Sage Journals: Discover world-class research

Abstract

Background: The fruiting body of Ganoderma sinense Zhao, Xu et Zhang iscertified as an authoritative medical material in the Chinese Pharmacopoeia 2020 edition and has been used as a crude drug for a long time. G sinense spores are crucial part of the fruiting body of G sinense and exhibited antitumor and immune-enhancing activities according to our previous reports. However, there were few studies about the chemical composition of G sinense spores. This study was aimed at the structure determination and antitumor effect of the sterols isolated from G sinense spores. Methods: Modern chromatographic methods were applied to isolate compounds from the ruptured spores of G sinense. Their chemical structures were elucidated by analyses of one-dimensional and two-dimensional ¹H nuclear magnetic resonance, and high-resolution electrospray ionization mass spectrometry data. The cytotoxicities of the isolated compounds against 3 tumor cell lines, human non-small cell lung cancer A549 and 95D cells, and human hepatocellular carcinoma HepG2 cells were measured by Cell Counting Kit-8. Results: A novel ergosterol derivative, (22E,24R)-3β,5β,6α,7α,14β-pentahydroxyergosta-8,22-dien-15-one (1), and 5 known sterols, (22E,24R)-3β,5α,9α,14β-tetrahydroxyergosta-7,22-dien-6-one (2), (22E,24R)-ergosta-7,9(11),22-triene-3β,5α,6β-triol (3), (22E,24R)-ergosta-7,9(11),22-triene-3β,5α,6β,14α-tetrol (4), β-daucosterine (5), and (22E,24R)-ergosta-7,22-diene-3β,5α,6β,9α-tetrol (6), were obtained from the ruptured spores of G sinense. Compound 1 exhibited cytotoxicity against A549 cells and HepG-2 cells with IC₅₀ values of 65.12 ± 4.76 and 97.34 ± 6.36 μM, respectively. Compounds 1, 2, and 4 were obtained from G sinense for the first time. Conclusion: This paper is a continuation of an investigation of the chemical ingredients from G sinense spores and their antitumor effect. Only compound 1 showed weak cytotoxicities against A549 cells and HepG-2 cells.

Keywords

spores ergosterol derivatives steroids cytotoxic activity antitumous effect

Introduction

Ganoderma sinense Zhao, Xu et Zhang is a member of the genus Ganoderma (lingzhi) which contains 181 species mainly distributed in the tropics and semitropics.^1,2 As 1 of 2 definitive raw drug materials of lingzhi reported in the Chinese Pharmacopoeia 2020, G sinense is well-known in East Asia and has been utilized extensively for health food and as a crude drug with a long history. However, few studies have been reported on the chemical constituents and pharmacological activities of G sinense, especially G sinense spores. Our earlier research reported that lipids obtained from G sinense spores exhibited antitumor and immune-enhancing properties^3,4 and described how to extract ergosterol ramification and elucidate its structure with their tumoricidal effect as well as antimetastatic activities.⁵ In a continuation of our investigation on the chemical ingredients from G sinense spores, our work reports the isolation and identification of a new compound, (22E,24R)-3β,5β,6α,7α,14β-pentahydroxyergosta-8,22-dien-15-one (1), and 4 known sterols, (22E,24R)-3β,5α,9α,14α-tetrahydroxyergosta-7,22-dien-6-one (2),⁶ (22E,24R)-ergosta-7,9(11),22-triene-3β,5α,6β-triol (3),⁷ (22E,24R)-ergosta-7,9(11),22-triene-3β,5α,6β,14α-tetrol (4),⁸ β-daucosterine (5),⁹ and (22E,24R)-ergosta-7,22-diene-3β,5α,6β,9α-tetrol (6)¹⁰ (Figure 1). Then, the toxic activities of the obtained compounds against 3 tumor cell lines, human non-small cell lung cancer A549 and 95D cells, and human hepatocellular carcinoma HepG2 cells were determined.

Figure 1.

Structures of the compounds isolated from Ganoderma sinense spores.

Results and Discussion

Compound 1 (Figure 1) behaves as a whitish powder. Its molecular formula, C₂₈H₄₄O₆, was confirmed by using high-resolution electrospray ionization mass spectrometry (HRESIMS). The ¹H nuclear magnetic resonance (NMR) spectrum showed 6 methyl protons at δ_H 1.06 (d, J = 6.7 Hz), 1.02 (s), 0.88 (d, J = 6.8 Hz), 0.79 (d, J = 6.8 Hz), 0.77 (d, J = 6.8 Hz) and 0.75 (s). The ¹H and ¹³C NMR spectra (Table 1) indicated that it contains groups such as 6 methyls, 6 methylenes, night methines (4 methines, 3 oxygenated methines and 2 sp² methines), and 7 quaternary carbons (2 oxygenated tertiary carbons, 3 sp² quaternary carbons, and 1 carbonyl carbon) (Figure S1-S4). Thus, its structure was presumed to be a C28 sterol based on the combined HRESIMS and NMR data (Figure 1).

Table 1.

¹H and ¹³C NMR Data for Compound 1 in DMSO-d6 (δ in ppm).

Position	δ_H^a	δ_C^b	HMBC
1	1.79, m	25.0	C-2, 3, 5, 10, 19
	1.51, m
2	1.63, m	30.4	C-1, 3, 4, 10
	1.11, m
3	3.94, m	65.0	C-1, 5
4	1.83, m	31.0	C-2, 6, 10, 19
	0.91, m
5		74.6
6	3.58, d (7.0)	74.8	C-4, 5, 7, 10
7	3.52, dt (7.0, 3.1)	73.9	C-6, 8, 9
8		125.0
9		144.4
10		44.2
11	2.20, m	22.5	C-8, 9, 10, 12, 13
	2.13, m
12	1.75, m	27.6	C-9, 11, 13, 14, 17, 18
12	1.47, m		C-9, 11, 13, 14, 17, 18
13		43.1
14		83.9
15		215.9
16	2.14, dd (19.1, 8.5)	38.0	C-13, 14, 15, 17, 20
	1.82, m
17	1.75, m	38.3	C-12, 13, 15, 16, 18, 20, 21, 22
18	0.75, s	15.7	C-12, 13, 14, 17
19	1.02, s	23.5	C-1, 5, 9, 10
20	2.10, m	38.1	C-13, 16, 17, 21, 22, 23
21	1.06, d (6.7)	20.0	C-17, 20, 22
22	5.20, dd (14.7, 6.2)	134.3	C-17, 20, 21, 23, 24
23	5.24, dd (14.7, 6.5)	132.6	C-20, 22, 24, 25, 28
24	1.44, m	32.5	C-22, 23, 25, 26, 27, 28
25	1.84, m	42.4	C-23, 24, 26, 27, 28
26	0.77, d (6.8)	19.2	C-24, 25, 27
27	0.79, d (6.7)	19.9	C-24, 25, 26
28	0.88, d (6.8)	17.8	C-23, 24, 25
3-OH	5.29, br s
5-OH	4.82, s		C-4, 5, 6
6-OH	4.47, br s
7-OH	5.76, d (4.7)		C-7, 8
14-OH	5.71, s		C-8, 13, 14

Abbreviations: NMR, nuclear magnetic resonance; HMBC, heteronuclear multiple bond correlation

600 MHz, coupling constants (J in Hz) are given in parentheses.

150 MHz.

The coupling constant 14.7 Hz confirmed the presence of an E-configuration for the double bond between C-22 (δ_C 134.3) and C-23 (δ_C 132.6). ¹H–¹H correlation spectroscopy (¹H-¹H COSY) and heteronuclear single quantum coherence identified 2 structural fragments, 1 from C-16 to C-28, and the other from C-1 to C-4 (Figure 2). In the heteronuclear multiple bond correlation spectrum, key relationships (Figure 2) were observed from methyl protons at δ_H 0.75 (H-18) to C-12, C-13, C-14, and C-17; H-16 to C-13, C-14, C-15 (δ_C 215.9), and C-17; hydroxyl protons at δ_H 5.71 (14-OH) to C-8 (δ_C 125.0), C-13, and C-14 (δ_C 83.9); H-11 to C-8 and C-9; H₃-19 (δ_H 1.02) to C-1 (δ_C 30.7), C-5 (δ_C 74.6), C-9 (δ_C 144.4), and C-10; and hydroxyl protons at δ_H 4.82 (5-OH) to C-4, C-5, C-6 (δ_C 74.8) and C-10. In addition, key relationships in the ¹H-¹H COSY spectrum were found between H-3 and hydroxyl protons at δ_H 5.29 (3-OH); H-6 and hydroxyl protons at δ_H 4.47 (6-OH); H-7 and hydroxyl protons at δ_H 5.76 (7-OH); and H-11 and H-12.

Figure 2.

(A) Structure of compound 1 with key HMBC and ¹H-¹H COSY correlations. (B) Key NOESY correlations and pyridine-induced deshielding effects for compound 1. Abbreviations: HMBC, heteronuclear multiple bond correlation; ¹H-¹H COSY, ¹H-¹H correlation spectroscopy; NOESY, nuclear overhauser⁃hauser-effect spectroscopy.

The relative configuration of compound 1 was determined through analysis of the nuclear overhauser⁃hauser-effect spectroscopy (NOESY) spectrum (Figure 2). The cross-peaks of H₃-19/H-6, H₃-19/5-OH, and H₃-18/14-OH in the NOESY spectrum indicated that 5-OH and 14-OH were all β-oriented while 6-OH were α-oriented. The ¹H NMR data in both CDCl₃ and C₅D₅N of compound 1 (Figure S2-3) were collected to further determine the relative configuration through pyridine-induced deshielding experiments.^11–13 The downfield shifts (Δδ + 0.19, + 0.65, + 0.79, + 0.29, + 0.25, respectively) were observed for H-3α, H-6β, H-7β, H₃-18, and H₃-19 in C₅D₅N compared with the ¹H NMR data in CDCl₃, indicating the configurations of the 3β,5β,6α,7α,14β-pentahydroxylated moieties (Figure 2). NOESY correlations of H₃-18/H-20 and H-17/H-21 indicated the 17β-oriented side chain and 20R configuration.^6,14 Additionally, fungal ergosteroids possess the 20R configuration because they are derived from squalene epoxide through a comparable biological synthesis method.¹⁵ The configuration of C-24 was assumed to be R because compound 1 was isolated from fungi and possesses a △²² unsaturated side chain.¹⁶ Thus compound 1 was assigned to be (22E,24R)-3β,5β,6α,7α,14β-pentahydroxyergosta-8,22-dien-15-one.

The 5 known compounds were identified through comparison of NMR data and ESI data with formerly reported compounds. Their structures were confirmed as (22E,24R)-3β,5α,9α,14β-tetrahydroxyergosta-7,22-dien-6-one (2),⁶ (22E,24R)-ergosta-7,9(11),22-triene-3β,5α,6β-triol (3),⁷ (22E,24R)-ergosta-7,9(11),22-triene-3β,5α,6β,14α-tetrol (4),⁸ β-daucosterine (5),⁹ and (22E,24R)-ergosta-7,22-diene-3β,5α,6β,9α-tetrol (6).¹⁰

Compound 1 exhibited the strongest cytotoxic activity against A549 cells and HepG2 cells with IC₅₀ values of 65.12 ± 4.76 and 97.34 ± 6.36 μM, respectively, while compounds 2-6 were considered inactive because of their high IC₅₀ values (Table S1). Doxorubicin, as a positive control, showed cellular toxicity against A549, 95D cells and HepG-2 cells with IC₅₀ values of 0.72 ± 0.15, 0.22 ± 0.04 and 3.56 ± 0.02 μM, respectively.

In summary, compounds 1, 2, and 4 were first obtained from G sinense, of which 1 was found to be new. The structures of 1 are unusual among natural sterols because of the cis A/B and C/D ring junctions. The cytotoxicity assay showed that compound 1 could inhibit the proliferation of A549 and HepG2 cells. Further investigations on the other chemical constituents from G sinense spores and their antitumor effects will be carried out to provide a reference for material base investigation and quality control of G sinense spores.

Experimental Section

General Procedures

Column chromatographies were mainly performed by a Buchi Sepacore® Chromatography system equipped with a C-605 pump controller and C-635 detector. Silica gel (200-300 or 300-400 mesh, Marine Chemical Factory, Qingdao), ODS-A (50 μm, YMC), ODS-AQ (50 μm, YMC), and Sephadex LH-20 (Pharmacia) were utilized for various column chromatographies. Optical rotations were recorded through an MCP 500 Modular Circular Polarimeter. ¹H NMR and ¹³C NMR spectra were obtained through Bruker AVANCE III HD 600 and 500 MHz spectrometers, using tetramethylsilane (TMS) as an internal standard. HRESIMS data were acquired by an LTQ Orbitrap Elite spectrometer.

Fungal Materials

G sinense spores were obtained in August 2020 from a Lingzhi base on a mountain of Fujian Province, cultivated by the Food Engineering Research Centre of the State Ministry of Education, Sun Yat-Sen University. They were authenticated by Dr Wenhua He. A voucher sample (HMAS 77207M) has been stored before.⁵

Extraction and Isolation

G sinense spore oil (1.21 kg) was extracted from sporoderm-broken spores (4.0 kg) by supercritical fluid with carbon dioxide, as described previously.¹⁷ The G sinense spore oil (1.21 kg) was further separated by silica gel column chromatography (SGCC) and eluted by ligroine, petroleum ether (60-90 °C)/ethyl acetate (9:1), petroleum ether/ethyl acetate (4:1), petroleum ether (60-90 °C)/ethyl acetate (3:2), and dichloromethane/methanol (1:1) to obtain 5 fractions (ZI-ZVI). Fraction ZVI (7.1 g) was chromatographed by SGCC and eluted with a dichloromethane/methanol gradient (99:1-1:1) to obtain 6 fractions (Z1–Z6). Z1 (3.1 g) was loaded on a reversed-phase SGCC eluted with an acetonitrile/water gradient (2:3-4:1) to afford Z1a (30 mg) and Z1b (35 mg). Z1a was chromatographed by SGCC eluted with a petroleum ether (60-90 °C)/ethanol gradient (19:1-93:7) and purified by recrystallization with acetonitrile to produce compound 1 (5 mg). Z1b was chromatographed by SGCC eluted with a petroleum ether (60-90 °C)/ ethyl acetate gradient (1:1-1:9) and purified by recrystallization with acetonitrile to produce compound 2 (6 mg). Z2 (1.0 g) was loaded on a reversed-phase SGCC eluted with an acetonitrile/water gradient (1:1-4:1) and further chromatographed by SGCC eluted with a petroleum ether (60-90 °C)/ethanol gradient (24:1-93:7) to obtain compound 3 (16 mg). Z4 (998 g) was loaded on an SGCC eluted with a petroleum ether/ethyl acetate gradient (1:1-1:99) to produce Z4a (31 mg) and Z4b (251 mg). Z4a was subjected to a reversed-phase SGCC and eluted with an acetonitrile/water gradient (2:3-3:2) to produce compound 4 (5 mg). Z4bs was chromatographed by SGCC eluted with a petroleum ether (60-90 °C)/ethanol gradient (93:7-17:3) to produce compound 5 (55 mg). Z5 (215 mg) was separated by SGCC and elutriated with a petroleum ether (60-90 °C)/ethanol gradient (47:3-9:1), followed by recrystallization with n-hexane/ethanol (9:1) to obtain compound 6 (45 mg).

Cytotoxicity Assay

The obtained ergosterol derivatives 1-6 were screened for their cytotoxic activities against A549, 95D, and HepG-2 cells, which were acquired from the Shanghai Cell Bank in China. Doxorubicin (Sigma) was utilized as a positive control. Cell toxicity was measured by Cell Counting Kit-8 (CCK-8, Beyotime), as described previously.⁵ The IC₅₀ data were obtained by GraphPad Prism 5.

Characterization of Compound 1

White solid; $[α]_{D}^{20} – 85.8$ (c 0.10, MeOH); HRESIMS m/z 459.3102 [M-H₂O + H]⁺ (calcd for C₂₈H₄₃O₅ [M-H₂O + H]⁺, 459.3109); ¹H NMR and ¹³C NMR (DMSO-d6) see Table 1; ¹H NMR (600 MHz, CDCl₃) δ 5.25 (1H, dd, J = 15.2, 8.4 Hz, H-23), 5.11 (1H, dd, J = 15.2, 8.4 Hz, H-22), 4.19 (1H, m, H-3), 3.92 (1H, d, J = 7.8 Hz, H-6), 3.78 (1H, m, H-7), 1.15 (3H, s, H-19), 1.12 (3H, d, J = 6.6 Hz, H-21), 0.91 (3H, d, J = 6.8 Hz, H-28), 0.90 (3H, s, H-18), 0.82 (3H, d, J = 6.7 Hz, H-27), 0.80 (3H, d, J = 6.8 Hz, H-26); ¹H NMR (600 MHz, C₅D₅N) δ 5.35 (1H, dd, J = 15.3, 7.1 Hz, H-23), 5.29 (1H, dd, J = 15.3, 7.9 Hz, H-22), 4.57 (2H, m, H-6 and H-7), 4.38 (1H, brs, H-3), 1.40 (3H, s, H-19), 1.23 (3H, d, J = 5.9 Hz, H-21), 1.19 (3H, s, H-18), 0.88 (3H, d, J = 6.8 Hz, H-28), 0.80 (3H, d, J = 6.0 Hz, H-27), 0.79 (3H, d, J = 6.0 Hz, H-26).

Supplemental Material

sj-docx-1-npx-10.1177_1934578X231171513 - Supplemental material for Polyhydroxy Sterols Isolated From Ganoderma sinense Spores and Their Cytotoxic Activities

Supplemental material, sj-docx-1-npx-10.1177_1934578X231171513 for Polyhydroxy Sterols Isolated From Ganoderma sinense Spores and Their Cytotoxic Activities by Danhong Lian, Xin Zhong, Lian Li, Li Gu, Yimei Zheng and Xin Liu in Natural Product Communications

Footnotes

Acknowledgments

The authors would like to thank Instrumental Analysis & Research Center, Sun Yat-Sen University, for NMR and HRESI measurements.

Declaration of Conflicting Interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This research was financially supported by the National Nature Science Foundation (81503333) of the People's Republic of China.

ORCID iD

Danhong Lian

Supplemental Material

Supplemental material for this article is available online.

References

Sun

Lebreton

Xing

, et al. Phylogenomics and comparative genomics highlight specific genetic features in Ganoderma species. J Fungi (Basel). 2022;8(3):311.

Sun

Xing

, et al. Species diversity, systematic revision and molecular phylogeny of Ganodermataceae (Polyporales, Basidiomycota) with an emphasis on Chinese collections. Stud Mycol. 2022;101:287-415.

Zhang

Zheng

Wang

Magnusson

Liu

. Lipid extract from completely sporoderm-broken germinating Ganoderma sinensis spores elicits potent antitumor immune responses in human macrophages. Phytother Res. 2009;23(6):844-850.

Lin

Zhu

, et al. Extract of Ganoderma sinensis spores induces cell cycle arrest of hepatoma cell via endoplasmic reticulum stress. Pharm Biol. 2021;59(1):704-714.

Lian

Zhong

Zheng

, et al. A new sterol from sporoderm-broken Ganoderma sinense spores and its anticancer activity. Nat Prod Commun. 2019;14(12):1-6.

Yaoita

Matsuki

Iijima

, et al. New sterols and triterpenoids from four edible mushrooms. Chem Pharm Bull (Tokyo). 2001;49(5):589-594.

Ishizuka

Yaoita

Kikuchi

. Sterol constituents from the fruit bodies of Grifola frondosa (FR.) S. F. Gray. Chem Pharm Bull (Tokyo). 1997;45(11):1756-1760.

Zang

Xiong

Zhai

, et al. Fomentarols A-D, sterols from the polypore macrofungus Fomes fomentarius. Phytochemistry. 2013;92:137-145.

Lee

Min

Choi

, et al. A new sesquiterpene lactone from Artemisia rubripes nakal. Arch Pharm Res. 2004;27(10):1016-1019.

10.

Bai

Pei

. Secondary metabolites from endophyte fungus Fusariums sp. LC-1. Chinese Pharmaceutical Journal. 2013;48(1):17-21.

11.

Liu

Chen

Wang

, et al. Cytotoxicity of lanostane-type triterpenoids and ergosteroids isolated from Omphalia lapidescens on MDA-MB-231 and HGC-27 cells. Biomed Pharmacother. 2019;118:109273.

12.

Shi

Huang

, et al. C(28) steroids from the fruiting bodies of Ganoderma resinaceum with potential anti-inflammatory activity. Phytochemistry. 2019;168:112109.

13.

Demarco

Farkas

Doddrell

, et al. Pyridine-induced solvent shifts in the nuclear magnetic resonance spectra of hydroxylic compounds. J Am Chem Soc. 1968;90:5480-5486.

14.

Qin

Gao

Zhang

, et al. Polyhydroxylated steroids from an endophytic fungus, Chaetomium globosum ZY-22 isolated from Ginkgo biloba. Steroids. 2009;74(9):786-790.

15.

Harrison

. The biosynthesis of triterpenoids, steroids, and carotenoids. Nat Prod Rep. 1988;5(4):387-415.

16.

Wright

JLC

Mcinnes

Shimizu

, et al. Identification of C-24 alkyl epimers of marine sterols by ¹³C nuclear magnetic resonance spectroscopy. Can J Chem. 1978;56:1898-1903.

17.

Liu

Wang

Yuan

. Pharmacological and anti-tumor activities of ganoderma spores processed by top-down approaches. J Nanosci Nanotechnol. 2005;5(12):2001-2013.

Supplementary Material

Please find the following supplemental material available below.

For Open Access articles published under a Creative Commons License, all supplemental material carries the same license as the article it is associated with.

For non-Open Access articles published, all supplemental material carries a non-exclusive license, and permission requests for re-use of supplemental material or any part of supplemental material shall be sent directly to the copyright owner as specified in the copyright notice associated with the article.

0.00 MB

5.32 MB