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New cytotoxic lanostanoid triterpenes from Ganoderma lingzhi
Phytochemistry Letters 17 (2016) 64–70
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New cytotoxic lanostanoid triterpenes from Ganoderma lingzhi Yhiya M. Amena,b , Qinchang Zhua , Mohamed S. Afifib , Ahmed F. Halimb , Ahmed Ashourb , Kuniyoshi Shimizua,* a Division of Systematic Forest and Forest Products Sciences, Department of Agro-Environmental Sciences, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 812-8581, Japan b Department of Pharmacognosy, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt
A R T I C L E I N F O
Article history: Received 8 April 2016 Received in revised form 25 June 2016 Accepted 6 July 2016 Available online xxx Keywords: Ganoderma lingzhi Lanostane triterpenes Cytotoxicity
A B S T R A C T
Further chemical investigation of the metabolites in the fruiting bodies of Ganoderma lingzhi resulted in isolation of eight triterpenes; two of them are new triterpene acid ethyl esters. Their structures were established based on spectroscopic studies and comparison with the known related compounds. The anticancer potential of the isolates were tested with an in vitro cytotoxic assay against five human cancer cell lines (MCF-7, HeLa, HCT-116, Caco-2 and HepG2) and two normal human cell lines (TIG-1 and HF19). Results showed that the new compounds have a strong to moderate selective cytotoxic activity against MCF-7 while they showed moderate to weak activity against HeLa cell line. Potent cytotoxic activities of some of the known isolated compounds are reported for the first time. ã 2016 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
1. Introduction Cancer is a leading cause of mortality in economically developed countries and the second leading cause of death in developing countries (Jemal et al., 2011). The number of new cancer cases reported annually worldwide will reach 15 million by 2020, with 70% of these cases occurring in developing countries (Kuete et al., 2013). Although chemotherapy is the most commonly used method to treat patients with cancer, the application of chemotherapeutic techniques is limited by their high cellular toxicity due to their lack of selectivity against cancer cells as well as the occurrence of other long-term side effects. Therefore, the ultimate goal of cancer chemotherapy is the development of new therapeutic selective agents that can kill malignant tumor cells or render them benign without affecting normal cells (Khazir et al., 2014). As a part of our continuing research for exploring the bioactive principles of Ganoderma lingzhi, we report the isolation of eight triterpenes including two new ones triterpene acid ethyl esters. The new compounds have a strong to moderate selective cytotoxic activity against MCF-7 while they showed moderate to weak activity against HeLa cell line without causing any cytotoxic activity to normal cell lines (TIG-1 and HF19). We reported also
some potent cytotoxic activities for some of the known isolated compounds for the first time. Ganoderma lingzhi is a well-known crude drug that has been used clinically in Oriental countries since ancient times due to its health promoting and tonic effects (Xia et al., 2014). During the past four decades, more than 400 secondary metabolites have been isolated from various Ganoderma species (Baby et al., 2015). The number of the new compounds identified from Ganodema increasing day after day. Triterpenoids are typical chemical constituents in G. lingzhi and possess important roles in pharmacological activities as anticancer (Chen and Zhong, 2009; Jiang et al., 2008; Liu et al., 2009; Wu et al., 2013, 2001; Zhao et al., 2015), antiviral (El-Mekkawy et al., 1998; Min et al., 1998; Zhu et al., 2015), anti-diabetic (Fatmawati et al., 2011; Zhao et al., 2015), antiandrogenic (Liu et al., 2006) and anti-inflammatory (Akihisa et al., 2007; Ko et al., 2008). Recently, we reported the isolation of a new lanostanoid triterpene, named lucidumol C with a potent, selective cytotoxicity against HCT-116 and remarkable cytotoxic activities against Caco-2, HepG2 and HeLa cell lines (Amen et al., 2016). Herein, we describe further, the structural elucidation and cytotoxicity of the isolated compounds against several cell lines besides their selectivity against two normal human fibroblast cell lines has been tested. 2. Results and discussion
* Corresponding author. E-mail address: [email protected] (K. Shimizu).
The chloroform fraction of the EtOH extract of G. lingzhi was subjected to chromatographic purification using Sephadex LH-20
http://dx.doi.org/10.1016/j.phytol.2016.07.024 1874-3900/ã 2016 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
Y.M. Amen et al. / Phytochemistry Letters 17 (2016) 64–70
Compound 1 was obtained as a white powder with [a]24D + 48.6 (c 0.22, MeOH). HR-ESI–MS spectrum gave a protonated molecular ion peak at m/z 541.3145 [M+H]+, calculated (541.3160) and a deprotonated molecular ion peak at m/z 539.3048 [MH], calculated (539.3014) in accordance with the molecular formula C32H44O7. It exhibited UV absorption at 247 nm. The 1H NMR
and repeated silica gel chromatographic steps, followed by reversed-phase column chromatography, to afford eight lanostanoid triterpenes, including two new compounds (1, 2). The known compounds (3–8) were identified by matching their spectral data with those of the corresponding ones and/or relevant compounds (Fig. 1).
21
22
18
11
28
30
7
4
O
COOCH2CH3
O COOCH2CH3
O O
27
15
9
26
O
13
1
O
24
20
O
19
O
OH
OH
O
OH
2
1
29
65
COOCH2CH3
COOCH3 O
O
O
O O
O
OH
OH
4
3
O
O COOCH2CH3
O
COOH
O
O
O
O O
O
O
OH
5
O
6
O
O COOH
O
O COOH
O
O
O
O O
OH
O O
O
8
7 Fig. 1. Structures of the isolated compounds (1–8).
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Y.M. Amen et al. / Phytochemistry Letters 17 (2016) 64–70
positions of unsaturation (D8,9) as follows: there were correlations observed between the methyl protons resonating at dH 1.43 (H3-30) and a carbon resonance at dc 142.4 beside correlations of H-7 with dc 142.4 thus placed dc 142.4 to C-9. The carbon resonance dc 159.9 was positioned to C-8 based on correlations of H-5 with dc 159.9 together with the HMBC cross-peak of H-7 with dc 159.9 supported by the other correlations of H-19 (1.24, s) with the same carbon resonance at dc 159.9. The above evidence indicated the presence of 8-ene structural fragment. The connectivity of a carbonyl group at C-11 was evidenced from the correlations of H-12 [(2.57 m, 3.04 d (J = 17.4)] with a carbonyl group at dc 199.6, thus assigned dc 199.6 to C-11. The HMBC correlations observed of H-12 with dc 159.9, confirmed the olefinic bond at C-8, C-9. The 13C NMR further revealed a carbon resonance at dc 216.9 which was carefully placed to C-15 based on a cross-peak in the HMBC spectrum, showed between dH 1.43 (s, H-30) with a carbonyl group at dc 216.9. The assignment of the carbon resonances of the side chain was again possible with the help of HMBC correlations. The position of the alkene in the side-chain was confirmed through correlations showed between the singlet peak at dH 2.14, assigned for H-21, with a carbon resonance at dc 155.8 together with a crosspeak between dH 2.87 (m), assigned for H-16, with the same carbon resonance at dc 155.8, thus placed carefully to C-20. Furthermore, HMBC showed cross-peaks between dH 3.24 (dd, 8.4, 18.6), assigned to H-17, to carbon resonances at dc 125.8 and 155.8, supported our previous conclusion. 1H NMR spectrum further
spectrum, analyzed with the aid of HMQC experiment, was indicative of five singlet methyl groups, at dH 0.86, 1.09, 1.13, 1.24 and 1.43, a doublet methyl resonance at dH 1.16 (J = 7.2), a multiplet methyl at dH 1.23, a vinyl methyl at dH 2.14, an oxymethine proton at dH 4.93 (dd, J = 7.8, 9.6) and one vinyl proton at dH 6.25 (s). The 13 C NMR spectrum, combined with HMQC experiment, confirmed the aforementioned moieties and displayed characteristic signals of eight methyl groups (dc 14.5, 17.4, 18.6, 19.6, 21.1, 21.2, 25.5 and 27.5), an oxygenated methine at dc 67.1, an a,b-unsaturated carbonyl at dc 159.9, 142.4 and 199.6, olefinic carbons at dc 125.8 and 155.8, ketone carbons at dc 200.7, 216.9 and 219.7, and a carboxyl carbonyl at dc 177.8. These data suggested an oxygenated lanostane-type triterpene with a conjugated enone (Xia et al., 2014). Unambiguous assignments for all the carbons could be made based on the correlations observed in HMBC spectrum (Fig. 2). In the HMBC spectrum, there were correlations between the proton signal at dH 1.60 assigned to H-5, and the carbon resonance at dc 219.7 and between the proton signals at dH 1.59 (m, H-1) and dH 2.88 (m, H-2) with the same carbon resonance at dc 219.7, thus established the signal dc 219.7 to C-3 of the skeleton. Furthermore, the proton signal of H-5 showed HMBC cross-peak with a carbon resonance at dc 67.1, thus established the attachment of a hydroxy group to C-7. The equatorial hydroxyl group at C-7 was deduced from the multiplicity of H-7 (dH 4.93, dd, J = 7.8, 9.6), which was further supported by the ROESY correlations observed from H-7 to H-5. Analysis of the HMBC spectrum confirmed the
O O O
O
O O
OH
1
O
O
O O O
O O
OH
2 Fig. 2. Selected HMBC correlations of 1 and 2.
O
Y.M. Amen et al. / Phytochemistry Letters 17 (2016) 64–70
revealed a down-field signal at dH 6.25, correlated through HMQC spectrum to a carbon resonance at dc 125.8 which is placed to C-22 based on HMBC correlations showed between H-21 (2.14, s) and dc 125.8. The remaining carbonyl group at dc 200.7, revealed by 13C NMR, was assigned to C-23 and confirmed by HMBC correlation of H-21 (2.14, s) with dc 200.7 together with correlations of dH 2.65 (m), 2.88 (m); assigned to H-24 and correlations of dH 2.91 (m), assigned to H-25 with the same carbon resonance at dc 200.7. The carboxylate moiety at dc 177.8, was assigned to C-26 based on correlations of H-24 [2.65 (m), 2.88 (m)], H-25 (2.91, m) and H-27 (1.16, d, J = 7.2) to the carbon resonance at dc 177.8. The presence of ethyl ester in compound 1 was confirmed by HMBC correlations of dH 4.11 (m)/dc 61.7 assigned for a methylene moiety with the carboxylate group at dc 177.8 and dc 14.5, assigned for a terminal methyl group, hence formed a carboxyethoxy side chain. Comparison of these spectroscopic data with those of methyl ganoderenate D (Shim et al., 2004), suggested that the skeleton should be the same except for the presence of carboxyethoxy side chain in 1 instead of carboxymethoxy side chain in methyl ganoderenate D. Hence, we proposed a name ethyl ganoderenate D to compound 1, which is a new natural product. Compound 2 was isolated as a white powder with [a]24D + 46.9 (c 0.13, MeOH). HR-ESI–MS spectrum showed a cationated molecular ion peak at m/z 621.2990 [M+Na]+, calculated (621.3040), and a deprotonated molecular ion peak at m/z 597.3068 [MH], calculated (597.3069) in agreement with the molecular formula C34H46O9. Its UV spectrum exhibited an absorption maximum at 247 nm. Comparison of the spectroscopic data of 2 with those of 1, revealed that the skeleton of 2 should be the same except for the protons [dH 5.78 (s), 2.08 (s)] and carbons (dc 80.6, 172.0, 21.1) resonances which were characteristic features of an acetoxyl group substituted on a carbon atom where two adjacent carbon atoms bear no proton (Cheng et al., 2010). The position of the acetoxyl group was further confirmed by a crosspeak between H-12 (5.78, s) and the carbon resonance at dc 172.0 in the HMBC spectrum, indicated that the acetoxyl group was located at C-12. Analysis of the ROESY experiment established the configuration at C-12 to be b for the acetoxy group, as there was no correlation observed between the singlet proton at dH 5.78 (H-12) with H-18 and H-19 considering the acetoxyl group was on the same face as Me-18 and Me-19 besides a correlation observed between H-12 and dH 1.71, assigned to H-5, confirmed the b-configuration of the acetoxy group at C-12. Comparison of the spectroscopic data with known compounds, compound 2 had almost the same chemical shifts as those of 7, except for an additional O-CH2CH3 group and upfielded esterified carboxylic carbon at dc 177.8. This evidence indicated that compound 2 was the ethyl esterified derivative of the known compound 12b-acetoxy-7b-hydroxy-3,11,15,23-tetraoxo-5a-lanosta-8,20dien-26-oic acid (7). Thus, compound 2 was characterized as 12b-acetoxy-7b-hydroxy-3,11,15,23-tetraoxo-5a-lanosta-8,20dien-26-oic acid ethyl ester; a new natural product. Since compounds 1 and 2 represent the ethyl esters of the corresponding ganoderic acids, the probability of being natural products might be suspected. To test for any direct esterification due to the long time contact with ethyl alcohol during the extraction process or to trans-esterification due to the repetitive use of ethyl acetate throughout the fractionation and purification steps, two of the isolated ganoderic acids, compounds 7 and 8 (Fig. 1) were selected and each was subjected to the method mentioned in the experimental section. Only the original unreacted acids were detected on the TLC layer without any evidence of the formation of their esterified compounds 2 or 5 respectively. The structures of the known compounds were identified by comparison of their spectroscopic data with those reported in the
67
literature as methyl lucidenate A (3) (Nishitoba et al., 1985; Xia et al., 2014), ethyl lucidenate A (4) (Li et al., 2013), 12b-acetoxy3,7,11,15,23-pentaoxo-5a-lanosta-8-en-26-oi acid ethyl ester (5) (Cheng et al., 2010), ganoderic acid GS-1 (6) (Sato et al., 2009), 12b-acetoxy-7b-hydroxy-3,11,15,23-tetraoxo-5a-lanosta-8,20dien-26-oic acid (7) (Cheng et al., 2010) and ganoderic acid F (8) (Hirotani and Furuya, 1986; Komoda et al., 1985). It is worth mentioning that this is the second report of ganoderic acid GS-1 in nature and the first report of it from G. lingzhi since the compound was isolated before from the fruiting bodies of G. sinense (Sato et al., 2009) (Table 1). The cytotoxicity of the isolated triterpenoids was evaluated against human breast carcinoma (MCF-7), human cervical carcinoma (HeLa), human colorectal carcinoma (HCT-116, Caco-2) and human liver carcinoma (HepG2). Selectivity of the compounds was evaluated against normal human fibroblast cells (TIG-1) and normal human fetal lung fibroblast cells (HF19). The results are summarized in Table 2. None of the compounds showed cytotoxicity to normal cell lines (TIG-1 and HF19), reflecting the selectivity of the compounds to the normal human cell lines. Regarding cytotoxicity to cancer cell lines, compounds 1 and 2 showed a strong to moderate selective cytotoxic activity against MCF-7 while they showed moderate to weak activity against HeLa cell line. We also report for the first time, the cytotoxic activity of some of the isolated known compounds against several cancer cell lines as shown in Table 2. Reviewing the literature, we found some reports discussing the cytotoxicity of some of the isolated compounds as follows: Li et al. (2013) evaluated methyl lucidenate A (3) and ethyl lucidenate A (4) against HepG2, the compounds showed moderate cytotoxic activity with IC50 values of 49.82 and Table 1 1 H (600 MHz) and Position
*
C NMR (150 MHz) spectral data of 1 and 2.*
1 1
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 OCH2CH3 OCH2CH3 OCOCH3 OCOCH3
13
H (J Hz)
1.59 (m) 2.88 (m)
1.60 (m) 1.31 br dd (7.2, 13.2) 4.93 dd (7.8, 9.6)
2.57 (m), 3.04 (d, 17.4)
2.87 (m) 3.24 dd (8.4, 18.6) 0.86 (s) 1.24 (s) 2.14 (s) 6.25 (s) 2.65 (m), 2.88 (m) 2.91 (m) 1.16 (d, 7.2) 1.13 (s) 1.09 (s) 1.43 (s) 4.11 (m) 1.23 (m)
Measured in CD3OD.
2 13
1
36.5 35.2 219.7 47.8 49.0 29.2 67.1 159.9 142.4 39.4 199.6 49.0 47.2 59.5 216.9 36.7 49.9 19.6 18.6 155.8 21.2 125.8 200.7 49.1 36.7 177.8 17.4 27.5 21.1 25.5 61.7 14.5
2.51 (m) 2.49 (m)
C
H (J Hz)
1.71 (m) 1.17 (m) 4.92 (m)
5.78 (s)
2.85 (m) 3.42 (m) 1.09 (s) 1.22 (s) 2.12 (s) 6.30 (s) 2.66 (m), 2.87 (m) 2.85 (m) 1.13 (d, 7.2) 1.10 (s) 1.09 (s) 1.37 (s) 4.11 (m) 1.17 (m) 2.08 (s)
13
C
35.2 35.15 219.7 47.8 49.0 27.5 67.3 160.0 142.4 39.3 194.0 80.6 47.5 60.4 210.7 36.8 51.3 20.9 18.6 155.9 21.1 126.4 200.1 49.1 36.8 177.8 17.4 27.1 21.5 25.3 61.7 17.4 172.0 21.1
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Table 2 Cytotoxic activities against MCF-7, HeLa, HCT-116, Caco-2, HepG2, TIG-1 and HF19.* Compounds
1 2 3 4 5 6 7 8 5-FU
IC50 (mM) MCF-7
HeLa
HCT-116
Caco-2
HepG2
TIG-1
HF19
35.9 7.5 71.7 41.7 62.3 1.2 80.8 >100 >100
89.9 31.8 >100 48.9 85.1 >100 >100 >100 19.6
92.6 >100 >100 33.9 69.9 40.1 >100 >100 65.4
>100 >100 >100 >100 >100 >100 >100 >100 63.6
>100 >100 >100 >100 >100 >100 >100 >100 >100
>100 >100 >100 >100 >100 >100 >100 >100 >100
>100 >100 >100 >100 >100 >100 >100 >100 >100
* The activity was shown as IC50 value, which was the concentration of the tested compound (mM) that decreased the number of viable cells by 50%. Results are expressed as the mean value of triplicate data points.
55.15 mg/mL, respectively by using the conventional MTT cell viability method. Our results are in contrast with these results, compounds 3 and 4 did not show any activity against HepG2 at a concentration 100 mM. Compound 3 was also recently evaluated against three cancer cell lines; A549 (non-small cell lung adenocarcinoma), MCF-7 and PC-3 (prostatic small cell carcinoma). The authors considered the compound inactive since it showed an IC50 value more than 50 mM (Nguyen et al., 2015). Our cytotoxicity result of compound 3 against MCF-7 matched well with this report since the compound showed an IC50 value of 71.7 mM. Regarding compounds 5 and 7, the only reported activity is the cytotoxicity against HeLa cell line (Cheng et al., 2010). In this report, compound 5 showed cytotoxic activity with an IC50 value of 63 mM while compound 7 was inactive at a concentration <300 mM. Our results matched well with this report since compound 5 showed cytotoxic activity against HeLa cell line with an IC50 value of 85.1 mM while compound 7 was inactive at a concentration 100 mM. Regarding compound 6, we are the first to report its cytotoxicity against MCF-7 and HCT-116 with IC50 values of 1.2 and 40.1 mM, respectively, Since the only reported activity of the compound was HIV-1 protease inhibition with an IC50 value of 58 mM (Sato et al., 2009). 3. Experimental 3.1. General experimental procedures Optical rotations were measured with a Jasco DIP-370 polarimeter. HPLC analysis was carried out using Inertsil ODS-3 column (5 mm, 4.6 150 mm, GL Science, Tokyo, Japan) attached to Agilent 1220 Infinity LC system, equipped with a binary solvent deliver system, an autosampler and a photodiode array detector monitored at 254, 280 nm, was used. 1H and 13C NMR spectra were obtained on a Bruker DRX 600 NMR spectrometer (Bruker Daltonics Inc., MA, USA) using TMS as an internal standard for chemical shifts. Chemical shifts (d) were expressed in ppm with reference to the TMS resonance. HR-ESI–MS data were determined using LC–MS–IT–TOF (Shimadzu, Tokyo, Japan). The instrument was fitted with an Inertsil ODS-3 column (5 mm, 4.6 150 mm, GL Science, Tokyo, Japan); using mobile phase composed of solvents A (water) and B (acetonitrile). The total flow rate was 0.5 mL/min. Based on the previous result of HPLC–PDA analysis, the LC chromatogram of compound 1 and 2 was obtained at UV 254 nm and 280 nm. Using isocratic elution of H2O—ACN (40:60), Rt of compound 1 was 15.9 min and that of compound 2 was 17.3 min. The MS instrument was operated using an ESI source in both positive and negative ionization modes with survey scans acquired from m/z 100–2000 for MS and m/z 50–1500 for MS/MS. The ionization parameters were as follows: probe voltage,
4.5 kV; nebulizer gas flow, 1.5 L/min; CDL temperature, 200 C; heat block temperature, 200 C. Dimethylsulfoxide (DMSO) and organic solvents were purchased from Wako Pure Chemical Industries (Osaka, Japan). Sephadex LH-20 was purchased from GE Healthcare (Uppsala, Sweden). Silica gel (75–120 mesh) and RP-18 silica gel (38–63 mm) were purchased from Wako Pure Chemical Industries (Osaka, Japan). Thin layer chromatography (TLC) silica gel 60 F254 were purchased from Merck Co., Darmstadt, Germany. Plates were developed in a solvent mixture of different organic solvents, and the developed chromatograms were visualized under 254 nm UV light and the spots were made visible by spraying with vanillin/H2SO4 reagent before warming in an oven preheated to 110 C for 5 min. 3.2. Fungal material The dried ethanolic extract of the fruiting bodies of G. lingzhi were kindly provided by Toyotanshien Co. Ltd. (3-1 Kitanijyonishi, Chuo-ku, Sapporo 060-0002, Japan). 3.3. Extraction and isolation The dried and ground fruiting bodies of G. lingzhi (100 kg) were extracted with 99.5% ethanol by Toyotanshien Co., Ltd. to give an extract (7.22 kg). The solvent free ethanolic extract was divided into parts. Each part was dissolved in the least amount of ethanol, diluted with distilled water in a separating funnel and then extracted successively with CHCl3 till exhaustion to get the CHCl3 fraction (totally 4.32 kg; 59.8% of the dried ethanolic extract). The process was repeated several times because of the large quantity of the ethanolic extract. A part of the CHCl3 fraction (2.16 kg) was subjected to chromatography on a silica gel column (70 16.5 cm). The column was gradient eluted with n-hexane–EtOAc (100:0 ! 0:100) then EtOAc–MeOH (100:0 ! 50:50), to give fractions (A-P). Fraction I (160 g) eluted with n-hexane: EtOAc (35: 65), was chromatographed again over silica gel column and gradient eluted with n-hexane–EtOAc (90:10 ! 0:100) to give 53 fractions. Fractions from 32 to 36 (39 g) was then chromatographed over silica gel column (75 4.5 cm) and gradient eluted with n-hexane-acetone (90: 10 ! 65: 35) to give 40 fractions, numbered as A. Fractions 21A-24A (4.2 gm) was chromatographed over silica gel column (25 6 cm) and gradient eluted with Dichloromethane (DCM)—MeOH (100:0 ! 96:4) to give 65 fractions. Fractions (41–45) (700 mg) were then chromatographed over Sephadex LH-20 column and isocratically eluted with DCM—MeOH (100:0 ! 96:4) to yield 12 fractions, of them fractions 4–8 (350 mg) were failed to be isolated by isocratic elution or gradient elution using H2O—MeOH over RP-18 silica gel column, so these fractions were chromatographed over RP-18 silica gel column (45 1.5 cm) and gradient eluted with H2O—ACN (55:45 ! 45:55), monitored by HPLC for careful follow-up of the fractions because of the close Rf values of the compounds on RP chromatograms, to yields compounds 1 (4 mg), 2 (3 mg), 3 (12.2 mg), 4 (10.8 mg) and 5 (9.5 mg). The Rt values of these compounds are as follows 9.2 min (3), 11.9 min (4), 15.9 min (1), 17.3 min (2) and 18.7 min (5) by using Inertsil ODS-3 column (5 mm, 4.6 150 mm) and a mobile phase composed of solvents A (water 40%) and B (acetonitrile 60%). The total flow rate was 0.5 mL/min. Fractions 34A-35A, 700 mg of these fractions were used in the isolation process of compounds 6, 7 and 8 by column chromatography over silica gel column (40 1.5 cm) to give 58 fractions. Fractions 19–23 (118 mg), were chromatographed over RP-18 silica gel column (35 1.5 cm) and isocratically eluted with H2O—ACN (65:35). Compound 6 (2 mg) was obtained in subfractions 9–11, compound 7 (4 mg) was obtained in subfractions 28–30 and compound 8 (9.6 mg) was obtained in subfractions 47–58.
Y.M. Amen et al. / Phytochemistry Letters 17 (2016) 64–70
To test for the natural occurrence of the ethyl ester compounds 2 and 5, we simulated the extraction process on the free acids 7 and 8 to check whether they will result in formation of 2 and 5, respectively or not, using a procedure reported by (Lee et al., 2011) with modifications. One mg of each of 7 and 8 was treated separately with ethyl alcohol for five days followed by distillation at 60 C. The residue obtained in each case, was then treated with ethyl acetate for another five days followed by evaporation of the solvent. RP-TLC of the residues of 7 and 8 on precoated silica gel plates GF254 using the solvent system H2O—ACN (4:6), revealed Rf values of 0.54 and 0.51, respectively exactly similar to the Rf values of 7 and 8, while compounds 2 and 5, showed Rf values of 0.3 and 0.27, respectively. 3.4. Cell culture All cell lines were obtained from Riken Bio resource Center of Japan (Ibaraki, Japan). The medium for culturing human breast cancer cells (MCF-7) and human cervical cancer cells (HeLa), is Eagle’s minimal essential medium (EMEM) (Wako Pure Chemical Industries, Osaka, Japan). The medium for culturing human hepatocellular cells (HepG2), human colorectal carcinoma cells (Caco-2), normal human fibroblast cells (TIG-1) and normal human fetal lung fibroblast cells (HF19), is Dulbecco’s Modified Eagle’s medium (DMEM) (Wako Pure Chemical Industries, Osaka, Japan). Human colorectal carcinoma cells (HCT-116) were cultured in McCoy’s 5A Medium (Wako Pure Chemical Industries, Osaka, Japan). All culture media were supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 U/mL streptomycin (Gibco BRL, Tokyo, Japan). Cytotoxicity to cancer and normal cell lines was determined as follows: cells were cultured in 96-well plates at densities of 2 104 cells/well, in a humid atmosphere of 5% (v/v) CO2 and 95% (v/v) air at 37 C. After 24 h, the cells were treated with six different concentrations of the isolated compounds (3.125, 6.25, 12.5, 25, 50, and 100 mM, dissolved in DMSO). 5-Fluorouracil was used as a positive control. After 72 h, cell viability was determined using WST-1 reagent as follows: 10 mL of WST-1 reagent was added to each well, followed by 2 h incubation at 37 C, after which the absorbance was measured at 450 nm using a Microplate Reader (Biotek, Winooski, VT, USA). 4. Conclusion In this contribution, eight triterpenoids were isolated from the fruiting bodies of Ganoderma lingzhi and two of them are new natural products. The chemical structures of the new compounds were elucidated on the basis of spectroscopic studies. All the compounds were assayed for their cytotoxicity against five cancer cell lines and two normal cell lines. The new compounds showed a strong to moderate cytotoxic activity against MCF-7 while they showed moderate to weak activity against HeLa cell line. None of the compounds showed cytotoxicity to normal cell lines (TIG-1 and HF19). For further clarification, we are continuing to investigate deeply the mechanism of cytotoxic activity of the potent compounds. Acknowledgments The Egyptian Government is acknowledged for the fellowship support to Yhiya M. Amen. We would like to express our gratitude to Prof. Tomofumi Miyamoto, graduate school of pharmaceutical sciences, Kyushu University, Japan, for carrying out the optical rotation measurement. We would like to thank Research and Education Support Center of the faculty of Agriculture, Kyushu University for supporting facilities for ESI-IT-TOF analysis. This work was performed under the Cooperative Research Program of
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“Network Joint Research Center for Materials and Devices”, Kyushu University. This work was supported by KAKENHI Grant Numbers 26660147 and 26304047. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phytol.2016. 07.024. References Akihisa, T., Nakamura, Y., Tagata, M., Tokuda, H., Yasukawa, K., Uchiyama, E., Suzuki, T., Kimura, Y., 2007. Anti-inflammatory and anti-tumor-promoting effects of triterpene acids and sterols from the fungus Ganoderma lucidum. Chem. Biodivers. 4, 224–231. doi:http://dx.doi.org/10.1002/cbdv.200790027. Amen, Y.M., Zhu, Q., Tran, H.-B., Afifi, M.S., Halim, A.F., Ashour, A., Mira, A., Shimizu, K., 2016. Lucidumol C, a new cytotoxic lanostanoid triterpene from Ganoderma lingzhi against human cancer cells. J. Nat. Med. doi:http://dx.doi.org/10.1007/ s11418-016-0976-2. Baby, S., Johnson, A.J., Govindan, B., 2015. Secondary metabolites from Ganoderma. 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Phytochemistry Letters journal homepage: www.elsevier.com/locate/phytol
New cytotoxic lanostanoid triterpenes from Ganoderma lingzhi Yhiya M. Amena,b , Qinchang Zhua , Mohamed S. Afifib , Ahmed F. Halimb , Ahmed Ashourb , Kuniyoshi Shimizua,* a Division of Systematic Forest and Forest Products Sciences, Department of Agro-Environmental Sciences, Graduate School of Bioresource and Bioenvironmental Sciences, Kyushu University, 812-8581, Japan b Department of Pharmacognosy, Faculty of Pharmacy, Mansoura University, Mansoura 35516, Egypt
A R T I C L E I N F O
Article history: Received 8 April 2016 Received in revised form 25 June 2016 Accepted 6 July 2016 Available online xxx Keywords: Ganoderma lingzhi Lanostane triterpenes Cytotoxicity
A B S T R A C T
Further chemical investigation of the metabolites in the fruiting bodies of Ganoderma lingzhi resulted in isolation of eight triterpenes; two of them are new triterpene acid ethyl esters. Their structures were established based on spectroscopic studies and comparison with the known related compounds. The anticancer potential of the isolates were tested with an in vitro cytotoxic assay against five human cancer cell lines (MCF-7, HeLa, HCT-116, Caco-2 and HepG2) and two normal human cell lines (TIG-1 and HF19). Results showed that the new compounds have a strong to moderate selective cytotoxic activity against MCF-7 while they showed moderate to weak activity against HeLa cell line. Potent cytotoxic activities of some of the known isolated compounds are reported for the first time. ã 2016 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
1. Introduction Cancer is a leading cause of mortality in economically developed countries and the second leading cause of death in developing countries (Jemal et al., 2011). The number of new cancer cases reported annually worldwide will reach 15 million by 2020, with 70% of these cases occurring in developing countries (Kuete et al., 2013). Although chemotherapy is the most commonly used method to treat patients with cancer, the application of chemotherapeutic techniques is limited by their high cellular toxicity due to their lack of selectivity against cancer cells as well as the occurrence of other long-term side effects. Therefore, the ultimate goal of cancer chemotherapy is the development of new therapeutic selective agents that can kill malignant tumor cells or render them benign without affecting normal cells (Khazir et al., 2014). As a part of our continuing research for exploring the bioactive principles of Ganoderma lingzhi, we report the isolation of eight triterpenes including two new ones triterpene acid ethyl esters. The new compounds have a strong to moderate selective cytotoxic activity against MCF-7 while they showed moderate to weak activity against HeLa cell line without causing any cytotoxic activity to normal cell lines (TIG-1 and HF19). We reported also
some potent cytotoxic activities for some of the known isolated compounds for the first time. Ganoderma lingzhi is a well-known crude drug that has been used clinically in Oriental countries since ancient times due to its health promoting and tonic effects (Xia et al., 2014). During the past four decades, more than 400 secondary metabolites have been isolated from various Ganoderma species (Baby et al., 2015). The number of the new compounds identified from Ganodema increasing day after day. Triterpenoids are typical chemical constituents in G. lingzhi and possess important roles in pharmacological activities as anticancer (Chen and Zhong, 2009; Jiang et al., 2008; Liu et al., 2009; Wu et al., 2013, 2001; Zhao et al., 2015), antiviral (El-Mekkawy et al., 1998; Min et al., 1998; Zhu et al., 2015), anti-diabetic (Fatmawati et al., 2011; Zhao et al., 2015), antiandrogenic (Liu et al., 2006) and anti-inflammatory (Akihisa et al., 2007; Ko et al., 2008). Recently, we reported the isolation of a new lanostanoid triterpene, named lucidumol C with a potent, selective cytotoxicity against HCT-116 and remarkable cytotoxic activities against Caco-2, HepG2 and HeLa cell lines (Amen et al., 2016). Herein, we describe further, the structural elucidation and cytotoxicity of the isolated compounds against several cell lines besides their selectivity against two normal human fibroblast cell lines has been tested. 2. Results and discussion
* Corresponding author. E-mail address: [email protected] (K. Shimizu).
The chloroform fraction of the EtOH extract of G. lingzhi was subjected to chromatographic purification using Sephadex LH-20
http://dx.doi.org/10.1016/j.phytol.2016.07.024 1874-3900/ã 2016 Phytochemical Society of Europe. Published by Elsevier Ltd. All rights reserved.
Y.M. Amen et al. / Phytochemistry Letters 17 (2016) 64–70
Compound 1 was obtained as a white powder with [a]24D + 48.6 (c 0.22, MeOH). HR-ESI–MS spectrum gave a protonated molecular ion peak at m/z 541.3145 [M+H]+, calculated (541.3160) and a deprotonated molecular ion peak at m/z 539.3048 [MH], calculated (539.3014) in accordance with the molecular formula C32H44O7. It exhibited UV absorption at 247 nm. The 1H NMR
and repeated silica gel chromatographic steps, followed by reversed-phase column chromatography, to afford eight lanostanoid triterpenes, including two new compounds (1, 2). The known compounds (3–8) were identified by matching their spectral data with those of the corresponding ones and/or relevant compounds (Fig. 1).
21
22
18
11
28
30
7
4
O
COOCH2CH3
O COOCH2CH3
O O
27
15
9
26
O
13
1
O
24
20
O
19
O
OH
OH
O
OH
2
1
29
65
COOCH2CH3
COOCH3 O
O
O
O O
O
OH
OH
4
3
O
O COOCH2CH3
O
COOH
O
O
O
O O
O
O
OH
5
O
6
O
O COOH
O
O COOH
O
O
O
O O
OH
O O
O
8
7 Fig. 1. Structures of the isolated compounds (1–8).
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Y.M. Amen et al. / Phytochemistry Letters 17 (2016) 64–70
positions of unsaturation (D8,9) as follows: there were correlations observed between the methyl protons resonating at dH 1.43 (H3-30) and a carbon resonance at dc 142.4 beside correlations of H-7 with dc 142.4 thus placed dc 142.4 to C-9. The carbon resonance dc 159.9 was positioned to C-8 based on correlations of H-5 with dc 159.9 together with the HMBC cross-peak of H-7 with dc 159.9 supported by the other correlations of H-19 (1.24, s) with the same carbon resonance at dc 159.9. The above evidence indicated the presence of 8-ene structural fragment. The connectivity of a carbonyl group at C-11 was evidenced from the correlations of H-12 [(2.57 m, 3.04 d (J = 17.4)] with a carbonyl group at dc 199.6, thus assigned dc 199.6 to C-11. The HMBC correlations observed of H-12 with dc 159.9, confirmed the olefinic bond at C-8, C-9. The 13C NMR further revealed a carbon resonance at dc 216.9 which was carefully placed to C-15 based on a cross-peak in the HMBC spectrum, showed between dH 1.43 (s, H-30) with a carbonyl group at dc 216.9. The assignment of the carbon resonances of the side chain was again possible with the help of HMBC correlations. The position of the alkene in the side-chain was confirmed through correlations showed between the singlet peak at dH 2.14, assigned for H-21, with a carbon resonance at dc 155.8 together with a crosspeak between dH 2.87 (m), assigned for H-16, with the same carbon resonance at dc 155.8, thus placed carefully to C-20. Furthermore, HMBC showed cross-peaks between dH 3.24 (dd, 8.4, 18.6), assigned to H-17, to carbon resonances at dc 125.8 and 155.8, supported our previous conclusion. 1H NMR spectrum further
spectrum, analyzed with the aid of HMQC experiment, was indicative of five singlet methyl groups, at dH 0.86, 1.09, 1.13, 1.24 and 1.43, a doublet methyl resonance at dH 1.16 (J = 7.2), a multiplet methyl at dH 1.23, a vinyl methyl at dH 2.14, an oxymethine proton at dH 4.93 (dd, J = 7.8, 9.6) and one vinyl proton at dH 6.25 (s). The 13 C NMR spectrum, combined with HMQC experiment, confirmed the aforementioned moieties and displayed characteristic signals of eight methyl groups (dc 14.5, 17.4, 18.6, 19.6, 21.1, 21.2, 25.5 and 27.5), an oxygenated methine at dc 67.1, an a,b-unsaturated carbonyl at dc 159.9, 142.4 and 199.6, olefinic carbons at dc 125.8 and 155.8, ketone carbons at dc 200.7, 216.9 and 219.7, and a carboxyl carbonyl at dc 177.8. These data suggested an oxygenated lanostane-type triterpene with a conjugated enone (Xia et al., 2014). Unambiguous assignments for all the carbons could be made based on the correlations observed in HMBC spectrum (Fig. 2). In the HMBC spectrum, there were correlations between the proton signal at dH 1.60 assigned to H-5, and the carbon resonance at dc 219.7 and between the proton signals at dH 1.59 (m, H-1) and dH 2.88 (m, H-2) with the same carbon resonance at dc 219.7, thus established the signal dc 219.7 to C-3 of the skeleton. Furthermore, the proton signal of H-5 showed HMBC cross-peak with a carbon resonance at dc 67.1, thus established the attachment of a hydroxy group to C-7. The equatorial hydroxyl group at C-7 was deduced from the multiplicity of H-7 (dH 4.93, dd, J = 7.8, 9.6), which was further supported by the ROESY correlations observed from H-7 to H-5. Analysis of the HMBC spectrum confirmed the
O O O
O
O O
OH
1
O
O
O O O
O O
OH
2 Fig. 2. Selected HMBC correlations of 1 and 2.
O
Y.M. Amen et al. / Phytochemistry Letters 17 (2016) 64–70
revealed a down-field signal at dH 6.25, correlated through HMQC spectrum to a carbon resonance at dc 125.8 which is placed to C-22 based on HMBC correlations showed between H-21 (2.14, s) and dc 125.8. The remaining carbonyl group at dc 200.7, revealed by 13C NMR, was assigned to C-23 and confirmed by HMBC correlation of H-21 (2.14, s) with dc 200.7 together with correlations of dH 2.65 (m), 2.88 (m); assigned to H-24 and correlations of dH 2.91 (m), assigned to H-25 with the same carbon resonance at dc 200.7. The carboxylate moiety at dc 177.8, was assigned to C-26 based on correlations of H-24 [2.65 (m), 2.88 (m)], H-25 (2.91, m) and H-27 (1.16, d, J = 7.2) to the carbon resonance at dc 177.8. The presence of ethyl ester in compound 1 was confirmed by HMBC correlations of dH 4.11 (m)/dc 61.7 assigned for a methylene moiety with the carboxylate group at dc 177.8 and dc 14.5, assigned for a terminal methyl group, hence formed a carboxyethoxy side chain. Comparison of these spectroscopic data with those of methyl ganoderenate D (Shim et al., 2004), suggested that the skeleton should be the same except for the presence of carboxyethoxy side chain in 1 instead of carboxymethoxy side chain in methyl ganoderenate D. Hence, we proposed a name ethyl ganoderenate D to compound 1, which is a new natural product. Compound 2 was isolated as a white powder with [a]24D + 46.9 (c 0.13, MeOH). HR-ESI–MS spectrum showed a cationated molecular ion peak at m/z 621.2990 [M+Na]+, calculated (621.3040), and a deprotonated molecular ion peak at m/z 597.3068 [MH], calculated (597.3069) in agreement with the molecular formula C34H46O9. Its UV spectrum exhibited an absorption maximum at 247 nm. Comparison of the spectroscopic data of 2 with those of 1, revealed that the skeleton of 2 should be the same except for the protons [dH 5.78 (s), 2.08 (s)] and carbons (dc 80.6, 172.0, 21.1) resonances which were characteristic features of an acetoxyl group substituted on a carbon atom where two adjacent carbon atoms bear no proton (Cheng et al., 2010). The position of the acetoxyl group was further confirmed by a crosspeak between H-12 (5.78, s) and the carbon resonance at dc 172.0 in the HMBC spectrum, indicated that the acetoxyl group was located at C-12. Analysis of the ROESY experiment established the configuration at C-12 to be b for the acetoxy group, as there was no correlation observed between the singlet proton at dH 5.78 (H-12) with H-18 and H-19 considering the acetoxyl group was on the same face as Me-18 and Me-19 besides a correlation observed between H-12 and dH 1.71, assigned to H-5, confirmed the b-configuration of the acetoxy group at C-12. Comparison of the spectroscopic data with known compounds, compound 2 had almost the same chemical shifts as those of 7, except for an additional O-CH2CH3 group and upfielded esterified carboxylic carbon at dc 177.8. This evidence indicated that compound 2 was the ethyl esterified derivative of the known compound 12b-acetoxy-7b-hydroxy-3,11,15,23-tetraoxo-5a-lanosta-8,20dien-26-oic acid (7). Thus, compound 2 was characterized as 12b-acetoxy-7b-hydroxy-3,11,15,23-tetraoxo-5a-lanosta-8,20dien-26-oic acid ethyl ester; a new natural product. Since compounds 1 and 2 represent the ethyl esters of the corresponding ganoderic acids, the probability of being natural products might be suspected. To test for any direct esterification due to the long time contact with ethyl alcohol during the extraction process or to trans-esterification due to the repetitive use of ethyl acetate throughout the fractionation and purification steps, two of the isolated ganoderic acids, compounds 7 and 8 (Fig. 1) were selected and each was subjected to the method mentioned in the experimental section. Only the original unreacted acids were detected on the TLC layer without any evidence of the formation of their esterified compounds 2 or 5 respectively. The structures of the known compounds were identified by comparison of their spectroscopic data with those reported in the
67
literature as methyl lucidenate A (3) (Nishitoba et al., 1985; Xia et al., 2014), ethyl lucidenate A (4) (Li et al., 2013), 12b-acetoxy3,7,11,15,23-pentaoxo-5a-lanosta-8-en-26-oi acid ethyl ester (5) (Cheng et al., 2010), ganoderic acid GS-1 (6) (Sato et al., 2009), 12b-acetoxy-7b-hydroxy-3,11,15,23-tetraoxo-5a-lanosta-8,20dien-26-oic acid (7) (Cheng et al., 2010) and ganoderic acid F (8) (Hirotani and Furuya, 1986; Komoda et al., 1985). It is worth mentioning that this is the second report of ganoderic acid GS-1 in nature and the first report of it from G. lingzhi since the compound was isolated before from the fruiting bodies of G. sinense (Sato et al., 2009) (Table 1). The cytotoxicity of the isolated triterpenoids was evaluated against human breast carcinoma (MCF-7), human cervical carcinoma (HeLa), human colorectal carcinoma (HCT-116, Caco-2) and human liver carcinoma (HepG2). Selectivity of the compounds was evaluated against normal human fibroblast cells (TIG-1) and normal human fetal lung fibroblast cells (HF19). The results are summarized in Table 2. None of the compounds showed cytotoxicity to normal cell lines (TIG-1 and HF19), reflecting the selectivity of the compounds to the normal human cell lines. Regarding cytotoxicity to cancer cell lines, compounds 1 and 2 showed a strong to moderate selective cytotoxic activity against MCF-7 while they showed moderate to weak activity against HeLa cell line. We also report for the first time, the cytotoxic activity of some of the isolated known compounds against several cancer cell lines as shown in Table 2. Reviewing the literature, we found some reports discussing the cytotoxicity of some of the isolated compounds as follows: Li et al. (2013) evaluated methyl lucidenate A (3) and ethyl lucidenate A (4) against HepG2, the compounds showed moderate cytotoxic activity with IC50 values of 49.82 and Table 1 1 H (600 MHz) and Position
*
C NMR (150 MHz) spectral data of 1 and 2.*
1 1
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 OCH2CH3 OCH2CH3 OCOCH3 OCOCH3
13
H (J Hz)
1.59 (m) 2.88 (m)
1.60 (m) 1.31 br dd (7.2, 13.2) 4.93 dd (7.8, 9.6)
2.57 (m), 3.04 (d, 17.4)
2.87 (m) 3.24 dd (8.4, 18.6) 0.86 (s) 1.24 (s) 2.14 (s) 6.25 (s) 2.65 (m), 2.88 (m) 2.91 (m) 1.16 (d, 7.2) 1.13 (s) 1.09 (s) 1.43 (s) 4.11 (m) 1.23 (m)
Measured in CD3OD.
2 13
1
36.5 35.2 219.7 47.8 49.0 29.2 67.1 159.9 142.4 39.4 199.6 49.0 47.2 59.5 216.9 36.7 49.9 19.6 18.6 155.8 21.2 125.8 200.7 49.1 36.7 177.8 17.4 27.5 21.1 25.5 61.7 14.5
2.51 (m) 2.49 (m)
C
H (J Hz)
1.71 (m) 1.17 (m) 4.92 (m)
5.78 (s)
2.85 (m) 3.42 (m) 1.09 (s) 1.22 (s) 2.12 (s) 6.30 (s) 2.66 (m), 2.87 (m) 2.85 (m) 1.13 (d, 7.2) 1.10 (s) 1.09 (s) 1.37 (s) 4.11 (m) 1.17 (m) 2.08 (s)
13
C
35.2 35.15 219.7 47.8 49.0 27.5 67.3 160.0 142.4 39.3 194.0 80.6 47.5 60.4 210.7 36.8 51.3 20.9 18.6 155.9 21.1 126.4 200.1 49.1 36.8 177.8 17.4 27.1 21.5 25.3 61.7 17.4 172.0 21.1
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Table 2 Cytotoxic activities against MCF-7, HeLa, HCT-116, Caco-2, HepG2, TIG-1 and HF19.* Compounds
1 2 3 4 5 6 7 8 5-FU
IC50 (mM) MCF-7
HeLa
HCT-116
Caco-2
HepG2
TIG-1
HF19
35.9 7.5 71.7 41.7 62.3 1.2 80.8 >100 >100
89.9 31.8 >100 48.9 85.1 >100 >100 >100 19.6
92.6 >100 >100 33.9 69.9 40.1 >100 >100 65.4
>100 >100 >100 >100 >100 >100 >100 >100 63.6
>100 >100 >100 >100 >100 >100 >100 >100 >100
>100 >100 >100 >100 >100 >100 >100 >100 >100
>100 >100 >100 >100 >100 >100 >100 >100 >100
* The activity was shown as IC50 value, which was the concentration of the tested compound (mM) that decreased the number of viable cells by 50%. Results are expressed as the mean value of triplicate data points.
55.15 mg/mL, respectively by using the conventional MTT cell viability method. Our results are in contrast with these results, compounds 3 and 4 did not show any activity against HepG2 at a concentration 100 mM. Compound 3 was also recently evaluated against three cancer cell lines; A549 (non-small cell lung adenocarcinoma), MCF-7 and PC-3 (prostatic small cell carcinoma). The authors considered the compound inactive since it showed an IC50 value more than 50 mM (Nguyen et al., 2015). Our cytotoxicity result of compound 3 against MCF-7 matched well with this report since the compound showed an IC50 value of 71.7 mM. Regarding compounds 5 and 7, the only reported activity is the cytotoxicity against HeLa cell line (Cheng et al., 2010). In this report, compound 5 showed cytotoxic activity with an IC50 value of 63 mM while compound 7 was inactive at a concentration <300 mM. Our results matched well with this report since compound 5 showed cytotoxic activity against HeLa cell line with an IC50 value of 85.1 mM while compound 7 was inactive at a concentration 100 mM. Regarding compound 6, we are the first to report its cytotoxicity against MCF-7 and HCT-116 with IC50 values of 1.2 and 40.1 mM, respectively, Since the only reported activity of the compound was HIV-1 protease inhibition with an IC50 value of 58 mM (Sato et al., 2009). 3. Experimental 3.1. General experimental procedures Optical rotations were measured with a Jasco DIP-370 polarimeter. HPLC analysis was carried out using Inertsil ODS-3 column (5 mm, 4.6 150 mm, GL Science, Tokyo, Japan) attached to Agilent 1220 Infinity LC system, equipped with a binary solvent deliver system, an autosampler and a photodiode array detector monitored at 254, 280 nm, was used. 1H and 13C NMR spectra were obtained on a Bruker DRX 600 NMR spectrometer (Bruker Daltonics Inc., MA, USA) using TMS as an internal standard for chemical shifts. Chemical shifts (d) were expressed in ppm with reference to the TMS resonance. HR-ESI–MS data were determined using LC–MS–IT–TOF (Shimadzu, Tokyo, Japan). The instrument was fitted with an Inertsil ODS-3 column (5 mm, 4.6 150 mm, GL Science, Tokyo, Japan); using mobile phase composed of solvents A (water) and B (acetonitrile). The total flow rate was 0.5 mL/min. Based on the previous result of HPLC–PDA analysis, the LC chromatogram of compound 1 and 2 was obtained at UV 254 nm and 280 nm. Using isocratic elution of H2O—ACN (40:60), Rt of compound 1 was 15.9 min and that of compound 2 was 17.3 min. The MS instrument was operated using an ESI source in both positive and negative ionization modes with survey scans acquired from m/z 100–2000 for MS and m/z 50–1500 for MS/MS. The ionization parameters were as follows: probe voltage,
4.5 kV; nebulizer gas flow, 1.5 L/min; CDL temperature, 200 C; heat block temperature, 200 C. Dimethylsulfoxide (DMSO) and organic solvents were purchased from Wako Pure Chemical Industries (Osaka, Japan). Sephadex LH-20 was purchased from GE Healthcare (Uppsala, Sweden). Silica gel (75–120 mesh) and RP-18 silica gel (38–63 mm) were purchased from Wako Pure Chemical Industries (Osaka, Japan). Thin layer chromatography (TLC) silica gel 60 F254 were purchased from Merck Co., Darmstadt, Germany. Plates were developed in a solvent mixture of different organic solvents, and the developed chromatograms were visualized under 254 nm UV light and the spots were made visible by spraying with vanillin/H2SO4 reagent before warming in an oven preheated to 110 C for 5 min. 3.2. Fungal material The dried ethanolic extract of the fruiting bodies of G. lingzhi were kindly provided by Toyotanshien Co. Ltd. (3-1 Kitanijyonishi, Chuo-ku, Sapporo 060-0002, Japan). 3.3. Extraction and isolation The dried and ground fruiting bodies of G. lingzhi (100 kg) were extracted with 99.5% ethanol by Toyotanshien Co., Ltd. to give an extract (7.22 kg). The solvent free ethanolic extract was divided into parts. Each part was dissolved in the least amount of ethanol, diluted with distilled water in a separating funnel and then extracted successively with CHCl3 till exhaustion to get the CHCl3 fraction (totally 4.32 kg; 59.8% of the dried ethanolic extract). The process was repeated several times because of the large quantity of the ethanolic extract. A part of the CHCl3 fraction (2.16 kg) was subjected to chromatography on a silica gel column (70 16.5 cm). The column was gradient eluted with n-hexane–EtOAc (100:0 ! 0:100) then EtOAc–MeOH (100:0 ! 50:50), to give fractions (A-P). Fraction I (160 g) eluted with n-hexane: EtOAc (35: 65), was chromatographed again over silica gel column and gradient eluted with n-hexane–EtOAc (90:10 ! 0:100) to give 53 fractions. Fractions from 32 to 36 (39 g) was then chromatographed over silica gel column (75 4.5 cm) and gradient eluted with n-hexane-acetone (90: 10 ! 65: 35) to give 40 fractions, numbered as A. Fractions 21A-24A (4.2 gm) was chromatographed over silica gel column (25 6 cm) and gradient eluted with Dichloromethane (DCM)—MeOH (100:0 ! 96:4) to give 65 fractions. Fractions (41–45) (700 mg) were then chromatographed over Sephadex LH-20 column and isocratically eluted with DCM—MeOH (100:0 ! 96:4) to yield 12 fractions, of them fractions 4–8 (350 mg) were failed to be isolated by isocratic elution or gradient elution using H2O—MeOH over RP-18 silica gel column, so these fractions were chromatographed over RP-18 silica gel column (45 1.5 cm) and gradient eluted with H2O—ACN (55:45 ! 45:55), monitored by HPLC for careful follow-up of the fractions because of the close Rf values of the compounds on RP chromatograms, to yields compounds 1 (4 mg), 2 (3 mg), 3 (12.2 mg), 4 (10.8 mg) and 5 (9.5 mg). The Rt values of these compounds are as follows 9.2 min (3), 11.9 min (4), 15.9 min (1), 17.3 min (2) and 18.7 min (5) by using Inertsil ODS-3 column (5 mm, 4.6 150 mm) and a mobile phase composed of solvents A (water 40%) and B (acetonitrile 60%). The total flow rate was 0.5 mL/min. Fractions 34A-35A, 700 mg of these fractions were used in the isolation process of compounds 6, 7 and 8 by column chromatography over silica gel column (40 1.5 cm) to give 58 fractions. Fractions 19–23 (118 mg), were chromatographed over RP-18 silica gel column (35 1.5 cm) and isocratically eluted with H2O—ACN (65:35). Compound 6 (2 mg) was obtained in subfractions 9–11, compound 7 (4 mg) was obtained in subfractions 28–30 and compound 8 (9.6 mg) was obtained in subfractions 47–58.
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To test for the natural occurrence of the ethyl ester compounds 2 and 5, we simulated the extraction process on the free acids 7 and 8 to check whether they will result in formation of 2 and 5, respectively or not, using a procedure reported by (Lee et al., 2011) with modifications. One mg of each of 7 and 8 was treated separately with ethyl alcohol for five days followed by distillation at 60 C. The residue obtained in each case, was then treated with ethyl acetate for another five days followed by evaporation of the solvent. RP-TLC of the residues of 7 and 8 on precoated silica gel plates GF254 using the solvent system H2O—ACN (4:6), revealed Rf values of 0.54 and 0.51, respectively exactly similar to the Rf values of 7 and 8, while compounds 2 and 5, showed Rf values of 0.3 and 0.27, respectively. 3.4. Cell culture All cell lines were obtained from Riken Bio resource Center of Japan (Ibaraki, Japan). The medium for culturing human breast cancer cells (MCF-7) and human cervical cancer cells (HeLa), is Eagle’s minimal essential medium (EMEM) (Wako Pure Chemical Industries, Osaka, Japan). The medium for culturing human hepatocellular cells (HepG2), human colorectal carcinoma cells (Caco-2), normal human fibroblast cells (TIG-1) and normal human fetal lung fibroblast cells (HF19), is Dulbecco’s Modified Eagle’s medium (DMEM) (Wako Pure Chemical Industries, Osaka, Japan). Human colorectal carcinoma cells (HCT-116) were cultured in McCoy’s 5A Medium (Wako Pure Chemical Industries, Osaka, Japan). All culture media were supplemented with 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 U/mL streptomycin (Gibco BRL, Tokyo, Japan). Cytotoxicity to cancer and normal cell lines was determined as follows: cells were cultured in 96-well plates at densities of 2 104 cells/well, in a humid atmosphere of 5% (v/v) CO2 and 95% (v/v) air at 37 C. After 24 h, the cells were treated with six different concentrations of the isolated compounds (3.125, 6.25, 12.5, 25, 50, and 100 mM, dissolved in DMSO). 5-Fluorouracil was used as a positive control. After 72 h, cell viability was determined using WST-1 reagent as follows: 10 mL of WST-1 reagent was added to each well, followed by 2 h incubation at 37 C, after which the absorbance was measured at 450 nm using a Microplate Reader (Biotek, Winooski, VT, USA). 4. Conclusion In this contribution, eight triterpenoids were isolated from the fruiting bodies of Ganoderma lingzhi and two of them are new natural products. The chemical structures of the new compounds were elucidated on the basis of spectroscopic studies. All the compounds were assayed for their cytotoxicity against five cancer cell lines and two normal cell lines. The new compounds showed a strong to moderate cytotoxic activity against MCF-7 while they showed moderate to weak activity against HeLa cell line. None of the compounds showed cytotoxicity to normal cell lines (TIG-1 and HF19). For further clarification, we are continuing to investigate deeply the mechanism of cytotoxic activity of the potent compounds. Acknowledgments The Egyptian Government is acknowledged for the fellowship support to Yhiya M. Amen. We would like to express our gratitude to Prof. Tomofumi Miyamoto, graduate school of pharmaceutical sciences, Kyushu University, Japan, for carrying out the optical rotation measurement. We would like to thank Research and Education Support Center of the faculty of Agriculture, Kyushu University for supporting facilities for ESI-IT-TOF analysis. This work was performed under the Cooperative Research Program of
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“Network Joint Research Center for Materials and Devices”, Kyushu University. This work was supported by KAKENHI Grant Numbers 26660147 and 26304047. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.phytol.2016. 07.024. References Akihisa, T., Nakamura, Y., Tagata, M., Tokuda, H., Yasukawa, K., Uchiyama, E., Suzuki, T., Kimura, Y., 2007. Anti-inflammatory and anti-tumor-promoting effects of triterpene acids and sterols from the fungus Ganoderma lucidum. Chem. Biodivers. 4, 224–231. doi:http://dx.doi.org/10.1002/cbdv.200790027. Amen, Y.M., Zhu, Q., Tran, H.-B., Afifi, M.S., Halim, A.F., Ashour, A., Mira, A., Shimizu, K., 2016. Lucidumol C, a new cytotoxic lanostanoid triterpene from Ganoderma lingzhi against human cancer cells. J. Nat. Med. doi:http://dx.doi.org/10.1007/ s11418-016-0976-2. Baby, S., Johnson, A.J., Govindan, B., 2015. Secondary metabolites from Ganoderma. 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