两种氮素形态对药用植物三叶青中黄酮类活性成分积累的影响及相关分子机制的初步探讨
|
摘要
以珍稀濒危药用植物三叶青(Tetrastigma hemsleyanum Diels et Gilg)为材料,通过遮荫条件下(30%自然光)的盆栽试验,研究了硝态氮(NO_3~--N)和铵态氮(NH_4~+-N)两种不同氮素形态对其根和叶中黄酮类活性成分含量的影响并探讨其机制。结果表明:NO_3~--N处理后的三叶青叶中原花青素B1、芦丁、山柰酚-3-O-芸香糖苷、槲皮素、异槲皮苷、山柰酚的含量比NH_4~+-N处理的高;其根中的原花青素B1、芦丁、山柰酚-3-O-芸香糖苷、槲皮素、异槲皮苷、山柰酚的含量比NH_4~+-N处理的高。进一步的研究发现NO_3~--N处理过的三叶青其根、叶中与黄酮类化合物合成相关的酶苯丙氨酸解氨酶(PAL)、查尔酮合成酶(CHS)转录与表达水平高于NH_4~+-N处理的三叶青。此外,NO_3~--N处理的三叶青叶、根中的PAL和CHS酶活性也高于NH_4~+-N处理。利用PAL特异性抑制剂AOA处理后,AOA能显著抑制PAL酶活性(P<0.05)并导致黄酮类成分含量极显著下降(P<0.01)。综上所述,施用NO_3~--N更有益于三叶青黄酮类化合物的生物合成,其主要是通过增加PAL代谢途径关键酶的基因表达量进而提高其酶活性来增强黄酮类化合物的合成。
The effects of two different nitrogen( N) forms( nitrate-N and ammonium-N) on the accumulation of flavonoids( procyanidin B1,rutin,kaempferol-3-O-rutinoside,quercetin,isoquercitrin and kaempferol) in the endangered medicinal plant Tetrastigma hemsleyanum( from Zhejiang Province) were examined through potted experiment under shady condition( 30% natural light),and its influence mechanism was also discussed. The flavonoids content was relatively higher in the root and leaf of T. hemsleyanum under nitrate-N supply than ammonium-N supply. The levels of transcription and expression of phenylalanine ammonia-lyase( PAL) and chalcone synthase( CHS) which are related to flavonoids synthesis were relatively higher in the root and leaf ofT. hemsleyanum under nitrate-N supply than ammonium-N supply. Besides,the enzyme activities of PAL and CHS were also higher in the root and leaf of T. hemsleyanum under nitrate-N supply than ammonium-N supply.The PAL-specific inhibitor( AOA) could significantly inhibit the enzyme activity of PAL( P < 0. 05) and lead to a very significant decrease in flavonoids content( P < 0. 01). In conclusion,nitrate-N supply is more beneficial to flavonoids synthesis in T. hemsleyanum,which mainly enhances the flavonoids synthesis by increasing the gene expression and activity of key enzymes in PAL metabolic pathway.
The effects of two different nitrogen( N) forms( nitrate-N and ammonium-N) on the accumulation of flavonoids( procyanidin B1,rutin,kaempferol-3-O-rutinoside,quercetin,isoquercitrin and kaempferol) in the endangered medicinal plant Tetrastigma hemsleyanum( from Zhejiang Province) were examined through potted experiment under shady condition( 30% natural light),and its influence mechanism was also discussed. The flavonoids content was relatively higher in the root and leaf of T. hemsleyanum under nitrate-N supply than ammonium-N supply. The levels of transcription and expression of phenylalanine ammonia-lyase( PAL) and chalcone synthase( CHS) which are related to flavonoids synthesis were relatively higher in the root and leaf ofT. hemsleyanum under nitrate-N supply than ammonium-N supply. Besides,the enzyme activities of PAL and CHS were also higher in the root and leaf of T. hemsleyanum under nitrate-N supply than ammonium-N supply.The PAL-specific inhibitor( AOA) could significantly inhibit the enzyme activity of PAL( P < 0. 05) and lead to a very significant decrease in flavonoids content( P < 0. 01). In conclusion,nitrate-N supply is more beneficial to flavonoids synthesis in T. hemsleyanum,which mainly enhances the flavonoids synthesis by increasing the gene expression and activity of key enzymes in PAL metabolic pathway.
引文
1.Feng Z Q,Hao W R,Lin X Y,et al.Antitumor activity of total flavonoids from Tetrastigma hemsleyanum Diels et Gilg is associated with the inhibition of regulatory T cells in mice[J].Onco Targets and Therapy,2014,7:947-956.
2 .Xu C J,Ding G Q,Fu J Y,et al.Immunoregulatory effects of ethyl-acetate fraction of extracts from Tetrastigma hemsleyanum Diels et.Gilg on immune functions of ICR mice[J].Biomedical and Environmental Sciences,2008,21(4):325-331.
3 .杨雄志,王翰华.三叶青提取物解热作用及对大白鼠下丘脑5-羟色胺、去甲肾上腺素、多巴胺含量的影响[J].长春中医药大学学报,2013,29(5):774-775,785.Yang X Z,Wang H H.Antipyretic effect of extracts of Tetrastigmae radix and effect on 5-hydroxytryptamine(5-HT),norepinephrine(NE),dopamine(DA)in hypothalamus rats[J].Journal of Changchun University of Traditional Chinese Medicine,2013,29(5):774-775,785.
4 .黄真,毛庆秋,魏佳平.三叶青提取物抗炎、镇痛及解热作用的实验研究[J].中国新药杂志,2005,14(7):861-864.Huang Z,Mao Q Q,WEI J P.Evaluation of anti-inflammatory,analgesic and antipyretic actions for the extracts from Radix tetrastigmae[J].Chinese Journal of New Drugs,2005,14(7):861-864.
5 .杨雄志,巫军.三叶青提取物抗乙肝病毒活性的研究[J].南京中医药大学学报,2009,25(4):294-296.Yang X Z,Wu J.A study of anti-HBV activity of extract of Radix tetrastigmae[J].Journal of Nanjing University of Traditional Chinese Medicine,2009,25(4):294-296.
6 .Xiong Y,Wu X W,Rao L Q.Tetrastigma hemsleyanum(Sanyeqing)root tuber extracts induces apoptosis in human cervical carcinoma He La cells[J].Journal of Ethnopharmacology,2015,165:46-53.
7 .Peng X,Zhang Y Y,Wang J,et al.Ethylacetate extract from Tetrastigma hemsleyanum induces apoptosis via the mitochondrial caspase-dependent intrinsic pathway in Hep G2 cells[J].Tumor Biology,2016,37(1):865-876.
8 .杨雄志.中药三叶青的药理作用与临床应用[J].吉林中医药,2009,29(6):517-518.Yang X Z.Pharmacological action and clinical application of Chinese Drug-Sanye Qing[J].Jilin Journal of Traditional Chinese Medicine,2009,29(6):517-518.
9 .Shao Q S,Deng Y M,SHEN H J,et al.Optimization of polysaccharides extraction from Tetrastigma hemsleyanum Diels et Gilg using response surface methodology[J].International Journal of Biological Macromolecules,2011,49(5):958-962.
10 .许文,傅志勤,林婧,等.HPLC-Q-TOF-MS和UPLC-Qq QMS的三叶青主要成分定性与定量研究[J].中国中药杂志,2014,39(22):4365-4372.Xu W,Fu Z Q,Lin J,et al.Qualitative and quantitative analysis of major constituents in Tetrastigma hemsleyanum by HPLC-Q-TOF-MS and UPLC-Qq Q-MS[J].China Journal of Chinese Materia Medica,2014,39(22):4365-4372.
11 .吴舟涛,朱玲燕,吴学谦,等.三叶青化学成分和抗肿瘤作用研究进展[J].中南药学,2017,15(3):319-324.Wu Z T,Zhu L Y,Wu X Q,et al.Research progress in the chemical compositions and anti-tumor effect of Tetrastigma hemsleyanum Diels et Gilg[J].Central South Pharmacy,2017,15(3):319-324.
12 .陈丽芸,郭素华.三叶青的化学成分及药理作用研究进展[J].浙江中医药大学学报,2012,36(12):1368-1370.Chen L Y,Guo S H.Progress in studies of chemical composition and pharmacological effects of Tetrastigmatis hemsleyani[J].Journal of Zhejiang Chinese Medical University,2012,36(12):1368-1370.
13 .Ibrahim M H,Jaafar H Z E,Rahmat A,et al.Involvement of nitrogen on flavonoids,glutathione,anthocyanin,ascorbic acid and antioxidant activities of Malaysian medicinal plant Labisia pumila Blume(Kacip Fatimah)[J].International Journal of Molecular Sciences,2012,13(1):393-408.
14 .Becker C,Urlic B,Jukicpika M J,et al.Nitrogen limited red and green leaf lettuce accumulate flavonoid glycosides,caffeic acid derivatives,and sucrose while losing chlorophylls,B-carotene and xanthophylls[J].PLo S One,2015,10 (11):e0142867.
15 .吕万祥,惠竹梅.不同形态氮素对“赤霞珠”葡萄果实品质的影响[J].北方园艺,2012,(14):5-8.LW X,Hui Z M.Effect of nitrogen forms on quality of‘Cabernet Sauvignon’grape[J].Northern Horticulture,2012,(14):5-8.
16 .Kovácik J,Klejdus B.Induction of phenolic metabolites and physiological changes in chamomile plants in relation to nitrogen nutrition[J].Food Chemistry,2014,142:334-341.
17 .Jones D H.Phenylalanine ammonia-lyase:regulation of its induction,and its role in plant development[J].Phytochemistry,1984,23(7):1349-1359.
18 .Petrussa E,Braidot E,Zancani M,et al.Plant flavonoidsbiosynthesis,transport and involvement in stress responses[J].International Journal of Molecular Sciences,2013,14(7):14950-14973.
19 .李青军,张炎,胡伟,等.氮素运筹对玉米干物质积累、氮素吸收分配及产量的影响[J].植物营养与肥料学报,2011,17(3):755-760.Li Q J,Zhang Y,Hu W,et al.Effects of nitrogen management on maize dry matter accumulation,nitrogen uptake and distribution and maize yield[J].Plant Nutrition and Fertilizer Science,2011,17(3):755-760.
20 .唐晓清,吕婷婷,施晟璐,等.氮素处理对不同种质菘蓝叶的氨基酸、总黄酮与矿质元素的影响[J].应用与环境生物学报,2017,23(2):374-378.Tang X Q,LüT T,Shi S L,et al.Effect of nitrogen treatment on amino acids,total flavonoids,and mineral elements of Isatis indigotica from different germplasms[J].Chinese Journal of Applied&Environmental Biology,2017,23(2):374-378.
21 .唐晓清,肖云华,赵雪玲,等.菘蓝根和叶的生物量与活性成分对氮素形态的响应[J].南京农业大学学报,2016,39(1):70-77.Tang X Q,Xiao Y H,Zhao X L,et al.Response of biomass and active components in roots and leaves of Isatis indigotica Fort.to nitrogen forms[J].Journal of Nanjing Agricultural University,2016,39(1):70-77.
22 .臧小云.氮素营养对荞麦生长及黄酮代谢的影响[D].南京:南京农业大学,2006.Zang X Y.Effect of nitrogen nutrition on growth and flavone metabolism in common buckwheat(F.esculentum Moench)[D].Nanjing:Nanjing Agricultural University,2006.
23 .朱孟炎,于博帆,陈华峰.外源硝态氮水平对长春花生理代谢的影响[J].植物研究,2016,36(4):535-541.Zhu M Y,Yu B F,Chen H F.Effects of nitrate nitrogen levels on physiological metabolism of Catharanthus roseus[J].Bulletin of Botanical Research,2016,36(4):535-541.
24 .Fallovo C,Schreiner M,Schwarz D,et al.Phytochemical changes induced by different nitrogen supply forms and radiation levels in two leafy Brassica species[J].Journal of Agricultural and Food Chemistry,2011,59(8):4198-4207.
25 .张朋,王康才,成明超,等.氮素形态对杭白菊生长及品质的影响[J].中国中药杂志,2014,39(17):3263-3268.Zhang P,Wang K C,Cheng M C,et al.Effects of nitrogen form on growth and quality of Chrysanthemums morifolium[J].China Journal of Chinese Materia Medica,2014,39(17):3263-3268.
26 .Cui X H,Murthy H N,Wu C H,et al.Adventitious root suspension cultures of Hypericum perforatum:effect of nitrogen source on production of biomass and secondary metabolites[J].In Vitro Cellular&Developmental BiologyPlant,2010,46(5):437-444.
2 .Xu C J,Ding G Q,Fu J Y,et al.Immunoregulatory effects of ethyl-acetate fraction of extracts from Tetrastigma hemsleyanum Diels et.Gilg on immune functions of ICR mice[J].Biomedical and Environmental Sciences,2008,21(4):325-331.
3 .杨雄志,王翰华.三叶青提取物解热作用及对大白鼠下丘脑5-羟色胺、去甲肾上腺素、多巴胺含量的影响[J].长春中医药大学学报,2013,29(5):774-775,785.Yang X Z,Wang H H.Antipyretic effect of extracts of Tetrastigmae radix and effect on 5-hydroxytryptamine(5-HT),norepinephrine(NE),dopamine(DA)in hypothalamus rats[J].Journal of Changchun University of Traditional Chinese Medicine,2013,29(5):774-775,785.
4 .黄真,毛庆秋,魏佳平.三叶青提取物抗炎、镇痛及解热作用的实验研究[J].中国新药杂志,2005,14(7):861-864.Huang Z,Mao Q Q,WEI J P.Evaluation of anti-inflammatory,analgesic and antipyretic actions for the extracts from Radix tetrastigmae[J].Chinese Journal of New Drugs,2005,14(7):861-864.
5 .杨雄志,巫军.三叶青提取物抗乙肝病毒活性的研究[J].南京中医药大学学报,2009,25(4):294-296.Yang X Z,Wu J.A study of anti-HBV activity of extract of Radix tetrastigmae[J].Journal of Nanjing University of Traditional Chinese Medicine,2009,25(4):294-296.
6 .Xiong Y,Wu X W,Rao L Q.Tetrastigma hemsleyanum(Sanyeqing)root tuber extracts induces apoptosis in human cervical carcinoma He La cells[J].Journal of Ethnopharmacology,2015,165:46-53.
7 .Peng X,Zhang Y Y,Wang J,et al.Ethylacetate extract from Tetrastigma hemsleyanum induces apoptosis via the mitochondrial caspase-dependent intrinsic pathway in Hep G2 cells[J].Tumor Biology,2016,37(1):865-876.
8 .杨雄志.中药三叶青的药理作用与临床应用[J].吉林中医药,2009,29(6):517-518.Yang X Z.Pharmacological action and clinical application of Chinese Drug-Sanye Qing[J].Jilin Journal of Traditional Chinese Medicine,2009,29(6):517-518.
9 .Shao Q S,Deng Y M,SHEN H J,et al.Optimization of polysaccharides extraction from Tetrastigma hemsleyanum Diels et Gilg using response surface methodology[J].International Journal of Biological Macromolecules,2011,49(5):958-962.
10 .许文,傅志勤,林婧,等.HPLC-Q-TOF-MS和UPLC-Qq QMS的三叶青主要成分定性与定量研究[J].中国中药杂志,2014,39(22):4365-4372.Xu W,Fu Z Q,Lin J,et al.Qualitative and quantitative analysis of major constituents in Tetrastigma hemsleyanum by HPLC-Q-TOF-MS and UPLC-Qq Q-MS[J].China Journal of Chinese Materia Medica,2014,39(22):4365-4372.
11 .吴舟涛,朱玲燕,吴学谦,等.三叶青化学成分和抗肿瘤作用研究进展[J].中南药学,2017,15(3):319-324.Wu Z T,Zhu L Y,Wu X Q,et al.Research progress in the chemical compositions and anti-tumor effect of Tetrastigma hemsleyanum Diels et Gilg[J].Central South Pharmacy,2017,15(3):319-324.
12 .陈丽芸,郭素华.三叶青的化学成分及药理作用研究进展[J].浙江中医药大学学报,2012,36(12):1368-1370.Chen L Y,Guo S H.Progress in studies of chemical composition and pharmacological effects of Tetrastigmatis hemsleyani[J].Journal of Zhejiang Chinese Medical University,2012,36(12):1368-1370.
13 .Ibrahim M H,Jaafar H Z E,Rahmat A,et al.Involvement of nitrogen on flavonoids,glutathione,anthocyanin,ascorbic acid and antioxidant activities of Malaysian medicinal plant Labisia pumila Blume(Kacip Fatimah)[J].International Journal of Molecular Sciences,2012,13(1):393-408.
14 .Becker C,Urlic B,Jukicpika M J,et al.Nitrogen limited red and green leaf lettuce accumulate flavonoid glycosides,caffeic acid derivatives,and sucrose while losing chlorophylls,B-carotene and xanthophylls[J].PLo S One,2015,10 (11):e0142867.
15 .吕万祥,惠竹梅.不同形态氮素对“赤霞珠”葡萄果实品质的影响[J].北方园艺,2012,(14):5-8.LW X,Hui Z M.Effect of nitrogen forms on quality of‘Cabernet Sauvignon’grape[J].Northern Horticulture,2012,(14):5-8.
16 .Kovácik J,Klejdus B.Induction of phenolic metabolites and physiological changes in chamomile plants in relation to nitrogen nutrition[J].Food Chemistry,2014,142:334-341.
17 .Jones D H.Phenylalanine ammonia-lyase:regulation of its induction,and its role in plant development[J].Phytochemistry,1984,23(7):1349-1359.
18 .Petrussa E,Braidot E,Zancani M,et al.Plant flavonoidsbiosynthesis,transport and involvement in stress responses[J].International Journal of Molecular Sciences,2013,14(7):14950-14973.
19 .李青军,张炎,胡伟,等.氮素运筹对玉米干物质积累、氮素吸收分配及产量的影响[J].植物营养与肥料学报,2011,17(3):755-760.Li Q J,Zhang Y,Hu W,et al.Effects of nitrogen management on maize dry matter accumulation,nitrogen uptake and distribution and maize yield[J].Plant Nutrition and Fertilizer Science,2011,17(3):755-760.
20 .唐晓清,吕婷婷,施晟璐,等.氮素处理对不同种质菘蓝叶的氨基酸、总黄酮与矿质元素的影响[J].应用与环境生物学报,2017,23(2):374-378.Tang X Q,LüT T,Shi S L,et al.Effect of nitrogen treatment on amino acids,total flavonoids,and mineral elements of Isatis indigotica from different germplasms[J].Chinese Journal of Applied&Environmental Biology,2017,23(2):374-378.
21 .唐晓清,肖云华,赵雪玲,等.菘蓝根和叶的生物量与活性成分对氮素形态的响应[J].南京农业大学学报,2016,39(1):70-77.Tang X Q,Xiao Y H,Zhao X L,et al.Response of biomass and active components in roots and leaves of Isatis indigotica Fort.to nitrogen forms[J].Journal of Nanjing Agricultural University,2016,39(1):70-77.
22 .臧小云.氮素营养对荞麦生长及黄酮代谢的影响[D].南京:南京农业大学,2006.Zang X Y.Effect of nitrogen nutrition on growth and flavone metabolism in common buckwheat(F.esculentum Moench)[D].Nanjing:Nanjing Agricultural University,2006.
23 .朱孟炎,于博帆,陈华峰.外源硝态氮水平对长春花生理代谢的影响[J].植物研究,2016,36(4):535-541.Zhu M Y,Yu B F,Chen H F.Effects of nitrate nitrogen levels on physiological metabolism of Catharanthus roseus[J].Bulletin of Botanical Research,2016,36(4):535-541.
24 .Fallovo C,Schreiner M,Schwarz D,et al.Phytochemical changes induced by different nitrogen supply forms and radiation levels in two leafy Brassica species[J].Journal of Agricultural and Food Chemistry,2011,59(8):4198-4207.
25 .张朋,王康才,成明超,等.氮素形态对杭白菊生长及品质的影响[J].中国中药杂志,2014,39(17):3263-3268.Zhang P,Wang K C,Cheng M C,et al.Effects of nitrogen form on growth and quality of Chrysanthemums morifolium[J].China Journal of Chinese Materia Medica,2014,39(17):3263-3268.
26 .Cui X H,Murthy H N,Wu C H,et al.Adventitious root suspension cultures of Hypericum perforatum:effect of nitrogen source on production of biomass and secondary metabolites[J].In Vitro Cellular&Developmental BiologyPlant,2010,46(5):437-444.