New Unsaturated Lactones and a Meroterpenoid from Ganoderma lucidum

Abstract

From the dried fruiting bodies of Ganoderma lucidum, 2 new unsaturated lactones, dayaolingzhilactones A and B (1 and 2), and 1 new meroterpenoid, dayaolingzhiol H (3), together with 10 known compounds (4-13), were isolated. Their chemical structures were identified by using spectroscopic data and calculated specific rotation. The inhibitory activities of compounds 1 and 2 toward acetylcholinesterase (AchE) were assessed in vitro with tacrine as the positive control. Both of them exhibited moderate AchE inhibitory activities at the concentration of 50 μM.

Ganoderma lucidum (Ganodermataceae), a fungus registered in the Pharmacopoeia of China and American Herbal Pharmacopoeia and Therapeutic Compendium, has been used in China for thousands of years for promoting health, longevity and spiritual growth. ¹ Currently, it is widely used for the treatment of bronchitis, allergies, hepatitis, hypertension, immunological disorders, neurasthenia, and diabetes. ^2,3 Previous chemical studies revealed that polysaccharides and triterpenoids are the 2 main pharmacologically active constituents in G. lucidum. ⁴ In addition, a few other structural types of compounds have also been reported sporadically such as alkaloids, steroids, lactones, and fatty acids. ⁴ As part of our chemical studies on Ganoderma genus, the title material was investigated, resulting in the isolation of 2 new unsaturated lactones (1 and 2), a new meroterpenoid (3), along with 10 known compounds (4-13) (Figure 1). Besides, the inhibitory activities of compounds 1 and 2 toward acetylcholinesterase (AchE) were assessed. Our efforts are described below.

Figure 1

Chemical structures of compounds 1 to 13.

Compound 1, a colorless oil, has the molecular formula C₁₅H₂₄O₅ based on the analysis of its HRESIMS m/z 283.1551 [M−H]^–, ¹³C NMR, and DEPT spectra, with 4 degrees of unsaturation. The ¹H NMR data (Table 1) of 1 show 2 methyls [δ _H 1.93 (3H, s, CH₃-15); 1.78 (3H, s, CH₃-14)]. The ¹³C NMR and DEPT spectra display 15 carbons including 2 methyls, 8 methylenes, and 5 nonprotonated carbons (1 ester carbonyl, 1 carboxylic acid, 2 sp² carbons, and 1 oxygenated sp³ carbon). The architecture of 1 was constructed by 2D NMR experiments. Interpretation of 1D and 2D NMR data discloses that the structure of compound 1 resembles that of 5-hydroxy-3,4-dimethyl-5-pentyl-2(5H)-furanone (4) ⁵ but differs in the structure of the side chain. The ¹H-¹H COSY (Figure 2) correlations of H₂-5/H₂-6/H₂-7 and H₂-10/H₂-11/H₂-12, together with the HMBC correlations of H₂-6/C-8, H₂-11/C-9, and H₂-11, H₂-12/C-13 (δ _C 178.4), suggest the length of the side chain, and the additional carboxylic acid group is at the terminal of the chain. In addition, HMBC correlations of H₃-14/C-2, C-3, H₃-15/C-2, C-3, C-4, and H₂-5/C-4 secured the presence of an unsaturated 5-membered lactone. Thus, the planar structure of 1 has been deduced. Finally, there is 1 chiral center in the structure of 1; its absolute configuration at C-4 in 1 was identified as 4R by comparing its specific rotation { ${\displaystyle [\alpha {]}_{\mathrm{D}}^{20}}$ –11.8 (c 0.17, MeOH)} with the reported { ${\displaystyle [\alpha {]}_{\mathrm{D}}^{23}}$ +5.4 (c 0.18, MeOH)} for 4S of sinularone I. ^6,7 This conclusion was further supported by the calculated optical values of model compounds (–54.0 for 4R, +54.0 for 4S) at B3LYP/6-311G(d,p) level in MeOH (see Supplementary Material 1, Table S1). Thus, the structure of 1, named dayaolingzhilactone A, was identified.

Table 1

¹H and ¹³C NMR Data of 1 to 3 (δ in ppm, J in Hz).

No.	1 a		No.	2 b		No.	3 b
	δH (J in Hz)	δ C		δH (J in Hz)	δ C		δH (J in Hz)	δ C
1		174.6	1		164.3	1		156.5
2		125.7	2	6.17 (d, 9.2)	114.0	2		120.4
3		160.4	3	7.48 (dd, 9.2, 6.9)	146.4	3	7.26 (d, 2.9)	115.4
4		109.2	4	6.26 (d, 6.9)	105.9	4		150.6
5	1.95 (m); 1.73 (m)	36.9	5		161.5	5	7.00 (dd, 8.9, 2.9)	125.8
6	1.24 (m); 1.15 (m)	24.1	6	6.14 overlap	121.3	6	6.78 (d, 8.9)	119.7
7	1.31 (overlap)	30.5	7	7.05 (dd, 15.3, 10.8)	137.0	1′		205.8
8	1.31 (overlap)	30.4	8	6.29 (dd, 16.2, 10.8)	132.0	2′	3.43 (dd, 17.8, 9.3)3.10 (dd, 17.8, 4.4)	41.4
9	1.31 (overlap)	30.3	9	6.11 overlap	137.7	3′	2.96 (m)	41.3
10	1.31 (overlap)	30.2	10	1.86 (d, 6.7)	18.7	4′	1.74 (m); 1.62 (m)	33.2
11	1.58 (m)	26.3				5′	2.10 (m)	26.5
12	2.26 (t, 7.2)	35.4				6′	5.16 (t, 7.2)	124.9
13		178.4				7′		136.8
14	1.78 (s)	8.2				8′	2.01 (m)	41.0
15	1.93 (s)	10.8				9′	2.16 (m)	27.1
16						10′	5.26 (t, 7.3)	128.5
17						11′		135.8
18						12′	4.05 (s)	61.4
19						13′	1.74 (s)	21.5
20						14′	1.62 (s)	16.1
						15′		179.5

^aRecorded at 400 (100) MHz in methanol-d ₄.

^bRecorded at 600 (150) MHz in methanol-d ₄.

Figure 2

Key COSY, HMBC, and ROESY correlations of 1 to 3.

Compound 2 possesses a molecular formula of C₁₀H₁₀O₂ based on its HRESIMS, ¹³C NMR, and DEPT data, with 6 degrees of unsaturation. The ¹H NMR spectrum of 2 (Table 1) shows signals assignable to 1 methyl [δ _H 1.86 (3H, d, J = 6.7 Hz, CH₃-10)] and 7 olefinic/aromatic protons [δ _H 7.48 (1H, dd, J = 9.2, 6.9 Hz, H-3), 7.05 (1H, dd, J = 15.3, 10.8 Hz, H-7), 6.29 (1H, dd, J = 16.2, 10.8 Hz, H-8), 6.26 (1H, d, J = 6.9 Hz, H-4), 6.17 (1H, d, J = 9.2 Hz, H-2), 6.14 (1H, overlap, H-6), 6.11 (1H, overlap, H-9)]. The ¹³C NMR and DEPT spectra display 10 carbons which are classified into 1 methyl, 7 sp² methines, and 2 nonprotonated carbons (1 ester carbonyl and 1 oxygenated sp² carbon), accounting for 5 degrees of unsaturation. The ¹H-¹H COSY (Figure 2) gives the correlations of H-2/H-3/H-4 and H-6/H-7/H-8/H-9/H₃-10. The remaining 1 degree of unsaturation could be attributed to the presence of a ring formed by the oxygen bridge between C-1 (δ _C 164.3) and C-5 (δ _C 161.5), which was further certified by the HMBC correlations of H-2, H-3/C-1 and H-4, H-6/C-5. The couple constants of J _6,7 value (15.3 Hz) and J _8,9 value (16.2 Hz) suggest the configurations of the double bonds Δ⁶⁽⁷⁾ and Δ⁸⁽⁹⁾ are both of trans-orientation. Thus, the structure of 2 was determined (Figure 1).

The molecular formula of compound 3 was determined to be C₂₁H₂₈O₆ (8 degrees of unsaturation) based on its HRESIMS, ¹³C NMR, and DEPT data. The ¹H NMR spectrum of 3 (Table 1) contains resonances for a typical ABX aromatic spin system [δ _H 7.26 (1H, d, J = 2.9 Hz, H-3), 7.00 (1H, dd, J = 8.9, 2.9 Hz, H-5), 6.78 (1H, d, J = 8.9 Hz, H-6)] and 2 olefinic protons [δ _H 5.26 (1H, t, J = 7.3 Hz, H-10′), 5.16 (1H, t, J = 7.2 Hz, H-6′)], suggesting the presence of a 1,2,4-trisubstituted aromatic ring and 2 double bonds in 3. The ¹³C NMR and DEPT spectra contain 21 carbons, attributed to 2 methyls, 6 methylenes (including 1 oxygenated), 6 methines, and 7 nonprotonated carbons (1 ketone carbonyl, 1 ester carbonyl, 2 sp² carbons, and 3 aliphatic carbons including 2 oxygenated). The ¹H-¹H COSY correlations of H₂-2′/H-3′/H₂-4′/H₂-5′/H-6′ and H₂-8′/H₂-9′/H-10′, together with the HMBC correlations of H₂-12′, H₃-13′/C-10′, C-11′, H₃-14′/C-6′, C-7′, C-8′, H-3′/C-1′ (δ _C 205.8), and H₂-2′, H₂-4′/C-15′ (δ _C 179.7), reveal the structure of the side chain from C-1′ to C-15′ in 3 as shown (Figure 1), and the HMBC correlations of H₂-2′/C-2 and H-3/C-1′ suggest the side chain is attached to C-2. For the geometry of the double bonds, the ROESY correlations of H-6′/H₂-8′ and H-10′/H₃-13′ indicate that the configurations of the Δ^6′(7′)and Δ^10′(11′)double bonds are both E-form. Thus, the planar structure of 3 was deduced. Compound 3 was proved to be optically pure by chiral HPLC analysis, and its absolute configuration was determined as 3′R by comparing its optical rotation (OR) { ${\displaystyle [\alpha {]}_{\mathrm{D}}^{20}}$ +17.2 (c 0.06, MeOH)} with { ${\displaystyle [\alpha {]}_{\mathrm{D}}^{25}}$ +10.5 (c 0.07, MeOH)} of applanatumol S. ^6,7

The known compounds were identified by comparing their spectroscopic data with the literature data. They are 5-hydroxy-3,4-dimethyl-5-pentyl-2(5H)-furanone (4), ⁵ 4-methoxy-6-styryl-2H-pyran-2-one (5), ⁸ ganoleucoin E (6), ⁹ ganoderal A (7), ¹⁰ ganoderol A (8), ¹¹ ganoderic acid S (9), ¹¹ ganoderic acid Y (10), ¹¹ ganoderol B (11), ¹¹ lumichrome (12), ¹² and cinnamic acid (13). ¹³

In this study, only the new compounds with the exception of 3 were evaluated for their inhibitory property toward AchE. It was found that both of them (50 µM) exhibit moderate inhibitory activities with the inhibition rate of 49.69% ± 0.63% and 35.45% ± 3.37%, respectively (tacrine as a positive control with the inhibition rate of 60.68% ± 2.85% at the concentration of 0.33 µM).

Experimental

General

ORs were recorded on a Jasco P-1020 digital polarimeter. UV spectra were measured on a Shimadzu UV2401PC spectrometer. CD spectra were measured on an Applied Photophysics Chirascan instrument. NMR spectra were measured on a Bruker AV 400, or 600 MHz spectrometer, with TMS as an internal standard. ESIMS and HRESIMS were measured on an Agilent 1290 UPLC/6540 Q-TOF instrument. RP-18 (40-60 µm, Daiso Co., Japan), MCI gel CHP 20P (75-150 µm, Tokyo, Japan), silica gel GF254 (80-100 mesh, Qingdao Marine Chemical Inc., China), and Sephadex LH-20 (Amersham Biosciences, Sweden) were used for column chromatography. Semipreparative HPLC was carried out using an Agilent 1200 liquid chromatograph (Agilent Technologies, Santa Clara, CA, USA). The columns used were YMC-Pack ODS-A (250 mm × 10 mm, i.d., 5 µm) and Daicel Chiralpak IC (250 mm × 10 mm, i.d., 5 µm).

Fungal Material

The fruiting bodies of G. lucidum were collected from Dayao County of Yunnan Province, China, in December 2015. The material was identified by Prof Zhu-Liang Yang at Kunming Institute of Botany, Chinese Academy of Sciences, and a voucher specimen (CHYX0596) has been deposited at the School of Pharmaceutical Sciences, Shenzhen University, since November 2017.

Extraction and Isolation

The dried material (4.0 kg) was soaked with 80% EtOH (20 L × 3 × 24 hours) to give a crude extract, which was extracted with EtOAc to afford an EtOAc-soluble extract (0.12 kg). The EtOAc extract was divided into 10 parts (Fr.1-Fr.10) by using an MCI gel CHP 20P column chromatography eluted with aqueous MeOH (40%-100%). Fr.5 (5.1 g) was passed through Sephadex LH-20 (MeOH) to yield 4 fractions (Fr.5.1-Fr.5.4). Fr.5.2 (0.4 g) was further separated by preparative HPLC (MeOH/H₂O, 60%) followed by semipreparative HPLC (ACN/H₂O, 40%) to give 6 (1.3 mg, t _R = 11.1 minutes, 3 mL/min). Fr.6 (3.7 g) was filtered by Sephadex LH-20 (MeOH) to give Fr.6.1 to Fr.6.6. Fr.6.3 (30.0 mg) was purified by semipreparative HPLC (ACN/H₂O, 48%) to yield 2 (1.3 mg, t _R = 14.3 minutes, 3 mL/min). Fr.6.4 (0.1 g) was first separated by preparative HPLC (MeOH/H₂O, 60%) and then purified by semipreparative HPLC (ACN/H₂O, 41%) to obtain 3 (2.6 mg, t _R = 23.7 minutes, 3 mL/min). Fr.6.5 (80.0 mg) was also first separated by preparative HPLC (MeOH/H₂O, 60%) to provide Fr.6.5.1 to Fr.6.5.7. Fr.6.5.2 (11.0 mg), Fr.6.5.4 (12.0 mg), and Fr.6.5.6 (8.0 mg) were further purified by semipreparative HPLC (ACN/H₂O, 45%, 42%, and 41%) to provide compounds 13 (8.8 mg, t _R = 19.2 minutes, 3 mL/min), 1 (2.5 mg, t _R = 9.7 minutes, 3 mL/min), and 4 (3.2 mg, t _R = 24.0 minutes, 3 mL/min), respectively. Fr.6.6 was submitted to semipreparative HPLC (MeOH/H₂O, 59%) to yield 12 (2.3 mg, t _R = 13.1 minutes, 3 mL/min). Fr.9 (36.0 g) was fractionated by Sephadex LH-20 (MeOH) to give Fr.9.1 to Fr.9.6. Fr.9.1 (50.0 mg) was then purified by Sephadex LH-20 (MeOH) followed by semipreparative HPLC (ACN/H₂O, 61%) to afford 5 (1.5 mg, t _R = 17.3 minutes, 3 mL/min). Fr.9.6 (6.4 g) was submitted to Sephadex LH-20 (MeOH) to give Fr.9.6.1 to Fr.9.6.4. Fr.9.6.2 (60.0 mg) was further separated by semipreparative HPLC (ACN/H₂O, 90%) to obtain 10 (10.5 mg, t _R = 16.3 minutes, 3 mL/min) and 11 (2.8 mg, t _R = 26.9 minutes, 3 mL/min). Fr.9.6.3 (35.0 mg) was also separated by semipreparative HPLC (ACN/H₂O, 95%) to afford 7 (10.4 mg, t _R = 20.4 minutes, 3 mL/min). Fr.9.6.4 (0.3 g) was first separated by preparative TLC (CHCl₃/MeOH/formic acid, 18:1:0.05) and then purified by semipreparative HPLC (ACN/H₂O, 91% and 90%) to produce compounds 8 (10.4 mg, t _R = 21.5 minutes, 3 mL/min) and 9 (8.3 mg, t _R = 21.3 minutes, 3 mL/min).

Compound 1

Colorless oil

${\displaystyle [\alpha {]}_{\mathrm{D}}^{20}}$ : –11.8 (c 0.17, MeOH)

UV (MeOH) λ _max (log ε): 207 (3.93) nm

ESIMS m/z: 283 [M–H]^–

HRESIMS m/z: 283.1551 [M–H]^– (calcd for C₁₅H₂₃O₅, 283.1551).

¹H and ¹³C NMR: Table 1

Compound 2

White powder

UV (MeOH) λ _max (log ε): 355 (3.80), 253 (3.86), 204 (3.68) nm

ESIMS m/z: 161 [M–H]^–

HRESIMS m/z: 161.0607 [M–H]^– (calcd for C₁₀H₉O₂, 161.0608)

¹H and ¹³C NMR: Table 1

Compound 3

Yellow gum

${\displaystyle [\alpha {]}_{\mathrm{D}}^{20}}$ : +17.2 (c 0.06, MeOH)

UV (MeOH) λ _max (log ε): 366 (3.62), 257 (3.88), 226 (4.22) nm

CD (MeOH): Δε ^{_{2 13}} –0.70, Δε ^{_{2 32}} +0.28

ESIMS m/z: 399 [M+Na]⁺

HRESIMS m/z 399.1779 [M+Na]⁺ (calcd for C₂₁H₂₈O₆Na, 399.1778)

¹H and ¹³C NMR: Table 1

Computational Methods

The OR calculation was performed by a Gaussian 09 program package. The conformational search generating low-energy conformers within a 6 kcal/mol was deduced by Conflex 7 to optimize the low-energy conformations. Then, the OR calculations were conducted at the B3LYP/6-311G(d,p) level in MeOH, and the results were processed using previously described methods. ¹⁴

Biological Evaluation

Inhibition of AchE was assayed using a microplate as previously described. ^15,16 The concentration of the compounds is 50 µM, and tacrine was used as a positive control with the concentration of 0.33 µM. The assay was done in triplicate and the results are expressed as mean ± SD.

Supplemental Material

Supplementary data - Supplemental material for New Unsaturated Lactones and a Meroterpenoid from Ganoderma lucidum

Supplemental material, Supplementary data, for New Unsaturated Lactones and a Meroterpenoid from Ganoderma lucidum by Qi-Hang Xin, Qi Luo, Ze-Hong Wu, Jiao-Jiao Zhang, Li-Zhong Liu, and Yong-Xian Cheng in Natural Product Communications