Three new diterpenoids isolated from Euphorbia nematocypha Hand.-Mazz and their anti-fungal activity

Abstract Three new diterpenoids, named nematocynine A-C (1-3), together with twelve known compounds (4-15) were isolated from the aerial part of Euphorbia nematocypha Hand.-Mazz (Hereinafter referred to E. nematocypha). Their structures were elucidated by detailed spectroscopic analysis and comparison with literature data. In addition, all the compounds were tested for their anti-candida albicans activities used alone or in combination with fluconazole against sensitive strain and resistant strain in vitro. Wherein only compound 11 shows weak activity against candida albicans resistant strain (MIC50 = 128.15 μg/mL) when used alone. Compounds 1, 4, 7, 8, 9, 10, 12, 13 and 15 in combination with fluconazole showed potent anti-fungal activities (MIC50 = 15 ± 5 μg/mL, FICI = 0.05 ± 0.04) against the Candida albicans resistant strain SC5314-FR. The synergistic effects were weaker against the Candida albicans resistant strain SC5314-FR when the compounds 2, 3, 5 and 14 were combined with fluconazole (FICI = 0.16 ± 0.06). Graphical Abstract


Introduction
Euphorbia nematocypha is a species of genus Euphorbia in the Euphorbiaceae family.Euphorbia (Euphorbiaceae family) is the largest genus of plants, with more than 2000 species worldwide (He et al. 2008).E. nematocypha is one of the earliest recorded species in the 'southern Yunnan herb' .E. nematocypha has been wildly used as traditional Chinese herbal medicine for water retention, cirrhotic ascites, traumatic haemorrhage and mange treatment (Dagang et al. 1992;Chen et al. 2014;Gan et al. 2022).Previous investigations indicated the existence of abundant diterpenoids (Xu et al. 2021) in plants of the genus Euphorbia.So far, More than 450 diterpenoids were reported from the genus Euphorbia (Xu et al. 2021).Diterpenoids from E. nematocypha have shown anticancer activity and antiproliferative effects as well as antivirus, anti-inflammatory, and antibacterial activities (Zheng et al. 1998;Liu et al. 2002;Valente et al. 2004;otuki et al. 2005;Luo and Wang 2006).To investigate the chemical constituents and search for new active diterpenoids of E. nematocypha, we conducted compounds isolation, structure elucidation and preliminary bioactivity evaluation.ultimately, three new and twelve known diterpenoids were isolated from E. nematocypha (Figure 1).This paper mainly reported the isolation, structural elucidation, and anti-fungal activities of these diterpenoids.

Results and discussion
After solvent extraction, ethanol extract of E. nematocypha was separated into organic phase and Aqueous phase fractions.The diterpenoid fraction by repeated column chromatography (CC), recrystallization and semi-preparative HPLC was separated, to afford three new diterpenoids (1-3) and twelve known diterpenoids (4-15).
Comparing their 13 C-NMR data, the major differences consisted in the position of the C-9 (δ C 49.0) of 3 and the C-9 (δ C 73.8) in excocarinols F(the hydroxyl group at C-9), indicating that C-9 of 3 was a methines.The HMBC cross-peaks from H-1 to C-2, C-3, C-5, C-9, C-10, C-20 and from H 3 -18 to C-3, C-4, C-5, C-19 demonstrated a six-membered ring with a hydroxyl group at C-2 and a carbonyl group at C-3.The HMBC cross-peaks from H 3 -17 to C-12, C-13, C-14, C-15 and from H-14 to C-7, C-9, C-12, C-17; along with one proton spin systems of H-9/H-11/H-12 in the CoSY spectrum, demonstrated a six-membered ring with a hydroxyl group at C-12 and a ethenyl group at C-13.The planar structure of 3 was also supported by the HMBC cross-peaks from H 3 -20 to C-1, C-5, C-9 and C-10, together with the CoSY correlations of H-5/H-6/H-7.The relative configuration of 3 was confirmed by the analysis of the NoESY spectra.The NoESY correlations of H 3 -19/H-5/H-9 indicated that CH 3 -19, H-5 and H-9 had an identical orientation; the absence of NoE correlation between H 3 -20/H-5 indicated that CH 3 -20 and H-5 had the opposite orientation.It was also supported by the NoE correlation H-5/H-9 and the absence of NoE correlation between H-9 /H 3 -20.And the correlations of H 3 -17/H-12/H-9 indicated that CH 3 -17/H-9 have opposite orientations to the hydroxyl group at C-12, the hydroxyl group at C-12 and ethenyl group at C-13 had an identical orientation.The orientation of CH 3 -20 (δ C 17.0) is β-CH 3 , because the chemical shift of β-CH 3 -20 (δ C 17.2-17.3)may be readily distinguished from that α-CH 3 -20 (δ C 18.2-22.4)when in the same annular structure (Abad et al. 2007;Yan et al. 2018;Wei et al. 2021).Based on the results described above, the structure of 3 was determined and named nematocynine C.

General experimental procedures
General HR-ESI-MS spectra were recorded on an Agilent_1100 HPLC/ToF spectrometer.CD spectra were obtained on a JASCoJ-810 spectrometers.optical rotation were measured by Autopol IV,Serial Number: 83650.uV spectra were confirmed on a Shimadzu uV2401PC spectrophotometer.oR spectra were confirmed on a Jasco P-1020 polarimeter.IR spectra were recorded on a Thermo Nicolet iS10 spectrophotometer.1D and 2D NMR spectra were recorded on a Bruker AM-400 HD (Bruker, Karlsruhe, Germany) or a Bruker-AVANCE III-600 (Bruker, Karlsruhe, Germany) with TMS as internal standard.shifts (δ) are expressed in ppm with reference to the solvent signals.Preparative high performance liquid chromatography (HPLC) was performed on Agilent 1260 Infinity II Preparative liquid chromatography system equipped with an automatic fraction collector.Semi-preparative HPLC were performed on an Agilent 1260 liquid chromatograph (column: Zorbax SB-C18, 250 × 9.4 mm, DAD detector).D-101 macroporous resin (Tianjin Haoju Technology Co.,Ltd.),silica gel (80-100 mesh, 100-200 mesh and 200-300 mesh; Qingdao Marine Chemical Inc.,Qingdao,China) and Sephadex LH-20 (Pharmacia) were used for Column chromatography (CC).Fractions were monitored by TLC and visualized by heating plates sprayed with 15% H 2 So 4 in EtoH.

Plant material
The aerial parts of E. nematocypha were collected in July 2020 in Xishuangbanna, Yunnan Province, China.The plant materials were identified by Prof. Yu Chen of the Kunming Institute of Botany, Chinese Academy of Sciences.A voucher specimen (No. 20200730) has been deposited in the Key Laboratory of Medicinal Chemistry for Natural Resource, Ministry of Education, Yunnan university, China.

Extraction and isolation
The dried aerial parts powder of E. nematocypha (5.0 kg) were extracted with ethanol (EtoH) for three times (each 25 L, 48h at room temperature).After removal of the solvent, the EtoH extract was suspended in water and partitioned with ethyl acetate (EtoAc) to give the corresponding EtoAc (EN1, 490 g) residue and water layer (EN2).

Anti-candida albicans activity assay
The antifungal activities of all the isolated compounds (1-15) in combination with fluconazole against the sensitive strain of candida albicans SC5314 and induced fluconazole-resistant candida albicans SC5314-FR were determined using a microdilution method.In short, the candida albicans solution was dilute with liquid sabouraud medium, the concentration of the fungal solution was counted and adjusted to 1*10 5 CFu/mL, and 96-well microdilution method was used, set and grouped into a compound group, a fluconazole group, and a compound + fluconazole group.The samples were added according to the gradient dilution and cultured in a constant temperature and humidity incubator at 37 °C for 24h.The optical density was measured with a microplate reader, and the inhibition ratio (MIC 50 ) and inhibition combination index (FICI) were calculated.FICI < 0.5 was considered synergistic, 0.5 ≤ FICI ≤ 4 was considered non interactive, and FICI > 4 was considered antagonistic.