Two New Eudesmane Sesquiterpenoids from the Flowers of Chrysanthemum indicum

1 Introduction

The plants of Asteraceae family, which contains 25 000-30 000 species belonging to over 1000 genera, have been considered as rich sources of eudesmane sesquiterpenoids [1]. Till now there have been over 1000 naturally occurring eudesmane sesquiterpenoids identified from the Asteraceae family, and these plant metabolites possessed diverse oxygenation and cleavage patterns [2]. Eudesmane-type sesquiterpenoids exhibit a wide range of biological activities, such as plant growth regulating, insect antifeedant, antifungal, anti-tumour, antibacterial, and antiviral activities [3-11]. Due to these, eudesmane-type sesquiterpenoids have long been the research focus of the phytochemists and pharmacologists.

The flowers of Chrysanthemum indicum has long been used a traditional heat-clearing and detoxifying herb in China. It is called Ye-ju-hua in the Chinese Pharmacopoeia [12]. The main usage of this traditional Chinese medicine was to treat inflammation, headache, and vertigo and for the preparation of a bitter tea used for antibacterial, antioxidant, and anti-inflammatory purposes [13]. It has long been our goal to find more novel sesquiterpenoids from traditional Chinese herbs [14-18]. As part of this program, an investigation of an aqueous EtOH extract of the flowers of C. indicum afforded 2 further new sesquiterpenoids (Fig. 1), 7-epi-eudesm-4(15), 11(13)-diene-1β, 3β-diol (1) and 7-epi-1β-hydroxy-β-eudesmol (2). In this paper we will report the procedures for the isolation and structure determination of the two new isolates.

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2 Results and Discussion

Sesquiterpenoid 1 was isolated as a white solid with a optical rotation value of [α]_D²⁰+5.0 (c 0.2, acetone), which suggested this compound could be a racemate. It was assigned a molecular formula of C₁₅H₂₄O₂ as determined based on an ion peak at m/z 219.1750 [M-H₂O+H]⁺ (calcd for C₁₅H₂₃O, 219.1743) in its positive HRESIMS, which suggested four degrees of unsaturation. The IR absorptions at 3384 and 1654 cm^-1 suggested the presence of hydroxy and olefinic functionalities, respectively. The ¹H and ¹³C NMR data of 1 revealed (Table 1) an angular methyl (δ_H 0.74, δ_C 9.9), a vinyl methyl (δ_H 1.70, δ_C 22.2), two oxymethines (δ_H 3.44, δ_C 77.1; δ_H 4.06, δ_C 70.4), and two terminal double bonds (δ_H 4.73, 5.11, δ_C 103.4, 151.6; δ_H 4.79, 4.91, δ_C 110.9, 146.4). These observations suggested that 1 possessed a eudesmane framework with two hydroxy groups and two terminal double bonds [14-18]. From the HMBC experiment (Fig. 2), the angular methyl (H₃-14) correlated with C-1, C-5, C-9 and C-10 (δ_C 77.1, 38.9, 32.3 and 40.5 respectively), indicating that one hydroxyl group was linked at C-1. Similarly, the exomethylene protons (H₂-15) correlated with C-3 and C-5 (δ_C 70.4 and 38.9), which clearly indicated that another hydroxyl group was located at C-3. In the NOE difference experiment, irradiation of H-1 (δ_H 3.44) enhanced the resonances of H-3 (0.81%), H-2α (1.47%), and H-5 (1.15%), but not H₃-14, indicating that H-1, H-3 and H-5 were all α-oriented (assuming that H₃-14 was β-oriented). Comparison of the carbon signal at C-7 (δ_C 38.4) with that of ligucyperonol (δ_C 45.1) [19] and β-dictyopterol (δ_C 45.3) [20], suggested a β-orientation of H-7. Finally sesquiterpenoid 1 was elucidated as 7-epi-eudesm-4(15), 11(13)-diene-1β, 3β-diol.Table 1 NMR Spectroscopic Data (in CDCl₃, 400 MHz) of Sesquiterpenoids 1 and 2

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Sesquiterpenoid 2 was obtained as a white solid with [α]_D²⁰+3.0 (c 0.35, acetone), which indicated it maybe exist as a racemate. Its molecular formula was assigned as C₁₅H₂₆O₂ on the basis of an ion at m/z 261.1815 [M+Na]⁺ (calcd for C₁₅H₂₆O₂Na, 261.1825) in the positive HRESIMS. Its IR absorptions at 3323 and 1645 cm^-1 indicated the occurrence of hydroxy and olefinic functionalities, respectively. The ¹H and ¹³C NMR data of 2 demonstrated three methyls (δ_H 1.11, 1.22, 1.32; δ_C 11.1, 29.2, 29.6), one oxymethine (δ_H 3.45; δ_C 79.3), one oxygenated tertiary carbon (δ_C 74.4), and one terminal double bond (δ_H 4.54, 4.78; δ_C 106.8, 149.3). These observations indicated 2 as an eudesmane sesquiterpenoid possessing two hydroxy groups and one terminal double bond [14-18]. Furthermore, its planer structure was constructed via HMBC experiment as shown in Fig. 2. From the NOE difference experiment, irradiation of H-1 (δ_H 3.45) enhanced the signals of H-3α (1.47%) and H-5 (1.11%) indicating that H-1 and H-5 was α-oriented. The chemical shifts of C-1/2/10 of compound 8 (δ_C 79.3/31.4/39.6 in CDCl₃), which were similar to those of β-dictyopterol (δ_C 79.3/31.5/40.2 in CDCl₃) [20], were used to determine the β-oriented Me-14. The β-orientation of H-7 was deduced from dramatic differences in chemical shifts of C-5/7/9 in compound 8 (δ_C 41.8/41.6/34.0 in CDCl₃) and 1β-hydroxy-β-eudesmol (δ_C 47.5/48.9/36.9 in CDCl₃) [21]. Thus, sesquiterpenoid 2 was established as 7-epi-1β-hydroxy-β-eudesmol.

3 Experimental Section

3.1 General

Optical rotations were measured on a Perkin-Elmer model 341 polarimeter with a 1 dm cell. IR spectra were obtained on a Nicolet NEXUS 670 FT-IR spectrometer. NMR spectra were recorded on a Bruker AVANCE Ш-400 spectrometer. Chemical shifts are given as δ (ppm) using TMS as internal standard. HRESIMS were carried out on a Bruker APEX Ⅱ mass spectrometer. Sephadex LH-20 (GE Healthcare Bio-science AB, Sweden), C₁₈ reversed-phase silica gel (YMC, ODS-A, AA120S50), Silica gel (200-300 mesh, Qingdao Marine Chemical factory, Qingdao, China.) were used for column chromatography (CC). TLC using silica GF₂₅₄ (10-40 μm) was detected at 254 nm and spots were visualized by spraying with 5% H₂SO₄ in EtOH (v/v) followed by heating.

3.2 Plant Material

The flowers of C. indicum was purchased from Huanghe Medicinal Material Market in Gansu in 2009 and identified by Associate Prof. H.Y. Qi of CAS Key Laboratory of Chemistry of Northwestern Plant Resources in LICP, where a voucher specimen (No. ZY2009C001) was deposited.

3.3 Extraction and Isolation

The air-dried flowers of C. indicum (2.0 kg) were pulverized and extracted with 95% EtOH (3×5 L) at 50 ℃ for 3 h each time. The combined extracts were evaporated to dryness under reduced pressure. The resulting residue was mixed with H₂O (1.5 L) to form a suspension and partitioned successively with petroleum ether (PE), EtOAc, and n-BuOH. The PE-soluble part (60 g) was subjected to silica gel CC with a gradient system of PE-EtOAc (60:1, 30:1, 15:1, 10:1, 7:1, 5:1, 3:1 and 1:1) to afford eight fractions (A-H) based on TLC analysis. Fractions F-G was purified over a column of Sephadex LH-20 using CHCl₃-MeOH (2:1) to give tho sub-fractions (F1 and G1) without pigments. Sub-fraction F1 was separated by silica gel CC eluted repeatedly with CHCl₃-EtOAc (25:1, 15:1 and 10:1), PE-EtOAc (10:1), CHCl₃-Me₂CO (15:1), and a column of C₁₈ reversed-phase silica gel (MeOH-H₂O, 70:30, 60:40), to give 2 (12.3 mg). Sub-fraction G1A was repeated purified by silica gel eluted with CHCl₃-Me₂CO (10:1) and PE-Me₂CO (8:1.5), and then over a C₁₈ reversed-phase silica gel eluted by (MeOH-H₂O, 60:40, 50:50) to yield 1 (16.1 mg).

3.4 Characteristic Data of Compounds

7-epi-eudesm-4(15), 11(13)-diene-1β, 3β-diol (1): White solid; [α]_D²⁰+5.0 (c 0.2, acetone); IR (KBr) ν_max: 3384, 2924, 1654, 1458, 1064, 803 cm^-1; ¹H and ¹³C NMR data, Table 1; HRESIMS: m/z 219.1750 [M-H₂O+H]⁺ (calcd for C₁₅H₂₃O, 219.1743).

7-epi-1β-hydroxy-β-eudesmol (2): White solid; [α]_D²⁰+3.0 (c 0.35, acetone); IR (KBr) ν_max: 3323, 2970, 1645, 1455, 1034, 884 cm^-1; ¹H and ¹³C NMR data, Table 1; HRESIMS: m/z 261.1815 [M+Na]⁺ (calcd for C₁₅H₂₆O₂Na, 261.1825).

Notes

Acknowledgements

This work was financially supported by National Natural Science Foundation of China (Nos. 81673325, 81711540311, and 21705156), the International Partnership Program of CAS (No. 153631KYSB20160004), Gansu Province Key International S & T Cooperation Project (No. 18YF1WA127), and CAS Pioneer Hundred Talents Program.

Compliance with Ethical Standards

Conflict of interest

The authors declare no competing financial interest.