一种炭角菌菌丝体生物活性的研究
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摘要
从一种牛肝菌Boletus sp.中分离得到一株炭角菌菌株,通过液体深层发酵获得菌丝体,并对其粗多糖石油醚和氯仿提取物进行了体外免疫力、抗氧化和抗肿瘤等活性评价。研究结果表明,此株炭角菌的菌丝体石油醚和氯仿提取物抗氧化和提高免疫活力的效果不明显,但炭角菌石油醚和氯仿提取物相对SPCA-1肿瘤细胞具有良好的抗肿瘤活性,100μg/m L石油醚和氯仿提取物细胞增殖的抑制率分别达到60.08%和68.41%,而50μg/m L石油醚和氯仿提取物可以分别诱导25.4%和26.3%的SPCA-1细胞发生早期凋亡,并引起P53、Caspase-3和Bax抗肿瘤标志蛋白表达上升和抗凋亡蛋白Bcl-2表达下降,表明两种提取物抗肿瘤效果是通过样品诱导细胞线粒体释放ROS来发挥的。同时,发现炭角菌可以激活细胞自身的保护性自噬,从而降低炭角菌的抗肿瘤活性。以上研究结果将为深入研究和开发以炭角菌为来源的抗肺癌药物提供科学依据。
The mycelium of a Xylaria sp. strain isolated from Boletus sp. was obtained through liquid submerged fermentation method. The immunomodulatory, antitumor, and antioxidant activities of the crude polysaccharide, the petroleum ether and chloroform extracts from the mycelium were evaluated. The result showed that the petroleum ether and chloroform extracts had good antitumor activity against SPCA-1 lung cells, and the antioxidant activity of the two extracts and the stimulation effect of crude polysaccharide on macrophages to release NO were not significant. Inhibitory rates of SPCA-1 were significantly increased by 60.0% and 68.41% respectively after the treatment by 100μg/m L petroleum ether extract and 100μg/m L chloroform extract of the Xylaria sp. strain. Flow cytometry result revealed that 50μg/m L petroleum ether extract and 50μg/m L chloroform extract of the Xylaria sp. strain brought about apoptosis of SPCA-1 cells and the apoptosis rates reached 25.4% and 26.3% respectively. Further investigation showed these extracts could increase expression of P53, caspase-3 and Bax, and decrease expression of anti- apoptotic protein Bcl-2. The anti-tumor activity of the petroleum ether and chloroform extracts had relation with ROS-triggered apoptosis. The protective autophagy of SPCA-1 was also activated by these extracts, and the inhibition of autophagy could stimulate inhibition effect of Xylaria sp. on SPCA-1 proliferation.
The mycelium of a Xylaria sp. strain isolated from Boletus sp. was obtained through liquid submerged fermentation method. The immunomodulatory, antitumor, and antioxidant activities of the crude polysaccharide, the petroleum ether and chloroform extracts from the mycelium were evaluated. The result showed that the petroleum ether and chloroform extracts had good antitumor activity against SPCA-1 lung cells, and the antioxidant activity of the two extracts and the stimulation effect of crude polysaccharide on macrophages to release NO were not significant. Inhibitory rates of SPCA-1 were significantly increased by 60.0% and 68.41% respectively after the treatment by 100μg/m L petroleum ether extract and 100μg/m L chloroform extract of the Xylaria sp. strain. Flow cytometry result revealed that 50μg/m L petroleum ether extract and 50μg/m L chloroform extract of the Xylaria sp. strain brought about apoptosis of SPCA-1 cells and the apoptosis rates reached 25.4% and 26.3% respectively. Further investigation showed these extracts could increase expression of P53, caspase-3 and Bax, and decrease expression of anti- apoptotic protein Bcl-2. The anti-tumor activity of the petroleum ether and chloroform extracts had relation with ROS-triggered apoptosis. The protective autophagy of SPCA-1 was also activated by these extracts, and the inhibition of autophagy could stimulate inhibition effect of Xylaria sp. on SPCA-1 proliferation.
引文
Cuervo AM,2004.Autophagy:in sickness and in health.Trends in Cell Biology,14(2):70-77
Dai YC,Yang ZL,2008.A revised checklist of medicinal fungi in China.Mycosystema,27(6):801-824(in Chinese)
Donadelli M,Dando I,Zaniboni T,Costanzo C,Dalla Pozza E,Scupoli MT,Scarpa A,Zappavigna S,Marra M,Abbruzzese A,Bifulco M,Caraglia M,Palmieri M,2011.Gemcitabine/cannabinoid combination triggers autophagy in pancreatic cancer cells through a ROS-mediated mechanism.Cell Death&Disease,2(4):e152
Gong QF,Wu SH,Tan NH,Chen ZH,2008b.Study on compounds with antioxidant and antitumor activity from fermental mycelium of Xylaria nigripes.Food Science and Technology,33(12):28-31(in Chinese)
Gong QF,Zhang YM,Tan NH,Chen ZH,2008a.Chemical constitutents in fermental mycelium of Xylaria nigripes.China Journal of Chinese Materia Medica,33(11):1269-1272(in Chinese)
Green LC,Wagner DA,Glowgowski J,Skipper PL,Wishnok JS,Tannenbaum SR,1982.Analysis of nirtate,nitrite and[15N]nitrate in biological fluids.Analytical Biochemistry,126(1):131-138
Guo AG,Wang ZY,1989.Autoxidation of pyrogallol-chemiluminescence assay for superoxide dismutase activity.Plant Physiology Communication,3:54-57(in Chinese)
Huang R,Xie XS,Fang XW,Ma KX,Wu SH,2015.Five new guaiane sesquiterpenes from the endophytic fungus Xylaria sp.YM 311647 of Azadirachta indica.Chemistry&Biodiversity,12(8):1281-1286
Jin YP,Yang ZJ,Luo MY,2015.Metabolites of endophytic fungus Xylaria sp.with biological activities.Chinese Pharmaceutical Journal,50(21):1853-1856(in Chinese)
Levine B,Klionsky DJ,2004.Development by self-digestion:molecular mechanisms and biological functions of autophagy.Developmental Cell,6(4):463-477
Li GD,Wu DQ,Li BY,2009.Research progresses on the role of cell autophagy in cancer.Cancer,28(4):445-448(in Chinese)
Li WJ,Chen Y,Nie SP,Xie MY,He M,Zhang SS,Zhu KX,2011.Ganoderma atrum polysaccharide induces anti-tumor activity via the mitochondrial apoptotic pathway related to activation of host immune response.Journal of Cellular Biochemistry,112(3):860-871
Li YX,Li HJ,1994.A novel species of Xylaria.Journal of Nanjing Agricultural University,17(3):145-147(in Chinese)
Lin HY,Juan SH,Shen SC,Hsu FL,Chen YC,2003.Inhibition of lipopolysaccharide-induced nitric oxide production by flavonoids in RAW264.7 macrophages involves heme oxygenase-1.Biochemical Pharmacology,66(9):1821-1832
Liu YF,Zhao Y,Yang Y,Tang QJ,Zhou S,Wu D,Feng N,Jia W,Zhang JS,2012.Enzymatic extraction of polysaccharide from Coprinus comatus fruit bodies.Acta Edulis Fungi,19(3):79-82(in Chinese)
Ma BJ,Yuan Y,Liu JK,2008.Chemical study on the fruiting bodies of Xylaria longipes.Natural Product Research and Development,20(1):63-65(in Chinese)
Ma C,Weng RA,Zhang P,2009.Advances on studies of Xylaria nigripes.Journal of Fungal Research,7(1):59-62(in Chinese)
Miao SS,Mao XH,Pei R,Miao SP,Xiang C,LüYJ,Yang XG,Sun J,Jia SS,Liu YP,2013.Lepista sordida polysaccharide induces apoptosis of Hep-2 cancer cells via mitochondrial pathway.International Journal of Biological Macromolecules,61:97-101
Shintani T,Klionsky DJ,2004.Autophagy in health and disease:a double-edged sword.Science,306(5698):990-995
Wang QB,Yang Y,Liu YF,Gao JH,Zhang JS,Tang QJ,Feng N,Zhou S,2010.Chemical composition and bioactivities of one-and two-year-old Phellinus baumii fruit bodies.Acta Edulis Fungi,17(2):71-75(in Chinese)
Wang WH,Fan H,Zhang JS,Yang Y,Zhao XL,Zhang K,Wu D,Yan MQ,Jia W,2015.Inhibition of MCF7 breast cancer cell proliferation and cell cycle disruption by ethanolic extracts of Phellinus baumii fruit bodies.Acta Edulis Fungi,22(2):46-50(in Chinese)
Wang XN,Huang WY,Liu JK,Tan RX,Li CY,2012.Chemical constituents in sporophore of Xylaria euglossa.Chinese Traditional and Herbal Drugs,43(12):2327-2332(in Chinese)
Wei H,Xu YM,Espinosa-Artiles P,Liu MX,Luo JG,U’Ren JM,Arnold AE,Gunatilaka AAL,2015.Sesquiterpenes and other constituents of Xylaria sp.NC1214.a fungal endophyte of the moss Hypnum sp.Phytochemistry,118:102-108
Wu GF,2001.A study on DPPH free-radical scavengers from Xylaria nigripes.Acta Microbiologica Sinica,41(3):363-366(in Chinese)
Wu W,Dai HQ,Bao L,Ren B,Lu JC,Luo YM,Guo LD,Zhang LX,Liu HW,2011.Isolation and structural elucidation of proline-containing cyclopentapeptides from an endolichenic Xylaria sp.Journal of Natural Products,74(5):1303-1308
Xu JT,1997.China medicinal fungi.United Press of Beijing and Medical University,Peking Union Medical College,Beijing.669-671(in Chinese)
Xu PP,You HC,Hu ZY,Li YY,Shen YM,2013.Two new pimarane-type diterpenes from the metabolites of Xylaria sp.290.Chinese Journal of Organic Chemistry,33(12):2618-2621(in Chinese)
Yan S,Li SF,Wu W,Zhao F,Bao L,Ding R,Gao H,Wen HA,Song FH,Liu HW,2011.Terpenoid and phenolic metabolites from the fungus Xylaria sp.associated with termite nests.Chemistry&Biodiversity,8(9):1689-1700
Yang XL,Liu JK,Luo DQ,Zhang S,2011.Chemical composition of black shank carbon.Research and Development of Natural Products,23(5):846-849(in Chinese)
Yu CC,Chiang PC,Lu PH,Kuo MT,Wen WC,Chen PN,Guh JH,2012.Antroquinonol,a natural ubiquinone derivative,induces a cross talk between apoptosis,autophagy and senescence in human pancreatic carcinoma cells.The Journal of Nutritional Biochemistry,23(8):900-907
Yu MY,Xu XY,Wei W,Jiang N,Luo X,2014.Protection of PC12 cells from hydrogen peroxide-induced injury by ethanolic extracts of Xylaria mycelium.Acta Edulis Fungi,21(4):49-52(in Chinese)
Zhang Q,Xiao J,Sun QQ,Qin JC,Pescitelli G,Gao JM,2014.Characterization of cytochalasins from the endophytic Xylaria sp.and their biological functions.Journal of Agricultural and Food Chemistry,62(45):10962-10969
Zhang ZY,Teruya K,Eto H,Shirahata S,2011.Fucoidan extract induces apoptosis in MCF-7 cells via a mechanism involving the ROS-dependent JNK activation and mitochondria-mediated pathways.PLo S One,6(11):e27441
戴玉成,杨祝良,2008.中国药用真菌名录及部分名称的修订.菌物学报,27(6):801-824
龚庆芳,张玉梅,谭宁华,陈作红,2008a.黑柄炭角菌发酵菌丝体的化学成分研究.中国中药杂志,33(11):1269-1272
龚庆芳,武守华,谭宁华,陈作红,2008b.黑柄炭角菌发酵菌丝中抗氧化及抗肿瘤活性的有效成分研究.食品科技,33(12):28-31
郭蔼光,王振镒,1989.邻苯三酚自氧化-化学发光法测定SOD活性.植物生理学报,3:54-57
金一平,杨志钧,罗敏玉,2015.植物内生真菌炭角菌属HCCB03890代谢产物及生物活性的研究.中国药学杂志,50(21):1853-1856
李国东,吴德全,李本义,2009.细胞自噬在肿瘤中作用的研究进展.癌症,28(4):445-448
李玉祥,李惠君,1994.炭角菌一新种.南京农业大学学报,17(3):145-147
刘艳芳,赵源,杨焱,唐庆九,周帅,吴迪,冯娜,贾薇,张劲松,2012.酶法辅助提取毛头鬼伞子实体粗多糖的研究.食用菌学报,19(3):79-82
麻兵继,阮元,刘吉开,2008.长柄炭角菌子实体化学成分研究.天然产物研究与开发,20(1):63-65
马橙,翁榕安,张平,2009.黑柄炭角菌的研究进展.菌物研究,7(1):59-62
王钦博,杨焱,刘艳芳,高君辉,张劲松,唐庆九,冯娜,周帅,2010.一年生和两年生桑黄子实体的成分和生物活性.食用菌学报,17(2):71-75
汪雯翰,樊华,张劲松,杨焱,赵香丽,张凯,吴迪,颜梦秋,贾薇,2015.桑黄醇提物对MCF7乳腺癌细胞活力和周期改变的影响.食用菌学报,22(2):46-50
王兴娜,黄午阳,刘吉开,谭仁祥,李春阳,2012.大炭角菌子实体化学成分的研究.中草药,43(12):2327-2332
吴根福,2001.黑柄炭角菌产生的DPPH自由基捕捉成分.微生物学报,41(3):363-366
徐锦堂,1997.中国药用真菌学.北京:北京医科大学,中国协和医科大学联合出版社.669-671
徐培培,尤怀成,胡志钰,李瑶瑶,沈月毛,2013.炭角菌Xylaria sp.290代谢产物中两个新的海松烷型二萜.有机化学,33(12):2618-2621
杨小龙,刘吉开,罗都强,张苏,2011.黑柄炭角菌的化学成分.天然产物研究与开发,23(5):846-849
余梦瑶,许晓燕,魏巍,江南,罗霞,2014.炭角菌菌丝体乙醇提取物对H2O2所致PC12细胞损伤的保护作用.食用菌学报,21(4):49-52
Dai YC,Yang ZL,2008.A revised checklist of medicinal fungi in China.Mycosystema,27(6):801-824(in Chinese)
Donadelli M,Dando I,Zaniboni T,Costanzo C,Dalla Pozza E,Scupoli MT,Scarpa A,Zappavigna S,Marra M,Abbruzzese A,Bifulco M,Caraglia M,Palmieri M,2011.Gemcitabine/cannabinoid combination triggers autophagy in pancreatic cancer cells through a ROS-mediated mechanism.Cell Death&Disease,2(4):e152
Gong QF,Wu SH,Tan NH,Chen ZH,2008b.Study on compounds with antioxidant and antitumor activity from fermental mycelium of Xylaria nigripes.Food Science and Technology,33(12):28-31(in Chinese)
Gong QF,Zhang YM,Tan NH,Chen ZH,2008a.Chemical constitutents in fermental mycelium of Xylaria nigripes.China Journal of Chinese Materia Medica,33(11):1269-1272(in Chinese)
Green LC,Wagner DA,Glowgowski J,Skipper PL,Wishnok JS,Tannenbaum SR,1982.Analysis of nirtate,nitrite and[15N]nitrate in biological fluids.Analytical Biochemistry,126(1):131-138
Guo AG,Wang ZY,1989.Autoxidation of pyrogallol-chemiluminescence assay for superoxide dismutase activity.Plant Physiology Communication,3:54-57(in Chinese)
Huang R,Xie XS,Fang XW,Ma KX,Wu SH,2015.Five new guaiane sesquiterpenes from the endophytic fungus Xylaria sp.YM 311647 of Azadirachta indica.Chemistry&Biodiversity,12(8):1281-1286
Jin YP,Yang ZJ,Luo MY,2015.Metabolites of endophytic fungus Xylaria sp.with biological activities.Chinese Pharmaceutical Journal,50(21):1853-1856(in Chinese)
Levine B,Klionsky DJ,2004.Development by self-digestion:molecular mechanisms and biological functions of autophagy.Developmental Cell,6(4):463-477
Li GD,Wu DQ,Li BY,2009.Research progresses on the role of cell autophagy in cancer.Cancer,28(4):445-448(in Chinese)
Li WJ,Chen Y,Nie SP,Xie MY,He M,Zhang SS,Zhu KX,2011.Ganoderma atrum polysaccharide induces anti-tumor activity via the mitochondrial apoptotic pathway related to activation of host immune response.Journal of Cellular Biochemistry,112(3):860-871
Li YX,Li HJ,1994.A novel species of Xylaria.Journal of Nanjing Agricultural University,17(3):145-147(in Chinese)
Lin HY,Juan SH,Shen SC,Hsu FL,Chen YC,2003.Inhibition of lipopolysaccharide-induced nitric oxide production by flavonoids in RAW264.7 macrophages involves heme oxygenase-1.Biochemical Pharmacology,66(9):1821-1832
Liu YF,Zhao Y,Yang Y,Tang QJ,Zhou S,Wu D,Feng N,Jia W,Zhang JS,2012.Enzymatic extraction of polysaccharide from Coprinus comatus fruit bodies.Acta Edulis Fungi,19(3):79-82(in Chinese)
Ma BJ,Yuan Y,Liu JK,2008.Chemical study on the fruiting bodies of Xylaria longipes.Natural Product Research and Development,20(1):63-65(in Chinese)
Ma C,Weng RA,Zhang P,2009.Advances on studies of Xylaria nigripes.Journal of Fungal Research,7(1):59-62(in Chinese)
Miao SS,Mao XH,Pei R,Miao SP,Xiang C,LüYJ,Yang XG,Sun J,Jia SS,Liu YP,2013.Lepista sordida polysaccharide induces apoptosis of Hep-2 cancer cells via mitochondrial pathway.International Journal of Biological Macromolecules,61:97-101
Shintani T,Klionsky DJ,2004.Autophagy in health and disease:a double-edged sword.Science,306(5698):990-995
Wang QB,Yang Y,Liu YF,Gao JH,Zhang JS,Tang QJ,Feng N,Zhou S,2010.Chemical composition and bioactivities of one-and two-year-old Phellinus baumii fruit bodies.Acta Edulis Fungi,17(2):71-75(in Chinese)
Wang WH,Fan H,Zhang JS,Yang Y,Zhao XL,Zhang K,Wu D,Yan MQ,Jia W,2015.Inhibition of MCF7 breast cancer cell proliferation and cell cycle disruption by ethanolic extracts of Phellinus baumii fruit bodies.Acta Edulis Fungi,22(2):46-50(in Chinese)
Wang XN,Huang WY,Liu JK,Tan RX,Li CY,2012.Chemical constituents in sporophore of Xylaria euglossa.Chinese Traditional and Herbal Drugs,43(12):2327-2332(in Chinese)
Wei H,Xu YM,Espinosa-Artiles P,Liu MX,Luo JG,U’Ren JM,Arnold AE,Gunatilaka AAL,2015.Sesquiterpenes and other constituents of Xylaria sp.NC1214.a fungal endophyte of the moss Hypnum sp.Phytochemistry,118:102-108
Wu GF,2001.A study on DPPH free-radical scavengers from Xylaria nigripes.Acta Microbiologica Sinica,41(3):363-366(in Chinese)
Wu W,Dai HQ,Bao L,Ren B,Lu JC,Luo YM,Guo LD,Zhang LX,Liu HW,2011.Isolation and structural elucidation of proline-containing cyclopentapeptides from an endolichenic Xylaria sp.Journal of Natural Products,74(5):1303-1308
Xu JT,1997.China medicinal fungi.United Press of Beijing and Medical University,Peking Union Medical College,Beijing.669-671(in Chinese)
Xu PP,You HC,Hu ZY,Li YY,Shen YM,2013.Two new pimarane-type diterpenes from the metabolites of Xylaria sp.290.Chinese Journal of Organic Chemistry,33(12):2618-2621(in Chinese)
Yan S,Li SF,Wu W,Zhao F,Bao L,Ding R,Gao H,Wen HA,Song FH,Liu HW,2011.Terpenoid and phenolic metabolites from the fungus Xylaria sp.associated with termite nests.Chemistry&Biodiversity,8(9):1689-1700
Yang XL,Liu JK,Luo DQ,Zhang S,2011.Chemical composition of black shank carbon.Research and Development of Natural Products,23(5):846-849(in Chinese)
Yu CC,Chiang PC,Lu PH,Kuo MT,Wen WC,Chen PN,Guh JH,2012.Antroquinonol,a natural ubiquinone derivative,induces a cross talk between apoptosis,autophagy and senescence in human pancreatic carcinoma cells.The Journal of Nutritional Biochemistry,23(8):900-907
Yu MY,Xu XY,Wei W,Jiang N,Luo X,2014.Protection of PC12 cells from hydrogen peroxide-induced injury by ethanolic extracts of Xylaria mycelium.Acta Edulis Fungi,21(4):49-52(in Chinese)
Zhang Q,Xiao J,Sun QQ,Qin JC,Pescitelli G,Gao JM,2014.Characterization of cytochalasins from the endophytic Xylaria sp.and their biological functions.Journal of Agricultural and Food Chemistry,62(45):10962-10969
Zhang ZY,Teruya K,Eto H,Shirahata S,2011.Fucoidan extract induces apoptosis in MCF-7 cells via a mechanism involving the ROS-dependent JNK activation and mitochondria-mediated pathways.PLo S One,6(11):e27441
戴玉成,杨祝良,2008.中国药用真菌名录及部分名称的修订.菌物学报,27(6):801-824
龚庆芳,张玉梅,谭宁华,陈作红,2008a.黑柄炭角菌发酵菌丝体的化学成分研究.中国中药杂志,33(11):1269-1272
龚庆芳,武守华,谭宁华,陈作红,2008b.黑柄炭角菌发酵菌丝中抗氧化及抗肿瘤活性的有效成分研究.食品科技,33(12):28-31
郭蔼光,王振镒,1989.邻苯三酚自氧化-化学发光法测定SOD活性.植物生理学报,3:54-57
金一平,杨志钧,罗敏玉,2015.植物内生真菌炭角菌属HCCB03890代谢产物及生物活性的研究.中国药学杂志,50(21):1853-1856
李国东,吴德全,李本义,2009.细胞自噬在肿瘤中作用的研究进展.癌症,28(4):445-448
李玉祥,李惠君,1994.炭角菌一新种.南京农业大学学报,17(3):145-147
刘艳芳,赵源,杨焱,唐庆九,周帅,吴迪,冯娜,贾薇,张劲松,2012.酶法辅助提取毛头鬼伞子实体粗多糖的研究.食用菌学报,19(3):79-82
麻兵继,阮元,刘吉开,2008.长柄炭角菌子实体化学成分研究.天然产物研究与开发,20(1):63-65
马橙,翁榕安,张平,2009.黑柄炭角菌的研究进展.菌物研究,7(1):59-62
王钦博,杨焱,刘艳芳,高君辉,张劲松,唐庆九,冯娜,周帅,2010.一年生和两年生桑黄子实体的成分和生物活性.食用菌学报,17(2):71-75
汪雯翰,樊华,张劲松,杨焱,赵香丽,张凯,吴迪,颜梦秋,贾薇,2015.桑黄醇提物对MCF7乳腺癌细胞活力和周期改变的影响.食用菌学报,22(2):46-50
王兴娜,黄午阳,刘吉开,谭仁祥,李春阳,2012.大炭角菌子实体化学成分的研究.中草药,43(12):2327-2332
吴根福,2001.黑柄炭角菌产生的DPPH自由基捕捉成分.微生物学报,41(3):363-366
徐锦堂,1997.中国药用真菌学.北京:北京医科大学,中国协和医科大学联合出版社.669-671
徐培培,尤怀成,胡志钰,李瑶瑶,沈月毛,2013.炭角菌Xylaria sp.290代谢产物中两个新的海松烷型二萜.有机化学,33(12):2618-2621
杨小龙,刘吉开,罗都强,张苏,2011.黑柄炭角菌的化学成分.天然产物研究与开发,23(5):846-849
余梦瑶,许晓燕,魏巍,江南,罗霞,2014.炭角菌菌丝体乙醇提取物对H2O2所致PC12细胞损伤的保护作用.食用菌学报,21(4):49-52