Anti-allergic prenylated hydroquinones and alkaloids from the fruiting body of Ganoderma calidophilum

Abstract

Six new prenylated hydroquinones named ganocalidin A–F (1–6) and two new compounds of ganocalicine A (7) and B (8), together with sixteen known compounds (9–24) were isolated from an EtOAc extract of the fruiting body of Ganoderma calidophilum. The new compound structures were elucidated on the basis of spectroscopic data analysis including 1D, 2D NMR and MS. Compounds 1 and 7 showed inhibitory activity effects on β-hexosaminidase activity (IC₅₀ 9.44 and 9.14 μM, respectively), and significantly reduced the production of IL-4 and LTB₄ by RBL-2H3 cells in response to antigen stimulation, suggesting that 1 and 7 possess anti-allergic activity.

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Introduction

Following changes to modern lifestyle and the ecological environment, the increasing prevalence of allergies has become a significant health problem worldwide, especially in westernized countries. Although a large number of allergy therapeutic agents have been introduced into clinical use, associated side effects are occasionally reported, such as dizziness, hypertrichosis, stomach upsets, and blurred vision.¹ Chinese medicinal herbs, as alternative therapies, have become a promising source for potential new anti-allergic drugs, and natural products have been proposed as anti-allergic drug candidates due to their great immunomodulatory efficacy and established safety records.² The Ganoderma species (Ganodermataceae), well known as a nostrum in Chinese myth, are widely used as functional foods and traditional medicine in Asian countries for a series of functions, such as nourishment, sedation, anti-inflammation, lowering blood pressure, and treating high blood fat and blood sugar (Z. B. Lin, 2007). Nowadays, there are a series of sterols, triterpenoids, and a few prenylated hydroquinones isolated from Ganoderma species with extensive biological and pharmacological activities, including cytotoxic (Huang, Liaw, Yang, Hseu, Kuo, Tsai, et al., 2012; Ma, Luo, Huang, Guo, Dai, & Zhao, 2013; X. Peng, Liu, Xia, Wang, Li, Deng, et al., 2015), hepatoprotective (L. Y. Liu, Chen, Liu, Wang, Kang, Li, et al., 2014; X. R. Peng, Liu, Han, Yuan, Luo, & Qiu, 2013), anti-inflammatory (Tung, Cuong, Hung, Lee, Woo, Choi, et al., 2013), and antioxidant (K. W. Lin, Chen, Yang, Wei, Hung, & Lin, 2013; Thang, Kuo, Hwang, Yang, Ngoc, Han, et al., 2013) properties. Ganoderma calidophilum is a rare wild fungi in the Ganodermataceae family, distributed in tropical areas of China, and its fruiting body of G. calidophilum is traditionally used in the treatment of allergic asthma, eczema, and allergic rhinitis, suggesting it may harbor anti-allergic natural components.^3,4 A series of sterols, triterpenoids, and prenylated hydroquinones isolated from Ganoderma species have been reported, possessing extensive biological and pharmacological activities.^5–7 However, the anti-allergic components of G. calidophilum are still unclear due to the lack of the natural resource. In order to investigate the anti-allergic components in G. calidophilum, the study of the chemical composition of G. calidophilum was carried out, and six new prenylated hydroquinones named ganocalidin A–F (1–6) and two alkaloids of ganocalicine A (7) and B (8) (Fig. 1) were obtained, along with twelve known compounds: (+)-fornicin C (9),⁸ jacareubin (10),⁹ ganodermadiol (11),¹⁰ ganoderone A (12),¹¹ lucidadiol (13),¹² ergosterol endoperoxide (14),¹³ 5α,6α-epoxy-(22E,24R)-ergosta-8,22-diene-3β-7α-diol (15),¹⁴ stellasterin (16),¹⁵ 5α,8α-dihydroxyl-(24R)-ergosta-6Z,22E-diene-3β-5α,8α-triol (17),¹⁶ 6-methoxyl-cerevisterol (18),¹⁷ 6-dehydrocerevisterol (19),¹⁸ 3,4-dihydroxybenzoic acid (20),¹⁹ 4-(2-hydroxy-4-methoxybutyl)-2-methoxyphenol (21),²⁰ 3-(3,4-dihydroxyphenyl) propanoic acid (22),²¹ 1-(4-hydroxy-3,5-dimethoxyphenyl) ethanone (23),²² and 1-(3,4-dihydroxyphenyl) ethanone (24)²³ were isolated. All of the known compounds were identified on the basis of detailed spectroscopic analysis in the literature. This study focused on the isolation and structural elucidation of the new compounds from G. calidophilum, and their anti-allergic activities were also evaluated.

Fig. 1 Structures of compounds 1–8.

Results and discussion

Structural elucidation of 1–8

Compound 1 was obtained as a colorless oil, and its molecular formula was assigned to be C₂₁H₂₄O₆ from its HREIMS (m/z 372.1570 [M]⁺, calcd for C₂₁H₂₄O₆, 372.1573) and NMR data (Table 1). The IR spectrum displayed the presence of hydroxyl (3408 cm⁻¹) and double bond (1595, 1419 cm⁻¹) absorptions. Analysis of its ¹³C NMR and DEPT spectra (Table 1) showed 21 carbon resonances, including one methyl, six methylenes (two oxygenated), six methines (six olefinic), and eight quaternary carbons (one carbonyls, six olefinic, and one ketal). The ¹³C NMR data of 1 were similar to those of fornicin B,⁸ a rare prenylated hydroquinone, except for additional shifts at δ_C 70.4 (d, C-12), 60.0 (d, C-13) and the absence of δ_C 25.8 (t, C-12), 17.7 (t, C-13), and 56.9 (q, –OMe) in fornicin B, indicating that C-12 and C-13 were oxygenated to be hydroxyls, and one of the hydroxyl substituted –OMe formed a C-1 ketal in compound 1. The HMBC (Fig. 2) correlations of 1 from H-12 [δ_H 4.29 (1H, d, J = 13.1 Hz) and 3.90 (1H, d, J = 13.1 Hz)], H-21 [δ_H 6.95 (1H, s)], and H-2 [δ_H 7.17 (1H, s)] to C-1 (δ_C 107.4 s) and from H-12 to C-10 (δ_C 137.1 d), and from H-13 [δ_H 4.49 (1H, d, J = 11.9 Hz) and 4.15 (1H, d, J = 11.9 Hz)] and H-9 [δ_H 2.46 (1H, m) and 2.14 (1H, m)] to C-11 (δ_C 133.4 s) confirmed this hypothesis. The other correlations in the ¹H–¹H COSY and HMBC spectrum further confirmed the atom connectivity in compound 1. The geometry of the double bonds as 2Z, 6E, and 10E, were proposed by NOE (Fig. 2) H-2 [δ_H 7.17 (1H, s)]/H-4 [δ_H 2.35 (2H, s)], H-6 [δ_H 4.94 (1H, dd, J = 9.0, 8.8 Hz)]/H-8 [δ_H 2.26 (1H, m) and 2.14 (1H, m)], and H-10 [δ_H 5.20 (1H, dd, J = 11.0, 10.4 Hz)]/H-12, respectively. Thus, the structure of compound 1 was assigned as shown, and named ganocalidin A.

Table 1 ¹H NMR and ¹³C NMR Data of 1–8^a

No	1^a		2^b		3^b		4^b		5^a		6^a		No	7^a		8^a
	δH multi, J (Hz)	δC	δH multi, J (Hz)	δC	δH multi, J (Hz)	δC	δH multi, J (Hz)	δC	δH multi, J (Hz)	δC	δH multi, J (Hz)	δC		δH multi, J (Hz)	δC	δH multi, J (Hz)	δC
a ^1H, ^13C NMR data measured at 500 and 125 MHz, respectively. ^a in CDCl3. ^b in CDCl3 + CD3OD.
1		107.4 s		204.6 s	5.07 d, 1H, 6.4	78.6 d		197.1 s		108.6 s		204.2 s	2		147.8 s		145.6 s
2	7.17 s, 1H	146.6 d	3.40 dd, 1H, 17.9, 9.1	40.5 t	7.26 d, 1H, 6.4	149.5 d	7.67 s, 1H	131.4 d	7.40 s, 1H	148.3 d	4.02 s, 2H	37.5 t	3		123.2 s		123.4 s
			3.05 dd, 1H, 17.9, 4.5
3		134.3 s	2.95 m, 1H	40.2 d		131.6 s		144.6 s		135.7 s		129.0 s	4	2.99 ddd, 1H, 16.8, 8.6, 3.7	28.1 t	2.99 t, 1H 7.4	30.3 t
														2.75 ddd, 1H, 16.8, 7.7, 6.4
4	2.35 m, 2H	25.2 t	1.72 m, 1H	32.4 t	2.05 m, 2H	26.8 t	2.64 t, 2H, 7.5	27.8 t	2.32 m, 2H	25.9 t	6.97 t, 1H, 7.2 z	145.5 d	5	2.34 m, 1H	32.2 t	2.20 tt, 2H, 7.5, 7.4	25.0 t
			1.58 m, 1H											2.24 m, 1H
5	2.34 m, 1H	23.4 t	2.08 m, 1H	25.9 t	2.42 m, 1H	25.4 t	2.18 dt, 2H, 7.5, 7.4	26.8 t	2.26 m, 2H	40.6 t	2.18 dd, 2H, 7.3, 7.2	28.8 t	6	4.86 dd, 1H, 6.3, 2.8	82.8 d	2.88 t, 1H, 7.5	30.2 t
	2.27 m, 1H		1.72 m, 1H		2.24 m, 1H
6	4.94 dd, 1H, 9.0, 8.8	126.1 d	4.94 dd, 1H, 9.0, 8.8	126.1 d	4.94 dd, 1H, 9.0, 8.8	126.1 d	5.09 t, 1H, 7.4	123.2 d	5.09 t, 1H, 7.2	124.1 d	5.13 t, 1H, 7.3	121.7 d	7	8.56 s, 1H	145.5 d	8.39 s, 1H	143.7 d
7		135.0 s		135.0 s		135.6 s		135.2 s		137.6 s		137.8 s	8		150.0 s		149.7 s
8	2.26 m, 1H	39.1 t	2.06 m, 2H	38.5 t	2.02 m, 2H	37.7 t	1.93 t, 2H, = 7.9	37.6 t	1.94 m, 2H	40.6 t	2.05 t, 2H, 7.6	39.1 t	9		138.3 s		140.2 s
	2.14 m, 1H
9	2.46 m, 1H	23.6 t	2.25 m, 1H	27.6 t	2.31 m, 1H	24.8 t	2.15 dt, 2H, 7.9, 7.3	27.2 t	2.11 m, 2H	26.9 t	2.22 dt, 2H, 7.6, 7.4	28.1 t	10	5.11 d, 1H, 13.7	65.4 t	5.16 s, 1H	65.6 t
	2.02 m, 1H		2.07 m, 1H		2.22 m, 1H									5.02 d, 1H, 13.7
10	5.20 dd, 1H, 11.0, 10.4	137.1 d	6.77 dd, 1H, 8.9, 2.4	143.2 d	6.71 m, 1H	143.2 d	6.77 t, 1H, 7.3	142.9 d	5.50 t, 1H, 7.3	130.2 d	6.68 t, 1H, 7.4	143.4 d	1′		123.6 s		123.7 s
11		133.4 s		128.0 s		127.1 s		127.1 s		139.2 s		127.5 s	2′		151.6 s		151.4 s
12	4.29 d, 1H, 13.1	70.4 t		171.3 s		171.0 s		170.7 s	4.07 s, 2H	65.6 t		170.7 s	3′	6.78 d, 1H, 8.7	119.0 d	6.87 d, 1H 8.6	118.4 d
	3.90 d, 1H, 13.1
13	4.49 d, 1H, 11.9	60.0 t	1.77 s, 3H	12.4 q	1.73 s, 3H	11.8 q	1.75 s, 3H	11.6 q	4.13 s, 2H	58.3 t	1.73 s, 3H	12.5 q	4′	6.74 dd, 1H, 8.7, 2.9	118.1 d	6.85 dd, 1H, 8.6, 2.3	118.2 d
	4.15 d, 1H, 11.9
14	1.61 s, 3H	15.0 q	1.59 s, 3H	16.1 q	1.55 s, 3H	15.5 q	1.55 s, 3H	15.3 q	1.48 s, 3H	16.1 q	1.59 s, 3H	16.3 q	5′		149.9 s		150.1 s
15		172.2 s		178.5 s		175.6 s		168.9 s		173.4 s		170.6 s	6′	7.58 d, 1H, 2.9	110.8 d	7.69 d, 1H 2.3	110.5 d
16		124.4 s		119.4 s		121.9 s		119.6 s		123.6 s		120.5 s	6-OMe	3.42 s, 3H	56.5 q
17		147.1 s		155.6 s		147.0 s		155.7 s		149.3 s		156.5 s
18	6.70 m, 1H	118.3 d	6.77 d, 1H, 8.9	119.1 d	6.64 d, 1H, 8.7	116.2 d	6.83 d, 1H, 8.9	118.6 d	6.67 m, 1H	118.4 d	6.74 d, 1H, 8.9	119.7 d
19	6.69 m, 1H	117.9 d	6.99 dd, 1H, 8.9, 2.9	125.4 d	6.59 d, 1H, 8.7	116.1 d	7.04 dd, 1H, 8.9, 2.9	125.4 d	6.66 m, 1H	118.2 d	6.95 dd, 1H, 8.9, 2.9	125.8 d
20		149.8 s		149.3 s		148.6 s		148.8 s		151.2 s		150.7 s
21	6.95 s, 1H	112.5 d	7.20 d, 1H, 2.9	114.8 d	6.49 s, 1H	112.0 d	7.14 d, 1H, 2.9	114.7 d	6.96 s, 1H	114.6 d	7.29 d, 1H, 2.9	115.6 d
OMe								3.26 s, 3H	52.2 q

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Fig. 2 Key HMBC and ¹H–¹H COSY correlations of 1–8 (HMBC →, ¹H–¹H COSY [thick line, graph caption]).

Compound 2 was assigned the molecular formula C₂₁H₂₆O₇ according to HRESIMS analysis (m/z 389.1597 [M − H]⁻, calcd for C₂₁H₂₅O₇, 389.1600). The ¹³C NMR (DEPT) spectroscopic data (Table 1) showed two methyls, five methylenes, six methines (five olefinic), and eight quaternary carbons (five olefinic, one ketone carbonyl, and two carboxyls). The ¹H and ¹³C NMR data of 2 were similar to those of 9, except for the shifts at δ_C 171.3 (s, C-12) in compound 2, and the substituent of δ_C 25.7 (q, C-12) in 9, revealed that the methyl group in C-12 was oxygenated to a carboxyl group in compound 2. The HMBC (Fig. 2) correlations of 2 from H-13 [δ_H 1.77 (3H, s)] to C-12 and C-11 (δ_C 128.0 s) further confirmed this hypothesis. The configuration of compound 2 was elucidated by ROESY experiments (Fig. 3) and was determined to be the same as that of compound 1 with E configurations for Δ^6,7 and Δ^10,11. The orientation of C-3 was difficult to determine. Therefore, the structure of compound 2 was elucidated as shown and named ganocalidin B.

Fig. 3 Key ROESY correlations of 1–7.

Compound 3 was defined with the molecular formula C₂₁H₂₄O₆ from HREIMS (m/z 372.1757 [M]⁺, calcd for C₂₁H₂₄O₆, 372.1572). The ¹³C NMR data (Table 1) of 3 were similar to those of fornicin B,⁴ except for the shifts at δ_C 78.6 (d, C-1) and 146.3 (s, C-12) in compound 3 instead of the responding carbons at δ_C 107.6 (s, C-1) and 25.8 (q, C-12) in fornicin B, and a methoxy carbon δ_C 56.9 (q, 1-OMe) was lost, indicating that the methoxy group at the ketal carbon C-1 was removed and the methyl group of C-12 was oxygenated to a carboxyl group in compound 3. The HMBC (Fig. 2) correlations of 3 from H-1 [δ_H 5.07 (1H, d, J = 6.4 Hz)] to C-3 (δ_C 131.6 s) and C-17 (δ_C 147.0 s), and from H-13 [δ_H 1.73 (3H, s)] to C-11 (δ_C 127.1 s) and C-12, together with the key ¹H–¹H COSY correlations H-1/H-2 [δ_H 7.26 (1H, d, J = 6.4 Hz)], confirmed this hypothesis. The other correlations in the HMBC and ¹H–¹H COSY spectrum further confirmed the atom connectivity in compound 3 (Fig. 2). The configuration of the skeleton in compound 3 was elucidated by ROESY experiments (Fig. 3) and was determined to be the same as that of compound 2, with a Z configuration for Δ^2,3 and E configurations for Δ^6,7 and Δ^10,11. The orientation of C-2 was difficult to determine. Thus, the structure of compound 3 was assigned as shown and named ganocalidin C.

Compound 4 was shown to have the molecular formula C₂₁H₂₄O₇ from HRESIMS (m/z 411.1413 [M + Na]⁺, calcd for C₂₁H₂₄O₇Na, 411.1420). Comparison of its 1D NMR data (Table 1) with 2 suggested that compound 4 had a similar structure to 2. The differences were additional shifts at δ_C 131.4 (d, C-2) and 144.6 (s, C-3) in 4 instead of shifts at δ_C 40.5 (t, C-2) and 40.2 (d, C-3) in compound 2, suggesting that a double bond was generated between C-2 and C-3 in 4. The key HMBC (Fig. 2) correlations of 4 from H-2 [δ_H 7.67 (1H, s)] to C-1 (δ_C 197.1 s), C-15 (δ_C 168.9 s) and C-17 (δ_C 155.7 s), and from H-5 [δ_H 2.18 (2H, dt, J = 7.5, 7.4 Hz)] to C-3 (δ_C 144.6 s) further supported the hypothesis. The other correlations in the HMBC and ¹H–¹H COSY spectrum (Fig. 2) further confirmed the atom connectivity in compound 4. The configurations of 2Z, 6E, and 10E in compound 4 were elucidated by NOE H-2/H-4 [δ_H 2.64 (2H, t, J = 7.5 Hz)], H-5/H-14 [δ_H 1.55 (3H, s)], H-6 [δ_H 5.09 (1H, t, J = 7.4 Hz)]/H-8 [δ_H 1.93 (2H, t, J = 7.9 Hz)], and H-9 [δ_H 2.15 (2H, dt, J = 7.9, 7.3 Hz)]/H-13 [δ_H 1.75 (3H, s)]. Thus, the structure of compound 7 was assigned as shown and named ganocalidin D.

Compound 5 was defined with the molecular formula C₂₂H₂₈O₇ from HRESIMS (m/z 403.1752 [M − H]⁻, calcd for C₂₂H₂₇O₇, 403.1757). The ¹H and ¹³C NMR data (Table 1) of 5 were similar to those of fornicin B,⁸ except for the signals at δ_C 65.6 (t, C-12) and 58.3 (t, C-13) in compound 5 replacing signals at δ_C 25.8 (s, C-12) and 17.7 (d, C-13) in fornicin B, meaning that the C-12 and C-13 were oxygenated to be methylol groups in compound 5. The HMBC (Fig. 2) correlations of 5 from H-12 [δ_H 4.07 (2H, s)] and H-13 [4.13 (2H, s)] to C-10 (δ_C 130.2 d) and C-11 (δ_C 139.2 d) confirmed this structural change. The other correlations in the HMBC and ¹H–¹H COSY spectrum (Fig. 2) further determined the atom connectivity in compound 5. The configurations of 2Z and 6E in compound 4 were elucidated by the ROESY experiments (Fig. 3). The orientation of C-2 was difficult to determine. Thus, the structure of compound 5 was assigned as shown and named ganocalidin E.

Compound 6 was formulated as C₂₁H₂₄O₃ from HRESIMS (m/z 387.1444 [M + Na]⁺, calcd for C₂₁H₂₃O₇, 387.1450). Compound 6 had similar ¹³C NMR data (Table 1) to those of 4, and the differences were shifts of δ_C 37.5 (t, C-2), 129.0 (s, C-3), and 145.5 (d, C-4) in 6, replacing shifts of δ_C 131.4 (d, C-2), 144.6 (s, C-3), and 27.8 (d, C-4) in 4, indicating that in compound 6, the double bond moved from C-2-C-3 to C-3-C-4. The HMBC correlations from H-2 [δ_H 4.02 (2H, s)] to C-15 (δ_C 170.6 s), C-16 (δ_C 120.5 s), and C-3 (δ_C 129.0 s), and from H-4 [δ_H 6.97 (H, t, J = 7.2 Hz)] to C-15, and the ¹H–¹H COSY correlations (Fig. 2) of H-4 with H-5 [δ_H 2.18 (2H, dd, J = 7.3, 7.2 Hz)] confirmed the hypothesis and the atom connectivity in compound 6. The configuration of compound 6 was determined to possess three trans-vinyl groups as shown in the structure by ROESY experiments (Fig. 3). Thus, the structure of compound 6 was assigned as shown and named ganocalidin F.

Compound 7 was assigned to be C₁₆H₁₅NO₃ by HREIMS (m/z 269.1051 [M]⁺, calcd for C₁₆H₁₅NO₃, 269.1051). Analysis of its ¹³C NMR and DEPT spectra (Table 1) showed 16 carbon resonances, including one methoxyl, three methylenes (one oxygenated), five methines (six olefinic and one oxygenated), and seven olefinic quaternary carbons. The ¹³C NMR data (Table 1) of 7 were similar to those of sinesine E, a rare ganoderma alkaloid, except for one additional shift at δ_C 56.5 (6-OMe) and the shift at δ_C 73.9 (d, C-6) replacing δ_C 82.8 (t, C-6) in sinesine E,²⁴ indicating that the hydroxyl group at C-6 was substituted by a methoxyl group in compound 7. The HMBC (Fig. 1) correlations of 7 from 6-OMe [δ_H 3.42 (3H, s)] to C-6 (δ_C 82.8 s) and ¹H–¹H COSY correlations from H-5 [δ_H 2.34 (1H, m) and 2.24 (1H, m)] to H-4 [δ_H 2.99 (1H, ddd, J = 16.8, 8.6, 3.7 Hz) and 2.75 (1H, ddd, J = 16.8, 7.7, 6.4 Hz)] and H-6 [δ_H 4.86 (1H, dd, J = 6.3, 2.8 Hz)] confirmed this hypothesis. The configuration of C-6 (R) was confirmed by comparing the NMR spectroscopy, specific rotation,²⁴ and the NOE correlation H-6/H-4β [δ_H 2.99 (1H, ddd, J = 16.8, 8.6, 3.7 Hz)]. Finally, compound 7 was determined and named ganocalicine A.

Compound 8 was defined with the molecular formula C₁₅H₁₃NO₂ from HREIMS (m/z 239.0940 [M]⁺, calcd for C₁₅H₁₃NO₂, 239.0946). Comparison of the similar ¹³C NMR data (Table 1) of 8 with those of 7 showed that the carbon signals at δ_C 30.2 (t, C-6) in 8 replaced those of δ_C 82.8 (d, C-6) and 56.6 (q, 6-OMe) in 7, indicating that the methoxy group at C-6 in 7 was removed in 8. This deduction was proved by the HMBC and ¹H–¹H COSY (Fig. 2) correlations in compound 8. Thus, the structure of compound 8 was assigned as shown and named ganocalicine B.

Bioactivity evaluation

In immediate-type hypersensitivity reactions, antigen-specific IgE binds to mast cells and basophils and is cross-linked by an allergen, which phosphorylates intracellular tyrosine kinase cascades and then triggers the release of preformed chemical mediators, and then results in the multiorgan symptoms of an allergy.²⁵ Antigen-stimulated RBL-2H3 cells have been used as principal models for the activation of mast cells and basophils in immediate allergic reactions to test the efficacy of new anti-allergy therapies by measuring the release of β-hexosaminidase, a well-established granule marker, as well as by analyzing the production of degranulation-independent mediators, such as leukotrienes and interleukin-4.²⁶ As shown in Fig. 4, RBL-2H3 cells sensitized with dinitrophenyl specific immunoglobulin E (DNP-IgE) and challenged with dinitrophenyl-bovine serum albumin (DNP-BSA) produced a significant release of β-hexosaminidase, and increased concentrations of IL-4 and LTB₄ (Fig. 4).

Fig. 4 Effect of compounds from G. calidophilum on the cell viability (a) and the release of β-hexosaminidase or IL-4 or LTB₄ (b–d) in DNP-BSA-activated RBL-2H3 cells. (a) Cytotoxic effects of compounds 1–8 on RBL-2H3 cells. The percentage survival of RBL-2H3 cells is shown following 24 h of incubation with 1–8 (10 μM). (b) The effects of compounds 1 and 7 on the release of β-hexosaminidase in DNP-BSA-activated RBL-2H3 cells. RBL-2H3 cells were seeded on a 24-well plate (1 × 10⁵ cells per well) in DMEM with 5% FBS at 37 °C, and incubated with DNP-IgE for 24 h. DNP-IgE-sensitized cells were exposed to 1 and 7 (5–10 μM) for 1 h, and then stimulated with DNP-BSA (1 μg ml⁻¹) for 1 h. The β-hexosaminidase activity was determined as described in the Experimental section. (c) Inhibition of IL-4 production by 1 and 7 in RBL-2H3 cells. (d) 1 and 7 decrease the secretion of LTB₄ in RBL-2H3 cells. Data are presented as the mean ± SD values of triplicate determinations. *P < 0.01, **P < 0.05, ***P < 0.001 versus DNP-BSA-treated group (n = 3).

Among the isolated compounds from G. calidophilum, the impact of the new compounds 1–8 on cell viability was firstly evaluated (each compound was evaluated between 1–10 μM), and no significant cytotoxicity was observed in the RBL-2H3 cells (Fig. 4a). Compounds 1–8 were further tested for their effects on the release of β-hexosaminidase in DNP-IgE-sensitized RBL-2H3 cells, and two compounds (1 and 7) showed higher inhibitory activities (IC₅₀ = 9.44 ± 1.95 and 9.14 ± 2.12 μM) than ketotifen fumarate, a positive control (IC₅₀ = 203.27 ± 10.81 μM) (Fig. 4b). However, no significant difference was observed for the inhibition of β-hexosaminidase by the other isolated compounds (2–6, 8) compared to the DNP-BSA-treated group. To investigate the effect of the compounds on the production of IL-4 and LTB₄ from activated mast cells, RBL-2H3 cells were sensitized with 1 μg ml⁻¹ DNP-IgE overnight and treated with 1–8 for 1 h, followed by treatment with DNP-BSA for 1.5 h. The result showed that treatment of sensitized RBL-2H3 with 1 and 7 (5–10 μM) significantly suppressed the production of the allergic cytokine IL-4 (Fig. 4). In addition, 1 and 7 (5–10 μM) also reduced the release of the lipid mediator LTB₄ in activated RBL-2H3, compared to untreated sensitized RBL-2H3 (Fig. 4).

These results implied that 1 and 7 might inhibit the activation of the FcεRI receptor-mediated intracellular signaling cascade molecules, which are responsible for allergic responses in antigen-stimulated RBL-2H3 cells. After the cross-linking of the IgE-bound FcεRI by an allergen, the intracellular spleen tyrosine kinase (Syk) becomes phosphorylated (p-Syk) to propagate downstream signaling. Activated Syk dependent signaling events increase the intracellular calcium level, which leads to mast cell degranulation, releasing a range of preformed and newly synthesized mediators that evoke a potent immune allergic response. While preformed mediators such as histamine, chemotactic factors, tryptases and chymases are stored in granules and released upon exocytosis, newly synthesized mediators such as prostaglandin, the leukotrienes and platelet activating factor are produced and secreted to the mucosal tissue, which provoke a variety of allergic symptoms. Therefore, by measuring the release of β-hexosaminidase, allergic cytokine and lipid mediator, it is revealed that compounds 1 and 7 from G. calidophilum are capable of inhibiting mast cell degranulation and the release of allergic mediators. This finding indicates that 1 and 7 are potential drugs to prevent or relieve allergic symptoms.

Conclusion

Mast cells play a critical role in immediate-type hypersensitivity reactions, and mast cell mediators are implicated in many different conditions including allergic rhinitis, conjunctivitis, asthma, and psoriasis.^27,28 Thus, there is intense interest in the development of agents which prevent mast cell mediator release or inhibit the actions of such mediators once released into the environment of the cell. As a complementary and alternative medicine for allergic asthma and rhinitis, the Ganoderma species (Ganodermataceae) is popularly prescribed in anti-allergic herbal formulae.² The pursuit of the compounds responsible for anti-allergic agents in G. calidophilum led to the isolation of six new prenylated hydroquinones and two new alkaloids, and anti-allergy assay results indicated that two compounds (1 and 7) are capable of inhibiting mast cell degranulation and the release of allergic cytokines, suggesting that they have potent anti-allergy abilities, which may aid drug development for anti-allergic treatment. The study of the bioactive components firstly clarified the structures of the prenylated hydroquinones and alkaloids in G. calidophilum, and revealed the chemical basis for the usage of G. calidophilum in anti-allergy applications.

Experimental section

General

Optical rotations were measured on a Cataceo Co. LTD Polar-L polarimeter. UV spectra were obtained on a Shimadzu UV-250 spectrometer. IR spectra were obtained on a Thermo Nicolet 380 spectrometer with KBr pellets. NMR spectra were recorded on a Bruker AV-500 spectrometer with TMS as an internal standard. ESIMS, HRESIMS, and HREIMS were recorded with a Micromass Autospec-Uitima-TOF, API QSTAR Pulsar 1, or a Waters Autospec Premier spectrometer. Silica gel (200–300 mesh, Qingdao Marine Chemical Inc., China), RP-18 (40–70 μm, Fuji Silysia Chemical Ltd., Japan) and Sephadex LH-20 (Amersham Biosciences, Sweden) were used for column chromatography. Semipreparative HPLC was performed on an Agilent 1100 liquid chromatograph with a Zorbax SB-C₁₈, ϕ 5 μm, 9.4 mm × 25 cm, column. Fractions were detected by TLC and spots were visualized by heating after spraying with 7% H₂SO₄ in ethanol.

Plant material

Fruiting bodies of Ganoderma calidophilum were collected in Qiongzhong country, Hainan Province, People’s Republic of China, and identified by Prof. X. L. Wu (Hainan University). A voucher specimen (HUANG00⁰⁷) was deposited at the Key Laboratory of Biology and Genetic Resources of Tropical Crops, Ministry of Agriculture, Institute of Tropical Bioscience and Biotechnology, Chinese Academy of Tropical Agriculture Sciences, Haikou, People’s Republic of China.

Extraction and isolation

The dried and powdered fruiting bodies of Daphne acutiloba (2.5 kg) were extracted with 95% EtOH under reflux four times (4 × 20 L). The extract was concentrated and suspended in water followed by extraction with EtOAc (3 × 4 L). The EtOAc extract (246 g) was separated by a silica gel column using a gradient solvent CHCl₃/MeOH (9 : 1–3 : 1 10 L) to afford fractions A–C. Fraction A was surrendered for a large quantity of oil with a TLC test. Fraction B (97 g) was separated by a silica gel column using a gradient solvent CHCl₃/MeOH (15 : 1–5 : 1) to afford fractions B1–B5. Fractions B1–B5 (1–5 g) were subjected to repeated RP-18 (MeOH/H₂O, 50%, 60%, 70%, 80%, 90%, and 100%), Saphadex LH-20 (MeOH) and semi-preparative HPLC (MeOH/H₂O, 37 : 63) to yield 9 (8.6 mg), 10 (72.3 mg) and 11 (2.1 g) from B1; 12 (7.6 mg), 14 (42.1 mg), 17 (8.9 mg), and 18 (20.2 mg) from B2; 13 (11.3 mg), 15 (24.6 mg), and 16 (24.0 mg) from B3; 17 (54.9 mg), 18 (34.6 mg), 19 (27.1 mg), and 20 (8.2 mg) from B4; and 1 (7.6 mg), 7 (15.2 mg), and 8 (16.2 mg) from B5, respectively; fraction C (36 g) was subjected to repeated RP-18 (MeOH/H₂O, 50%, 60%, 70%, 80%, 90%, and 100%), Saphadex LH-20 (MeOH) and semi-preparative HPLC (MeOH/H₂O, 37 : 63) to yield 2 (5.9 mg), 3 (5.1 mg), 4 (7.6 mg), 5 (9.8 mg), 6 (25.2 mg), 21 (68.4 mg), 22 (5.9 mg), 23 (3.8 mg), 24 (6.7 mg).

Ganocalidin A (1). Orange oil; [α]^30.1_D −7.70 (c 0.10, MeOH); UV (MeOH) λ_max (log ε) 191 (5.16), 192 (5.16), 197 (5.16), 272 (4.39) nm; IR (KBr) ν_max 3408, 2922, 1595, 1419, 1118, 1040 cm⁻¹; ¹H and ¹³C NMR data see Table 1; ESIMS negative m/z [M − H]⁻ 371 (100); HREIMS m/z [M]⁺ 372.1570 (calcd for C₂₁H₂₄O₆, 372.1573).

Ganocalidin B (2). Orange oil; [α]^18.3_D −5.60 (c 0.30, MeOH); UV (MeOH) λ_max (log ε) 191 (5.21), 203 (5.01), 269 (4.67), 357 (3.86), 360 (3.86) nm; IR (KBr) ν_max 3346, 2920, 1651, 1645, 1634, 1372, 1159, 1110, 1060, 1005, 559 cm⁻¹; ¹H and ¹³C NMR data see Table 1; ESIMS positive m/z [M + Na]⁺ 413 (100); HREIMS negative m/z [M − H]⁻ 389.1597 (calcd for C₂₁H₂₅O₇, 389.1600).

Ganocalidin C (3). Orange oil; [α]^30.1_D −0.24 (c 0.17, MeOH); UV (MeOH) λ_max (log ε) 194 (4.76), 196 (4.76), 203 (4.48), 205 (4.47), 210 (4.45), 291 (3.70), 372 (3.43) nm; IR (KBr) ν_max 3422, 2924, 2360, 1639, 1027, 410 cm⁻¹; ¹H and ¹³C NMR data see Table 1; EIMS m/z [M]⁻ 372 (20), 204 (20), 137 (100); HREIMS m/z [M]⁺ 372.1574 (calcd for C₂₁H₂₄O₆, 372.1573).

Ganocalidin D (4). Orange oil; UV (MeOH) λ_max (log ε) 197 (5.38), 272 (4.54), 387 (3.84) nm; IR (KBr) ν_max 3386, 2923, 1677, 1413, 1373, 1162, 1112, 1059, 1025, 560 cm⁻¹; ¹H and ¹³C NMR data see Table 1; ESIMS positive m/z [M + Na]⁺ 411 (100); HRESIMS positive m/z [M + Na]⁺ 411.1413 (calcd for C₂₁H₂₄O₇Na, 411.1420).

Ganocalidin E (5). Orange oil; [α]^30.1_D −13.03 (c 0.40, MeOH); UV (MeOH) λ_max (log ε) 193 (5.40), 200 (4.52), 291 (3.84), 306 (3.86), 309 (3.86), 354 (3.73), 356 (3.73), 366 (3.71) nm; IR (KBr) ν_max 3384, 3926, 1598, 1417, 1116, 1030, 551, 408 cm⁻¹; ¹H and ¹³C NMR data see Table 1; ESIMS positive m/z [M + Na]⁺ 427 (100); HRESIMS negative m/z [M − H]⁻ 403.1752 (calcd for C₂₂H₂₇O₇, 403.1757).

Ganocalidin F (6). Orange oil; [α]^30.1_D −9.50 (c 0.60, MeOH); UV (MeOH) λ_max (log ε) 194 (5.21), 197 (5.21), 216 (4.67), 297 (3.45), 362 (3.87) nm; IR (KBr) ν_max 3351, 2922, 1637, 1424, 1372, 1160, 1110, 1057, 557, 428 cm⁻¹; ¹H and ¹³C NMR data see Table 1; ESIMS positive m/z [M + Na]⁺ 411 (100); HRESIMS negative m/z [M − H]⁻ 387.1444 (calcd for C₂₁H₂₃O₇, 387.1450).

Ganocalicine A (7). Colorless powder; [α]^30.1_D −12.20 (c 0.45, MeOH); UV (MeOH) λ_max (log ε) 195 (5.13), 201 (4.86), 212 (4.63), 236 (4.37), 274 (4.28), 307 (3.77), 343 (4.05) nm; IR (KBr) ν_max 3415, 2924, 1641, 1600, 1124, 1034, 442 cm⁻¹; ¹H and ¹³C NMR data see Table 1; ESIMS positive m/z [M + H]⁺ 270 (100); HREIMS m/z [M]⁺ 269.1051 (calcd for C₁₆H₁₅NO₃, 269.1052).

Ganocalicine B (8). Colorless powder; UV (MeOH) λ_max (log ε) 194 (5.04), 200 (4.62), 202 (4.61), 208 (4.58), 213 (4.58), 274 (4.26), 343 (4.20) nm; IR (KBr) ν_max 3415, 2922, 1636, 1110, 1026, 410 cm⁻¹; ¹H and ¹³C NMR data see Table 1; ESIMS positive m/z [M + H]⁺ 240 (100); HREIMS m/z [M]⁺ 239.0940 (calcd for C₁₅H₁₃NO₂, 239.0946).

Cell culture

Rat basophilic leukemia (RBL-2H3) cells from the cell bank of Shanghai Science Academy were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% (v/v) FBS, 100 U per mL penicillin, and 100 μg mL⁻¹ streptomycin at 37 °C in a humidified atmosphere of 5% CO₂.

Cell viability test

An MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide) assay was conducted to examine cell viability. RBL-2H3 cells (5 × 10⁵ cells per well, 100 μL per well) were plated into a 96-well plate. After 24 h of incubation, cells were incubated with 1–8 (1–10 μM) for 3 hours, and the medium was replaced with MTT solution (250 μg mL⁻¹) and incubated at 37 °C for 4 h. The medium was carefully discarded and formazan was resuspended in 200 μL of dimethyl sulfoxide (DMSO). The absorbance was measured at 490 nm using a microplate reader. Values measured from untreated cells were considered to represent 100% viability. The assay was repeated three times, and each concentration was set in triplicate.

β-Hexosaminidase release activity

RBL-2H3 cells were seeded in a 24-well plate (5 × 10⁵ cells per well), and sensitized with dinitrophenol (DNP)-specific IgE (1 μg mL⁻¹) at 37 °C overnight. The IgE-sensitized cells were preincubated with compounds 1–8 for 30 min, and then stimulated with DNP-BSA for 2 h. To measure the activity of β-hexosaminidase released from the cells, the cultured media were centrifuged (17 000 g, 10 min) at 4 °C. The supernatant (50 μL) was mixed with 50 μL of 0.1 M sodium citrate buffer (pH 4.5) containing 10 mM 4-nitrophenyl N-acetyl-β-D-glucosaminide in a 96-well plate, and then incubated for 90 min at 37 °C. The absorbance was measured at 405 nm after terminating the reaction by addition of 0.2 M glycine (pH 10.0). The inhibition percentage of β-hexosaminidase released from RBL-2H3 cells was calculated using the following equation, and the IC₅₀ values were determined by GraphPad prism 5.01 software graphically: anti-degranulation activity (%) = [1 − (T − N)/(C − N)] × 100%. The control (C) was DNP-BSA (+), test sample (−); test (T) was DNP-BSA (+), test sample (+); and the normal (N) was DNP-BSA (−), test sample (−). Ketotifen fumarate was used as a reference compound. The inhibition assay was repeated three times, and each concentration was set in triplicate.

Determination of IL-4 and LTB₄

To measure the levels of interleukin-4 (IL-4) and leukotriene B₄ (LTB₄) in the cultured media, all cultured media were centrifuged at 4 °C, and the samples were stored at −80 °C until assay. IL-4 and LTB₄ were quantified using an ELISA kit according to the manufacturer’s instructions.

Statistical analyses

The data were analyzed using a one-way ANOVA followed by Dunnett’s multiple comparison test with GraphPad Prism software (GraphPad Prism version 5.01 for Windows, San Diego, CA, USA). The values are expressed as mean ± SD. Differences with p < 0.05 were considered significant.

Acknowledgements

This work was supported by grants from the Natural Science Foundation of China (31300294, 81370494, 81403160), Special Fund for Agro-Scientific Research in the Public Interest (201303117), National Support Science and Technology Subject (2013BAI11B04), Fundamental Scientific Research Funds for CATAS (ITBB2015ZD02), Natural Science Foundation of Hainan Province (214039), and the Shenzhen Innovation of Science and Technology Commission (No. ZDSYS201506050935272, JCYJ20150403091931191, YLWS20140609102809065, 201401099).

Notes and references

1. M. Furue, H. Terao, W. Rikihisa, K. Urabe, N. Kinukawa, Y. Nose and T. Koga, Br. J. Dermatol., 2003, 148, 128–133.
2. X. M. Li and L. Brown, J. Allergy Clin. Immunol., 2009, 123, 297–306.
3. Coloured Illustrations of Ganodermataceae of China, ed. X. L. Wu and Y. C. Dai, Science Press, Beijing, 2005, pp. 19–22.
4. X. L. Wu, X. L. Mao, G. E. Tuli, B. Song, T. H. Li, Y. X. Zhao, S. L. Chen, L. K. Zeng, S. Z. Huang, T. C. Wen and C. Y. Deng, Medicinal Fungi of China, Science Press, Beijing, 2013, pp. 330–331.
5. Q. Y. Ma, Y. Luo, S. Z. Huang, Z. K. Guo, H. F. Dai and Y. X. Zhao, J. Asian Nat. Prod. Res., 2013, 15, 1214–1219.
6. X. R. Peng, J. Q. Liu, Z. H. Han, X. X. Yuan, H. R. Luo and M. H. Qiu, Food Chem., 2013, 141, 920–926.
7. H. C. Huang, C. C. Liaw, H. L. Yang, Y. C. Hseu, H. T. Kuo, Y. C. Tsai, S. C. Chien, S. Amagaya, Y. C. Chen and Y. H. Kuo, Phytochemistry, 2012, 84, 177–183.
8. X. M. Niu, S. H. Li, H. D. Sun and C. T. Che, J. Nat. Prod., 2006, 69, 1364–1365.
9. F. F. Zhong, Y. Chen, P. Wang, H. J. Feng and G. Z. Yang, Chin. J. Chem., 2009, 27, 74–80.
10. M. Arisawa, A. Fujita, M. Saga, H. Fukumura, T. Hayashi, M. Shimizu and N. Morita, J. Nat. Prod., 1986, 49, 621–625.
11. T. H. J. Niedermeyer, U. Lindequist, R. Mentel, D. Goerdes, E. Schmidt, K. Thurow and M. Lalk, J. Nat. Prod., 2005, 68, 1728–1731.
12. A. G. Gonzalez, F. Leon, A. Rivera, C. M. Munoz and J. Bermejo, J. Nat. Prod., 1999, 62, 1700–1701.
13. G. Y. Li, B. G. Li, G. Y. Liu and G. L. Zhang, Chin. J. Appl. Environ. Biol., 2005, 11, 67–70.
14. H. C. Kwon, S. D. Zee, S. Y. Cho, S. U. Choi and K. R. Lee, Arch. Pharmacal Res., 2002, 25, 851–855.
15. M. P. Zimmerman and C. Djerassi, J. Am. Chem. Soc., 1991, 113, 3530–3533.
16. J. T. Feng and Y. P. Shi, Pharmazie, 2005, 60, 464–467.
17. X. Luo, F. Li, P. B. Shinde, J. Hong, C. O. Lee, K. S. Im and J. H. Jung, J. Nat. Prod., 2006, 69, 1760–1768.
18. S. P. Yang, J. Xu and J. M. Yue, Chin. J. Chem., 2003, 21, 1390–1394.
19. F. Yin, L. Cheng and F. C. Lou, Chin. J. Nat. Med., 2004, 2, 149–151.
20. M. Mutz, K. J. Barr and J. Gestwicki, Application: WO Pat., 2009-US6600, 2010077317, 2010.
21. N. Wang, J. H. Wang, J. Cheng and X. Li, J. Shenyang Pharm. Univ., 2003, 20, 425–427.
22. X. Zhang, H. Gao, N. L. Wang and X. S. Yao, J. Chin. Med. Mater., 2006, 37, 652–655.
23. M. T. Alonso, E. Brunet, O. Juanes and J. C. Rodriguez-Ubis, J. Photochem. Photobiol., A, 2002, 147, 113–125.
24. J. Q. Liu, C. F. Wang, X. R. Peng and M. H. Qiu, Nat. Prod. Bioprospect., 2011, 1, 93–96.
25. S. H. He, H. Y. Zhang, X. N. Zeng, D. Chen and P. C. Yang, Acta Pharmacol. Sin., 2013, 34, 1270–1283.
26. E. Passante and N. Frankish, J. Photochem. Photobiol., A, 2009, 58, 737–745.
27. J. Kalesnikoff and S. J. Galli, Nat. Immunol., 2008, 9, 1215–1223.
28. S. N. Abraham and A. L. St John, Nat. Rev. Immunol., 2010, 10, 440–452.

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Footnotes

† Electronic supplementary information (ESI) available: The HPLC fingerprint of the ethanol extract, spectra of ¹H and ¹³C NMR, MS, HSQC, and HMBC of 1–8. See DOI: 10.1039/c6ra01466f

‡ These authors contributed equally to this work.

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