Chinese Journal of Natural Medicines 2014, 12(9): 06850688
Chinese Journal of Natural Medicines
Mycophenolic acid derivatives from cultures of the mushroom Laetiporus sulphureu FAN Qiong-Ying1, YIN Xia2, 3, LI Zheng-Hui2, LI Yan2, LIU Ji-Kai2, FENG Tao2, ZHAO Bao-Hua1 1
College of Life Science, Hebei Normal University, Shijiazhuang 050024, China;
2
State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of
Sciences, Kunming 650204, China; 3
University of Chinese Academy of Sciences, Beijing 100049, China Available online September 2014
[ABSTRACT] AIM: To investigate the chemical constituents of the cultures of Laetiporus sulphureus (Bull.) Murrill. METHOD: Compounds were isolated and purified by various chromatographic techniques. The structure of the new compound was determined by interpretation of MS and 1D-, 2D-NMR spectroscopic data, while the known compounds were identified by comparison of their data with those reported. RESULTS: Three mycophenolic acid derivatives, 6-((2E, 6E)-3, 7-dimethyldeca-2, 6-dienyl)-7-hydroxy-5-methoxy-4-methylphtanlan-1-one (1), 6-((2E, 6E)-3, 7, 11-trimethyldedoca-2, 6, 10-trienyl)-5, 7-dihydroxy-4-methylphtanlan-1-one (2), and 6-((2E, 6E)-3, 7, 11-trimethyldedoca-2, 6, 10-trienyl)-7-hydroxy-5-methoxy-4-methylphtanlan-1-one (3) were isolated. CONCLUSION: Among them, compound 1 was new, and compound 2 exhibited moderate cytotoxicity against HL-60, SMMC-7721, A-549, and MCF-7 cells, with IC50 values of 39.1, 31.1, 27.4, and 35.7 μmol·L−1, respectively. [KEY WORDS] Laetiporus sulphureus; Mycophenolic acid derivatives; Cytotoxicity
[CLC Number] R284.1
[Document code] A
[Article ID] 2095-6975(2014)09-0685-04
Introduction Laetiporus sulphureus (Bull.) Murrill, belonging to the ecological group xylotrophs, was reported to exhibit antioxidative [1], antibacterial activities [1-2], cytotoxic [3], and dopamine D2 receptor agonistic activities [3]. Previous chemical studies have revealed the presence of lanostanoid triterpenoids [3], pigments [4], and benzofurans [5]. In continuation of [Received on] 01-Jul.-2013 [Research funding] This project was supported by the National Basic Research Program of China (973 Program, No. 2009CB 522300), and the National Natural Sciences Foundation of China (Nos. 30830113, U1132607). [*Corresponding author] FENG Tao: Prof., Tel: 86-871-65216327; Fax: 86-871-65219934; E-mail: [email protected]; ZHAO Bao-Hua: Prof., Tel: 86-311-80789712; E-mail: zhaobaohua86178@ sohu.com These authors have no conflict of interest to declare. Published by Elsevier B.V. All rights reserved
research aimed at the discovery of biologically active secondary metabolites of higher fungi [6-13], cultures of L. sulphureus were studied. As a result, three mycophenolic acid analogues (1–3) with isoprene polyene side chains were obtained. Mycophenolic acid, a 150 year odyssey, has a great reputation from antibiotic, antitumor to immunosuppressant activities [14], and analogues and derivatives of mycophenolic acid were widely studied for their chemical diversity and promising activity. All of these compounds were evaluated for cytotoxic activities to determine if the changes in the side chain had effects on the bioactivities.
Results and Discussion Compound 1 had the molecular formula C22H30O5 on the basis of HR-EI-MS ([M]+, 374.209 5, Calcd. for C22H30O5, 374.209 3). The IR absorption bands at 3 428 and 1 737 cm−1 indicated OH and ester functional groups. Preliminary analysis of the 1H NMR data (Table 1) revealed an aromatic methyl group at δ 2.14 (s), a methoxy group at δ 3.77 (s), and an OCH2
– 685 –
Fan Qiong-Ying, et al. / Chin J Nat Med, 2014, 12(9): 685688
to that of 6-((2E, 6E)-3, 7, 11-trimethyldedoca-2, 6, 10-trienyl) -7-hydroxy-5-methoxy-4-methylphtanlan-1-one (3) [17], except that the double bond between C-10 and C-11 in 3 was cleaved, producing a hydroxymethyl at C-10 in 1. This conclusion was supported by the HMBC correlations from δH 3.58 (2H, t, J = 6.4 Hz, H-10) to δC 30.5 (t, C-9). In addition, the HMBC correlations from δH 2.14 (3H, s, Me-Ar) to C-4, C-3a and C-5, from δH 3.77 (3H, s, OMe) to C-5, and from H-1΄ to C-5, C-6 and C-7 permitted all the substituting groups on the phenyl ring to be located. Detailed analysis of the HMBC spectrum suggested that other units of 1 were the same as those of 3 (Fig. 2). The double bonds at C-2(3) and C-6(7) were determined to be E-form according to the ROESY correlations of H-11/H-1 and H-12/H-5. Thus, the structure of 1 was established as 6-((2E, 6E)-3, 7-dimethyldeca-2, 6-dienyl)- 7-hydroxy-5-methoxy-4-methylphtanlan-1-one.
Fig. 1 Structures of compounds 1–3 Table 1
1
H- and 13C NMR data of 1 (J in Hz)
Carbon
1 δH
1 3
δC mult. 172.8 s
5.19 (1H, s)
3a
69.9 t 143.9 s
4
116.6 s
5
163.5 s
6
122.5 s
7
153.6 s
7a
106.3 s
1
3.38 (2H, d, 7.0)
22.6 t
2
5.18 (1H, overlap)
122.0 d
Two known analogues were identified as 6-((2E, 6E)-3, 7, 11-trimethyldedoca-2, 6, 10-trienyl)-5, 7-dihydroxy-4-methylphtanlan-1-one (2) [18] and 6-((2E, 6E)-3, 7, 11-trimethyldedoca-2, 6, 10-trienyl)-7-hydroxy-5-methoxy-4-methylphtanlan-1-one (3) [17] by comparison of their spectroscopic data with those reported. Compounds 1–3 were evaluated for their cytotoxicity against five human cancer cell lines. Compound 2 exhibited moderate inhibitory effects against several human cancer cell lines (Table 2).
135.5 s
3 4
Fig. 2 Key 1H-1H COSY, HMBC and ROESY correlations for 1
1.99 (2H, m)
39.4 t
5
2.07 (2H, m)
26.1 t
6
5.09 (1H, m)
124.5 d 134.5 s
7 8
2.01 (2H, m)
35.8 t
9
1.63 (2H, m)
30.5 t
10
3.58 (2H, t, 6.4)
62.6 t
11
1.77 (3H, s)
16.0 q
12
1.57 (3H, s)
15.8 q
CH3-Ar
2.14 (3H, s)
11.5 q
OCH3
3.77 (3H, s)
60.9 q
Table 2 Cytotoxicity of compound 2 (IC50, μmol·L−1) Compound
HL-60
SMMC-7721
A-549
MCF-7
2
39.1
31.1
27.4
35.7
1.3
3.4
7.3
16.1
Cisplatin a
group at δ 5.19 (s). The 13C NMR (DEPT) data (Table 1) displayed four CH3 including an aromatic and a methoxy group, seven CH2 (two oxygenated), two CH, and nine quaternary carbons (one lactone carbonyl), indicating the existence of a full substituted aromatic ring. In the HMBC spectrum (Fig. 2), the correlations from δH 5.19 (1H, s, H-3) to δC 172.8 (s, C-1), 143.9 (s, C-3a), and 106.3 (s, C-7a) indicated the existence of an α, β-unsaturated-γ-lactone, showing a similar nucleus as that of mycophenolic acid [15-16]. Further comparison of the NMR data suggested that compound 1 had a similar structure
a
SW480 > 40 14.7
Positive control
Experimental General experimental procedures Column chromatography (CC): silica gel (200–300 mesh, Qingdao Marine Chemical Ltd., People’s Republic of China), Rp-18 gel (20–45 μm, Fuji Silysia Chemical Ltd., Japan) and Sephadex LH-20 (Amersham Biosciences, Sweden). UV and IR spectra were obtained on a Shimadzu UV2401PC (Shimadzu, Kyoto, Japan) and a Bruker Tensor 27 FT-IR spectrometer with KBr pellets (Bruker, Karlsruher, Germany). 1D-and
– 686 –
CHEN You-Hua, et al. / Chin J Nat Med, 2014, 12(9): 685688
2D-NMR spectra were obtained on Bruker AM-400, DRX-500, or AV-600 spectrometers with tetramethylsilane (TMS) as an internal standard at room temperature, J in Hz. HR-EI-MS: Water Autospec Premier P776. Preparative HPLC was performed on an Agilent 1100 liquid chromatography system (Agilent Technologies, Santa Clara, CA, USA) equipped with a Zorbax SB-C18 column (21.2 mm × 150 mm). Fractions were monitored by TLC. Spots were visualized by heating silica gel plates immersed in Vanillin-H2SO4 in ethanol. Fungal material and cultivation conditions The fungus Laetiporus sulphureus (Bull.) Murrill. was collected from Changbai Mountain, Jilin Province, China, in 2006. The fungus was identified by Prof. DAI Yu-Cheng at Beijing Forestry University. The culture medium consisted of glucose (5%), peptone from porcine meat (0.15%), yeast powder (0.5%), KH2PO4 (0.5%) and MgSO4. Fermentation was carried out on a shaker at 160 rpm for 25 days. Extraction and isolation The culture broth (21 L) was filtered, and the filtrate was extracted three times with ethyl acetate (V/V, 1 : 1), while the mycelium was extracted three times with CHCl3–MeOH (1 : 1, 4 L). The EtOAc layer, together with the mycelium extract, was concentrated under reduced pressure to give a crude extract (8.5 g). This residue was separated over silica gel column by gradient elution with CHCl3–MeOH (1 : 0 to 0 : 1; 2 L) to obtain six fractions (Frs. 1–6). Fr. 2 (700 mg) was separated over CC (Sephadex LH-20; CHCl3–MeOH, 1 : 1, 700 mL) to give three fractions (Fr. 2.1–Fr. 2.3). The separation of Fr. 2.1 (80 mg) over CC (silica gel; CHCl3–Me2CO, 100 : 1, 500 mL) yielded 2 (10 mg). Fr. 2.3 (85 mg) was separated by semipreparative HPLC (MeCN–H2O, 3 : 1 to 9 : 1, 10 mL·min-1 in 15 min) to give 3 (21 mg). Fr. 3 (135 mg) was subjected to CC (Rp-18 gel; MeOH–H2O, 1 : 1 to 1 : 0, 300 mL), followed by semipreparative HPLC (MeCN–H2O, 1 : 1 to 4 : 1, 10 mL·min−1 in 10 min) to yield 1 (9.6 mg). Cytotoxicity assay Human myeloid leukemia HL-60, hepatocellular carcinoma SMMC-7721, lung cancer A-549 cells, breast cancer MCF-7, and colon cancer SW480 cell lines were used in the cytotoxicity assay. All the cell lines were cultured in RPMI-1640 or DMEM medium (Hyclone, Logan, UT, USA), supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA) in 5% CO2 at 37 C. The cytotoxicity assay was performed according to the MTT (3-(4, 5-dimethylthiazol2-yl)-2, 5-diphenyl tetrazolium bromide) method in 96-well microplates [19]. Briefly, 100 µL adherent cells were seeded into each well of 96-well cell culture plates and allowed to adhere for 12 h before drug addition, while suspended cells were seeded immediately before drug addition with initial density of 1 × 105 cells/mL. Each tumor cell line was exposed to the test compound dissolved in DMSO at concentrations of 0.0625, 0.32, 1.6, 8, and 40 μmol·L−1 in triplicate
for 48 h, with cisplatin (Sigma, St. Louis, MO, USA) as a positive control (IC50 values: see Table 2). After compound treatment, cell viability was detected and a cell growth curve was plotted. IC50 values were calculated by Reed and Muench’s method [20].
Identification 6-((2E, 6E)-3, 7-Dimethyldeca-2, 6-dienyl)-7-hydroxy5-methoxy-4-methylphtanlan-1-one (1), colorless oil; IR (KBr) max: 3 428, 2 937, 1 737, 1 621, 1 455, 1 135 cm−1; UV max (MeOH) nm (log ) 347 (2.91), 304 (3.60), 206 (5.01); 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 150 MHz) data, see Table 1; HR-EI-MS m/z 374.209 5 ([M]+, Calcd. for 374.209 3).
References [1]
[2]
[3]
[4]
[5]
[6]
[7] [8]
[9]
[10]
[11]
[12] [13]
[14]
– 687 –
Karaman M, Jovin E, Malbasa R, et al. Medicinal and edible lignicolous fungi as natural sources of antioxidative and antibacterial agents [J]. Phytother Res, 2010, 24 (10): 1473-1481. Kim SE, Lee IK, Jung YA, et al. Mushrooms collected from Deogyu mountain, Muju, Korea and their antioxidant activity [J]. Mycobiology, 2012, 40 (2): 134-137. Zjawiony JK. Biologically active compounds from Aphyllophorales (Polypore) fungi [J]. J Nat Prod, 2004, 67 (2): 300-310. Davoli P, Mucci A, Schenetti L,et al. Laetiporic acids, a family of non-carotenoid polyene pigments from fruit-bodies and liquid cultures of Laetiporus sulphureus (Polyporales, Fungi) [J]. Phytochemistry, 2005, 66 (7): 817-823. Yoshikawa K, Bando S, Arihara S, et al. A benzofuran glycoside and an acetylenic acid from the fungus Laetiporus sulphureus var. miniatus [J]. Chem Pharm Bull, 2001, 49 (3): 327-329. Ding JH, Feng T, Li ZH, et al. Trefolane A, a sesquiterpenoid with a new skeleton from cultures of the basidiomycete Tremella foliacea [J]. Org Lett, 2012, 14 (18): 4976-4978. Jiang MY, Feng T, Liu JK. N-Containing compound of macromycetes [J]. Nat Prod Rep, 2011, 28 (3): 783-808. Jiang MY, Li Y, Wang F, et al. Isoprenylated cyclohexanoids from the basidimycete Hexagonia speciosa [J]. Phytochemistry, 2011, 72 (9): 923-928. Liu LY, Li ZH, Dong ZJ, et al. Two novel fomannosane-type sesquiterpenoids from the culture of the basidiomycete Agrocybe salicacola [J]. Nat Prod Bioprospect, 2012, 2 (3): 130-132. Wang F, Zhou DS, Wei GZ, et al. Chlorantholides A-F, eudesmane-type sesquiterpene lactones from Chloranthus elatior [J]. Phytochemistry, 2012, 77: 312-317. Yin X, Feng T, Li ZH, et al. Chemical investigation on the cultures of the fungus Xylaria carpophila [J]. Nat Prod Bioprospect, 2011, 1 (2): 75-80. Zhou ZY, Liu JK. Pigments of fungi (macromycetes) [J]. Nat Prod Rep, 2010, 27 (11): 1531-1570. Zhou ZY, Shi GQ, Fontaine R, et al. Evidence for the natural toxin from the mushroom Trogia venenata as a cause of suden unexpected death in yunnan province, China [J]. Angew Chem Int Ed, 2012, 51 (10): 2368-2370. Bentley R. Mycophenolic acid: a one hundred year odyssey from antibiotic to immunosuppressant [J]. Chem Rev, 2000, 100 (10): 3801-3825.
Fan Qiong-Ying, et al. / Chin J Nat Med, 2014, 12(9): 685688
[15] Danheiser RL, Gee SK, Perez JJ. Total synthesis of mycophenolic acid [J]. J Am Chem Soc, 1985, 108 (4): 806-810. [16] Habib E, León F, Bauer JD, et al. Mycophenolic derivatives from Eupenicillium parvum [J]. J Nat Prod, 2008, 71 (11): 1915-1918. [17] Doerfler DL, Ernst LA, Campbell IM. Biosynthetic inter- mediates en route to mycophenolic acid in Penicillium brevi- compactum [J]. J Chem Soc Chem Commun, 1980, (8): 329-330.
[18] Canonica L, Rindone B, Scolastico C. Synthesis of myco- phenolic acid analogs through prenylation of 5, 7-dihydroxyphthalides [J]. Tetrahedron Lett, 1971, 12 (28): 2689- 2690. [19] Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays [J]. J Immunol Methods, 1983, 65 (1-2): 55-63. [20] Reed LJ, Muench H. A simple method of estimating fifty percent endpoints [J]. Am J Hygiene, 1938, 27 (3): 493-497.
Cite this article as: FAN Qiong-Ying, YIN Xia, LI Zheng-Hui, LI Yan, LIU Ji-Kai, FENG Tao, ZHAO Bao-Hua. Mycophenolic acid derivatives from cultures of the mushroom Laetiporus sulphureu [J]. Chinese Journal of Natural Medicines, 2014, 12(9): 685-688
– 688 –
Chinese Journal of Natural Medicines
Mycophenolic acid derivatives from cultures of the mushroom Laetiporus sulphureu FAN Qiong-Ying1, YIN Xia2, 3, LI Zheng-Hui2, LI Yan2, LIU Ji-Kai2, FENG Tao2, ZHAO Bao-Hua1 1
College of Life Science, Hebei Normal University, Shijiazhuang 050024, China;
2
State Key Laboratory of Phytochemistry and Plant Resources in West China, Kunming Institute of Botany, Chinese Academy of
Sciences, Kunming 650204, China; 3
University of Chinese Academy of Sciences, Beijing 100049, China Available online September 2014
[ABSTRACT] AIM: To investigate the chemical constituents of the cultures of Laetiporus sulphureus (Bull.) Murrill. METHOD: Compounds were isolated and purified by various chromatographic techniques. The structure of the new compound was determined by interpretation of MS and 1D-, 2D-NMR spectroscopic data, while the known compounds were identified by comparison of their data with those reported. RESULTS: Three mycophenolic acid derivatives, 6-((2E, 6E)-3, 7-dimethyldeca-2, 6-dienyl)-7-hydroxy-5-methoxy-4-methylphtanlan-1-one (1), 6-((2E, 6E)-3, 7, 11-trimethyldedoca-2, 6, 10-trienyl)-5, 7-dihydroxy-4-methylphtanlan-1-one (2), and 6-((2E, 6E)-3, 7, 11-trimethyldedoca-2, 6, 10-trienyl)-7-hydroxy-5-methoxy-4-methylphtanlan-1-one (3) were isolated. CONCLUSION: Among them, compound 1 was new, and compound 2 exhibited moderate cytotoxicity against HL-60, SMMC-7721, A-549, and MCF-7 cells, with IC50 values of 39.1, 31.1, 27.4, and 35.7 μmol·L−1, respectively. [KEY WORDS] Laetiporus sulphureus; Mycophenolic acid derivatives; Cytotoxicity
[CLC Number] R284.1
[Document code] A
[Article ID] 2095-6975(2014)09-0685-04
Introduction Laetiporus sulphureus (Bull.) Murrill, belonging to the ecological group xylotrophs, was reported to exhibit antioxidative [1], antibacterial activities [1-2], cytotoxic [3], and dopamine D2 receptor agonistic activities [3]. Previous chemical studies have revealed the presence of lanostanoid triterpenoids [3], pigments [4], and benzofurans [5]. In continuation of [Received on] 01-Jul.-2013 [Research funding] This project was supported by the National Basic Research Program of China (973 Program, No. 2009CB 522300), and the National Natural Sciences Foundation of China (Nos. 30830113, U1132607). [*Corresponding author] FENG Tao: Prof., Tel: 86-871-65216327; Fax: 86-871-65219934; E-mail: [email protected]; ZHAO Bao-Hua: Prof., Tel: 86-311-80789712; E-mail: zhaobaohua86178@ sohu.com These authors have no conflict of interest to declare. Published by Elsevier B.V. All rights reserved
research aimed at the discovery of biologically active secondary metabolites of higher fungi [6-13], cultures of L. sulphureus were studied. As a result, three mycophenolic acid analogues (1–3) with isoprene polyene side chains were obtained. Mycophenolic acid, a 150 year odyssey, has a great reputation from antibiotic, antitumor to immunosuppressant activities [14], and analogues and derivatives of mycophenolic acid were widely studied for their chemical diversity and promising activity. All of these compounds were evaluated for cytotoxic activities to determine if the changes in the side chain had effects on the bioactivities.
Results and Discussion Compound 1 had the molecular formula C22H30O5 on the basis of HR-EI-MS ([M]+, 374.209 5, Calcd. for C22H30O5, 374.209 3). The IR absorption bands at 3 428 and 1 737 cm−1 indicated OH and ester functional groups. Preliminary analysis of the 1H NMR data (Table 1) revealed an aromatic methyl group at δ 2.14 (s), a methoxy group at δ 3.77 (s), and an OCH2
– 685 –
Fan Qiong-Ying, et al. / Chin J Nat Med, 2014, 12(9): 685688
to that of 6-((2E, 6E)-3, 7, 11-trimethyldedoca-2, 6, 10-trienyl) -7-hydroxy-5-methoxy-4-methylphtanlan-1-one (3) [17], except that the double bond between C-10 and C-11 in 3 was cleaved, producing a hydroxymethyl at C-10 in 1. This conclusion was supported by the HMBC correlations from δH 3.58 (2H, t, J = 6.4 Hz, H-10) to δC 30.5 (t, C-9). In addition, the HMBC correlations from δH 2.14 (3H, s, Me-Ar) to C-4, C-3a and C-5, from δH 3.77 (3H, s, OMe) to C-5, and from H-1΄ to C-5, C-6 and C-7 permitted all the substituting groups on the phenyl ring to be located. Detailed analysis of the HMBC spectrum suggested that other units of 1 were the same as those of 3 (Fig. 2). The double bonds at C-2(3) and C-6(7) were determined to be E-form according to the ROESY correlations of H-11/H-1 and H-12/H-5. Thus, the structure of 1 was established as 6-((2E, 6E)-3, 7-dimethyldeca-2, 6-dienyl)- 7-hydroxy-5-methoxy-4-methylphtanlan-1-one.
Fig. 1 Structures of compounds 1–3 Table 1
1
H- and 13C NMR data of 1 (J in Hz)
Carbon
1 δH
1 3
δC mult. 172.8 s
5.19 (1H, s)
3a
69.9 t 143.9 s
4
116.6 s
5
163.5 s
6
122.5 s
7
153.6 s
7a
106.3 s
1
3.38 (2H, d, 7.0)
22.6 t
2
5.18 (1H, overlap)
122.0 d
Two known analogues were identified as 6-((2E, 6E)-3, 7, 11-trimethyldedoca-2, 6, 10-trienyl)-5, 7-dihydroxy-4-methylphtanlan-1-one (2) [18] and 6-((2E, 6E)-3, 7, 11-trimethyldedoca-2, 6, 10-trienyl)-7-hydroxy-5-methoxy-4-methylphtanlan-1-one (3) [17] by comparison of their spectroscopic data with those reported. Compounds 1–3 were evaluated for their cytotoxicity against five human cancer cell lines. Compound 2 exhibited moderate inhibitory effects against several human cancer cell lines (Table 2).
135.5 s
3 4
Fig. 2 Key 1H-1H COSY, HMBC and ROESY correlations for 1
1.99 (2H, m)
39.4 t
5
2.07 (2H, m)
26.1 t
6
5.09 (1H, m)
124.5 d 134.5 s
7 8
2.01 (2H, m)
35.8 t
9
1.63 (2H, m)
30.5 t
10
3.58 (2H, t, 6.4)
62.6 t
11
1.77 (3H, s)
16.0 q
12
1.57 (3H, s)
15.8 q
CH3-Ar
2.14 (3H, s)
11.5 q
OCH3
3.77 (3H, s)
60.9 q
Table 2 Cytotoxicity of compound 2 (IC50, μmol·L−1) Compound
HL-60
SMMC-7721
A-549
MCF-7
2
39.1
31.1
27.4
35.7
1.3
3.4
7.3
16.1
Cisplatin a
group at δ 5.19 (s). The 13C NMR (DEPT) data (Table 1) displayed four CH3 including an aromatic and a methoxy group, seven CH2 (two oxygenated), two CH, and nine quaternary carbons (one lactone carbonyl), indicating the existence of a full substituted aromatic ring. In the HMBC spectrum (Fig. 2), the correlations from δH 5.19 (1H, s, H-3) to δC 172.8 (s, C-1), 143.9 (s, C-3a), and 106.3 (s, C-7a) indicated the existence of an α, β-unsaturated-γ-lactone, showing a similar nucleus as that of mycophenolic acid [15-16]. Further comparison of the NMR data suggested that compound 1 had a similar structure
a
SW480 > 40 14.7
Positive control
Experimental General experimental procedures Column chromatography (CC): silica gel (200–300 mesh, Qingdao Marine Chemical Ltd., People’s Republic of China), Rp-18 gel (20–45 μm, Fuji Silysia Chemical Ltd., Japan) and Sephadex LH-20 (Amersham Biosciences, Sweden). UV and IR spectra were obtained on a Shimadzu UV2401PC (Shimadzu, Kyoto, Japan) and a Bruker Tensor 27 FT-IR spectrometer with KBr pellets (Bruker, Karlsruher, Germany). 1D-and
– 686 –
CHEN You-Hua, et al. / Chin J Nat Med, 2014, 12(9): 685688
2D-NMR spectra were obtained on Bruker AM-400, DRX-500, or AV-600 spectrometers with tetramethylsilane (TMS) as an internal standard at room temperature, J in Hz. HR-EI-MS: Water Autospec Premier P776. Preparative HPLC was performed on an Agilent 1100 liquid chromatography system (Agilent Technologies, Santa Clara, CA, USA) equipped with a Zorbax SB-C18 column (21.2 mm × 150 mm). Fractions were monitored by TLC. Spots were visualized by heating silica gel plates immersed in Vanillin-H2SO4 in ethanol. Fungal material and cultivation conditions The fungus Laetiporus sulphureus (Bull.) Murrill. was collected from Changbai Mountain, Jilin Province, China, in 2006. The fungus was identified by Prof. DAI Yu-Cheng at Beijing Forestry University. The culture medium consisted of glucose (5%), peptone from porcine meat (0.15%), yeast powder (0.5%), KH2PO4 (0.5%) and MgSO4. Fermentation was carried out on a shaker at 160 rpm for 25 days. Extraction and isolation The culture broth (21 L) was filtered, and the filtrate was extracted three times with ethyl acetate (V/V, 1 : 1), while the mycelium was extracted three times with CHCl3–MeOH (1 : 1, 4 L). The EtOAc layer, together with the mycelium extract, was concentrated under reduced pressure to give a crude extract (8.5 g). This residue was separated over silica gel column by gradient elution with CHCl3–MeOH (1 : 0 to 0 : 1; 2 L) to obtain six fractions (Frs. 1–6). Fr. 2 (700 mg) was separated over CC (Sephadex LH-20; CHCl3–MeOH, 1 : 1, 700 mL) to give three fractions (Fr. 2.1–Fr. 2.3). The separation of Fr. 2.1 (80 mg) over CC (silica gel; CHCl3–Me2CO, 100 : 1, 500 mL) yielded 2 (10 mg). Fr. 2.3 (85 mg) was separated by semipreparative HPLC (MeCN–H2O, 3 : 1 to 9 : 1, 10 mL·min-1 in 15 min) to give 3 (21 mg). Fr. 3 (135 mg) was subjected to CC (Rp-18 gel; MeOH–H2O, 1 : 1 to 1 : 0, 300 mL), followed by semipreparative HPLC (MeCN–H2O, 1 : 1 to 4 : 1, 10 mL·min−1 in 10 min) to yield 1 (9.6 mg). Cytotoxicity assay Human myeloid leukemia HL-60, hepatocellular carcinoma SMMC-7721, lung cancer A-549 cells, breast cancer MCF-7, and colon cancer SW480 cell lines were used in the cytotoxicity assay. All the cell lines were cultured in RPMI-1640 or DMEM medium (Hyclone, Logan, UT, USA), supplemented with 10% fetal bovine serum (Hyclone, Logan, UT, USA) in 5% CO2 at 37 C. The cytotoxicity assay was performed according to the MTT (3-(4, 5-dimethylthiazol2-yl)-2, 5-diphenyl tetrazolium bromide) method in 96-well microplates [19]. Briefly, 100 µL adherent cells were seeded into each well of 96-well cell culture plates and allowed to adhere for 12 h before drug addition, while suspended cells were seeded immediately before drug addition with initial density of 1 × 105 cells/mL. Each tumor cell line was exposed to the test compound dissolved in DMSO at concentrations of 0.0625, 0.32, 1.6, 8, and 40 μmol·L−1 in triplicate
for 48 h, with cisplatin (Sigma, St. Louis, MO, USA) as a positive control (IC50 values: see Table 2). After compound treatment, cell viability was detected and a cell growth curve was plotted. IC50 values were calculated by Reed and Muench’s method [20].
Identification 6-((2E, 6E)-3, 7-Dimethyldeca-2, 6-dienyl)-7-hydroxy5-methoxy-4-methylphtanlan-1-one (1), colorless oil; IR (KBr) max: 3 428, 2 937, 1 737, 1 621, 1 455, 1 135 cm−1; UV max (MeOH) nm (log ) 347 (2.91), 304 (3.60), 206 (5.01); 1H NMR (CDCl3, 400 MHz) and 13C NMR (CDCl3, 150 MHz) data, see Table 1; HR-EI-MS m/z 374.209 5 ([M]+, Calcd. for 374.209 3).
References [1]
[2]
[3]
[4]
[5]
[6]
[7] [8]
[9]
[10]
[11]
[12] [13]
[14]
– 687 –
Karaman M, Jovin E, Malbasa R, et al. Medicinal and edible lignicolous fungi as natural sources of antioxidative and antibacterial agents [J]. Phytother Res, 2010, 24 (10): 1473-1481. Kim SE, Lee IK, Jung YA, et al. Mushrooms collected from Deogyu mountain, Muju, Korea and their antioxidant activity [J]. Mycobiology, 2012, 40 (2): 134-137. Zjawiony JK. Biologically active compounds from Aphyllophorales (Polypore) fungi [J]. J Nat Prod, 2004, 67 (2): 300-310. Davoli P, Mucci A, Schenetti L,et al. Laetiporic acids, a family of non-carotenoid polyene pigments from fruit-bodies and liquid cultures of Laetiporus sulphureus (Polyporales, Fungi) [J]. Phytochemistry, 2005, 66 (7): 817-823. Yoshikawa K, Bando S, Arihara S, et al. A benzofuran glycoside and an acetylenic acid from the fungus Laetiporus sulphureus var. miniatus [J]. Chem Pharm Bull, 2001, 49 (3): 327-329. Ding JH, Feng T, Li ZH, et al. Trefolane A, a sesquiterpenoid with a new skeleton from cultures of the basidiomycete Tremella foliacea [J]. Org Lett, 2012, 14 (18): 4976-4978. Jiang MY, Feng T, Liu JK. N-Containing compound of macromycetes [J]. Nat Prod Rep, 2011, 28 (3): 783-808. Jiang MY, Li Y, Wang F, et al. Isoprenylated cyclohexanoids from the basidimycete Hexagonia speciosa [J]. Phytochemistry, 2011, 72 (9): 923-928. Liu LY, Li ZH, Dong ZJ, et al. Two novel fomannosane-type sesquiterpenoids from the culture of the basidiomycete Agrocybe salicacola [J]. Nat Prod Bioprospect, 2012, 2 (3): 130-132. Wang F, Zhou DS, Wei GZ, et al. Chlorantholides A-F, eudesmane-type sesquiterpene lactones from Chloranthus elatior [J]. Phytochemistry, 2012, 77: 312-317. Yin X, Feng T, Li ZH, et al. Chemical investigation on the cultures of the fungus Xylaria carpophila [J]. Nat Prod Bioprospect, 2011, 1 (2): 75-80. Zhou ZY, Liu JK. Pigments of fungi (macromycetes) [J]. Nat Prod Rep, 2010, 27 (11): 1531-1570. Zhou ZY, Shi GQ, Fontaine R, et al. Evidence for the natural toxin from the mushroom Trogia venenata as a cause of suden unexpected death in yunnan province, China [J]. Angew Chem Int Ed, 2012, 51 (10): 2368-2370. Bentley R. Mycophenolic acid: a one hundred year odyssey from antibiotic to immunosuppressant [J]. Chem Rev, 2000, 100 (10): 3801-3825.
Fan Qiong-Ying, et al. / Chin J Nat Med, 2014, 12(9): 685688
[15] Danheiser RL, Gee SK, Perez JJ. Total synthesis of mycophenolic acid [J]. J Am Chem Soc, 1985, 108 (4): 806-810. [16] Habib E, León F, Bauer JD, et al. Mycophenolic derivatives from Eupenicillium parvum [J]. J Nat Prod, 2008, 71 (11): 1915-1918. [17] Doerfler DL, Ernst LA, Campbell IM. Biosynthetic inter- mediates en route to mycophenolic acid in Penicillium brevi- compactum [J]. J Chem Soc Chem Commun, 1980, (8): 329-330.
[18] Canonica L, Rindone B, Scolastico C. Synthesis of myco- phenolic acid analogs through prenylation of 5, 7-dihydroxyphthalides [J]. Tetrahedron Lett, 1971, 12 (28): 2689- 2690. [19] Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays [J]. J Immunol Methods, 1983, 65 (1-2): 55-63. [20] Reed LJ, Muench H. A simple method of estimating fifty percent endpoints [J]. Am J Hygiene, 1938, 27 (3): 493-497.
Cite this article as: FAN Qiong-Ying, YIN Xia, LI Zheng-Hui, LI Yan, LIU Ji-Kai, FENG Tao, ZHAO Bao-Hua. Mycophenolic acid derivatives from cultures of the mushroom Laetiporus sulphureu [J]. Chinese Journal of Natural Medicines, 2014, 12(9): 685-688
– 688 –
