粗毛纤孔菌三萜类化合物的诱导合成及其抗氧化功能分析
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摘要
为探索外源诱导剂对粗毛纤孔菌生物合成三萜类化合物及其抗氧化功能的影响,采用茉莉酸甲酯、水杨酸、油酸、水曲柳煎汁、Mn~(2+)、Fe~(2+)、Cu~(2+)7种诱导剂在不同浓度条件下对粗毛纤孔菌菌丝体生长以及胞内三萜类化合物生物合成情况进行研究,并测定粗毛纤孔菌三萜类化合物的抗氧化功能。结果表明,7种诱导剂均显著促进粗毛纤孔菌菌丝体生长及三萜类化合物合成,其中油酸效果最为显著,在第0天加入3%油酸可得到菌丝体量为(7.146±0.131)g/L,菌丝体中三萜含量为(56.377±3.178)mg/g,三萜总量为402.870 mg/L,与不加入诱导剂的对照组相比三萜总量增加了143.74%。抗氧化能力实验表明,经过7种诱导剂得到的三萜类化合物的抗氧化能力也存在差异,其中油酸诱导的粗毛纤孔菌三萜类化合物抗氧化效果最好,总还原力为1.342,1,1-二苯基-2-三硝基苯肼自由基清除率IC_(50)为0.099 mg/mL,羟自由基清除率IC_(50)为1.294 mg/mL,超氧阴离子自由基清除率IC_(50)为0.001 mg/mL,与对照组相比各自由基清除率分别提高了79.48%、66.71%、28.10%及29.22%。本研究为诱导剂在粗毛纤孔菌三萜类化合物生物合成上应用以及开发新的抗氧化剂提供科学数据。
This study aimed to explore the effect of exogenous inducers on the biosynthesis and antioxidant properties of triterpenoids in Inonotus hispidus. We used seven inducers at different concentrations including methyl jasmonate(MeJA),salicylic acid(SA), oleic acid, willow branch decoction, Mn~(2+), Fe~(2+) and Cu~(2+). The results showed that all the seven inducers signi?cantly promoted the mycelial growth of I. hispidus and the biosynthesis of triterpenoids, oleic acid being the most effective among these. A mycelial biomass yield of(7.146 ± 0.131) g/L was obtained by the addition of 3% oleic acid at the beginning of fermentation. The content of triterpenoids in the mycelia was(56.377 ± 3.178) mg/g, and the total production of triterpenoids was 402.870 mg/L, which was increased by 143.74% compared with the control group without any inducer added. The antioxidant tests showed that the antioxidant capacity of triterpenoids varied with inducer type. Among these inducers, oleic acid induced triterpenoids with the strongest antioxidant effect; the total reducing power was 1.342, and the IC_(50) values for scavenging of 1,1-diphenyl-2-picrylhydrazyl(DPPH), hydroxyl and superoxide anion radicals were 0.099, 1.294 and 0.001 mg/mL, respectively. The total reducing power and the free radical scavenging rates were increased by 79.48%, 66.71%, 28.10% and 29.22% compared with those of the control group, respectively. The results can provide scienti?c data for the application of inducers in the biosynthesis of triterpenoids and hence the development of new antioxidants.
This study aimed to explore the effect of exogenous inducers on the biosynthesis and antioxidant properties of triterpenoids in Inonotus hispidus. We used seven inducers at different concentrations including methyl jasmonate(MeJA),salicylic acid(SA), oleic acid, willow branch decoction, Mn~(2+), Fe~(2+) and Cu~(2+). The results showed that all the seven inducers signi?cantly promoted the mycelial growth of I. hispidus and the biosynthesis of triterpenoids, oleic acid being the most effective among these. A mycelial biomass yield of(7.146 ± 0.131) g/L was obtained by the addition of 3% oleic acid at the beginning of fermentation. The content of triterpenoids in the mycelia was(56.377 ± 3.178) mg/g, and the total production of triterpenoids was 402.870 mg/L, which was increased by 143.74% compared with the control group without any inducer added. The antioxidant tests showed that the antioxidant capacity of triterpenoids varied with inducer type. Among these inducers, oleic acid induced triterpenoids with the strongest antioxidant effect; the total reducing power was 1.342, and the IC_(50) values for scavenging of 1,1-diphenyl-2-picrylhydrazyl(DPPH), hydroxyl and superoxide anion radicals were 0.099, 1.294 and 0.001 mg/mL, respectively. The total reducing power and the free radical scavenging rates were increased by 79.48%, 66.71%, 28.10% and 29.22% compared with those of the control group, respectively. The results can provide scienti?c data for the application of inducers in the biosynthesis of triterpenoids and hence the development of new antioxidants.
引文
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[10]冯杰,冯娜,贾薇,等.氮源对灵芝菌丝体液态深层发酵合成灵芝三萜的影响[J].菌物学报,2016, 35(6):722-733. DOI:10.13346/j.mycosystema. 150143.
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[12]田雪梅,张国利,宋爱荣.液体发酵法筛选樟芝优良诱变新菌株的研究[J].菌物学报,2012, 31(6):900-908. DOI:10.13346/j.mycosystema.2012.06.009.
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[16]王艳,尹静,马泓思,等.钙离子在介导SA诱导白桦悬浮细胞三萜合成途径中的作用[J].北京林业大学学报,2014, 36(2):51-58.DOI:10.13332/j.cnki.jbfu.2014.02.013.
[17]姚强,高兴喜,宫志远,等.不同脂肪酸对灵芝液体发酵的影响[J].食用菌学报,2010, 17(3):55-59. DOI:1005-9873(2010)03-0055-05.
[18] BALAZOVA A, URDOVA J, BILKA F, et al. Evaluation of manganese chloride's effect on biosynthetic properties of in vitro cultures of Eschscholzia californica Cham.[J]. 2018, 23(4):971.DOI:10.3390/molecules23040971.
[19]徐红云.粗毛纤孔菌发酵、三萜的提取与分离纯化研究[D].哈尔滨:东北林业大学,2013.
[20] LI J, LIN X J, TONG J H, et al. ITS sequence analysis of antrodia cinnamomea and total triterpenoid detection of liquid fermentation mycelium[J].Southwest China Journal of Agricultural Sciences, 2015, 28(5):2649-2654. DOI:1001-4829(2015)28:6<2649:NZZIXL>2.0.TX;2-K.
[21] BENZIE I, STRAIN J. The ferric reducing ability of plasma(FRAP)as a measure of"antioxidant power":the FRAP assay[J]. Analytical Biochemistry. 1996, 239(1):70-76. DOI:10.1006/abio. 1996.0292.
[22] OZGEN M, REESE R N, TULIO A Z, et al. Modified 2,2-azinobis-3-ethylbenzothiazoline-6-sulfonic acid(ABTS)method to measure antioxidantcapacity of selected small fruits and comparison to ferric reducing antioxidant power(FRAP)and 2,2'-diphenyl-lpicrylhydrazyl(DPPH)methods[J]. Journal of Agricultural&Food Chemistry, 2006, 54(4):1151-1157. DOI:10.1021/jf051960d.
[23] XIONG S L,LI A,HUANG N,et al. Antioxidant and immunoregulatory activity of different polysaccharide fractions from tuber of Ophiopogon japonicus[J]. Carbohydrate Polymers, 2011,86(3):1273-1280. DOI:10.1016/j.carbpol.2011.06.025.
[24] MARKLUND S, MARKLAMD G. Involvement of superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase[J]. European Journal of Biochemistry, 2005,43(7):469-474.
[25] EWA K, EWA B, ANNA L, et al. Methyl jasmonate as a control factor of the synthase squalene gene promoter and ginsenoside production in American ginseng hairy root cultured in shake flasks and a nutrient sprinkle bioreactor[J]. Industrial Crops&Products, 2018, 115:182-193.DOI:10.1016/j.indcrop.2018.02.036.
[26]王康宇,于丽莉,张美萍,等.茉莉酸甲酯调控下人参发状根皂苷合成相关基因表达的研究[J].中国中药杂志,2017, 42(12):2269-2275.DOI:10.19540/j.cnki.cjcmm.2017.0106.
[27]任春林,尹静,潘亚婕,等.水杨酸对白桦悬浮细胞中齐墩果酸积累及防御酶活性的影响[J].中草药,2012, 43(5):972-977.
[28]冯杰,冯娜,杨焱,等.通气量对灵芝菌丝体液态深层发酵合成灵芝三萜的影响[J].天然产物研究与开发,2015, 27(9):1564-1570.DOI:10.16333/j.1001-6880.2015.09.010.
[29]孙冰沁.基于油酸诱导的高产三萜灵芝菌丝体发酵及生物合成途径中关键酶编码基因研究[D].杭州:浙江大学,2017.
[30] EMR R, STEWART G G.The effects of increased magnesium and calcium concentrations on yeast fermentation performance in high gravity worts[J]. Journal of the Institute of Brewing, 1997, 103(5):287-291.
[31]杨慧,张美彦,宋春艳,等.铁、锌、钙离子在三种食用菌菌丝体中的富集及对其生长的影响[J].食用菌学报,2017, 24(2):27-33.DOI:10.16488/j.cnki.1005-9873.2017.02.07.
[32] YA J T, LI W Z. Improvement of ganoderic acid and ganoderma polysaccharide biosynthesis by Ganoderma lucidum fermentation under the inducement of Cu2+[J]. Biotechnology Progress, 2010, 26(2):417-423. DOI:10.1002/btpr.352.
[2]XIAO S L,TIAN Z Y,WANG Y F,et al.Recent progress in the antiviral activity and mechanism study of pentacyclic triterpenoids and their derivatives[J].Medicinal Research Reviews,2018,38(3):951-976.
[3]李德海,杜令娟,康宁,等.提取技术对粗毛纤孔菌三萜类化合物制备及体外降血脂作用的影响[J].食品科学,2018,39(10):291-297.DOI:10.7506/spkx1002-6630-201810044.
[4]昝立峰,包海鹰.粗毛纤孔菌的研究进展[J].食用菌学报,2011,18(1):78-82.DOI:10.16488/j.cnki.1005-9873.2011.01.008.
[5] REN Q, LU X, HAN J, et al. Triterpenoids and phenolics from the fruiting bodies of Inonotus hispidus and their activations of melanogenesis and tyrosinase[J]. Chinese Chemical Letters, 2017, 28(5):1052-1056. DOI:1001-8417(2017)28:5<1052:TAPFIF>2.0.TX;2-B.
[6] ZAN L F, BAO H Y, BAU T, et al. Chemical composition in fruiting body of Inonotus hispidus(Hymenochaetales,Basidiomycota)[J]. Mygosystema, 2013, 32(1):150-156. DOI:1672-6472(2013)32:1<150:CMQKJZ>2.0.TX;2-8.
[7] ZHANG Y, BAO H Y. A comparative study on antitumor activities of polysaccharides from four species of polyporaceae[J]. Mygosystema, 2014,33(1):114-120. DOI:1672-6472(2014)33:1<114:SZDKJZ>2.0.TX;2-7.
[8] SONG Y X, WANG H, ZHANG D H, et al. Artificial cultivation test of inonotus hispidus parasitizing on mulberry[J]. Acta Sericologica Sinica, 2016,42(6):1085-1091. DOI:0257-4799(2016)42:6<1085:JSS SDC>2.0.TX;2-O.
[9]王婷,包海鹰,图力古尔,等.寄生于蒙古黄榆上的粗毛纤孔菌生物学特性及驯化栽培[J].菌物学报,2016, 35(6):694-704.DOI:10.13346/j.mycosystema. 150078.
[10]冯杰,冯娜,贾薇,等.氮源对灵芝菌丝体液态深层发酵合成灵芝三萜的影响[J].菌物学报,2016, 35(6):722-733. DOI:10.13346/j.mycosystema. 150143.
[11]翟双星,冯杰,冯娜,等.灵芝三萜液态深层发酵的研究进展[J].农学学报,2018, 8(1):118-124.
[12]田雪梅,张国利,宋爱荣.液体发酵法筛选樟芝优良诱变新菌株的研究[J].菌物学报,2012, 31(6):900-908. DOI:10.13346/j.mycosystema.2012.06.009.
[13]徐红云,王占斌.响应面优化粗毛纤孔菌三萜提取工艺的研究[J].食品工业科技,2013, 34(23):228-232; 236. DOI:10.13386/j.issn1002-0306.2013.23.031.
[14]韦朝阳,贺亮,邵双双,等.外源添加物对食药用菌液体发酵影响的研究进展[J].食品科学,2015, 36(7):245-250. DOI:10.7506/spkx 1002-6630-201507045.
[15]陈林,崔培梧,鲁耀邦,等.茉莉酸甲酯对茯苓三萜生物合成的调控研宄[J].湖南中医药大学学报,2017, 37(6):606-610. DOI:10.3969/j.issn.1674-070X.2017.06.007.
[16]王艳,尹静,马泓思,等.钙离子在介导SA诱导白桦悬浮细胞三萜合成途径中的作用[J].北京林业大学学报,2014, 36(2):51-58.DOI:10.13332/j.cnki.jbfu.2014.02.013.
[17]姚强,高兴喜,宫志远,等.不同脂肪酸对灵芝液体发酵的影响[J].食用菌学报,2010, 17(3):55-59. DOI:1005-9873(2010)03-0055-05.
[18] BALAZOVA A, URDOVA J, BILKA F, et al. Evaluation of manganese chloride's effect on biosynthetic properties of in vitro cultures of Eschscholzia californica Cham.[J]. 2018, 23(4):971.DOI:10.3390/molecules23040971.
[19]徐红云.粗毛纤孔菌发酵、三萜的提取与分离纯化研究[D].哈尔滨:东北林业大学,2013.
[20] LI J, LIN X J, TONG J H, et al. ITS sequence analysis of antrodia cinnamomea and total triterpenoid detection of liquid fermentation mycelium[J].Southwest China Journal of Agricultural Sciences, 2015, 28(5):2649-2654. DOI:1001-4829(2015)28:6<2649:NZZIXL>2.0.TX;2-K.
[21] BENZIE I, STRAIN J. The ferric reducing ability of plasma(FRAP)as a measure of"antioxidant power":the FRAP assay[J]. Analytical Biochemistry. 1996, 239(1):70-76. DOI:10.1006/abio. 1996.0292.
[22] OZGEN M, REESE R N, TULIO A Z, et al. Modified 2,2-azinobis-3-ethylbenzothiazoline-6-sulfonic acid(ABTS)method to measure antioxidantcapacity of selected small fruits and comparison to ferric reducing antioxidant power(FRAP)and 2,2'-diphenyl-lpicrylhydrazyl(DPPH)methods[J]. Journal of Agricultural&Food Chemistry, 2006, 54(4):1151-1157. DOI:10.1021/jf051960d.
[23] XIONG S L,LI A,HUANG N,et al. Antioxidant and immunoregulatory activity of different polysaccharide fractions from tuber of Ophiopogon japonicus[J]. Carbohydrate Polymers, 2011,86(3):1273-1280. DOI:10.1016/j.carbpol.2011.06.025.
[24] MARKLUND S, MARKLAMD G. Involvement of superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase[J]. European Journal of Biochemistry, 2005,43(7):469-474.
[25] EWA K, EWA B, ANNA L, et al. Methyl jasmonate as a control factor of the synthase squalene gene promoter and ginsenoside production in American ginseng hairy root cultured in shake flasks and a nutrient sprinkle bioreactor[J]. Industrial Crops&Products, 2018, 115:182-193.DOI:10.1016/j.indcrop.2018.02.036.
[26]王康宇,于丽莉,张美萍,等.茉莉酸甲酯调控下人参发状根皂苷合成相关基因表达的研究[J].中国中药杂志,2017, 42(12):2269-2275.DOI:10.19540/j.cnki.cjcmm.2017.0106.
[27]任春林,尹静,潘亚婕,等.水杨酸对白桦悬浮细胞中齐墩果酸积累及防御酶活性的影响[J].中草药,2012, 43(5):972-977.
[28]冯杰,冯娜,杨焱,等.通气量对灵芝菌丝体液态深层发酵合成灵芝三萜的影响[J].天然产物研究与开发,2015, 27(9):1564-1570.DOI:10.16333/j.1001-6880.2015.09.010.
[29]孙冰沁.基于油酸诱导的高产三萜灵芝菌丝体发酵及生物合成途径中关键酶编码基因研究[D].杭州:浙江大学,2017.
[30] EMR R, STEWART G G.The effects of increased magnesium and calcium concentrations on yeast fermentation performance in high gravity worts[J]. Journal of the Institute of Brewing, 1997, 103(5):287-291.
[31]杨慧,张美彦,宋春艳,等.铁、锌、钙离子在三种食用菌菌丝体中的富集及对其生长的影响[J].食用菌学报,2017, 24(2):27-33.DOI:10.16488/j.cnki.1005-9873.2017.02.07.
[32] YA J T, LI W Z. Improvement of ganoderic acid and ganoderma polysaccharide biosynthesis by Ganoderma lucidum fermentation under the inducement of Cu2+[J]. Biotechnology Progress, 2010, 26(2):417-423. DOI:10.1002/btpr.352.