朱红栓菌提取物抗氧化、抗肿瘤活性评价及相关化学成分分析
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摘要
采用石油醚、乙酸乙酯、乙醇浸提朱红栓菌Trametes cinnabarina子实体干粉,得到不同极性提取物;采用清除DPPH自由基、羟自由基、超氧阴离子自由基能力,测定提取物的体外抗氧化活性;MTT法检测提取物对人肝癌细胞株Hep G2细胞增殖的抑制作用。结果表明,朱红栓菌石油醚、乙酸乙酯、乙醇提取物均具有一定的抗氧化、抗肿瘤活性;各提取物在浓度为4–5mg/m L时,对DPPH自由基、羟自由基和超氧阴离子自由基清除能力大小依次为乙酸乙酯提取物>乙醇提取物>石油醚提取物;乙酸乙酯提取物对3种自由基的最高清除率分别为60.23%、74.49%、63.84%。各提取物对人肝癌细胞株Hep G2细胞增殖抑制作用大小依次为乙酸乙酯提取物>乙醇提取物>石油醚提取物;乙酸乙酯提取物的抑制率最高达55.93%。采用硅胶和凝胶等柱色谱方法结合核磁、波谱和质谱等技术对乙酸乙酯提取物的化学组分进行分析,共分离纯化出11种化合物,分别鉴定为:麦角甾醇(1),邻苯二甲酸二丁酯(2),对羟基苯甲酸甲酯(3),麦角甾-7,22,二烯-3-酮(4),1-[(12E,16E)-12,16-二十碳二烯酰基]-2-[(E,E)-7,11-十八碳二烯酰基]-3-硬脂酰基甘油(5),cinnabarin(6),过氧麦角甾醇(7),尿嘧啶(8),甘露醇(9),腺嘌呤核苷(10),豆甾-7,22-二烯-3β,5α,6β-三醇(11)。除化合物6外均为首次从朱红栓菌子实体中分离得到。研究结果为开发利用朱红栓菌子实体提供了科学依据。
Different polarity extract was prepared from Trametes cinnabarina fruiting body powder by petroleum ether, ethyl acetate and ethanol. The antioxidant activities of three kinds of the extract were evaluated by scavenging free radical ability of 1,1-diphenyl-2-picrylhydrazyl(DPPH), hydroxyl radical and superoxide anion radical. The results indicated that the extract possessed antioxidant activity in vitro. In terms of antioxidant activity, the ethyl acetate extract is the best among the three kinds of extract. The highest scavenging rate of DPPH radical, hydroxyl radical and superoxide anion radical was 60.23%, 74.49% and 63.84%, respectively. The anti-tumor activities of different extract were evaluated by MTT method via measuring inhibition rate of cell proliferation of Hep G2 cell lines. The results showed that all kinds of extract displayed anti-tumor activities in vitro. The anti-tumor activity of ethyl acetate extract is the best, compared with ethanol extract and petroleum ether extract. The highest inhibition rate of the ethyl acetate extract to Hep G2 cells was 55.93%. The chemical constituents of the ethyl acetate extract were isolated and purified using silica gel column chromatography. NMR and MS spectral analysis were used to identify the structure of obtained compounds. Eleven kinds of compounds were finally isolated and identified as follows: ergosterol(1), phthalic acid dibutyl ester(2), methyl 4-hydroxybenzoate(3), ergosta-7,22-dien-3-one(4), 1-[(12 E,16 E)-12,16-eicosadienoyl]-2-[(E,E)-7,11-octadecadienoyl]-3-stearoylglycerol(5), cinnabarin(6), ergosterol peroxide(7), uracil(8), mannitol(9), adenosine(10) and(22 E, 24 S)-24-methyl-5α-choleata-7, 22-diene-3β, 5, 6β-triol(11). All compounds except for cinnabarin were isolated from the fruiting body of T. cinnabarina for the first time. The results provide scientific basis for development and utilization of fruiting body of T. cinnabarina.
Different polarity extract was prepared from Trametes cinnabarina fruiting body powder by petroleum ether, ethyl acetate and ethanol. The antioxidant activities of three kinds of the extract were evaluated by scavenging free radical ability of 1,1-diphenyl-2-picrylhydrazyl(DPPH), hydroxyl radical and superoxide anion radical. The results indicated that the extract possessed antioxidant activity in vitro. In terms of antioxidant activity, the ethyl acetate extract is the best among the three kinds of extract. The highest scavenging rate of DPPH radical, hydroxyl radical and superoxide anion radical was 60.23%, 74.49% and 63.84%, respectively. The anti-tumor activities of different extract were evaluated by MTT method via measuring inhibition rate of cell proliferation of Hep G2 cell lines. The results showed that all kinds of extract displayed anti-tumor activities in vitro. The anti-tumor activity of ethyl acetate extract is the best, compared with ethanol extract and petroleum ether extract. The highest inhibition rate of the ethyl acetate extract to Hep G2 cells was 55.93%. The chemical constituents of the ethyl acetate extract were isolated and purified using silica gel column chromatography. NMR and MS spectral analysis were used to identify the structure of obtained compounds. Eleven kinds of compounds were finally isolated and identified as follows: ergosterol(1), phthalic acid dibutyl ester(2), methyl 4-hydroxybenzoate(3), ergosta-7,22-dien-3-one(4), 1-[(12 E,16 E)-12,16-eicosadienoyl]-2-[(E,E)-7,11-octadecadienoyl]-3-stearoylglycerol(5), cinnabarin(6), ergosterol peroxide(7), uracil(8), mannitol(9), adenosine(10) and(22 E, 24 S)-24-methyl-5α-choleata-7, 22-diene-3β, 5, 6β-triol(11). All compounds except for cinnabarin were isolated from the fruiting body of T. cinnabarina for the first time. The results provide scientific basis for development and utilization of fruiting body of T. cinnabarina.
引文
Bondet V,Brand-Williams W,Berset C,1997.Kinetics and mechanisms of antioxidant activity using the DPPH·free radical method.LWT-Food Science and Technology,30(6):609-615
Borel JF,Kis ZL,1991.The discovery and development of cyclosporine(sandimmune).Transplantation Proceedings,23:1867-1874
Chen SY,1997.Chinese medicinal mycology.Beijing Medical University&China Union Medical University Unionsverlag,Beijing.1-282(in Chinese)
Clough JM,1993.The strobilurin fungicides from mushroom to molecule to market.Natural Product Report,56:277-278
Dias DA,Urban S,2009.HPLC and NMR studies of phenoxazone alkaloids from Pycnoporus cinnabarinus.Natural Product Communications,4(4):489
Dixson DD,Boddy CN,Doyle RP,2011.Reinvestigation of coenzyme Q10 isolation from Sporidiobolus johnsonii.Chemistry&Biodiversity,8(6):1033-1051
Ferrari M,Fornasiero MC,Isetta AM,1990.MTT colorimetric assay for testing macrophage cytotoxic activity in vitro.Journal of Immunological Methods,131(2):165-172
Govindan S,Johnson EER,Christopher J,Shanmugam J,Thirumalairaj V,Gopalan J,2016.Antioxidant and anti-aging activities of polysaccharides from Calocybe indica var.APK2.Experimental and Toxicologic Pathology,68(6):329-334
Guo XL,2013.Study on extraction and characteristics of pigment from Trametes sanguinea(syn.Pycnoporus sanguineus).Master Thesis,Heilongjiang University,Harbin.23-24(in Chinese)
Ham SS,Kima SH,Moona SY,Chungb MJ,Cuic CB,Hand EK,Chungd CK,Choee M,2008.Antimutagenic effects of subfractions of Chaga mushroom(Inonotus obliquus)extract.Mutation Research/Genetic Toxicology and Environmental Mutagenesis,672(1):55-59
Huang NL,1998.Colored illustrations of macrofungi(mushrooms)of China.China Agricultural Press,Beijing.76(in Chinese)
Jiang HQ,Wang JP,Liu YH,Gong LL,Liu HY,2008.Separation and identification of chemical compositions from Bidens bipinnata L.Food and Drug,10(5):15-17(in Chinese)
Kobori M,Yoshida M,Ohnishi-Kameyama M,Shinmoto H,2007.Ergosterol peroxide from an edible mushroom suppresses inflammatory responses in RAW264.7macrophages and growth of HT29 colon adenocarcinoma cells.British Journal of Pharmacology,150:209-219
Lee IK,Jung JY,Seok SJ,Kim WG,Yun BS,2006.Free radical scavengers from the medicinal mushroom Inonotus xeranticus and their proposed biogenesis.Bioorganic&Medicinal Chemistry Letters,16(21):5621-5624
Liu JK,2007.Biologically active substances from higher fungi.Bulletin of National Natural Science Foundation of China,2:69-70(in Chinese)
Liu LJ,Cao L,Jiang YP,Pan Y,2009.Study on the toxicity and analgesic and anti-inflammatory effects of semen strychni before and after being fermented by Trametes cinnabarina.Journal of Nanjing University of Traditional Chinese Medicine,25(3):205-208(in Chinese)
Lv JH,Li ZM,Lu YJ,Cao K,Zhang JX,Wang LA,2017.Study on chemical constituents of mycelium of Trametes cinnabarina.Modern Chinese Medicine,19(11):1537-1540(in Chinese)
Lv ZM,Jiang YT,Wu LJ,Liu K,2008.Chemical constituents from dried sorophore of cultured Cordyceps militaris.China Journal of Chinese Materia Medica,33(24):2914-2917(in Chinese)
Ma WG,Li CX,Wang DZ,Yang CY,1994.Ergosterol peroxides from Cryptoporus volvatus.Acta Botanica Yunnanica,16(2):196-200
Miyaoka H,Nishijima S,Mitome H,Yamada Y,2000.Three new scalarane sesterterpenoids from the Okinawan sponge Hyrtios erectus.Journal of Natural Products,63(10):1369-1372
Najafabad AM,Jamei R,2014.Free radical scavenging capacity and antioxidant activity of methanolic and ethanolic extracts of plum(Prunus domestica L.)in both fresh and dried samples.Avicenna Journal of Phytomedicine,4(4):343-353
Poon A,Sawynok J,1998.Antinociception by adenosine analogs and inhibitors of adenosine metabolism in an inflammatory thermal hyperalgesia model in the rat.Pain,74(2-3):235-245
Qi CC,Chen GY,Chen WH,Song XP,Han CR,2013.Chemical constituents from roots of Ficus auriculata.Chinese Traditional and Herbal Drugs,44(22):3127-3130(in Chinese)
R?secke J,K?nig WA,2000.Constituents of various wood-rotting basidiomycetes.Phytochemistry,54(6):603-610
Samsel M,Dzierzbicka K,2011.Therapeutic potential of adenosine analogues and conjugates.Pharmacological Reports,63(3):601-617
Sheng L,Zhu GL,Tong QY,2014.Effect of uracil on pullulan production by Aureobasidium pullulans CGMCC1234.Carbohydrate Polymers,101(30):435-437
Smania EFA,Smania JA,Loguercio-Leite C,1998.Cinnabarin synthesis by Pycnoporus sanguineus strains and antimicrobial activity against bacteria from food products.Revista de Microbiologia,29(4):317
Song MJ,Bao HY,Bau T,Li Y,2015.Antitumor activity and structure-activity relationship of four steroids from Fomitiporia ellipsoidea.Mycosystema,34(2):293-300(in Chinese)
Yan Z,T ian Y,Ma YP,Fu XC,Zhang ZX,Wang JH,2010.Study on the chemical constituents of from the roots of Tripterygium wilfordii.Modern Chinese Medicine,12(1):23-24,32(in Chinese)
Yilmaz N,Dulger H,Kiymaz N,Yilmaz C,Gudu BO,Demir I,2007.Activity of mannitol and hypertonic saline therapy on the oxidant and antioxidant system during the acute term after traumatic brain injury in the rats.Brain Research,1164:132-135
Zhao DD,Li DY,Wei JX,Hua HM,Li ZL,2012.Isolation and structural identification of the chemical constituents from the flowers of Xanthoceras sorbifolia Bunge.Journal of Shenyang Pharmaceutical University,7:514-518(in Chinese)
Zhu F,Lu WH,Chen X,Wu JS,2014.Isolation and identification of two phenoxazone alkaloids from Trametes cinnabarina(Jacq.)Fr.Natural Product Research and Development,26:538-360(in Chinese)
Zhu MH,Chen P,Wang Q,Li Y,2016.Research on chemical constituents of the fruiting bodies of Pleurotus ostreatus.Edible and Medicinal Mushrooms,24(4):242-246,261(in Chinese)
陈士瑜,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.282
郭夏立,2013.血红栓菌的色素提取及其特性研究.哈尔滨:黑龙江大学硕士论文.23-24
黄年来,1998.中国大型真菌原色图鉴.北京:中国农业出版社.76
蒋海强,王建平,刘玉红,巩丽丽,刘红燕,2008.鬼针草化学成分的分离和鉴定.食品与药品,10(5):15-17
刘吉开,2007.高等真菌化学成分及其生物活性的研究.中国科学基金,2:69-70
刘亮镜,曹亮,蒋亚平,潘扬,2009.马钱子经朱红栓菌发酵前后毒性及镇痛、抗炎作用的实验研究.南京中医药大学学报,25(3):205-208
吕建华,李忠民,鲁玉佳,曹坤,张金秀,王立安,2017.朱红栓菌菌丝体的化学成分研究.中国现代中药,19(11):1537-1540
吕子明,姜永涛,吴立军,刘珂,2008.人工蛹虫草子实体化学成分研究.中国中药杂志,33(24):2914-2917
祁翠翠,陈光英,陈文豪,宋小平,韩长日,2013.大果榕根的化学成分研究.中草药,44(22):3127-3130
宋明杰,包海鹰,图力古尔,李玉,2015.椭圆嗜蓝孢孔菌中四种甾类化合物的抗肿瘤活性及构效关系分析.菌物学报,34(2):293-300
严振,田洋,马跃平,傅晓春,张振学,王金辉,2010.雷公藤根化学成分研究.中国现代中药,12(1):23-24,32
赵丹丹,李丹毅,魏金霞,华会明,李占林,2012.文冠果花化学成分的分离与鉴定.沈阳药科大学学报,7:514-518
朱峰,卢卫红,陈忻,吴静姝,2014.朱红栓菌中两个吩噁嗪酮类生物碱的分离与鉴定.天然产物研究与开发,26:538-360
朱明洪,陈屏,王琦,李玉,2016.平菇子实体化学成分的分析研究.食药用菌,24(4):242-246,261
Borel JF,Kis ZL,1991.The discovery and development of cyclosporine(sandimmune).Transplantation Proceedings,23:1867-1874
Chen SY,1997.Chinese medicinal mycology.Beijing Medical University&China Union Medical University Unionsverlag,Beijing.1-282(in Chinese)
Clough JM,1993.The strobilurin fungicides from mushroom to molecule to market.Natural Product Report,56:277-278
Dias DA,Urban S,2009.HPLC and NMR studies of phenoxazone alkaloids from Pycnoporus cinnabarinus.Natural Product Communications,4(4):489
Dixson DD,Boddy CN,Doyle RP,2011.Reinvestigation of coenzyme Q10 isolation from Sporidiobolus johnsonii.Chemistry&Biodiversity,8(6):1033-1051
Ferrari M,Fornasiero MC,Isetta AM,1990.MTT colorimetric assay for testing macrophage cytotoxic activity in vitro.Journal of Immunological Methods,131(2):165-172
Govindan S,Johnson EER,Christopher J,Shanmugam J,Thirumalairaj V,Gopalan J,2016.Antioxidant and anti-aging activities of polysaccharides from Calocybe indica var.APK2.Experimental and Toxicologic Pathology,68(6):329-334
Guo XL,2013.Study on extraction and characteristics of pigment from Trametes sanguinea(syn.Pycnoporus sanguineus).Master Thesis,Heilongjiang University,Harbin.23-24(in Chinese)
Ham SS,Kima SH,Moona SY,Chungb MJ,Cuic CB,Hand EK,Chungd CK,Choee M,2008.Antimutagenic effects of subfractions of Chaga mushroom(Inonotus obliquus)extract.Mutation Research/Genetic Toxicology and Environmental Mutagenesis,672(1):55-59
Huang NL,1998.Colored illustrations of macrofungi(mushrooms)of China.China Agricultural Press,Beijing.76(in Chinese)
Jiang HQ,Wang JP,Liu YH,Gong LL,Liu HY,2008.Separation and identification of chemical compositions from Bidens bipinnata L.Food and Drug,10(5):15-17(in Chinese)
Kobori M,Yoshida M,Ohnishi-Kameyama M,Shinmoto H,2007.Ergosterol peroxide from an edible mushroom suppresses inflammatory responses in RAW264.7macrophages and growth of HT29 colon adenocarcinoma cells.British Journal of Pharmacology,150:209-219
Lee IK,Jung JY,Seok SJ,Kim WG,Yun BS,2006.Free radical scavengers from the medicinal mushroom Inonotus xeranticus and their proposed biogenesis.Bioorganic&Medicinal Chemistry Letters,16(21):5621-5624
Liu JK,2007.Biologically active substances from higher fungi.Bulletin of National Natural Science Foundation of China,2:69-70(in Chinese)
Liu LJ,Cao L,Jiang YP,Pan Y,2009.Study on the toxicity and analgesic and anti-inflammatory effects of semen strychni before and after being fermented by Trametes cinnabarina.Journal of Nanjing University of Traditional Chinese Medicine,25(3):205-208(in Chinese)
Lv JH,Li ZM,Lu YJ,Cao K,Zhang JX,Wang LA,2017.Study on chemical constituents of mycelium of Trametes cinnabarina.Modern Chinese Medicine,19(11):1537-1540(in Chinese)
Lv ZM,Jiang YT,Wu LJ,Liu K,2008.Chemical constituents from dried sorophore of cultured Cordyceps militaris.China Journal of Chinese Materia Medica,33(24):2914-2917(in Chinese)
Ma WG,Li CX,Wang DZ,Yang CY,1994.Ergosterol peroxides from Cryptoporus volvatus.Acta Botanica Yunnanica,16(2):196-200
Miyaoka H,Nishijima S,Mitome H,Yamada Y,2000.Three new scalarane sesterterpenoids from the Okinawan sponge Hyrtios erectus.Journal of Natural Products,63(10):1369-1372
Najafabad AM,Jamei R,2014.Free radical scavenging capacity and antioxidant activity of methanolic and ethanolic extracts of plum(Prunus domestica L.)in both fresh and dried samples.Avicenna Journal of Phytomedicine,4(4):343-353
Poon A,Sawynok J,1998.Antinociception by adenosine analogs and inhibitors of adenosine metabolism in an inflammatory thermal hyperalgesia model in the rat.Pain,74(2-3):235-245
Qi CC,Chen GY,Chen WH,Song XP,Han CR,2013.Chemical constituents from roots of Ficus auriculata.Chinese Traditional and Herbal Drugs,44(22):3127-3130(in Chinese)
R?secke J,K?nig WA,2000.Constituents of various wood-rotting basidiomycetes.Phytochemistry,54(6):603-610
Samsel M,Dzierzbicka K,2011.Therapeutic potential of adenosine analogues and conjugates.Pharmacological Reports,63(3):601-617
Sheng L,Zhu GL,Tong QY,2014.Effect of uracil on pullulan production by Aureobasidium pullulans CGMCC1234.Carbohydrate Polymers,101(30):435-437
Smania EFA,Smania JA,Loguercio-Leite C,1998.Cinnabarin synthesis by Pycnoporus sanguineus strains and antimicrobial activity against bacteria from food products.Revista de Microbiologia,29(4):317
Song MJ,Bao HY,Bau T,Li Y,2015.Antitumor activity and structure-activity relationship of four steroids from Fomitiporia ellipsoidea.Mycosystema,34(2):293-300(in Chinese)
Yan Z,T ian Y,Ma YP,Fu XC,Zhang ZX,Wang JH,2010.Study on the chemical constituents of from the roots of Tripterygium wilfordii.Modern Chinese Medicine,12(1):23-24,32(in Chinese)
Yilmaz N,Dulger H,Kiymaz N,Yilmaz C,Gudu BO,Demir I,2007.Activity of mannitol and hypertonic saline therapy on the oxidant and antioxidant system during the acute term after traumatic brain injury in the rats.Brain Research,1164:132-135
Zhao DD,Li DY,Wei JX,Hua HM,Li ZL,2012.Isolation and structural identification of the chemical constituents from the flowers of Xanthoceras sorbifolia Bunge.Journal of Shenyang Pharmaceutical University,7:514-518(in Chinese)
Zhu F,Lu WH,Chen X,Wu JS,2014.Isolation and identification of two phenoxazone alkaloids from Trametes cinnabarina(Jacq.)Fr.Natural Product Research and Development,26:538-360(in Chinese)
Zhu MH,Chen P,Wang Q,Li Y,2016.Research on chemical constituents of the fruiting bodies of Pleurotus ostreatus.Edible and Medicinal Mushrooms,24(4):242-246,261(in Chinese)
陈士瑜,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.282
郭夏立,2013.血红栓菌的色素提取及其特性研究.哈尔滨:黑龙江大学硕士论文.23-24
黄年来,1998.中国大型真菌原色图鉴.北京:中国农业出版社.76
蒋海强,王建平,刘玉红,巩丽丽,刘红燕,2008.鬼针草化学成分的分离和鉴定.食品与药品,10(5):15-17
刘吉开,2007.高等真菌化学成分及其生物活性的研究.中国科学基金,2:69-70
刘亮镜,曹亮,蒋亚平,潘扬,2009.马钱子经朱红栓菌发酵前后毒性及镇痛、抗炎作用的实验研究.南京中医药大学学报,25(3):205-208
吕建华,李忠民,鲁玉佳,曹坤,张金秀,王立安,2017.朱红栓菌菌丝体的化学成分研究.中国现代中药,19(11):1537-1540
吕子明,姜永涛,吴立军,刘珂,2008.人工蛹虫草子实体化学成分研究.中国中药杂志,33(24):2914-2917
祁翠翠,陈光英,陈文豪,宋小平,韩长日,2013.大果榕根的化学成分研究.中草药,44(22):3127-3130
宋明杰,包海鹰,图力古尔,李玉,2015.椭圆嗜蓝孢孔菌中四种甾类化合物的抗肿瘤活性及构效关系分析.菌物学报,34(2):293-300
严振,田洋,马跃平,傅晓春,张振学,王金辉,2010.雷公藤根化学成分研究.中国现代中药,12(1):23-24,32
赵丹丹,李丹毅,魏金霞,华会明,李占林,2012.文冠果花化学成分的分离与鉴定.沈阳药科大学学报,7:514-518
朱峰,卢卫红,陈忻,吴静姝,2014.朱红栓菌中两个吩噁嗪酮类生物碱的分离与鉴定.天然产物研究与开发,26:538-360
朱明洪,陈屏,王琦,李玉,2016.平菇子实体化学成分的分析研究.食药用菌,24(4):242-246,261