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

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 (5–25); and 12 known compounds (26–37) 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 (21–26) 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.


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].
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.
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 Compound 2 showed a molecular formula of C 30 H 50 O 5 based on 13 C-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 13 C-NMR spectrum of 1 was replaced by the signal (δ C 216.4) for a carbonyl group in the 13 C-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 C 30 H 44 O 5 based on 13 C-NMR data ( Table 2) and positive ion mode HRESIMS, which showed a pseudomolecular ion peak at m/z 507.3084 [M + Na] + (calcd. for C 30 H 44 NaO 5 , 507.3086). The 1 H-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 13 C-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 sp 3 methylenes, three sp 3 methines, and seven sp 3 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.
Based on 13 C-NMR data (Tables 5 and 6) and HRESIMS, the molecular formulae of glinusopposides I-K (13)(14)(15) (Tables 5 and 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) (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
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)(22)(23)(24)(25)(26) or oxidation (3) of 3-OH was helpful in increasing the activity based on a comparison of the MIC 50 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.

General Experimental Procedures
This part can be found in the Supplementary Materials.

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.

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 H 2 O 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 CHCl 3 to yield the CHCl 3 -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).

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 (MIC 50 ) was calculated by the Reed-Muench method [26].

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.
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).