude extract (650.1 g), which was suspended in H2O and then extracted with petroleum ether. The water-soluble phase was adjusted to pH 1−2 with 1% HCl and then partitioned with EtOAc to afford the EtOAc-soluble extract (B, 130.0 g). The aqueous phase was basified with 5% NaOH solution to pH 9−10 and then extracted with CHCl3 to yield the CHCl3-soluble extract (A, 25.2 g). The aqueous phase was extracted with n-butanol to yield the n-butanol-soluble extract (C, 142.0 g).
The CHCl3 extract (A, 25.2 g) was subjected to silica gel column chromatography (CC, CH2Cl2-MeOH, 50:1→0:1, v/v) to yield four main fractions A1–A4. Fraction A1 (931.1 mg) was subjected to reversed phase (RP-C18) silica gel CC eluted with MeOH-H2O (30%→100%). The 30% MeOH-eluted part (86.3 mg) was separated on a Sephadex LH-20 CC (MeOH) and purified by semipreparative HPLC (Welch Ultimate AQ-C18, MeOH-H2O, 18:82, 0.8 mL/min) to yield 36 (6.0 mg, tR = 56.431 min). The 60% MeOH-eluted part (399.8 mg) was separated on a Sephadex LH-20 CC (MeOH) to yield 1 (4.9 mg), 2 (4.7 mg), and 31 (24.6 mg) recrystallized from MeOH, as well as 37 (0.4 mg) recrystallized from CH2Cl2. The 80% MeOH-eluted part (127.3 mg) was purified by Sephadex LH-20 CC (CH2Cl2-MeOH, 1:1) and semipreparative HPLC (Agilent Zorbax SB-C18, MeOH-H2O (containing 0.05% TFA), 67:33, 2 mL/min) to obtain 11 (1.1 mg, tR = 36.803 min) and 9 (5.7 mg, tR = 43.850 min). Fraction A2 (2.1 g) was separated on an RP-18 silica gel CC eluted with MeOH-H2O (30%→100%). The 70% MeOH-eluted part (96.8 mg) was purified by silica gel CC (CH2Cl2-MeOH-H2O, 300:10:1) to yield 8 (24.3 mg) recrystallized from MeOH. Fraction A3 (2.5 g) was separated on an RP-18 silica gel CC eluted with MeOH-H2O (20%→100%). The 60% MeOH-eluted part (965.5 mg) was purified by silica gel CC (EtOAc-MeOH, 20:1) to yield two main subfractions (A3-1 and A3-2). The subfraction A3-1 (91.8 mg) was purified by silica gel CC (CH2Cl2-MeOH, 10:1) and semipreparative HPLC (Agilent Zorbax SB-C18, CH3CN-H2O, 35:65, 2 mL/min) to yield 17 (1.0 mg, tR = 28.240 min). The subfraction A3-2 (192.0 mg) was purified by silica gel CC (CH2Cl2-MeOH-H2O, 80:10:1) to yield two further subfractions (A3-2-1 and A3-2-2). The subfraction A3-2-1 (33.1 mg) was purified by semipreparative HPLC (Agilent Zorbax SB-C18, MeCN-H2O, 30:70, 2 mL/min) to yield 34 (16.6 mg, tR = 31.175 min) and 16 (7.6 mg, tR = 45.162 min). The subfraction A3-2-2 (7.0 mg) was purified by semipreparative HPLC (Welch Ultimate AQ-C18, MeCN-H2O, 30:70, 1 mL/min) to yield 35 (1.4 mg, tR = 7.176 min). Fraction A4 (3.0 g) was separated on an RP-C18 silica gel CC eluted with MeOH-H2O (20%→100%) to yield two further subfractions. The 40% MeOH-eluted part (217.9 mg) was purified by silica gel CC (CH2Cl2-MeOH-H2O, 70:10:1) to yield 32 (38.4 mg). The 50% MeOH-eluted part (494.4 mg) was recrystallized from MeOH to yield 33 (290.3 mg).
The part of EtOAc extract (B, 27.0 g) was separated on an RP-18 silica gel CC eluted with MeOH-H2O (5%→100%) to yield five main fractions (B1–B5). The 50% MeOH-eluted part (B1, 2.7 g) was purified by silica gel CC (CH2Cl2-MeOH, 50:1→30:1) to afford 30 (26.8 mg). The 60% MeOH-eluted part (B2, 545.7 mg) was purified by silica gel CC (CH2Cl2-MeOH, 30:1) to yield 12 (5.5 mg). The 70% MeOH-eluted part (B3, 2.5 g) was purified by silica gel CC (CH2Cl2-MeOH, 30:1→20:1) to afford 28 (11.1 mg) and three main subfractions (B3-1–B3-3). Subfraction B3-1 (29.0 mg) was purified by semipreparative HPLC (Agilent Zorbax SB-C18, MeCN-H2O (containing 0.05% TFA), 47:53, 2 mL/min) and further by semipreparative HPLC (Agilent Zorbax SB-C18, MeOH-H2O (containing 0.05% TFA), 75:25, 2 mL/min) to yield 13 (3.9 mg, tR = 30.891 min) and 5 (2.1 mg, tR = 41.804 min). Subfraction B3-2 (91.7 mg) was purified by silica gel CC (CH2Cl2-MeOH, 30:1) and semipreparative HPLC (Agilent Zobrax SB-C18, MeCN-H2O (containing 0.05% TFA), 50:50, 2 mL/min) to yield 7 (5.3 mg, tR = 14.414 min) and 15 (5.3 mg, tR = 16.844 min). Subfraction B3-3 (459.4 mg) was purified by silica gel CC (CH2Cl2-MeOH, 30:1) and semipreparative HPLC (Agilent Zorbax SB-C18, MeCN-H2O, 45:55, 2 mL/min) to yield 21 (5.7 mg, tR = 12.996 min), 6 (5.9 mg, tR = 16.490 min), and 14 (3.6 mg, tR = 17.736 min). The 80% MeOH-eluted part (B4, 1.1 g) was purified by silica gel CC (CH2Cl2-MeOH, 30:1) to yield two main subfractions (B4-1 and B4-2). Subfraction B4-1 (97.3 mg) was purified by semipreparative HPLC (Agilent Zorbax SB-C18, MeCN-H2O (containing 0.05% TFA), 45:55, 2 mL/min) to afford 24 (18.3 mg, tR = 42.714 min), 26 (29.0 mg, tR = 51.174 min), and 10 (1.6 mg, tR = 62.511 min). Subfraction B4-2 (153.1 mg) was purified by semipreparative HPLC (Agilent Zorbax SB-C18, MeCN-H2O (containing 0.05% TFA), 43:57, 2 mL/min) to yield 25 (12.4 mg, tR = 37.244 min), 22 (42.6 mg, tR = 45.406 min), 23 (16.2 mg, tR = 66.231 min), and a mixture. The mixture was purified by semipreparative HPLC (Agilent Eclipse XDB-C18, MeCN-H2O (containing 0.05% TFA), 40:60, 1 mL/min) to yield 20 (3.5 mg, tR = 18.741 min). The 90% MeOH-eluted part (B5, 1.1 g) was separated on a Sephadex LH-20 CC (MeOH) to yield two main subfractions (B5-1 and B5-2). Subfraction B5-1 (535.3 mg) was purified by silica gel CC (CH2Cl2-MeOH, 30:1) and semipreparative HPLC (Agilent Zorbax SB-C18, MeCN/H2O (containing 0.05% TFA), 49:51, 2 mL/min) to yield 19 (3.9 mg, tR = 43.926 min). Subfraction B5-2 (244.5 mg) was purified by silica gel CC (petroleum ether-EtOAc, 2:1→1:1) to yield two further subfractions (B5-2-1 and B5-2-2). Subfraction B5-2-1 (52.9 mg) was purified by semipreparative HPLC (Agilent Zorbax SB-C18, MeCN-H2O (containing 0.05% TFA), 60:40, 2 mL/min) to yield 3 (7.6 mg, tR = 35.867 min), 4 (3.5 mg, tR = 47.842 min), and 27 (21.0 mg, tR = 53.439 min). Subfraction B5-2-2 (20.2 mg) was purified by semipreparative HPLC (Agilent Zorbax SB-C18, MeCN-H2O (containing 0.05% TFA), 70:30, 2 mL/min) to yield 29 (0.7 mg, tR = 15.194 min) and 18 (3.1 mg, tR =16.328 min).

3.4. Spectroscopic and Physical Data

3β,12β,16β,21β,22-Pentahydroxyhopane (1). Colorless blocks (Me-H2O, 10:1); [ α ] D 25 −18 (c 0.05, MeOH); UV (MeOH) λmax (logε) 203 (3.34) nm; ECD (c 0.05, MeOH) λmaxε) 240 (+0.35), 226 (−0.36), 198 (+1.15) nm; 1H and 13C-NMR data, see Table 1; ESIMS m/z 515 [M + Na]+; HRESIMS m/z 515.3718 [M + Na]+ (calcd for C30H52NaO5, 515.3712).
Crystal data for 1: C30H52O5, M = 524.75, a = 7.8358(3) Å, b = 17.7389(6) Å, c = 20.4841(7) Å, α = 90°, β = 90°, γ = 90°, V = 2847.26(17) Å3, T = 100.(2) K, space group P212121, Z = 4, μ(Cu Kα) = 0.653 mm−1, 51,068 reflections measured, 5642 independent reflections (Rint = 0.0514). The final R1 values were 0.0363 (I > 2σ(I)). The final wR(F2) values were 0.1081 (I > 2σ(I)). The final R1 values were 0.0371 (all data). The final wR(F2) values were 0.1102 (all data). The goodness of fit on F2 was 0.998. Flack parameter = 0.02(5). The supplementary crystallographic data can be obtained free of charge from the Cambridge Crystallographic Data Centre (CCDC) (deposition number CCDC 1917520) via http://www.ccdc.cam.ac.uk.
12β,16β,21β,22-Tetrahydroxyhopan-3-one (2). White powder; [ α ] D 25 +2 (c 0.12, MeOH); UV (MeOH) λmax (logε) 280 (1.98), 219 (2.73), 203 (2.91) nm; ECD (c 0.12, MeOH) λmaxε) 289 nm (+0.17), 232 (+0.28), 208 (–0.29) nm; IR νmax (KBr) 3443, 3427, 1690, 1452, 1385, 1084, 1047, 879 cm−1; 1H and 13C-NMR data, see Table 1; ESIMS m/z 513 [M + Na]+; HRESIMS m/z 513.3551 [M + Na]+ (calcd for C30H50NaO5, 513.3556).
The 3-Oxo-olean-12-ene-28,30-dioic acid (3). White powder; [ α ] D 25 +59 (c 0.26, MeOH); UV (MeOH) λmax (logε) 371 (1.73), 252 (2.75), 239 (2.72) nm; 1H and 13C-NMR data, see Table 2; ESIMS m/z 507 [M + Na]+; HRESIMS m/z 507.3084 [M + Na]+ (calcd for C30H44NaO5, 507.3086).
The 3β-Hydroxyoleana-11,13(18)-diene-28,29-dioic acid 29-methyl ester (4). White powder; [ α ] D 26 –7 (c 0.15, MeOH); UV (MeOH) λmax (logε) 250 (2.65), 242 (2.59) nm; ECD (c 0.09, MeOH) λmaxε) 250 (–3.92) nm; 1H and 13C-NMR data, see Table 2; ESIMS m/z 521 [M + Na]+; HRESIMS m/z 521.3234 [M + Na]+ (calcd for C31H46NaO5, 521.3237).
Glinusopposide A (5). White powder; [ α ] D 26 –8 (c 0.05, MeOH); 1H and 13C-NMR data, see Table 3; ESIMS m/z 613 [M + Na]+; HRESIMS: m/z 613.4068 [M + Na]+ (calcd for C35H58NaO7, 613.4080).
Glinusopposide B (6). White powder; [ α ] D 25 –20 (c 0.1, MeOH); 1H and 13C-NMR data, see Table 3; ESIMS m/z 759 [M + Na]+; HRESIMS m/z 759.4650 [M + Na]+ (calcd for C41H68NaO11, 759.4659).
Glinusopposide C (7). White powder; [ α ] D 26 –6 (c 0.35, MeOH); 1H and 13C-NMR data, see Table 3; ESIMS m/z 759 [M + Na]+; HRESIMS m/z 759.4656 [M + Na]+ (calcd for C41H68NaO11, 759.4659).
Glinusopposide D (8). White powder; [ α ] D 21 –36 (c 0.16, pyridine); UV (MeOH) λmax (logε) 275 (2.77), 245 (3.02), 204 (3.59) nm; ECD (c 0.099, MeOH) λmaxε) 284 (+1.00), 249 (–0.17), 218 (+1.03), 197 (–1.11) nm; 1H and 13C-NMR data, see Table 4; ESIMS m/z 833 [M + K]+, 817 [M + Na]+; HRESIMS m/z 817.4709 [M + Na]+ (calcd for C43H70NaO13, 817.4714).
Glinusopposide E (9). White powder; [ α ] D 20 –32 (c 0.09, MeOH); UV (MeOH) λmax (logε) 415 (2.39), 206 (4.09) nm; ECD (c 0.065, MeOH) λmaxε) 284 (+0.48), 206 (+0.73), 200 (–1.80) nm; IR νmax (KBr) 3442, 3428, 1677, 1644, 1449, 1431, 1385, 1202, 1144, 1086, 1047, 879 cm−1; 1H and 13C-NMR data, see Table 4; ESIMS m/z 843 [M + Na]+; HRESIMS m/z 843.4867 [M + Na]+ (calcd for C45H72NaO13, 843.4871).
Glinusopposide F (10). White solid; [ α ] D 25 –31 (c 0.04, MeOH); 1H and 13C-NMR data, see Table 4; ESIMS m/z 885 [M + Na]+; HRESIMS m/z 885.4930 [M + Na]+ (calcd for C47H74NaO14, 885.4976).
Glinusopposide G (11). White powder; [ α ] D 20 –7 (c 0.15, MeOH); UV (MeOH) λmax (logε) 252 (3.21), 206 (3.81) nm; ECD (c 0.078, MeOH) λmaxε) 201 (–1.59), 197 (+1.29) nm; IR νmax (KBr) 3448, 3427, 1639, 1447, 1383, 1084, 1046, 879 cm−1; 1H and 13C-NMR data, see Table 4; ESIMS m/z 829 [M + Na]+; HRESIMS m/z 829.4711 [M + Na]+ (calcd for C44H70NaO13, 829.4714).
Glinusopposide H (12). White powder; [ α ] D 25 +7 (c 0.28, MeOH); 1H and 13C-NMR data, see Table 5; ESIMS m/z 641 [M + Na]+; HRESIMS m/z 641.4021 [M + Na]+ (calcd for C36H58NaO8, 641.4024).
Glinusopposide I (13). White powder; [ α ] D 25 –10 (c 0.07, MeOH); 1H and 13C-NMR data, see Table 5; ESIMS m/z 611 [M + Na]+; HRESIMS m/z 611.3898 [M + Na]+ (calcd for C35H56NaO7, 611.3924).
Glinusopposide J (14). White powder; [ α ] D 25 –22 (c 0.11, MeOH); 1H and 13C-NMR data, see Table 5; ESIMS: m/z 757 [M + Na]+; HRESIMS m/z 757.4486 [M + Na]+ (calcd for C41H66NaO11, 757.4503).
Glinusopposide K (15). White powder; [ α ] D 25 –16 (c 0.13, MeOH); 1H and 13C-NMR data, see Table 6; ESIMS m/z 757 [M + Na]+; HRESIMS m/z 757.4510 [M+ Na]+ (calcd for C41H66NaO11, 757.4503).
Glinusopposide L (16). White powder; [ α ] D 25 –25 (c 0.1, MeOH); UV (MeOH) λmax (logε) 203 (3.82) nm; ECD (c 0.076, MeOH) λmaxε) 196 (–6.89) nm; IR νmax (KBr) 3425, 1632, 1454, 1385, 1129, 1094, 1047, 974 cm−1; 1H and 13C-NMR data, see Table 6; ESIMS m/z 731 [M + Na]+; HRESIMS m/z 731.4353 [M + Na]+ (calcd for C39H64NaO11, 731.4346).
Glinusopposide M (17). White powder; [ α ] D 20 –25 (c 0.05, MeOH); UV (MeOH) λmax (logε) 203 (3.54) nm; ECD (c 0.05, MeOH) λmaxε) 206 (–4.38), 196 (+4.32) nm; IR νmax (KBr) 3443, 3426, 1639, 1453, 1421, 1384, 1084, 1047, 879 cm−1; 1H and 13C-NMR data, see Table 6; ESIMS m/z 731 [M + Na]+; HRESIMS m/z 731.4347 [M + Na]+ (calcd for C39H64NaO11, 731.4346).
Glinusopposide N (18). White powder; [ α ] D 26 –7 (c 0.18, MeOH); UV (MeOH) λmax (logε) 250 (3.76), 213 (3.38) nm; 1H and 13C-NMR data, see Table 7; ESIMS m/z 567 [M + Na]+; HRESIMS m/z 567.3655 [M + Na]+ (calcd for C33H52NaO6, 567.3656).
Glinusopposide O (19). White powder; [ α ] D 25 –14 (c 0.13, MeOH); UV (MeOH) λmax (logε) 250 (3.76), 218 (3.43) nm; 1H and 13C-NMR data, see Table 7; ESIMS m/z 729 [M + K]+, 713 [M + Na]+; HRESIMS m/z 713.4200 [M + Na]+ (calcd for C39H62NaO10, 713.4241).
Glinusopposide P (20). White powder; [ α ] D 25 –39 (c 0.1, MeOH); UV (MeOH) λmax (logε) 250 (4.07), 217 (3.68) nm; 1H and 13C-NMR data, see Table 7; ESIMS m/z 857 [M + Na]+; HRESIMS m/z 857.4300 [M + Na]+ (calcd for C44H66NaO15, 857.4299).
Glinusopposide Q (21). White powder; [ α ] D 25 +40 (c 0.13, MeOH); 1H and 13C-NMR data, see Table 8; ESIMS m/z 726 [M + Na]+; HRESIMS m/z 726.4201 [M + Na]+ (calcd for C39H61NNaO10, 726.4193).
Glinusopposide R (22). White powder; [ α ] D 25 +10 (c 0.15, MeOH); ECD (c 0.078, MeOH) λmaxε) 221 (–1.44) nm; 1H and 13C-NMR data, see Table 8; ESIMS m/z 859 [M + Na]+; HRESIMS m/z 859.4452 [M + Na]+ (calcd for C44H68NaO15, 859.4450).
Glinusopposide S (23). White powder; [ α ] D 25 +8 (c 0.1, MeOH); 1H and 13C-NMR data, see Table 8; ESIMS m/z 873 [M + Na]+; HRESIMS m/z 873.4606 [M + Na]+ (calcd for C45H70NaO15, 873.4607).
Glinusopposide T (24). White powder; [ α ] D 24 +71 (c 0.22, MeOH); 1H and 13C-NMR data, see Table 9; ESIMS m/z 655 [M + Na]+; HRESIMS m/z 655.3820 [M + Na]+ (calcd for C36H56NaO9, 655.3822).
Glinusopposide U (25). White powder; [ α ] D 25 +11 (c 0.1, MeOH); 1H and 13C-NMR data, see Table 9; ESIMS m/z 801 [M + Na]+; HRESIMS m/z 801.4393 [M + Na]+ (calcd for C42H66NaO13, 801.4396).
The 3β-O-(6-O-Methyl-β-d-glucuronopyranosyl)-olean-12-ene-28,30-dioic acid 30-methyl ester (26). White powder; [ α ] D 25 +66 (c 0.12, MeOH); 1H and 13C-NMR data, see Table 9; ESIMS m/z 713 [M + Na]+; HRESIMS m/z 713.3871 [M + Na]+ (calcd for C38H58NaO11, 713.3877).

3.5. Acid Hydrolysis and Sugar Analysis

3.5.1. Acid Hydrolysis of 31 and Acetylation of Xylose

Compound 31 (15.5 mg) was dissolved in 2 M HCl (1 mL) and stirred at 90 °C for 4 h. After cooling, the solution was evaporated to dryness under reduced pressure. The reaction mixture was purified by silica gel column chromatography (CH2Cl2-MeOH-H2O, 500:10:1, 300:10:1, 200:10:1) to afford xylose (1.9 mg). The sugar was dissolved in pyridine (0.1 mL) and acetic anhydride (0.1 mL) and stirred for 21 h at room temperature. Then, water (5 mL) was added to the reaction mixture, followed by extraction with EtOAc (5 mL). The organic layer was dried under reduced pressure to yield 1,2,3,4-tetra-O-acetyl-d-xylopyranose (0.9 mg), which was identified based on its 1H-NMR spectrum and optical rotation value: [ α ] D 21 −31 (c 0.08, CHCl3) [21].

3.5.2. Acid Hydrolysis of the Saponin Mixture and Acetylation of Rhamnose

The n-butanol-soluble part (20.0 g) was subjected to D101 resin column chromatography, eluted using water (discarded) and 60% EtOH to yield the saponin mixture (4.0 g). The latter (1.0 g) was dissolved in 2 M HCl (3 mL) and stirred at 90 °C for 5 h. The reaction mixture was dried and purified by silica gel column chromatography (CH2Cl2-MeOH-H2O, 500:10:1, 200:10:1, 100:10:1) to yield rhamnose (97.9 mg) and glucose (9.4 mg). The glucose was identified as d-glucose based on its 1H-NMR spectrum and optical rotation value: [ α ] D 19 +40 (c 0.22, H2O) [22]. The rhamnose (97.9 mg) was dissolved in pyridine (0.1 mL) and acetic anhydride (0.1 mL) and stirred for 21 h at room temperature. Then, water (5 mL) was added to the reaction mixture, followed by extraction with EtOAc (5 mL). The organic layer was dried under reduced pressure and purified by silica gel column chromatography (petroleum ether-EtOAc, 50:1) to yield 1,2,3,4-tetra-O-acetyl-α-l-rhamnopyranose (1.4 mg), which was identified based on its 1H-NMR spectrum and optical rotation value: [ α ] D 21 −27 (c 0.14, CHCl3) [23].

3.6. Antimicrobial Assays

The fungi strains T. rubrum ATCC 4438 and M. gypseum CBS118893) were purchased from the Institute of Dermatology and Hospital for Skin Diseases, Chinese Academy of Medical Sciences. An antifungal assay was performed according to modified versions of the clinical and laboratory standards institute (CLSI), formerly national committee for clinical laboratory standards (NCCLS) methods, as described previously [24,25]. Terbinafine hydrochloride was used as a positive control. The 50% minimum inhibitory concentration (MIC50) was calculated by the Reed-Muench method [26].

4. Conclusions

In this study, four new triterpenoids (14), 21 new triterpenoids glycosides (525), and 12 known compounds were isolated from G. oppositifolius. However, we cannot exclude the possibility that some of the isolated compounds might be artifacts resulted from the extraction treatment, for example compound 23 might be artifacts of ethanol extraction. The triterpenoids and their glycosides were hopane-type and oleanane-type which have been proofed to be existing in this plant [9,13]. Four compounds including glinusopposide B (6), glinusopposide Q (21), glinusopposide T (24), and glinusopposide U (25) showed considerable inhibitory activities against M. gypseum and T. rubrum. According to the study of SARs, sugars at 3-hydroxy, 30-carboxymethyl group, and the double bond at C-12 play a key role in oleanane type compounds for antifungal activities. The SARs of hopane type compounds for antifungal activities remain for further research. This study provides a scientific evidence of traditional practice on applying G. oppositifolius to treat dermatophytosis.

Supplementary Materials

The following are available online. General experimental procedures; Figures S1–S198: 1D and 2D-NMR and HRESIMS spectra of compounds 126, structures of known compounds (2637), and key 2D-NMR correlations of 2, 611, and 1325.

Author Contributions

Y.W. and X.Y. designed the research; D.Z. performed the research; J.Y. identified the plant of study; M.M.S. and T.N.O. provided the material and traditional knowledge; D.Z. and Y.W. analyzed the data and wrote the paper. Y.W., X.Y., and Y.F. revised the manuscript. X.-N.L. provided the data of single-crystal X-ray diffraction. All authors have read and approved the final manuscript.

Funding

This study was supported by the Southeast Asia Biodiversity Research Institute, Chinese Academy of Sciences (no. 2015CASEABRIRG001 and Y4ZK111B01), the National Natural Science Foundation of China (no. 31670338), and the International Partnership Program of Chinese Academy of Sciences (no. 153631KYSB20160004).

Acknowledgments

We thank to Xue Bai, at Kunming Institute of Botany, Chinese Academy of Sciences, for the antifungal assay.

Conflicts of Interest

The authors declare no conflict of interest.

References

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Sample Availability: Samples of the compounds 8, 31, 33, and 34 are available from the authors.
Figure 1. Chemical structures of compounds 126 from Glinus oppositifolius.
Figure 1. Chemical structures of compounds 126 from Glinus oppositifolius.
Molecules 24 02206 g001
Figure 2. Key 2D-NMR correlations of 1, 35, 12, and 26.
Figure 2. Key 2D-NMR correlations of 1, 35, 12, and 26.
Molecules 24 02206 g002
Figure 3. X-ray crystallographic structure of 1.
Figure 3. X-ray crystallographic structure of 1.
Molecules 24 02206 g003
Table 1. 1H (500 MHz) and 13C (126 MHz) NMR data of 1 and 2 in Pyridine-d5 (δ in ppm, J in Hz).
Table 1. 1H (500 MHz) and 13C (126 MHz) NMR data of 1 and 2 in Pyridine-d5 (δ in ppm, J in Hz).
1 2
No.δHδCδHδC
11.68 m
0.97 m
39.11.79 m
1.31 m
39.4
21.81 m28.32.48 m
2.42 m
34.4
33.44 br t (8.3)78.0 216.4
4 39.5 47.4
50.78 dd (12.0, 1.7)55.71.28 m54.8
61.51 m
1.33 m
18.91.37 m
1.31 m
20.0
71.43 m
1.21 m
33.61.38 m
1.19 m
32.7
8 45.1 45.2
91.39 m49.31.39 m48.4
10 37.3 36.8
112.11 m
1.64 m
33.22.04 m
1.65 m
33.4
124.23 m69.14.20 m69.0
131.84 d (10.7)56.41.84 d (10.8)56.5
14 41.8 41.7
151.93 dd (12.7, 4.2)
1.71 m
46.01.92 dd (12.6, 4.3)
1.70 m
45.9
164.48 m66.84.48 m66.7
172.41 d (11.7)73.82.41 d (11.6)73.8
18 47.5 47.5
192.60 m
2.14 m
43.12.60 m
2.13 m
43.1
202.05 m
1.95 m
37.82.07 m
1.96 m
37.8
21 85.7 85.7
22 75.5 75.5
231.21 s28.71.11 s26.6
241.01 s16.31.00 s21.3
250.82 s16.10.81 s15.6
260.97 s17.00.95 s16.6
271.13 s19.51.10 s19.4
281.19 s17.31.19 s17.3
291.56 s26.61.56 s26.7
301.61 s27.31.61 s27.4
3-OH5.78 br s
12-OH5.32 d (6.0) 5.38 d (6.9)
Table 2. 1H and 13C-NMR data of 3 and 4 (δ in ppm, J in Hz).
Table 2. 1H and 13C-NMR data of 3 and 4 (δ in ppm, J in Hz).
3 (Pyridine-d5) 4 (Methanol-d4) 4 (Pyridine-d5)
No.δH (500 MHz)δC (126 MHz)δH (600 MHz)δC (151 MHz)δH (500 MHz)δC (126 MHz)
11.65 m
1.30 m
39.11.92 m
1.05 m
39.41.88 m
1.07 m
38.5
22.51 m
2.37 m
34.41.69 m
1.64 m
27.91.94 m
1.89 m
28.1
3 216.33.18 dd (11.7, 4.9)79.83.49 dd (10.6, 5.0)78.1
4 47.5 40.1 39.6
51.32 m55.40.84 br d (12.1)56.40.91 dd (12.2, 1.8)55.3
61.35 m19.81.63 m
1.46 m
19.61.61 m
1.44 m
18.8
71.46 m
1.31 m
32.71.35 m33.71.31 m32.9
8 39.7 42.2 41.2
91.70 m47.21.99 br s56.02.06 br s54.9
10 36.9 38.0 37.1
111.88 m23.85.72 br d (11.2)129.05.78 br d (10.5)127.8
125.72 br t (3.3)122.86.33 dd (11.2, 2.8)126.26.63 dd (10.5, 2.6)125.7
13 144.8 139.8 138.3 a
14 42.2 43.6 42.7
152.18 m28.51.72 m
1.08 m
26.21.97 m
1.08 m
25.6
16α
16β
2.19 m
2.08 m
24.01.71 m
1.95 m
33.81.78 m
2.25 m
33.1
17 46.4 49.3 a 48.8
183.63 dd (13.7, 4.0)43.5 130.8 131.2 a
19α
19β
1.92 dd (13.7, 13.7)
2.50 m
43.12.85 dd (14.5, 1.7)
2.17 d (14.5)
35.93.18 d (15.2)
2.78 d (15.2)
35.6
20 44.1 44.8 43.9
212.41 m
1.47 m
31.21.83 m
1.57 m
33.02.28 m
1.78 m
32.5
222.41 m
2.08 m
34.82.29 m
1.41 m
35.42.67 ddd (13.8, 3.5, 3.5)
1.52 m
35.0
231.14 s26.60.98 s28.61.24 s28.6
240.99 s21.60.78 s15.91.03 s16.1
250.86 s14.90.94 s18.80.97 s18.4
261.00 s17.30.81 s17.31.07 s17.0
271.30 s26.11.00 s20.31.09 s20.1
28 180.1 179.9 DAS b
291.43 s29.11.11 s20.41.28 s20.2
30 179.5 180.4 178.5
30-OMe 3.67 s52.63.60 s51.8
a Detected by HMBC. b Disappeared signal.
Table 3. 1H and 13C-NMR data of 5–7 in Pyridine-d5 (δ in ppm, J in Hz).
Table 3. 1H and 13C-NMR data of 5–7 in Pyridine-d5 (δ in ppm, J in Hz).
5 6 7
No.δH (600 MHz)δC (151 MHz)δH (500 MHz)δC (126 MHz)δH (600 MHz)δC (151 MHz)
11.70 m
0.97 m
39.31.63 m
0.90 m
39.11.67 m
0.94 m
39.3
22.21 m
1.91 m
27.42.13 m
1.88 m
27.02.14 m
1.87 m
27.3
33.38 dd (11.7, 4.4)89.13.29 dd (11.8, 4.2)88.63.31 dd (11.9, 4.4)89.3
4 40.1 39.7 40.1
50.79 br d (11.9)56.20.71 br d (9.3)56.00.76 br d (12.0)56.2
61.51 m
1.33 m
19.11.46 m
1.33 m
18.61.50 m
1.32 m
19.0
71.39 m
1.19 m
34.01.33 m
1.15 m
33.51.40 m
1.19 m
34.0
8 43.8 43.4 43.8
91.40 m50.01.35 m49.61.40 m50.0
10 37.4 37.0 37.4
112.12 m
1.64 m
33.52.07 m
1.60 m
33.02.10 m
1.63 m
33.5
124.20 m69.24.17 m68.74.20 m69.1
131.72 d (10.7)56.91.68 d (11.1)56.51.72 overlapped57.0
14 42.1 41.6 42.1
151.51 m
1.20 m
34.81.48 m
1.17 m
34.41.52 m
1.21 m
34.8
161.44 m20.21.42 m19.81.46 m20.2
171.82 dd (12.1, 2.9)56.31.78 dd (11.9, 2.9)55.91.82 dd (12.0, 2.8)56.3
18 45.8 45.4 45.8
192.58 m
1.68 m
45.22.55 m
1.65 m
44.82.58 m
1.68 m
45.2
202.17 m
1.68 m
36.22.14 m
1.65 m
35.72.17 m
1.68 m
36.2
21 54.5 54.0 54.5
22 213.0 212.6 213.0
231.33 s28.61.25 s28.01.27 s28.5
241.01 s17.21.17 s16.90.97 s17.2
250.82 s16.50.79 s16.20.81 s16.5
260.99 s17.50.95 s17.00.99 s17.5
271.05 s18.41.01 s18.01.05 s18.4
281.12 s17.21.09 s16.81.12 s17.2
291.22 s21.51.19 s21.01.22 s21.5
302.19 s25.92.16 s25.42.19 s25.9
1′4.87 d (7.6)108.24.83 d (7.3)106.24.77 d (7.5)107.8
2′4.05 dd (8.6, 7.6)76.04.24 dd (8.2, 7.3)78.04.04 dd (8.6, 7.5)75.9
3′4.20 m79.14.18 m79.74.32 dd (8.8, 8.8)83.5
4′4.26 m71.74.15 m71.64.17 m70.2
5′4.41 dd (11.3, 5.2)
3.80 dd (11.3, 10.5)
67.64.33 m
3.71 dd (10.5, 9.9)
67.04.36 m
3.74 dd (11.2, 10.3)
67.4
1″ 6.54 d (0.9)102.06.30 br s103.2
2″ 4.88 m72.54.82 dd (3.4, 1.3)73.1
3″ 4.69 dd (9.5, 3.1)72.64.62 dd (9.3, 3.4)73.2
4″ 4.35 m74.24.37 dd (9.3, 9.3)74.6
5″ 4.77 m69.85.01 m70.4
6″ 1.70 d (6.2)18.81.71 d (6.2)19.1
12-OH 5.23 d (6.2)
2″-OH 6.69 br s
4″-OH 6.72 br s
Table 4. 1H and 13C-NMR data of 811 in Pyridine-d5 (δ in ppm, J in Hz).
Table 4. 1H and 13C-NMR data of 811 in Pyridine-d5 (δ in ppm, J in Hz).
8 9 10 11
No.δH (500 MHz)δC (126 MHz)δH (500 MHz)δC (126 MHz)δH 800 MHzδC (201 MHz)δH (800 MHz)δC (201 MHz)
11.62 m
0.85 m
38.81.63 m
0.86 m
38.81.41 m
0.66 m
38.61.64 m
0.87 m
38.8
22.03 m
1.80 m
26.82.04 m
1.81 m
26.81.98 m
1.76 m
26.62.05 m
1.82 m
26.8
33.25 dd (11.8, 4.4)88.83.26 dd (11.8, 4.4)88.83.22 dd (11.9, 4.6)88.83.29 dd (11.9, 4.0)88.8
4 39.6 39.6 39.5 39.6
50.69 d (11.7)55.70.69 br d (11.8)55.70.64 m55.50.70 br d (12.5)55.7
61.43 m
1.27 m
18.61.44 m
1.27 m
18.61.41 m
1.23 m
18.41.41 m
1.31 m
18.6
71.40 m
1.21 m
33.61.41 m
1.22 m
33.61.35 m
1.17 m
33.31.42 m
1.23 m
33.6
8 45.7 45.7 45.8 45.7
91.33 m49.11.34 m49.11.28 m48.51.35 m49.1
10 36.9 36.9 36.8 36.9
112.07 m
1.62 m
33.02.09 m
1.63 m
33.01.98 m
1.37 m
28.12.09 m
1.63 m
33.0
124.19 m68.64.21 m68.65.47 m72.24.20 m68.6
131.79 d (10.7)55.81.79 d (10.5)55.81.90 d (11.5)52.41.79 d (10.6)55.8
14 41.8 41.8 41.6 41.7
151.87 m
1.69 m
45.71.88 m
1.70 m
45.71.83 m
1.64 m
45.21.88 m
1.70 m
45.6
164.12 m65.54.11 m65.54.06 m65.04.12 m65.5
172.28 d (11.3)63.72.28 d (11.4)63.72.23 d (11.5)63.32.28 d (11.7)63.7
18 47.1 47.1 46.4 47.1
192.61 m
1.87 m
45.82.63 m
1.90 m
45.81.83 m
1.68 m
44.82.62 m
1.89 m
45.7
202.05 m
1.74 m
37.62.06 m
1.74 m
37.62.04 m
1.66 m
37.52.06 m
1.73 m
37.6
21 53.6 53.6 53.4 53.6
22 214.9 215.0 214.6 214.9
231.22 s28.01.22 s28.01.21 s27.91.25 s28.0
240.89 s16.60.90 s16.60.88 s16.60.94 s16.7
250.76 s16.00.76 s16.00.72 s15.90.77 s16.0
260.98 s17.00.99 s17.00.93 s16.80.99 s17.0
271.11 s19.21.11 s19.21.08 s18.91.12 s19.1
281.21 s17.81.21 s17.81.03 s17.71.21 s17.8
291.66 s21.01.66 s21.01.61 s20.91.66 s21.0
302.34 s26.32.34 s26.32.37 s26.32.35 s26.3
1′4.74 d (7.7)107.24.76 d (7.5)107.24.75 d (7.5)107.24.83 d (7.3)106.9
2′4.00 m75.94.00 dd (8.3, 7.5)75.84.01 dd (8.4, 7.5)75.84.08 m75.0
3′4.40 m78.34.43 m79.04.43 m79.04.35 m83.9
4′5.28 ddd (9.7, 9.7, 5.5)71.35.38 overlapped71.05.39 m70.95.44 m70.7
5′4.30 m
3.58 dd (11.3, 11.0)
63.14.35 m
3.63 dd (11.3 10.0)
63.24.38 m
3.65 m
63.24.35 m
3.68 m
63.3
1″6.26 d (1.2)102.66.24 br s102.86.25 br s102.95.25 d (7.8)106.9
2″4.69 br s72.44.74 dd (3.2, 1.4)72.54.72 m72.53.99 t (7.8)75.8
3″4.45 m72.74.45 m72.64.44 m72.64.14 m78.4
4″4.30 t (9.3)73.94.30 t (9.4)73.94.29 dd (9.5, 9.5)74.04.15 m71.0
5″4.43 m70.04.40 m70.04.40 m70.14.31 m
3.70 m
67.6
6″1.70 d (6.2)18.81.68 d (6.3)18.91.69 d (6.2)18.9
1′′′ 170.4 165.9 165.9 165.9
2′′′2.15 s21.16.02 dq (15.6, 1.6)122.76.04 br d (14.6)122.85.99 br d (15.5)123.1
3′′′ 7.06 m146.07.07 m145.97.09 m145.4
4′′′ 1.66 d (6.8)17.81.61 dd (7.0, 1.5)17.81.61 br d (6.9)17.8
1′′′′ 170.4
2′′′′ 2.15 s21.9
12-OH5.34 d (6.1)
16-OH5.49 d (5.0)
2′-OH7.68 d (6.1)
2″-OH6.80 br s
4″-OH6.80 br s
Table 5. 1H and 13C-NMR data of 1214 in Pyridine-d5 (δ in ppm, J in Hz).
Table 5. 1H and 13C-NMR data of 1214 in Pyridine-d5 (δ in ppm, J in Hz).
12 13 14
No.δH (500 MHz)δC (126 MHz)δH (600 MHz)δC (151 MHz)δH (600 MHz)δC (151 MHz)
11.61 m
0.84 m
39.01.72 m
0.98 m
39.51.68 m
0.95 m
39.7
21.78 m28.32.21 m
1.90 m
27.42.17 m
1.92 m
27.5
33.41 m78.13.37 dd (11.8, 4.5)89.03.31 dd (11.9, 4.2)88.9
4 39.5 40.1 40.1
50.69 m56.00.75 br d (10.8)56.30.71 br d (11.5)56.6
61.49 m
1.28 m
18.81.51 m
1.33 m
19.01.47 m
1.31 m
19.0
71.27 m34.21.32 m34.61.29 m34.6
8 46.8 47.2 47.2
91.22 m48.41.40 m49.51.37 m49.5
10 37.6 37.6 37.5
112.37 m
1.48 m
27.22.09 m
1.66 m
33.52.07 m
1.65 m
33.5
124.37 m74.54.04 m69.74.04 m69.7
131.87 d (11.2)54.01.92 d (11.1)56.21.91 d (11.1)56.2
14 41.5 41.9 41.9
151.92 m
1.29 m
43.01.99 dd (11.6. 6.2)
1.38 m
43.71.97 dd (11.7, 6.1)
1.36 m
43.7
164.84 m76.04.93 m76.64.92 m76.6
17 152.5 153.0 153.0
18 46.0 46.4 46.4
192.88 m
2.54 m
53.73.15 m
2.70 m
54.63.15 m
2.70 ddd (13.6, 8.2, 2.6)
54.5
202.37 m
2.04 m
25.62.50 m
2.20 m
26.12.50 m
2.21 m
26.1
21 146.8 147.5 147.5
22 84.2 84.5 84.5
231.20 s28.71.31 s28.51.25 s28.3
240.99 s16.40.99 s17.21.19 s17.4
250.79 s16.60.85 s17.00.85 s17.1
260.88 s16.51.02 s16.91.01 s16.9
270.94 s15.91.07 s16.21.06 s16.2
281.67 s22.21.59 s23.01.59 s23.0
291.44 s29.21.49 s29.21.49 s29.2
301.44 s28.61.47 s29.51.47 s29.5
1′5.19 d (7.5)100.14.86 d (7.5)108.24.85 d (7.4)106.6
2′4.07 m75.94.05 m76.04.27 dd (8.3, 7.4)78.4
3′4.37 m78.84.20 dd (8.7, 8.7)79.14.20 dd (8.7, 8.3)80.1
4′4.26 dd (9.5, 9.5)72.44.26 m71.74.17 m72.0
5′4.05 m78.44.39 dd (11.3, 5.2)
3.79 dd (11.3, 10.7)
67.64.34 dd (11.4, 4.8)
3.73 dd (11.4, 9.9)
67.4
6′4.57 dd (11.7, 2.2)
4.36 m
63.4
1″ 6.58 br s102.4
2″ 4.90 dd (3.4, 1.4)72.9
3″ 4.71 dd (9.4, 3.4)73.0
4″ 4.38 dd (9.3, 9.3)74.6
5″ 4.80 m70.2
6″ 1.73 d (6.2)19.2
12-OH 5.46 br s
Table 6. 1H and 13C-NMR data of 1517 in Pyridine-d5 (δ in ppm, J in Hz).
Table 6. 1H and 13C-NMR data of 1517 in Pyridine-d5 (δ in ppm, J in Hz).
15 16 17
No.δH (600 MHz)δC (151 MHz)δH (500 MHz)δC (126 MHz)δH (800 MHz)δC (201 MHz)
11.69 m
0.95 m
39.51.64 m
0.90 m
38.81.63 m
0.91 m
39.0
22.14 m
1.87 m
27.32.11 m
1.83 m
26.82.10 m
1.83 m
26.9
33.30 dd (11.9, 4.3)89.23.27 dd (11.8, 4.4)88.83.26 dd (12.0, 4.3)88.8
4 40.1 39.6 39.6
50.73 br d (11.3)56.30.72 br d (11.9)55.70.73 br d (11.7)55.9
61.48 m
1.31 m
19.01.47 m
1.30 m
18.61.44 m
1.30 m
18.6
71.31 m34.61.45 m
1.27 m
33.51.40 m
1.31 m
33.6
8 47.2 45.4 44.8
91.39 m49.51.38 m49.21.47 m49.6
10 37.5 36.9 37.0
112.09 m
1.65 m
33.52.07 m
1.61 m
33.12.08 m
1.62 m
33.4
124.04 m69.74.19 m69.34.08 m69.6
131.92 d (11.1)56.21.77 d (10.9)53.91.91 m55.1
14 47.2 41.8 41.7
152.00 m
1.38 m
43.71.87 dd (12.5, 4.2)
1.73 m
45.11.92 m
1.87 m
44.1
164.93 m76.64.32 m67.24.99 overlapped68.1
17 153.02.14 d (10.8)62.2 143.9
18 46.4 45.4 52.6
193.15 m
2.70 m
54.62.53 m
1.78 m
43.22.58 m
2.20 m
46.5
202.50 m
2.21 m
26.12.42 m29.92.54 m
2.18 m
38.1
21 147.5 152.2 128.5
22 84.56.06 t (2.0)
5.14 t (2.0)
106.12.33 s16.0
231.25 s28.41.23 s28.01.21 s28.0
240.95 s17.20.93 s16.70.92 s16.7
250.83 s17.00.78 s16.00.79 s16.4
261.02 s16.91.01 s17.00.98 s16.5
271.07 s16.21.16 s19.11.25 s17.5
281.59 s23.01.06 s16.01.39 s20.6
291.49 s29.2
301.47 s29.5
1′4.76 d (7.5)107.84.74 d (7.4)107.44.73 d (7.5)107.4
2′4.03 m75.94.01 m75.44.00 m75.4
3′4.32 dd (8.9, 8.9)83.54.30 t (8.9)83.04.29 t (8.8)83.0
4′4.16 m70.24.14 m69.74.13 m69.7
5′4.35 m
3.73 dd (11.2, 10.4)
67.44.32 m
3.72 dd (11.3, 10.2)
67.04.33 m
3.71 dd (11.2, 10.3)
66.9
1″6.30 br s103.36.26 d (1.2)102.96.26 br s102.8
2″4.82 dd (3.3, 1.5)73.14.78 br s72.64.78 br s72.6
3″4.62 dd (9.4, 3.3)73.24.60 br d (9.1)72.84.59 br d (8.7)72.8
4″4.37 dd (9.4, 9.4)74.64.34 m74.24.33 m74.2
5″5.00 m70.44.98 overlapped70.04.97 overlapped70.0
6″1.71 d (6.2)19.21.68 d (6.2)18.71.68 d (6.1)18.7
12-OH 5.27 d (6.5) 5.20 d (6.2)
16-OH 5.72 d (5.9) 6.03 d (5.5)
2′-OH 7.29 d (6.0) 7.26 d (6.0)
4′-OH 6.79 d (5.9) 6.76 d (5.6)
2″-OH 6.74 br s 6.71 br s
3″-OH 6.47 br s 6.43 br s
4″-OH 6.74 br s 6.71 br s
Table 7. 1H and 13C-NMR data of 1820 in Pyridine-d5 (δ in ppm, J in Hz).
Table 7. 1H and 13C-NMR data of 1820 in Pyridine-d5 (δ in ppm, J in Hz).
18 19 20
No.δH (600 MHz)δC (151 MHz)δH (500 MHz)δC (126 MHz)δH (J in Hz) (600 MHz)δC (151 MHz)
11.69 m
0.98 m
39.21.61 m
0.90 m
38.81.70 m
0.89 m
38.5
22.21 m
1.91 m
27.42.10 m
1.82 m
26.92.14 m
1.87 m
27.0
33.39 dd (11.9, 4.3)89.13.27 dd (11.9, 4.4)88.83.32 dd (11.9, 4.5)89.8
4 40.2 39.7 40.1
50.84 m56.50.77 overlapped56.00.79 br d (12.1)55.6
61.57 m
1.42 m
19.11.54 m
1.37 m
18.61.52 m
1.33 m
18.8
71.52 m33.91.48 m33.51.28 m33.2
8 41.8 41.3 41.6
91.50 m50.21.45 m49.71.98 br s55.2
10 37.7 37.3 37.1
112.18 m
1.66 m
34.32.14 m
1.61 m
33.95.69 br d (10.1)128.2
124.33 m69.44.28 m69.06.60 dd (10.1, 2.9)126.2
132.17 d (11.1)53.32.13 d (11.1)52.8 138.7
14 47.5 47.0 43.1
155.76 d (10.3)135.55.72 d (10.3)135.11.96 m
1.09 m
26.0
166.42 d (10.3)120.46.39 d (10.3)120.02.26 m
1.78 m
33.5
17 141.9 141.5 49.2
18 48.6 48.2 131.4
192.59 m
2.24 m
45.32.55 m
2.21 m
44.93.17 d (14.5)
2.77 d (14.5)
36.0
202.60 m
2.14 m
36.82.55 m
2.09 m
36.3 44.3
21 131.8 131.42.31 m
1.79 m
32.9
221.73 s14.41.70 s14.02.66 m
1.52 m
35.3
231.35 s28.61.25 s28.01.25 s28.2
241.02 s17.20.94 s16.70.89 s16.8
250.81 s16.30.76 s15.90.84 s18.7
261.03 s19.70.99 s19.31.02 s17.4
271.34 s19.51.30 s19.01.11 s20.5
281.32 s20.41.29 s20.0 179.1
29 1.27 s20.7
30 178.9
30-Me 3.60 s52.2
1′4.88 d (7.7)108.24.74 d (7.5)107.44.92 d (7.9)107.6
2′4.05 dd (8.4, 7.7)76.04.00 dd (8.4, 7.5)75.44.07 dd (8.7, 7.9)76.2
3′4.20 dd (8.8, 8.4)79.14.28 m83.04.45 dd (9.3, 8.7)82.3
4′4.26 m71.74.12 m69.74.41 dd (9.3, 9.3)71.9
5′4.40 dd (11.0, 5.1)
3.80 dd (11.0, 10.4)
67.64.32 m
3.70 dd (11.0, 10.5)
67.04.58 d (9.3)77.6
6′ 171.3
6′-OMe 3.79 s52.7
1″ 6.27 br s102.86.33 br s103.4
2″ 4.79 br s72.64.76 dd (3.3, 1.4)73.0
3″ 4.59 dd (9.3, 3.0)72.84.57 dd (9.3, 3.3)73.2
4″ 4.34 dd (9.3, 9.3)74.24.35 dd (9.3, 9.3)74.6
5″ 4.97 m69.95.08 m70.3
6″ 1.67 d (6.1)18.71.71 d (6.1)19.1
Table 8. 1H and 13C-NMR data of 2123 in Pyridine-d5 (δ in ppm, J in Hz).
Table 8. 1H and 13C-NMR data of 2123 in Pyridine-d5 (δ in ppm, J in Hz).
21 22 23
No.δH (500 MHz)δC (126 MHz)δH (500 MHz)δC (126 MHz)δH (500 MHz)δC (126 MHz)
11.35 m
0.82 m
38.61.36 m
0.82 m
38.51.37 m
0.83 m
38.5
22.19 m
1.76 m
26.42.04 m
1.77 m
26.62.07 m
1.78 m
26.6
33.25 dd (11.9, 4.4)89.23.28 overlapped89.43.27 overlapped89.3
4 39.3 39.5 39.5
50.75 overlapped55.80.75 overlapped55.70.74 overlapped55.7
61.49 m
1.28 m
18.61.46 m
1.25 m
18.41.46 m
1.26 m
18.4
71.46 m
1.27 m
33.31.44 m
1.26 m
33.21.45 m
1.26 m
33.2
8 39.7 42.1 42.1
91.60 m48.11.61 m48.01.62 dd (8.6, 8.6)48.0
10 37.0 36.9 36.9
111.85 m23.8 1.85 m23.71.85 m23.7
125.58 br s122.55.59 br s123.15.59 dd (3.4, 3.4)123.1
13 145.5 144.5 144.5
14 42.2 39.7 39.7
152.21 m28.62.12 m
1.18 m
28.42.13 m
1.27 m
28.4
162.08 m24.22.12 m
2.02 m
23.92.13 m
2.03 m
23.9
17 46.4 46.2 46.2
183.36 dd (13.5, 3.6)43.63.29 overlapped43.43.29 overlapped43.4
192.26 m
1.82 m
43.22.25 br d (13.2)
1.81 m
42.72.25 br d (12.0)
1.82 m
42.7
20 44.3 44.2 44.2
212.16 m
1.45 m
31.22.19 m
1.46 m
30.92.19 br d (13.5)
1.47 m
30.9
222.06 m
1.96 m
34.92.08 m
1.97 m
34.62.09 m
1.98, m
34.6
231.19 s28.21.24 s28.11.23 s28.1
240.98 s17.00.90 s16.90.90 s16.9
250.75 s15.40.75 s15.40.76 s15.4
260.89 br s17.60.95 s17.30.95 s17.3
271.31 s26.21.31 s26.21.31 s26.2
28 DAP a 179.9 179.9
291.22 s28.71.23 s28.51.23 s28.5
30 177.4 177.2 177.2
30-OMe3.63 s51.73.65 s51.83.65 s51.7
1′5.05 d (8.2)105.04.88 d (7.8)107.14.88 d (7.9)107.2
2′4.58 m58.14.34 m74.14.05 dd (8.4, 7.9)75.8
3′4.41 dd (9.3, 8.7)76.34.42 dd (9.0, 9.0)81.94.44 dd (8.9, 8.4)81.9
4′4.18 dd (9.3, 9.3)72.74.37 m71.44.41 dd (9.4, 8.9)71.4
5′3.97 m78.44.56 d (9.4)77.24.55 d (9.4)77.2
6′4.57 m
4.37 m
63.0 170.8 170.3
6′-OMe, 3.77 s52.24.29 m61.4
OEt 1.19 t (7.1)14.3
1″ 170.36.30 br s102.96.33 d (1.2)102.8
2″2.15 s23.84.75 m72.54.77 dd (3.4, 1.2)72.6
3″ 4.55 m72.74.56 dd (9.2, 3.4)72.7
4″ 4.34 m74.14.36 dd (9.2, 9.2)74.1
5″ 5.05 m69.95.08 m69.8
6″ 1.69 d (6.1)18.61.69 d (6.2)18.7
NH8.94 d (9.0)
a Disappeared.
Table 9. 1H- and 13C-NMR data of 2426 in Pyridine-d5 (δ in ppm, J in Hz).
Table 9. 1H- and 13C-NMR data of 2426 in Pyridine-d5 (δ in ppm, J in Hz).
24 25 26
No.δH (500 MHz)δC (126 MHz)δH (500 MHz)δC (126 MHz)δH (500 MHz)δC (126 MHz)
11.51 m
0.97 m
38.81.47 m
0.93 m
38.71.39 m
0.85 m
38.6
22.16 m
1.87 m
26.82.09 m
1.81 m
26.72.13 m
1.83 m
26.6
33.35 dd (11.7, 4.4)88.73.28 overlapped88.83.36 dd (11.6, 4.3)89.2
4 39.6 39.5 39.6
50.82 overlapped55.90.79 overlapped55.80.79 overlapped55.8
61.49 m
1.28 m
18.51.47 m
1.27 m
18.51.47 m
1.26 m
18.5
71.47 m
1.28 m
33.21.46 m
1.27 m
33.21.45 m
1.27 m
33.2
8 39.7 42.0 39.7
91.67 dd (9.0, 8.7)48.11.65 m48.01.63 dd (9.0, 8.6)48.0
10 37.1 37.0 37.0
111.89 m23.81.88 m23.71.87 m23.7
125.60 br t (3.1)123.25.60 dd (3.4, 3.4)123.15.60 br t (3.3)123.1
13 144.5 144.4 144.5
14 42.1 39.7 42.1
152.13 m
1.19 m
28.42.13 m
1.27 m
28.42.13 m
1.20 m
28.4
162.13 m
2.03 m
23.92.12 m
2.02 m
23.82.12 m
2.03 m
23.9
17 46.2 46.2 46.2
183.30 dd (13.6, 3.6)43.43.29 overlapped43.43.29 dd (13.6, 3.9)43.4
192.26 m
1.82 dd (13.6, 13.6)
42.72.26 br d (13.5)
1.82 dd (13.5, 13.5)
42.72.26 m
1.82 m
42.7
20 44.2 44.2 44.2
212.19 m
1.46 m
30.92.19 br d (13.1)
1.46 m
30.82.20 m
1.46 m
30.9
222.10 m
1.98 m
34.62.09 m
1.98 m
34.52.09 m
1.98 m
34.6
231.30 s28.21.24 s28.11.30 s28.2
240.97 s17.00.92 s16.90.95 s17.0
250.82 s15.50.80 s15.50.78 s15.5
260.97 s17.40.96 s17.30.95 s17.4
271.30 s26.21.30 s26.11.30 s26.2
28 179.9 179.9 179.9
291.23 s28.51.23 s28.51.23 s28.5
30 177.2 177.2 177.2
30-OMe3.65 s51.83.64 s51.73.65 s51.8
1′4.83 d (7.6)107.84.73 d (7.5)107.45.00 d (7.8)107.3
2′4.01 dd (8.7, 7.6)75.64.00 dd (8.8, 7.5)75.44.09 dd (9.0, 7.8)75.4
3′4.17 dd (8.7, 8.7)78.74.29 dd (8.8, 8.8)82.94.28 dd (9.0, 9.0)77.9
4′4.23 m71.34.13 m69.74.48 dd (9.0, 9.7)73.3
5′4.38 dd (11.2, 5.1)
3.78 dd (11.2, 10.2)
67.24.34 m
3.73 dd (10.6, 10.2)
66.94.61 d (9.7)77.3
6′ 170.9
6′-OMe 3.73 s52.1
1″ 6.27 d (1.2)102.7
2″ 4.79 dd (3.3, 1.2)72.6
3″ 4.60 dd (9.3, 3.3)72.7
4″ 4.34 dd (9.3, 9.3)74.1
5″ 4.98 m69.9
6″ 1.67 d (6.2)18.7
Table 10. Antifungal Effects of Compounds from G. oppositifolius against Microsporum gypseum and Trichophyton rubrum a.
Table 10. Antifungal Effects of Compounds from G. oppositifolius against Microsporum gypseum and Trichophyton rubrum a.
CompoundMIC50 (μM) ± SD
M. gypseumT. rubrum
1105.0 ± 0.6>300
3128.1 ± 1.4>300
67.1 ± 1.214.3 ± 2.1
12260.1 ± 2.3>300
1546.8 ± 0.1>300
16120.7 ± 1.4>300
1829.3 ± 3.4>300
1934.9 ± 1.2>300
216.7 ± 2.113.4 ± 1.1
2240.3 ± 0.5>300
2339.9 ± 1.2>300
246.8 ± 3.211.9 ± 0.3
2511.1 ± 2.413.0 ± 1.3
2622.0 ± 0.9>300
terbinafine hydrochloride (positive control)0.008 ± 0.3731.647 ± 0.101
a Compounds 2, 47, 911, 13, 14, 17, 20, and 2737 were inactive (MIC50 > 300 μM).

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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. https://doi.org/10.3390/molecules24122206

AMA Style

Zhang D, Fu Y, Yang J, Li X-N, San MM, Oo TN, Wang Y, Yang X. Triterpenoids and Their Glycosides from Glinus Oppositifolius with Antifungal Activities against Microsporum Gypseum and Trichophyton Rubrum. Molecules. 2019; 24(12):2206. https://doi.org/10.3390/molecules24122206

Chicago/Turabian Style

Zhang, Dongdong, Yao Fu, Jun Yang, Xiao-Nian Li, Myint Myint San, Thaung Naing Oo, Yuehu Wang, and Xuefei Yang. 2019. "Triterpenoids and Their Glycosides from Glinus Oppositifolius with Antifungal Activities against Microsporum Gypseum and Trichophyton Rubrum" Molecules 24, no. 12: 2206. https://doi.org/10.3390/molecules24122206

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