黄顶菊中噻吩类和酚酸类物质的研究
|
摘要
本文对外来入侵植物黄顶菊中噻吩类及酚酸类组分的提取工艺、含量测定及分离纯化进行了综合的研究,为黄顶菊资源化利用提供了数据支持。
实验研究了噻吩类及酚酸类组分的提取工艺,通过含量测定确定各有效成分的最佳提取工艺。提取噻吩类工艺为：石油醚,料液比1：10,65℃下回流提取一次,时间1h。提取酚酸类工艺为：碱水提取,pH=10的NaOH溶液,料液比1：15,95℃下回流提取两次,每次1h,滤液用1mol·L-1的HCl调pH值为3.
高速逆流色谱法(HSCCC)是一种无需要任何固态支持介质的液液分配色谱分离支持,由于不使用固态支持介质,避免了因不可逆吸附而引起的样品损失、失活、变性等。高速逆流色谱法已广泛应用于天然产物有效成分的分离,本实验用半制备型高速逆流色谱优化选择的两相溶剂体系为：正己烷-乙腈(1：1,v/v),从265.6mg黄顶菊提取粗品中分离纯化出2.2mg 5-(3-丁烯-1-炔基)-2,2'联二噻吩,5.2mgα-三联噻吩和4.3mg 5-(3-戊烯-1-炔基)-2,2'联二噻吩三种单体,纯度分别为90.2%,99.9%和92.1%,并通过EI-MS,1H NMR和13C NMR光谱数据分析确认其结构。其中化合物5-(3-戊烯-1-炔基)-2,2'联二噻吩首次从黄顶菊中发现并分离得到。
采用培养皿滤纸法对黄顶菊中化感物质进行了部分生物测试,对其化感作用进行了初步研究。
In this article, the extraction techniques of thiophenes and phenolic acids from Flaveria bidentis (L) Kuntze were investigated, including determination, the optimum solution in extraction, separation and purification. The data was supported in order to make use of this invasive plant.
This thesis carried out a study on the extraction techniques of thiophenes and phenolic acids. The optimum extraction techniques were determined by the results of detection. The optimum extraction condition of thiophenes by refluxing is as follows:petroleum ether, ratio of material and fluid:1:10(w/v), once refluxing under 65℃, extracting time:1h. The optimum extraction condition of phenolic acids by refluxing is as follows:solution of NaOH at pH=10, ratio of material and fluid:1:15(w/v), twice refluxing under 95℃, extracting time:1h, filtrate was adjusted to pH=3 using 1mol·L-1 HCl.
High-speed counter-current chromatography (HSCCC), being a support-free liquid-liquid partition method, eliminates irreversible adsorption of sample onto the solid support, and has been widely used in preparative separation of natural products. In present study, the two phase solvent system of HSCCC composed of hexane-acetonitrile=1:1(v/v), which has been selected by semi-preparative HSCCC.2.2 mg 5-(3-buten-1-ynyl)-2,2'-bithienyl,5.2 mg a-terthienyl and 4.3 mg 5-(3-penten-1-ynyl)-2,2'-bithienyl with the purity of 90.2%,99.9% and 92.1% respectively, were produced from 265.6 mg crude extract, and 5-(3-penten-1-ynyl)-2,2'-bithienyl was isolated from Flaveria bidentis (L) Kuntze for the first time. The structures of the separated compounds were identified by EI-MS,1HNMR and 13CNMR.
By using Petri dishes filter method, part of bioassay of allelochemicals in Flaveria bidentis (L) Kuntze were proceeded in order to study its allelopathy.
实验研究了噻吩类及酚酸类组分的提取工艺,通过含量测定确定各有效成分的最佳提取工艺。提取噻吩类工艺为：石油醚,料液比1：10,65℃下回流提取一次,时间1h。提取酚酸类工艺为：碱水提取,pH=10的NaOH溶液,料液比1：15,95℃下回流提取两次,每次1h,滤液用1mol·L-1的HCl调pH值为3.
高速逆流色谱法(HSCCC)是一种无需要任何固态支持介质的液液分配色谱分离支持,由于不使用固态支持介质,避免了因不可逆吸附而引起的样品损失、失活、变性等。高速逆流色谱法已广泛应用于天然产物有效成分的分离,本实验用半制备型高速逆流色谱优化选择的两相溶剂体系为：正己烷-乙腈(1：1,v/v),从265.6mg黄顶菊提取粗品中分离纯化出2.2mg 5-(3-丁烯-1-炔基)-2,2'联二噻吩,5.2mgα-三联噻吩和4.3mg 5-(3-戊烯-1-炔基)-2,2'联二噻吩三种单体,纯度分别为90.2%,99.9%和92.1%,并通过EI-MS,1H NMR和13C NMR光谱数据分析确认其结构。其中化合物5-(3-戊烯-1-炔基)-2,2'联二噻吩首次从黄顶菊中发现并分离得到。
采用培养皿滤纸法对黄顶菊中化感物质进行了部分生物测试,对其化感作用进行了初步研究。
In this article, the extraction techniques of thiophenes and phenolic acids from Flaveria bidentis (L) Kuntze were investigated, including determination, the optimum solution in extraction, separation and purification. The data was supported in order to make use of this invasive plant.
This thesis carried out a study on the extraction techniques of thiophenes and phenolic acids. The optimum extraction techniques were determined by the results of detection. The optimum extraction condition of thiophenes by refluxing is as follows:petroleum ether, ratio of material and fluid:1:10(w/v), once refluxing under 65℃, extracting time:1h. The optimum extraction condition of phenolic acids by refluxing is as follows:solution of NaOH at pH=10, ratio of material and fluid:1:15(w/v), twice refluxing under 95℃, extracting time:1h, filtrate was adjusted to pH=3 using 1mol·L-1 HCl.
High-speed counter-current chromatography (HSCCC), being a support-free liquid-liquid partition method, eliminates irreversible adsorption of sample onto the solid support, and has been widely used in preparative separation of natural products. In present study, the two phase solvent system of HSCCC composed of hexane-acetonitrile=1:1(v/v), which has been selected by semi-preparative HSCCC.2.2 mg 5-(3-buten-1-ynyl)-2,2'-bithienyl,5.2 mg a-terthienyl and 4.3 mg 5-(3-penten-1-ynyl)-2,2'-bithienyl with the purity of 90.2%,99.9% and 92.1% respectively, were produced from 265.6 mg crude extract, and 5-(3-penten-1-ynyl)-2,2'-bithienyl was isolated from Flaveria bidentis (L) Kuntze for the first time. The structures of the separated compounds were identified by EI-MS,1HNMR and 13CNMR.
By using Petri dishes filter method, part of bioassay of allelochemicals in Flaveria bidentis (L) Kuntze were proceeded in order to study its allelopathy.
引文
[1]万方浩,郭建英,王德辉,McNeely JA防治外来入侵物种生物多样性与外来入侵物种管理国际研讨会论文集[C].北京：中国环境科学出版社,2002：77-102.
[2]高贤明,唐廷贵,梁宇.外来植物黄顶菊的入侵警报及防控对策[J].生物多样性,2004,12(2)：274-279.
[3]陈艳,刘坤,张国良,谭万忠.外来入侵杂草黄顶菊生物活性及化学成分研究进展[J].杂草科学,2007,(4)：1-3.
[4]戴素雅.黄顶菊的危害及防除技术[J].植物保护,2006,(10)：1
[5]Green W M, de Billot M C, Joffe T, et al. Indigenous plants and weeds on the Makhathini flats as refuge hosts to maintain bollworm population susceptibility to transgenic cotton (BollgardTM) [J]. African Entomology,2003,11(1):21-29.
[6]Broussalis A M, Ferraro G E, Martino V S. Argentine plants as potential source of insecticidal compounds [J]. Journal of Ethnopharmacology,1999,67:219-233.
[7]Ricciardi A, Esquivel G Plantas de posible utilidad et el control de insectos [J]. Anales SAIPA, 1986,7:40-64.
[8]Bardon A, Borkosky S, Ybarra M I, et al. Bioactive plants from Argentina and Bolivia [J]. Fitoterapia,2007,78(3):227-231.
[9]Ferraro G, Broussalis A, Martino V, et al. Argentine medicinal plant:antiviral screening [J]. International symposium on medicinal and aromatic plants,1992,306:239-244.
[10]李香菊,张米茹,李咏军,等.黄顶菊水提取液对植物种子发芽及胚根伸长的化感作用研究[J].杂草科学,2007(4)：15-19.
[11]张金林,刘颖超,庞民,等.黄顶菊提取物除草剂乳油及其制备工艺[P]：CN,200710061859.3.2007-04-30.
[12]王朋,梁文举,孔垂华,等.外来杂草入侵的化学机制[J].应用生态学报,2004,15(4)700-707.
[13]宋启示,何明,唐建淮,等.紫茎泽兰的互感潜力[J].植物生态学报,2000,24(3)：362-365.
[14]万方浩,王韧.恶性害草豚草的生物学及生态学特征[J].杂草学报,1990,4(1)：45-48.
[15]郭琼霞,陈颖,沈荔花,等.加拿大一枝黄花对豆类和蔬菜的化感作用研究[J].检验检疫科学,2006,16(6)：10-12.
[16]周文杰.黄顶菊对小麦种子化感作用的初步研究[J].衡水学院学报,2008,10(1)：60-64.
[17]O J Pereyra de Santiago, H R Julliani. Isolation of Quercetin 3,7,3',4'-Tetrasulphate from Flaveria bidentis L. Otto. Kuntze [J]. Experientia,1972,28:380.
[18]陈艳,刘坤,张国良,等.外来入侵杂草黄顶菊生物活性及化学成分研究进展[J].2007,4：1-3.
[19]金文荣.华东蓝刺头化学成分及其生物活性研究[D].浙江：浙江大学,2008.
[20]Hudson J. B., Towers G H. N. Therapeutic potential of plant photosensitizers [J]. Pharmacology and Therapeutics,1991,49:188-222.
[21]Cho K., Ryw M., ChinK., et al. Thiophene compounds as antivirus, anti-inflammatory and immunoregulatory agents [P]. JP:9502,665,1995-01-06.
[22]Agnese A M, Nunez Montoya S C, Espinar L A, et al. Chemotaxonomic Features in Argentinian Species of Flaveria [J]. Biochemical Systematics and Ecology,1999,27:739-742.
[23]Vladimir, C, Jitka, V., Lenka, K. et al. Phototoxic activity of a thiophene polyacetylene from Leuzea carthamoides [J]. Fitoterapia,2006,77:194-199.
[24]万树青,徐汉虹,赵善欢,等.光活化多炔类化合物对蚊幼虫的毒力[J].昆虫学报,2000,43(3)：264-270.
[25]杨淑娟,万树青,蒋志胜,等.两种光敏化合物光诱导稗草细胞产氧自由基电子自旋共振的检测与分析[J].植物保护学报,2004,31(3)：294-298.
[26]J.B. Hudson, L.Harris, A. Teeple, et al. The anti-HIV activity of the phytochemical a-terthienyl [J]. Antiviral Research,1993,20:33-43.
[27]兰志银,丘昌隆.格氏试剂交叉偶联反应制备α-三联噻吩[J].化学试剂,1995,17(5)：289-290.
[28]张囡,杜丽丽,王冬,等.中药酚酸类成分的研究进展[J].中国现代中药,2006,8(2)：25-28.
[29]王媚,吴皓.中药有机酸类成分的提取分享及测定方法研究进展[J].南京中医药大学学报,2004,20(3)：190-192.
[30]宫静静,谢静,沈平孃,等.高速逆流色谱技术在中药及天然产物领域中的应用[J].中国现代中药,2007,9(1)：33-35.
[31]曹学丽.高速逆流色谱分离技术及应用[M].北京：化学工业出版社,2005：1.
[32]张天佑.逆流色谱技术[M].北京：北京科学技术出版社,1991：1.
[33]Alain B, Billardello B. Test to evaluate countercurrent chromatographs [J]. J.Chromatogr.A, 2000,90(2):32.
[34]Tsao R, Deng Z Y. Separation procedures for naturally occurring antioxidant phytochemicals [J]. J.Chromatogr.B, Analytical Technologies in the Biomedical and Life Sciences,2004,8(12):85.
[35]魏芸,张天佑,吴克友.高速逆流色谱法对刺五加有效成分刺五加苷的分离制备[J].色谱,2002,20(6)：543-545.
[36]刘迪,陈雪峰,宋晓宇.高速逆流色谱技术应用现状[J].食品与机械,2006,22(2)：98-101.
[37]Ito Y, et al. High-speed preparative countercurrent chromatography with a coil planet entrlfuge [J]. J.Chromatogr.A,1982,244-247.
[38]Ou Yang X K, Jin M C, He C H. Preparative separation of four major alkaloids from medicional plant of Tripterygium Wilfordii Hook Fusing high-speed counter-current chromatography [J]. Sep.Purif.Technol,2007,56:319-324.
[39]Xie Q Q, Wei Y, Zhang G L. Separation of flavonol glycosides from Flaveria bidentis (L.) Kuntze by high-speed counter-current chromatography [J]. Sep. Purif. Technol.,2010,72:229-233.
[40]Wei Y, Gao Y L, Zhang K, et al. Isolation of caffeic acid from eupatorium adenophorum spreng by high-speed countercurrent chromatography and synthesis of caffeic acid-intercalated layered double hydroxide [J]. J. Liq. Chromatogr. R. T.2010,33(6):837-845.
[41]Lu H T, Jiang Y, Chen F. Application of preparative high-speed counter-current chromatography for separation of chlorogenic acid from Flos Lonicerae [J]. J.Chromatogr.A,2004,1026:185.
[42]Yao S, Luo J G, Huang X F, et al. Application of preparative high-speed counter-current chromatography/preparative high-performance liquid chromatography mode in rapid separation of saponins [J]. J.Chromatogr.B,2008,01:047.
[43]Yoichi S, Yoko H, Chao Z M,et al.Application of high-speed counter-current chromatography to the separation of coumarin and related compounds [J]. J.Chromatogr.A,1997,759:47.
[44]Wei Y, Zhang T Y, Ito Y. Counter-current chromatographic separation of glycoprotein components from Morchella esculenta (L) with a polymer phase system by a cross-axis coil planet centrifuge [J]. J.Chromatogr.A,2001,917:347.
[45]Yoichi S,Naomi M,Heisaburo S,et al.Purification of lactic acid dehydrogenase from crude bovineheart extract by pH-peak focusing counter-current chromatography [J]. J.Chromatogr.B, Analytical Technologies in the Biomedical and Life Sciences,2002,776:183.
[46]戴德舜,王义明,罗国安.高速逆流色谱研究进展[J].分析化学,2001,29(5)：586.
[47]伍方勇,戴德舜,王义明,等.高速逆流色谱与质谱联用在中药分析中的应用[J].高等学校化学学报,2002,23(9)：1698.
[48]曾任森.化感作用研究中的生物测定方法综述[J].应用生态学报,1999,10(1)：123-126.
[49]施月红,谷文祥.生化他感作用研究中的生物测定方法[J].生态科学,1998,17(1)：84-89.
[2]高贤明,唐廷贵,梁宇.外来植物黄顶菊的入侵警报及防控对策[J].生物多样性,2004,12(2)：274-279.
[3]陈艳,刘坤,张国良,谭万忠.外来入侵杂草黄顶菊生物活性及化学成分研究进展[J].杂草科学,2007,(4)：1-3.
[4]戴素雅.黄顶菊的危害及防除技术[J].植物保护,2006,(10)：1
[5]Green W M, de Billot M C, Joffe T, et al. Indigenous plants and weeds on the Makhathini flats as refuge hosts to maintain bollworm population susceptibility to transgenic cotton (BollgardTM) [J]. African Entomology,2003,11(1):21-29.
[6]Broussalis A M, Ferraro G E, Martino V S. Argentine plants as potential source of insecticidal compounds [J]. Journal of Ethnopharmacology,1999,67:219-233.
[7]Ricciardi A, Esquivel G Plantas de posible utilidad et el control de insectos [J]. Anales SAIPA, 1986,7:40-64.
[8]Bardon A, Borkosky S, Ybarra M I, et al. Bioactive plants from Argentina and Bolivia [J]. Fitoterapia,2007,78(3):227-231.
[9]Ferraro G, Broussalis A, Martino V, et al. Argentine medicinal plant:antiviral screening [J]. International symposium on medicinal and aromatic plants,1992,306:239-244.
[10]李香菊,张米茹,李咏军,等.黄顶菊水提取液对植物种子发芽及胚根伸长的化感作用研究[J].杂草科学,2007(4)：15-19.
[11]张金林,刘颖超,庞民,等.黄顶菊提取物除草剂乳油及其制备工艺[P]：CN,200710061859.3.2007-04-30.
[12]王朋,梁文举,孔垂华,等.外来杂草入侵的化学机制[J].应用生态学报,2004,15(4)700-707.
[13]宋启示,何明,唐建淮,等.紫茎泽兰的互感潜力[J].植物生态学报,2000,24(3)：362-365.
[14]万方浩,王韧.恶性害草豚草的生物学及生态学特征[J].杂草学报,1990,4(1)：45-48.
[15]郭琼霞,陈颖,沈荔花,等.加拿大一枝黄花对豆类和蔬菜的化感作用研究[J].检验检疫科学,2006,16(6)：10-12.
[16]周文杰.黄顶菊对小麦种子化感作用的初步研究[J].衡水学院学报,2008,10(1)：60-64.
[17]O J Pereyra de Santiago, H R Julliani. Isolation of Quercetin 3,7,3',4'-Tetrasulphate from Flaveria bidentis L. Otto. Kuntze [J]. Experientia,1972,28:380.
[18]陈艳,刘坤,张国良,等.外来入侵杂草黄顶菊生物活性及化学成分研究进展[J].2007,4：1-3.
[19]金文荣.华东蓝刺头化学成分及其生物活性研究[D].浙江：浙江大学,2008.
[20]Hudson J. B., Towers G H. N. Therapeutic potential of plant photosensitizers [J]. Pharmacology and Therapeutics,1991,49:188-222.
[21]Cho K., Ryw M., ChinK., et al. Thiophene compounds as antivirus, anti-inflammatory and immunoregulatory agents [P]. JP:9502,665,1995-01-06.
[22]Agnese A M, Nunez Montoya S C, Espinar L A, et al. Chemotaxonomic Features in Argentinian Species of Flaveria [J]. Biochemical Systematics and Ecology,1999,27:739-742.
[23]Vladimir, C, Jitka, V., Lenka, K. et al. Phototoxic activity of a thiophene polyacetylene from Leuzea carthamoides [J]. Fitoterapia,2006,77:194-199.
[24]万树青,徐汉虹,赵善欢,等.光活化多炔类化合物对蚊幼虫的毒力[J].昆虫学报,2000,43(3)：264-270.
[25]杨淑娟,万树青,蒋志胜,等.两种光敏化合物光诱导稗草细胞产氧自由基电子自旋共振的检测与分析[J].植物保护学报,2004,31(3)：294-298.
[26]J.B. Hudson, L.Harris, A. Teeple, et al. The anti-HIV activity of the phytochemical a-terthienyl [J]. Antiviral Research,1993,20:33-43.
[27]兰志银,丘昌隆.格氏试剂交叉偶联反应制备α-三联噻吩[J].化学试剂,1995,17(5)：289-290.
[28]张囡,杜丽丽,王冬,等.中药酚酸类成分的研究进展[J].中国现代中药,2006,8(2)：25-28.
[29]王媚,吴皓.中药有机酸类成分的提取分享及测定方法研究进展[J].南京中医药大学学报,2004,20(3)：190-192.
[30]宫静静,谢静,沈平孃,等.高速逆流色谱技术在中药及天然产物领域中的应用[J].中国现代中药,2007,9(1)：33-35.
[31]曹学丽.高速逆流色谱分离技术及应用[M].北京：化学工业出版社,2005：1.
[32]张天佑.逆流色谱技术[M].北京：北京科学技术出版社,1991：1.
[33]Alain B, Billardello B. Test to evaluate countercurrent chromatographs [J]. J.Chromatogr.A, 2000,90(2):32.
[34]Tsao R, Deng Z Y. Separation procedures for naturally occurring antioxidant phytochemicals [J]. J.Chromatogr.B, Analytical Technologies in the Biomedical and Life Sciences,2004,8(12):85.
[35]魏芸,张天佑,吴克友.高速逆流色谱法对刺五加有效成分刺五加苷的分离制备[J].色谱,2002,20(6)：543-545.
[36]刘迪,陈雪峰,宋晓宇.高速逆流色谱技术应用现状[J].食品与机械,2006,22(2)：98-101.
[37]Ito Y, et al. High-speed preparative countercurrent chromatography with a coil planet entrlfuge [J]. J.Chromatogr.A,1982,244-247.
[38]Ou Yang X K, Jin M C, He C H. Preparative separation of four major alkaloids from medicional plant of Tripterygium Wilfordii Hook Fusing high-speed counter-current chromatography [J]. Sep.Purif.Technol,2007,56:319-324.
[39]Xie Q Q, Wei Y, Zhang G L. Separation of flavonol glycosides from Flaveria bidentis (L.) Kuntze by high-speed counter-current chromatography [J]. Sep. Purif. Technol.,2010,72:229-233.
[40]Wei Y, Gao Y L, Zhang K, et al. Isolation of caffeic acid from eupatorium adenophorum spreng by high-speed countercurrent chromatography and synthesis of caffeic acid-intercalated layered double hydroxide [J]. J. Liq. Chromatogr. R. T.2010,33(6):837-845.
[41]Lu H T, Jiang Y, Chen F. Application of preparative high-speed counter-current chromatography for separation of chlorogenic acid from Flos Lonicerae [J]. J.Chromatogr.A,2004,1026:185.
[42]Yao S, Luo J G, Huang X F, et al. Application of preparative high-speed counter-current chromatography/preparative high-performance liquid chromatography mode in rapid separation of saponins [J]. J.Chromatogr.B,2008,01:047.
[43]Yoichi S, Yoko H, Chao Z M,et al.Application of high-speed counter-current chromatography to the separation of coumarin and related compounds [J]. J.Chromatogr.A,1997,759:47.
[44]Wei Y, Zhang T Y, Ito Y. Counter-current chromatographic separation of glycoprotein components from Morchella esculenta (L) with a polymer phase system by a cross-axis coil planet centrifuge [J]. J.Chromatogr.A,2001,917:347.
[45]Yoichi S,Naomi M,Heisaburo S,et al.Purification of lactic acid dehydrogenase from crude bovineheart extract by pH-peak focusing counter-current chromatography [J]. J.Chromatogr.B, Analytical Technologies in the Biomedical and Life Sciences,2002,776:183.
[46]戴德舜,王义明,罗国安.高速逆流色谱研究进展[J].分析化学,2001,29(5)：586.
[47]伍方勇,戴德舜,王义明,等.高速逆流色谱与质谱联用在中药分析中的应用[J].高等学校化学学报,2002,23(9)：1698.
[48]曾任森.化感作用研究中的生物测定方法综述[J].应用生态学报,1999,10(1)：123-126.
[49]施月红,谷文祥.生化他感作用研究中的生物测定方法[J].生态科学,1998,17(1)：84-89.