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

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 δ_H 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 δ_H 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, δ_C 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 (δ_C 78.0) for an oxygenated methine in the ¹³C-NMR spectrum of 1 was replaced by the signal (δ_C 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 δ_H 1.43 (s), 1.30 (s), 1.14 (s), 1.00 (s), 0.99 (s), and 0.86 (s) and one olefinic group at δ_H 5.72. The ¹³C-NMR data of 3 (Table 2) indicated the presence of six methyl groups, two carboxylic carbons at δ_C 180.1 and 179.5, one carbonyl carbon at δ_C 216.3, and two olefinic carbons (one quaternary at δ_C 144.8 and one methane at δ_C 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 δ_H 1.11 (s), 1.00 (s), 0.98 (s), 0.94 (s), 0.81 (s), and 0.78 (s); a methoxy group at δ_H 3.67 (s); and a disubstituted double bond at δ_H 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 δ_C 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 [δ_H 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 (8–11) 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 δ_H 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 9–11 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 [δ_H 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 (δ_C 152.5 and 146.8), and one β-glucopyranose [δ_H 5.19 (d, J = 7.6 Hz); δ_C 100.1, 78.8, 78.4, 75.9, 72.4, and 63.4]. In addition to the signals for the sugar, signals (δ_C 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 (δ_C 76.0) and C-22 (δ_C 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 (13–15) were deduced to be C₃₅H₅₆O₇, C₄₁H₆₆O₁₁, and C₄₁H₆₆O₁₁, respectively. Comparison of the NMR data of 13–15 (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 13–15 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 [δ_H 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); δ_C 103.4, 74.6, 73.2, 73.0, 70.3, and 19.1], and a 6-O-methyl-β-glucuronopyranosyl group [δ_H 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); δ_C 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 (21–25) 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 δ_H 8.94 (d, J = 9.0 Hz) and 2.15 (s), along with δ_C 170.3 and 23.8 manifested the presence of an acetylamino unit which was further confirmed by the HMBC correlations from δ_H 2.15 (H-2″) to δ_C 170.3 (C-1″) and from δ_H 8.94 (NH) to δ_C 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 δ_C 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 (δ_C 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 21–25 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 (1–37) 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 (21–26) or oxidation (3) of 3-OH was helpful in increasing the activity based on a comparison of the MIC₅₀ values of 3 and 21–26 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, t_R = 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, t_R = 36.803 min) and 9 (5.7 mg, t_R = 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, t_R = 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, t_R = 31.175 min) and 16 (7.6 mg, t_R = 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, t_R = 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, t_R = 30.891 min) and 5 (2.1 mg, t_R = 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, t_R = 14.414 min) and 15 (5.3 mg, t_R = 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, t_R = 12.996 min), 6 (5.9 mg, t_R = 16.490 min), and 14 (3.6 mg, t_R = 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, t_R = 42.714 min), 26 (29.0 mg, t_R = 51.174 min), and 10 (1.6 mg, t_R = 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, t_R = 37.244 min), 22 (42.6 mg, t_R = 45.406 min), 23 (16.2 mg, t_R = 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, t_R = 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, t_R = 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, t_R = 35.867 min), 4 (3.5 mg, t_R = 47.842 min), and 27 (21.0 mg, t_R = 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, t_R = 15.194 min) and 18 (3.1 mg, t_R =16.328 min).

3.4. Spectroscopic and Physical Data

3β,12β,16β,21β,22-Pentahydroxyhopane (1). Colorless blocks (Me-H₂O, 10:1); _[α]D25 −18 (c 0.05, MeOH); UV (MeOH) λ_max (logε) 203 (3.34) nm; ECD (c 0.05, MeOH) λ_max (Δε) 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) ų, T = 100.(2) K, space group P212121, Z = 4, μ(Cu Kα) = 0.653 mm⁻¹, 51,068 reflections measured, 5642 independent reflections (R_int = 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]D25 +2 (c 0.12, MeOH); UV (MeOH) λ_max (logε) 280 (1.98), 219 (2.73), 203 (2.91) nm; ECD (c 0.12, MeOH) λ_max (Δε) 289 nm (+0.17), 232 (+0.28), 208 (–0.29) nm; IR ν_max (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]D25 +59 (c 0.26, MeOH); UV (MeOH) λ_max (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]D26 –7 (c 0.15, MeOH); UV (MeOH) λ_max (logε) 250 (2.65), 242 (2.59) nm; ECD (c 0.09, MeOH) λ_max (Δε) 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]D26 –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]D25 –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]D26 –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]D21 –36 (c 0.16, pyridine); UV (MeOH) λ_max (logε) 275 (2.77), 245 (3.02), 204 (3.59) nm; ECD (c 0.099, MeOH) λ_max (Δε) 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]D20 –32 (c 0.09, MeOH); UV (MeOH) λ_max (logε) 415 (2.39), 206 (4.09) nm; ECD (c 0.065, MeOH) λ_max (Δε) 284 (+0.48), 206 (+0.73), 200 (–1.80) nm; IR ν_max (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]D25 –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]D20 –7 (c 0.15, MeOH); UV (MeOH) λ_max (logε) 252 (3.21), 206 (3.81) nm; ECD (c 0.078, MeOH) λ_max (Δε) 201 (–1.59), 197 (+1.29) nm; IR ν_max (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]D25 +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]D25 –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]D25 –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]D25 –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]D25 –25 (c 0.1, MeOH); UV (MeOH) λ_max (logε) 203 (3.82) nm; ECD (c 0.076, MeOH) λ_max (Δε) 196 (–6.89) nm; IR ν_max (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]D20 –25 (c 0.05, MeOH); UV (MeOH) λ_max (logε) 203 (3.54) nm; ECD (c 0.05, MeOH) λ_max (Δε) 206 (–4.38), 196 (+4.32) nm; IR ν_max (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]D26 –7 (c 0.18, MeOH); UV (MeOH) λ_max (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]D25 –14 (c 0.13, MeOH); UV (MeOH) λ_max (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]D25 –39 (c 0.1, MeOH); UV (MeOH) λ_max (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]D25 +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]D25 +10 (c 0.15, MeOH); ECD (c 0.078, MeOH) λ_max (Δε) 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]D25 +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]D24 +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]D25 +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]D25 +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]D21 −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]D19 +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]D21 −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 (1−4), 21 new triterpenoids glycosides (5−25), 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.

	1		2
No.	δH	δC	δH	δC
1	1.68 m 0.97 m	39.1	1.79 m 1.31 m	39.4
2	1.81 m	28.3	2.48 m 2.42 m	34.4
3	3.44 br t (8.3)	78.0		216.4
4		39.5		47.4
5	0.78 dd (12.0, 1.7)	55.7	1.28 m	54.8
6	1.51 m 1.33 m	18.9	1.37 m 1.31 m	20.0
7	1.43 m 1.21 m	33.6	1.38 m 1.19 m	32.7
8		45.1		45.2
9	1.39 m	49.3	1.39 m	48.4
10		37.3		36.8
11	2.11 m 1.64 m	33.2	2.04 m 1.65 m	33.4
12	4.23 m	69.1	4.20 m	69.0
13	1.84 d (10.7)	56.4	1.84 d (10.8)	56.5
14		41.8		41.7
15	1.93 dd (12.7, 4.2) 1.71 m	46.0	1.92 dd (12.6, 4.3) 1.70 m	45.9
16	4.48 m	66.8	4.48 m	66.7
17	2.41 d (11.7)	73.8	2.41 d (11.6)	73.8
18		47.5		47.5
19	2.60 m 2.14 m	43.1	2.60 m 2.13 m	43.1
20	2.05 m 1.95 m	37.8	2.07 m 1.96 m	37.8
21		85.7		85.7
22		75.5		75.5
23	1.21 s	28.7	1.11 s	26.6
24	1.01 s	16.3	1.00 s	21.3
25	0.82 s	16.1	0.81 s	15.6
26	0.97 s	17.0	0.95 s	16.6
27	1.13 s	19.5	1.10 s	19.4
28	1.19 s	17.3	1.19 s	17.3
29	1.56 s	26.6	1.56 s	26.7
30	1.61 s	27.3	1.61 s	27.4
3-OH	5.78 br s
12-OH	5.32 d (6.0)		5.38 d (6.9)

	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)
1	1.65 m 1.30 m	39.1	1.92 m 1.05 m	39.4	1.88 m 1.07 m	38.5
2	2.51 m 2.37 m	34.4	1.69 m 1.64 m	27.9	1.94 m 1.89 m	28.1
3		216.3	3.18 dd (11.7, 4.9)	79.8	3.49 dd (10.6, 5.0)	78.1
4		47.5		40.1		39.6
5	1.32 m	55.4	0.84 br d (12.1)	56.4	0.91 dd (12.2, 1.8)	55.3
6	1.35 m	19.8	1.63 m 1.46 m	19.6	1.61 m 1.44 m	18.8
7	1.46 m 1.31 m	32.7	1.35 m	33.7	1.31 m	32.9
8		39.7		42.2		41.2
9	1.70 m	47.2	1.99 br s	56.0	2.06 br s	54.9
10		36.9		38.0		37.1
11	1.88 m	23.8	5.72 br d (11.2)	129.0	5.78 br d (10.5)	127.8
12	5.72 br t (3.3)	122.8	6.33 dd (11.2, 2.8)	126.2	6.63 dd (10.5, 2.6)	125.7
13		144.8		139.8		138.3 a
14		42.2		43.6		42.7
15	2.18 m	28.5	1.72 m 1.08 m	26.2	1.97 m 1.08 m	25.6
16α 16β	2.19 m 2.08 m	24.0	1.71 m 1.95 m	33.8	1.78 m 2.25 m	33.1
17		46.4		49.3 a		48.8
18	3.63 dd (13.7, 4.0)	43.5		130.8		131.2 a
19α 19β	1.92 dd (13.7, 13.7) 2.50 m	43.1	2.85 dd (14.5, 1.7) 2.17 d (14.5)	35.9	3.18 d (15.2) 2.78 d (15.2)	35.6
20		44.1		44.8		43.9
21	2.41 m 1.47 m	31.2	1.83 m 1.57 m	33.0	2.28 m 1.78 m	32.5
22	2.41 m 2.08 m	34.8	2.29 m 1.41 m	35.4	2.67 ddd (13.8, 3.5, 3.5) 1.52 m	35.0
23	1.14 s	26.6	0.98 s	28.6	1.24 s	28.6
24	0.99 s	21.6	0.78 s	15.9	1.03 s	16.1
25	0.86 s	14.9	0.94 s	18.8	0.97 s	18.4
26	1.00 s	17.3	0.81 s	17.3	1.07 s	17.0
27	1.30 s	26.1	1.00 s	20.3	1.09 s	20.1
28		180.1		179.9		DAS b
29	1.43 s	29.1	1.11 s	20.4	1.28 s	20.2
30		179.5		180.4		178.5
30-OMe			3.67 s	52.6	3.60 s	51.8

a Detected by HMBC. b Disappeared signal.

	5		6		7
No.	δH (600 MHz)	δC (151 MHz)	δH (500 MHz)	δC (126 MHz)	δH (600 MHz)	δC (151 MHz)
1	1.70 m 0.97 m	39.3	1.63 m 0.90 m	39.1	1.67 m 0.94 m	39.3
2	2.21 m 1.91 m	27.4	2.13 m 1.88 m	27.0	2.14 m 1.87 m	27.3
3	3.38 dd (11.7, 4.4)	89.1	3.29 dd (11.8, 4.2)	88.6	3.31 dd (11.9, 4.4)	89.3
4		40.1		39.7		40.1
5	0.79 br d (11.9)	56.2	0.71 br d (9.3)	56.0	0.76 br d (12.0)	56.2
6	1.51 m 1.33 m	19.1	1.46 m 1.33 m	18.6	1.50 m 1.32 m	19.0
7	1.39 m 1.19 m	34.0	1.33 m 1.15 m	33.5	1.40 m 1.19 m	34.0
8		43.8		43.4		43.8
9	1.40 m	50.0	1.35 m	49.6	1.40 m	50.0
10		37.4		37.0		37.4
11	2.12 m 1.64 m	33.5	2.07 m 1.60 m	33.0	2.10 m 1.63 m	33.5
12	4.20 m	69.2	4.17 m	68.7	4.20 m	69.1
13	1.72 d (10.7)	56.9	1.68 d (11.1)	56.5	1.72 overlapped	57.0
14		42.1		41.6		42.1
15	1.51 m 1.20 m	34.8	1.48 m 1.17 m	34.4	1.52 m 1.21 m	34.8
16	1.44 m	20.2	1.42 m	19.8	1.46 m	20.2
17	1.82 dd (12.1, 2.9)	56.3	1.78 dd (11.9, 2.9)	55.9	1.82 dd (12.0, 2.8)	56.3
18		45.8		45.4		45.8
19	2.58 m 1.68 m	45.2	2.55 m 1.65 m	44.8	2.58 m 1.68 m	45.2
20	2.17 m 1.68 m	36.2	2.14 m 1.65 m	35.7	2.17 m 1.68 m	36.2
21		54.5		54.0		54.5
22		213.0		212.6		213.0
23	1.33 s	28.6	1.25 s	28.0	1.27 s	28.5
24	1.01 s	17.2	1.17 s	16.9	0.97 s	17.2
25	0.82 s	16.5	0.79 s	16.2	0.81 s	16.5
26	0.99 s	17.5	0.95 s	17.0	0.99 s	17.5
27	1.05 s	18.4	1.01 s	18.0	1.05 s	18.4
28	1.12 s	17.2	1.09 s	16.8	1.12 s	17.2
29	1.22 s	21.5	1.19 s	21.0	1.22 s	21.5
30	2.19 s	25.9	2.16 s	25.4	2.19 s	25.9
1′	4.87 d (7.6)	108.2	4.83 d (7.3)	106.2	4.77 d (7.5)	107.8
2′	4.05 dd (8.6, 7.6)	76.0	4.24 dd (8.2, 7.3)	78.0	4.04 dd (8.6, 7.5)	75.9
3′	4.20 m	79.1	4.18 m	79.7	4.32 dd (8.8, 8.8)	83.5
4′	4.26 m	71.7	4.15 m	71.6	4.17 m	70.2
5′	4.41 dd (11.3, 5.2) 3.80 dd (11.3, 10.5)	67.6	4.33 m 3.71 dd (10.5, 9.9)	67.0	4.36 m 3.74 dd (11.2, 10.3)	67.4
1″			6.54 d (0.9)	102.0	6.30 br s	103.2
2″			4.88 m	72.5	4.82 dd (3.4, 1.3)	73.1
3″			4.69 dd (9.5, 3.1)	72.6	4.62 dd (9.3, 3.4)	73.2
4″			4.35 m	74.2	4.37 dd (9.3, 9.3)	74.6
5″			4.77 m	69.8	5.01 m	70.4
6″			1.70 d (6.2)	18.8	1.71 d (6.2)	19.1
12-OH			5.23 d (6.2)
2″-OH			6.69 br s
4″-OH			6.72 br s

	8		9		10		11
No.	δH (500 MHz)	δC (126 MHz)	δH (500 MHz)	δC (126 MHz)	δH 800 MHz	δC (201 MHz)	δH (800 MHz)	δC (201 MHz)
1	1.62 m 0.85 m	38.8	1.63 m 0.86 m	38.8	1.41 m 0.66 m	38.6	1.64 m 0.87 m	38.8
2	2.03 m 1.80 m	26.8	2.04 m 1.81 m	26.8	1.98 m 1.76 m	26.6	2.05 m 1.82 m	26.8
3	3.25 dd (11.8, 4.4)	88.8	3.26 dd (11.8, 4.4)	88.8	3.22 dd (11.9, 4.6)	88.8	3.29 dd (11.9, 4.0)	88.8
4		39.6		39.6		39.5		39.6
5	0.69 d (11.7)	55.7	0.69 br d (11.8)	55.7	0.64 m	55.5	0.70 br d (12.5)	55.7
6	1.43 m 1.27 m	18.6	1.44 m 1.27 m	18.6	1.41 m 1.23 m	18.4	1.41 m 1.31 m	18.6
7	1.40 m 1.21 m	33.6	1.41 m 1.22 m	33.6	1.35 m 1.17 m	33.3	1.42 m 1.23 m	33.6
8		45.7		45.7		45.8		45.7
9	1.33 m	49.1	1.34 m	49.1	1.28 m	48.5	1.35 m	49.1
10		36.9		36.9		36.8		36.9
11	2.07 m 1.62 m	33.0	2.09 m 1.63 m	33.0	1.98 m 1.37 m	28.1	2.09 m 1.63 m	33.0
12	4.19 m	68.6	4.21 m	68.6	5.47 m	72.2	4.20 m	68.6
13	1.79 d (10.7)	55.8	1.79 d (10.5)	55.8	1.90 d (11.5)	52.4	1.79 d (10.6)	55.8
14		41.8		41.8		41.6		41.7
15	1.87 m 1.69 m	45.7	1.88 m 1.70 m	45.7	1.83 m 1.64 m	45.2	1.88 m 1.70 m	45.6
16	4.12 m	65.5	4.11 m	65.5	4.06 m	65.0	4.12 m	65.5
17	2.28 d (11.3)	63.7	2.28 d (11.4)	63.7	2.23 d (11.5)	63.3	2.28 d (11.7)	63.7
18		47.1		47.1		46.4		47.1
19	2.61 m 1.87 m	45.8	2.63 m 1.90 m	45.8	1.83 m 1.68 m	44.8	2.62 m 1.89 m	45.7
20	2.05 m 1.74 m	37.6	2.06 m 1.74 m	37.6	2.04 m 1.66 m	37.5	2.06 m 1.73 m	37.6
21		53.6		53.6		53.4		53.6
22		214.9		215.0		214.6		214.9
23	1.22 s	28.0	1.22 s	28.0	1.21 s	27.9	1.25 s	28.0
24	0.89 s	16.6	0.90 s	16.6	0.88 s	16.6	0.94 s	16.7
25	0.76 s	16.0	0.76 s	16.0	0.72 s	15.9	0.77 s	16.0
26	0.98 s	17.0	0.99 s	17.0	0.93 s	16.8	0.99 s	17.0
27	1.11 s	19.2	1.11 s	19.2	1.08 s	18.9	1.12 s	19.1
28	1.21 s	17.8	1.21 s	17.8	1.03 s	17.7	1.21 s	17.8
29	1.66 s	21.0	1.66 s	21.0	1.61 s	20.9	1.66 s	21.0
30	2.34 s	26.3	2.34 s	26.3	2.37 s	26.3	2.35 s	26.3
1′	4.74 d (7.7)	107.2	4.76 d (7.5)	107.2	4.75 d (7.5)	107.2	4.83 d (7.3)	106.9
2′	4.00 m	75.9	4.00 dd (8.3, 7.5)	75.8	4.01 dd (8.4, 7.5)	75.8	4.08 m	75.0
3′	4.40 m	78.3	4.43 m	79.0	4.43 m	79.0	4.35 m	83.9
4′	5.28 ddd (9.7, 9.7, 5.5)	71.3	5.38 overlapped	71.0	5.39 m	70.9	5.44 m	70.7
5′	4.30 m 3.58 dd (11.3, 11.0)	63.1	4.35 m 3.63 dd (11.3 10.0)	63.2	4.38 m 3.65 m	63.2	4.35 m 3.68 m	63.3
1″	6.26 d (1.2)	102.6	6.24 br s	102.8	6.25 br s	102.9	5.25 d (7.8)	106.9
2″	4.69 br s	72.4	4.74 dd (3.2, 1.4)	72.5	4.72 m	72.5	3.99 t (7.8)	75.8
3″	4.45 m	72.7	4.45 m	72.6	4.44 m	72.6	4.14 m	78.4
4″	4.30 t (9.3)	73.9	4.30 t (9.4)	73.9	4.29 dd (9.5, 9.5)	74.0	4.15 m	71.0
5″	4.43 m	70.0	4.40 m	70.0	4.40 m	70.1	4.31 m 3.70 m	67.6
6″	1.70 d (6.2)	18.8	1.68 d (6.3)	18.9	1.69 d (6.2)	18.9
1′′′		170.4		165.9		165.9		165.9
2′′′	2.15 s	21.1	6.02 dq (15.6, 1.6)	122.7	6.04 br d (14.6)	122.8	5.99 br d (15.5)	123.1
3′′′			7.06 m	146.0	7.07 m	145.9	7.09 m	145.4
4′′′			1.66 d (6.8)	17.8	1.61 dd (7.0, 1.5)	17.8	1.61 br d (6.9)	17.8
1′′′′						170.4
2′′′′					2.15 s	21.9
12-OH	5.34 d (6.1)
16-OH	5.49 d (5.0)
2′-OH	7.68 d (6.1)
2″-OH	6.80 br s
4″-OH	6.80 br s

	12		13		14
No.	δH (500 MHz)	δC (126 MHz)	δH (600 MHz)	δC (151 MHz)	δH (600 MHz)	δC (151 MHz)
1	1.61 m 0.84 m	39.0	1.72 m 0.98 m	39.5	1.68 m 0.95 m	39.7
2	1.78 m	28.3	2.21 m 1.90 m	27.4	2.17 m 1.92 m	27.5
3	3.41 m	78.1	3.37 dd (11.8, 4.5)	89.0	3.31 dd (11.9, 4.2)	88.9
4		39.5		40.1		40.1
5	0.69 m	56.0	0.75 br d (10.8)	56.3	0.71 br d (11.5)	56.6
6	1.49 m 1.28 m	18.8	1.51 m 1.33 m	19.0	1.47 m 1.31 m	19.0
7	1.27 m	34.2	1.32 m	34.6	1.29 m	34.6
8		46.8		47.2		47.2
9	1.22 m	48.4	1.40 m	49.5	1.37 m	49.5
10		37.6		37.6		37.5
11	2.37 m 1.48 m	27.2	2.09 m 1.66 m	33.5	2.07 m 1.65 m	33.5
12	4.37 m	74.5	4.04 m	69.7	4.04 m	69.7
13	1.87 d (11.2)	54.0	1.92 d (11.1)	56.2	1.91 d (11.1)	56.2
14		41.5		41.9		41.9
15	1.92 m 1.29 m	43.0	1.99 dd (11.6. 6.2) 1.38 m	43.7	1.97 dd (11.7, 6.1) 1.36 m	43.7
16	4.84 m	76.0	4.93 m	76.6	4.92 m	76.6
17		152.5		153.0		153.0
18		46.0		46.4		46.4
19	2.88 m 2.54 m	53.7	3.15 m 2.70 m	54.6	3.15 m 2.70 ddd (13.6, 8.2, 2.6)	54.5
20	2.37 m 2.04 m	25.6	2.50 m 2.20 m	26.1	2.50 m 2.21 m	26.1
21		146.8		147.5		147.5
22		84.2		84.5		84.5
23	1.20 s	28.7	1.31 s	28.5	1.25 s	28.3
24	0.99 s	16.4	0.99 s	17.2	1.19 s	17.4
25	0.79 s	16.6	0.85 s	17.0	0.85 s	17.1
26	0.88 s	16.5	1.02 s	16.9	1.01 s	16.9
27	0.94 s	15.9	1.07 s	16.2	1.06 s	16.2
28	1.67 s	22.2	1.59 s	23.0	1.59 s	23.0
29	1.44 s	29.2	1.49 s	29.2	1.49 s	29.2
30	1.44 s	28.6	1.47 s	29.5	1.47 s	29.5
1′	5.19 d (7.5)	100.1	4.86 d (7.5)	108.2	4.85 d (7.4)	106.6
2′	4.07 m	75.9	4.05 m	76.0	4.27 dd (8.3, 7.4)	78.4
3′	4.37 m	78.8	4.20 dd (8.7, 8.7)	79.1	4.20 dd (8.7, 8.3)	80.1
4′	4.26 dd (9.5, 9.5)	72.4	4.26 m	71.7	4.17 m	72.0
5′	4.05 m	78.4	4.39 dd (11.3, 5.2) 3.79 dd (11.3, 10.7)	67.6	4.34 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 s	102.4
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 m	70.2
6″					1.73 d (6.2)	19.2
12-OH					5.46 br s

	15		16		17
No.	δH (600 MHz)	δC (151 MHz)	δH (500 MHz)	δC (126 MHz)	δH (800 MHz)	δC (201 MHz)
1	1.69 m 0.95 m	39.5	1.64 m 0.90 m	38.8	1.63 m 0.91 m	39.0
2	2.14 m 1.87 m	27.3	2.11 m 1.83 m	26.8	2.10 m 1.83 m	26.9
3	3.30 dd (11.9, 4.3)	89.2	3.27 dd (11.8, 4.4)	88.8	3.26 dd (12.0, 4.3)	88.8
4		40.1		39.6		39.6
5	0.73 br d (11.3)	56.3	0.72 br d (11.9)	55.7	0.73 br d (11.7)	55.9
6	1.48 m 1.31 m	19.0	1.47 m 1.30 m	18.6	1.44 m 1.30 m	18.6
7	1.31 m	34.6	1.45 m 1.27 m	33.5	1.40 m 1.31 m	33.6
8		47.2		45.4		44.8
9	1.39 m	49.5	1.38 m	49.2	1.47 m	49.6
10		37.5		36.9		37.0
11	2.09 m 1.65 m	33.5	2.07 m 1.61 m	33.1	2.08 m 1.62 m	33.4
12	4.04 m	69.7	4.19 m	69.3	4.08 m	69.6
13	1.92 d (11.1)	56.2	1.77 d (10.9)	53.9	1.91 m	55.1
14		47.2		41.8		41.7
15	2.00 m 1.38 m	43.7	1.87 dd (12.5, 4.2) 1.73 m	45.1	1.92 m 1.87 m	44.1
16	4.93 m	76.6	4.32 m	67.2	4.99 overlapped	68.1
17		153.0	2.14 d (10.8)	62.2		143.9
18		46.4		45.4		52.6
19	3.15 m 2.70 m	54.6	2.53 m 1.78 m	43.2	2.58 m 2.20 m	46.5
20	2.50 m 2.21 m	26.1	2.42 m	29.9	2.54 m 2.18 m	38.1
21		147.5		152.2		128.5
22		84.5	6.06 t (2.0) 5.14 t (2.0)	106.1	2.33 s	16.0
23	1.25 s	28.4	1.23 s	28.0	1.21 s	28.0
24	0.95 s	17.2	0.93 s	16.7	0.92 s	16.7
25	0.83 s	17.0	0.78 s	16.0	0.79 s	16.4
26	1.02 s	16.9	1.01 s	17.0	0.98 s	16.5
27	1.07 s	16.2	1.16 s	19.1	1.25 s	17.5
28	1.59 s	23.0	1.06 s	16.0	1.39 s	20.6
29	1.49 s	29.2
30	1.47 s	29.5
1′	4.76 d (7.5)	107.8	4.74 d (7.4)	107.4	4.73 d (7.5)	107.4
2′	4.03 m	75.9	4.01 m	75.4	4.00 m	75.4
3′	4.32 dd (8.9, 8.9)	83.5	4.30 t (8.9)	83.0	4.29 t (8.8)	83.0
4′	4.16 m	70.2	4.14 m	69.7	4.13 m	69.7
5′	4.35 m 3.73 dd (11.2, 10.4)	67.4	4.32 m 3.72 dd (11.3, 10.2)	67.0	4.33 m 3.71 dd (11.2, 10.3)	66.9
1″	6.30 br s	103.3	6.26 d (1.2)	102.9	6.26 br s	102.8
2″	4.82 dd (3.3, 1.5)	73.1	4.78 br s	72.6	4.78 br s	72.6
3″	4.62 dd (9.4, 3.3)	73.2	4.60 br d (9.1)	72.8	4.59 br d (8.7)	72.8
4″	4.37 dd (9.4, 9.4)	74.6	4.34 m	74.2	4.33 m	74.2
5″	5.00 m	70.4	4.98 overlapped	70.0	4.97 overlapped	70.0
6″	1.71 d (6.2)	19.2	1.68 d (6.2)	18.7	1.68 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

	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)
1	1.69 m 0.98 m	39.2	1.61 m 0.90 m	38.8	1.70 m 0.89 m	38.5
2	2.21 m 1.91 m	27.4	2.10 m 1.82 m	26.9	2.14 m 1.87 m	27.0
3	3.39 dd (11.9, 4.3)	89.1	3.27 dd (11.9, 4.4)	88.8	3.32 dd (11.9, 4.5)	89.8
4		40.2		39.7		40.1
5	0.84 m	56.5	0.77 overlapped	56.0	0.79 br d (12.1)	55.6
6	1.57 m 1.42 m	19.1	1.54 m 1.37 m	18.6	1.52 m 1.33 m	18.8
7	1.52 m	33.9	1.48 m	33.5	1.28 m	33.2
8		41.8		41.3		41.6
9	1.50 m	50.2	1.45 m	49.7	1.98 br s	55.2
10		37.7		37.3		37.1
11	2.18 m 1.66 m	34.3	2.14 m 1.61 m	33.9	5.69 br d (10.1)	128.2
12	4.33 m	69.4	4.28 m	69.0	6.60 dd (10.1, 2.9)	126.2
13	2.17 d (11.1)	53.3	2.13 d (11.1)	52.8		138.7
14		47.5		47.0		43.1
15	5.76 d (10.3)	135.5	5.72 d (10.3)	135.1	1.96 m 1.09 m	26.0
16	6.42 d (10.3)	120.4	6.39 d (10.3)	120.0	2.26 m 1.78 m	33.5
17		141.9		141.5		49.2
18		48.6		48.2		131.4
19	2.59 m 2.24 m	45.3	2.55 m 2.21 m	44.9	3.17 d (14.5) 2.77 d (14.5)	36.0
20	2.60 m 2.14 m	36.8	2.55 m 2.09 m	36.3		44.3
21		131.8		131.4	2.31 m 1.79 m	32.9
22	1.73 s	14.4	1.70 s	14.0	2.66 m 1.52 m	35.3
23	1.35 s	28.6	1.25 s	28.0	1.25 s	28.2
24	1.02 s	17.2	0.94 s	16.7	0.89 s	16.8
25	0.81 s	16.3	0.76 s	15.9	0.84 s	18.7
26	1.03 s	19.7	0.99 s	19.3	1.02 s	17.4
27	1.34 s	19.5	1.30 s	19.0	1.11 s	20.5
28	1.32 s	20.4	1.29 s	20.0		179.1
29					1.27 s	20.7
30						178.9
30-Me					3.60 s	52.2
1′	4.88 d (7.7)	108.2	4.74 d (7.5)	107.4	4.92 d (7.9)	107.6
2′	4.05 dd (8.4, 7.7)	76.0	4.00 dd (8.4, 7.5)	75.4	4.07 dd (8.7, 7.9)	76.2
3′	4.20 dd (8.8, 8.4)	79.1	4.28 m	83.0	4.45 dd (9.3, 8.7)	82.3
4′	4.26 m	71.7	4.12 m	69.7	4.41 dd (9.3, 9.3)	71.9
5′	4.40 dd (11.0, 5.1) 3.80 dd (11.0, 10.4)	67.6	4.32 m 3.70 dd (11.0, 10.5)	67.0	4.58 d (9.3)	77.6
6′						171.3
6′-OMe					3.79 s	52.7
1″			6.27 br s	102.8	6.33 br s	103.4
2″			4.79 br s	72.6	4.76 dd (3.3, 1.4)	73.0
3″			4.59 dd (9.3, 3.0)	72.8	4.57 dd (9.3, 3.3)	73.2
4″			4.34 dd (9.3, 9.3)	74.2	4.35 dd (9.3, 9.3)	74.6
5″			4.97 m	69.9	5.08 m	70.3
6″			1.67 d (6.1)	18.7	1.71 d (6.1)	19.1

	21		22		23
No.	δH (500 MHz)	δC (126 MHz)	δH (500 MHz)	δC (126 MHz)	δH (500 MHz)	δC (126 MHz)
1	1.35 m 0.82 m	38.6	1.36 m 0.82 m	38.5	1.37 m 0.83 m	38.5
2	2.19 m 1.76 m	26.4	2.04 m 1.77 m	26.6	2.07 m 1.78 m	26.6
3	3.25 dd (11.9, 4.4)	89.2	3.28 overlapped	89.4	3.27 overlapped	89.3
4		39.3		39.5		39.5
5	0.75 overlapped	55.8	0.75 overlapped	55.7	0.74 overlapped	55.7
6	1.49 m 1.28 m	18.6	1.46 m 1.25 m	18.4	1.46 m 1.26 m	18.4
7	1.46 m 1.27 m	33.3	1.44 m 1.26 m	33.2	1.45 m 1.26 m	33.2
8		39.7		42.1		42.1
9	1.60 m	48.1	1.61 m	48.0	1.62 dd (8.6, 8.6)	48.0
10		37.0		36.9		36.9
11	1.85 m	23.8	1.85 m	23.7	1.85 m	23.7
12	5.58 br s	122.5	5.59 br s	123.1	5.59 dd (3.4, 3.4)	123.1
13		145.5		144.5		144.5
14		42.2		39.7		39.7
15	2.21 m	28.6	2.12 m 1.18 m	28.4	2.13 m 1.27 m	28.4
16	2.08 m	24.2	2.12 m 2.02 m	23.9	2.13 m 2.03 m	23.9
17		46.4		46.2		46.2
18	3.36 dd (13.5, 3.6)	43.6	3.29 overlapped	43.4	3.29 overlapped	43.4
19	2.26 m 1.82 m	43.2	2.25 br d (13.2) 1.81 m	42.7	2.25 br d (12.0) 1.82 m	42.7
20		44.3		44.2		44.2
21	2.16 m 1.45 m	31.2	2.19 m 1.46 m	30.9	2.19 br d (13.5) 1.47 m	30.9
22	2.06 m 1.96 m	34.9	2.08 m 1.97 m	34.6	2.09 m 1.98, m	34.6
23	1.19 s	28.2	1.24 s	28.1	1.23 s	28.1
24	0.98 s	17.0	0.90 s	16.9	0.90 s	16.9
25	0.75 s	15.4	0.75 s	15.4	0.76 s	15.4
26	0.89 br s	17.6	0.95 s	17.3	0.95 s	17.3
27	1.31 s	26.2	1.31 s	26.2	1.31 s	26.2
28		DAP a		179.9		179.9
29	1.22 s	28.7	1.23 s	28.5	1.23 s	28.5
30		177.4		177.2		177.2
30-OMe	3.63 s	51.7	3.65 s	51.8	3.65 s	51.7
1′	5.05 d (8.2)	105.0	4.88 d (7.8)	107.1	4.88 d (7.9)	107.2
2′	4.58 m	58.1	4.34 m	74.1	4.05 dd (8.4, 7.9)	75.8
3′	4.41 dd (9.3, 8.7)	76.3	4.42 dd (9.0, 9.0)	81.9	4.44 dd (8.9, 8.4)	81.9
4′	4.18 dd (9.3, 9.3)	72.7	4.37 m	71.4	4.41 dd (9.4, 8.9)	71.4
5′	3.97 m	78.4	4.56 d (9.4)	77.2	4.55 d (9.4)	77.2
6′	4.57 m 4.37 m	63.0		170.8		170.3
6′-OMe,			3.77 s	52.2	4.29 m	61.4
OEt					1.19 t (7.1)	14.3
1″		170.3	6.30 br s	102.9	6.33 d (1.2)	102.8
2″	2.15 s	23.8	4.75 m	72.5	4.77 dd (3.4, 1.2)	72.6
3″			4.55 m	72.7	4.56 dd (9.2, 3.4)	72.7
4″			4.34 m	74.1	4.36 dd (9.2, 9.2)	74.1
5″			5.05 m	69.9	5.08 m	69.8
6″			1.69 d (6.1)	18.6	1.69 d (6.2)	18.7
NH	8.94 d (9.0)

a Disappeared.

	24		25		26
No.	δH (500 MHz)	δC (126 MHz)	δH (500 MHz)	δC (126 MHz)	δH (500 MHz)	δC (126 MHz)
1	1.51 m 0.97 m	38.8	1.47 m 0.93 m	38.7	1.39 m 0.85 m	38.6
2	2.16 m 1.87 m	26.8	2.09 m 1.81 m	26.7	2.13 m 1.83 m	26.6
3	3.35 dd (11.7, 4.4)	88.7	3.28 overlapped	88.8	3.36 dd (11.6, 4.3)	89.2
4		39.6		39.5		39.6
5	0.82 overlapped	55.9	0.79 overlapped	55.8	0.79 overlapped	55.8
6	1.49 m 1.28 m	18.5	1.47 m 1.27 m	18.5	1.47 m 1.26 m	18.5
7	1.47 m 1.28 m	33.2	1.46 m 1.27 m	33.2	1.45 m 1.27 m	33.2
8		39.7		42.0		39.7
9	1.67 dd (9.0, 8.7)	48.1	1.65 m	48.0	1.63 dd (9.0, 8.6)	48.0
10		37.1		37.0		37.0
11	1.89 m	23.8	1.88 m	23.7	1.87 m	23.7
12	5.60 br t (3.1)	123.2	5.60 dd (3.4, 3.4)	123.1	5.60 br t (3.3)	123.1
13		144.5		144.4		144.5
14		42.1		39.7		42.1
15	2.13 m 1.19 m	28.4	2.13 m 1.27 m	28.4	2.13 m 1.20 m	28.4
16	2.13 m 2.03 m	23.9	2.12 m 2.02 m	23.8	2.12 m 2.03 m	23.9
17		46.2		46.2		46.2
18	3.30 dd (13.6, 3.6)	43.4	3.29 overlapped	43.4	3.29 dd (13.6, 3.9)	43.4
19	2.26 m 1.82 dd (13.6, 13.6)	42.7	2.26 br d (13.5) 1.82 dd (13.5, 13.5)	42.7	2.26 m 1.82 m	42.7
20		44.2		44.2		44.2
21	2.19 m 1.46 m	30.9	2.19 br d (13.1) 1.46 m	30.8	2.20 m 1.46 m	30.9
22	2.10 m 1.98 m	34.6	2.09 m 1.98 m	34.5	2.09 m 1.98 m	34.6
23	1.30 s	28.2	1.24 s	28.1	1.30 s	28.2
24	0.97 s	17.0	0.92 s	16.9	0.95 s	17.0
25	0.82 s	15.5	0.80 s	15.5	0.78 s	15.5
26	0.97 s	17.4	0.96 s	17.3	0.95 s	17.4
27	1.30 s	26.2	1.30 s	26.1	1.30 s	26.2
28		179.9		179.9		179.9
29	1.23 s	28.5	1.23 s	28.5	1.23 s	28.5
30		177.2		177.2		177.2
30-OMe	3.65 s	51.8	3.64 s	51.7	3.65 s	51.8
1′	4.83 d (7.6)	107.8	4.73 d (7.5)	107.4	5.00 d (7.8)	107.3
2′	4.01 dd (8.7, 7.6)	75.6	4.00 dd (8.8, 7.5)	75.4	4.09 dd (9.0, 7.8)	75.4
3′	4.17 dd (8.7, 8.7)	78.7	4.29 dd (8.8, 8.8)	82.9	4.28 dd (9.0, 9.0)	77.9
4′	4.23 m	71.3	4.13 m	69.7	4.48 dd (9.0, 9.7)	73.3
5′	4.38 dd (11.2, 5.1) 3.78 dd (11.2, 10.2)	67.2	4.34 m 3.73 dd (10.6, 10.2)	66.9	4.61 d (9.7)	77.3
6′						170.9
6′-OMe					3.73 s	52.1
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 m	69.9
6″			1.67 d (6.2)	18.7

Compound	MIC50 (μM) ± SD
	M. gypseum	T. rubrum
1	105.0 ± 0.6	>300
3	128.1 ± 1.4	>300
6	7.1 ± 1.2	14.3 ± 2.1
12	260.1 ± 2.3	>300
15	46.8 ± 0.1	>300
16	120.7 ± 1.4	>300
18	29.3 ± 3.4	>300
19	34.9 ± 1.2	>300
21	6.7 ± 2.1	13.4 ± 1.1
22	40.3 ± 0.5	>300
23	39.9 ± 1.2	>300
24	6.8 ± 3.2	11.9 ± 0.3
25	11.1 ± 2.4	13.0 ± 1.3
26	22.0 ± 0.9	>300
terbinafine hydrochloride (positive control)	0.008 ± 0.373	1.647 ± 0.101

a Compounds 2, 4–7, 9–11, 13, 14, 17, 20, and 27–37 were inactive (MIC50 > 300 μM).

Supplementary Materials

The following are available online. General experimental procedures; Figures S1-S198: 1D and 2D-NMR and HRESIMS spectra of compounds 1-26, structures of known compounds (26-37), and key 2D-NMR correlations of 2, 6-11, and 13-25.

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.

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