深裂竹根七化学成分与生物活性研究
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摘要
深裂竹根七[Disporopsis pernyi (Hua) Diels]为百合科竹根七属植物,主要分布于南亚海拔500-2500m地区。深裂竹根七长期以来作为一种民间中药,用于治‘疗虚汗、慢性咳嗽、妇女产后虚弱和月经不调。本文对深裂竹根七的化学成分、提取工艺、含量测定方法、体外抗菌和抗氧化活性进行了初步研究。
采用硅胶柱色谱、Sephadex-LH20柱色谱、制备薄层色谱等分离手段从深裂竹根七根70%乙醇提取物中分离,并通过理化性质、IR、MS、NMR等波谱手段,对以下7个化合物进行了结构确证：硬脂酸、棕榈酸、芦丁、木犀草素、白桦脂醇、葡萄糖和水杨酸。其中硬脂酸、棕榈酸和白桦脂醇为首次从深裂竹根七中分离得到。
对深裂竹根七多酚成分进行了高效液相色谱串联飞行时间质谱法(HPLC-Q-TOF-MS)定性研究：采用Diamonsil Ci8柱,乙腈-0.1%甲酸水溶液(pH2.4)进行梯度洗脱,流速为1.0mL·min-1,采用负离子模式检测。利用对照品、文献对照等方法确证了深裂竹根七6种酚类成分。建立气相色谱串联质谱法(GC-MS)对深裂竹根七挥发性成分进行研究。采用岛津QP5050A气质联用仪,HP-5石英毛细管柱(30m×0.25mm;0.25μm),首次鉴定了深裂竹根七12种挥发性成分。
建立空气-乙炔火焰原子吸收分光光度法(FAAS)测定深裂竹根七中的10种元素含量。样品采用硝酸-高氯酸(4：1,v/v),在电热板上程序升温消解后测定。结果表明,药材中Na、Mg、K、Cu、Zn和Ca元素广泛地存在于深裂竹根七药材中,而As、Cr、Mo和Pb元素含量均低于限量。该方法简单、快速,可以作为深裂竹根七中Na、Mg、K、Cu、Zn、Ca、As、Cr、Mo和Pb的元素测定方法。使用UPLC法,测定了深裂竹根七中芦丁、木犀草素、槲皮素和白桦脂酸的含量。
采用纸片法和试管逐级稀释法,对深裂竹根七中的挥发油、总皂苷、总多酚和总多糖进行了抗菌活性的考察。以卡那霉素和氟康唑为阳性对照,通过对抑菌圈大小的测定,最低抑菌浓度(MIC)和最低杀菌浓度(MBC)的测定,系统地评价了深裂竹根七中四种提取物抗菌活性。在4种微生物实验中,结果总多酚显较强的抗菌活性,其他提取物未显示明显的抗菌活性。
采用两种经典的抗氧化活性实验方法(DPPH法和FRAP法)进行深裂竹根七4种提取物的抗氧化活性评价。以2,6-二叔丁基-4-甲基苯酚(BHT)为阳性对照,结果深裂竹根七中挥发油、总皂苷、总多酚和总多糖都具有一定的抗氧化活性,其抗氧化活性强弱顺序为：挥发油<多糖<皂苷<多酚 比较了深裂竹根七及其混淆品(玉竹、竹节参、吉祥草和开口箭)在挥发油、抗菌和抗氧化活性方面的异同点。结果表明虽然这五种药材在挥发油成分上有一定的相似之处,但仍存在一定差别。在五种中药甲醇提取物中,开口箭的甲醇提取物显示抗菌活性,而其他四种中药甲醇提取物未显示抗菌活性。用DPPH法和FRAP法评价五种中药甲醇提取物的抗氧化活性,结果这五种中药甲醇提取物都具有一定的抗氧化活性,其抗氧化活性强弱顺序为：深裂竹根七<玉竹<竹节参<吉祥草<开口箭。
本研究采用HPLC-Q-TOF-MS、GC-MS、UV和FAAS等多种分析方法和手段,对深裂竹根七中的化学成分进行了定性和定量研究,考察了深裂竹根七的抗氧化和抗菌活性,对深裂竹根七药材的药效物质基础研究、质量控制和药理作用进行了初步地探索和研究。
Disporopsis pernyi (Hua) Diels which belongs to Disporopsis genus of Liliaceae family, mainly grows in south Asia up to an altitude of500-2500m. Disporopsis pernyi (Hua) Diels has been widely used in folk medicine for the treatment of abnormal sweating, chronic cough, women postpartum weakness and irregular menstrual etc. The chemical constituents, extraction process, the quality control, antimicrobial and antioxidant ability of Disporopsis pernyi (Hua) Diels were investigated in this study.
Nine chemical constituents were isolated from70%ethanol extraction of the root of Disporopsis pernyi (Hua) Diels, by using silica gel column chromatography, Sephadex-LH20column chromatography. The structures of following7compounds (Stearic acid, Palmitic acid, Rutin, Luteolin, Betulin, Salicylic acid, Glucose) were fully elucidated by chemical, physical and spectroscopic methods (MS, UV, IR and NMR). Among them, Stearic acid, Palmitic acid and Betulin were isolated from Disporopsis pernyi (Hua) Diels for the first time.
High performance liquid chromatography quadrupole of flight time mass spectrometry (HPLC-Q-TOF-MS) was applied to study the phenols components of Disporopsis pernyi (Hua) Diels. The analysis was performed on a Diamonsil C18column using gradient elution with the mobile phase consisting of0.1%formic acid in acetonitrile and water (pH2.4) and the flow rate was1.0mL·min-1. The detection was performed under anion pattern detection mode. Standards, references and other methods were used to identify the5phenols compounds of Disporopsis pernyi (Hua) Diels. Gas chromatography tandem mass spectrometry (GC-MS) method was developed to study the volatile compounds of Disporopsis pernyi (Hua) Diels. The essential oil analysis was carried out on SHIMADZU QP5050A series instrument coupled with HP-5MS capillary column (30m×0.25mm; film thickness0.25μm). Twelve volatile compositions of Disporopsis pernyi were identified for the first time.
The content of10elements were determined by Flame Atomic Absorption Spectrometry (FAAS). The samples needed to be digested in nitric acid-perchloric acid (4:1, v/v) on the heating pad before measured. The result shows that Na, Mg, K, Cu, Zn and Ca are widely existed in Disporopsis pernyi (Hua) Diels, while the content of As, Cr, Mo and Pb are all lower than limit content. This method was found to be simple, rapid, accurate and good for the determination of Na, Mg, K, Cu, Zn, Ca, As, Cr, Mo and Pb in Disporopsis pernyi.
Polyphenols were quantified by ultra-performance liquid chromatography (UPLC). A total of4polyphenolic compounds found in extract were identified and presented:rutin, luteolin, quercetin, betulinic acid.
Disk diffusion method and gradually tube dilution method were used to investigate the antibacterial ability of essential oil, total saponins, total polyphenols and total polysaccharide. Through the determing the size of inhibition zone and MIC, the antibacterial ability of essential oil, total saponins, total polyphenols and total polysaccharide of Disporopsis pernyi (Hua) Diels was evaluated systematically. The antimicrobial activity of the four extracts was evaluated against4microorganisms. The total polyphenols of Disporopsis pernyi showed antimicrobial activities comparable with standard antibiotics, while the other extracts did not showed antimicrobial activity.
The antioxidant potential was evaluated using following in vitro methods:FRAP and DPPH radical. The antioxidant activity of samples was compared with the activity of BHT. Results showed that all extracts possessed concentration-dependent antioxidant activity. The antioxidant activities were in descending order:BHT> total polyphenols> total saponins> total polysaccharide> essential oil.
To indicates the differences between composition compounds of essential oil, in vitro antimicrobial and antioxidant activity of Disporopsis pernyi and those of its adulterants(Polygonatum odoratum, Panax japonicus, Recineckea carnea and Tupistra chinensis). Although these herbs have similarities in composition, there were remarkable differences between them. The methanol extract of Tupistra chinensis showed antimicrobial activities against the tested microorganisms in the disc diffusion test. The antioxidant potential was evaluated using following in vitro methods:Ferric reducing antioxidant power (FRAP) and2,2-diphenyl-l-picrylhydrazil (DPPH) radical. Results showed that all extracts possessed concentration-dependent antioxidant activity. The abilities of antioxidant activities were in descending order:BHT>Tupistra chinensis>Recineckea carnea> Panax japonicus>Polygonatum odoratum>Disporopsis pernyi.
In conclusion, the constituents of the Disporopsis pernyi (Hua) Diels were investigated by HPLC-Q-TOF-MS, GC-MS, FAAS, UV and various analytical techniques. The the antioxidant and antimicrobial activities of Disporopsis pernyi and those of its adulterants were determined. This study provided useful information for research of chemical constituents study, quality control and biological activity of Disporopsis pernyi (Hua) Diels.
采用硅胶柱色谱、Sephadex-LH20柱色谱、制备薄层色谱等分离手段从深裂竹根七根70%乙醇提取物中分离,并通过理化性质、IR、MS、NMR等波谱手段,对以下7个化合物进行了结构确证：硬脂酸、棕榈酸、芦丁、木犀草素、白桦脂醇、葡萄糖和水杨酸。其中硬脂酸、棕榈酸和白桦脂醇为首次从深裂竹根七中分离得到。
对深裂竹根七多酚成分进行了高效液相色谱串联飞行时间质谱法(HPLC-Q-TOF-MS)定性研究：采用Diamonsil Ci8柱,乙腈-0.1%甲酸水溶液(pH2.4)进行梯度洗脱,流速为1.0mL·min-1,采用负离子模式检测。利用对照品、文献对照等方法确证了深裂竹根七6种酚类成分。建立气相色谱串联质谱法(GC-MS)对深裂竹根七挥发性成分进行研究。采用岛津QP5050A气质联用仪,HP-5石英毛细管柱(30m×0.25mm;0.25μm),首次鉴定了深裂竹根七12种挥发性成分。
建立空气-乙炔火焰原子吸收分光光度法(FAAS)测定深裂竹根七中的10种元素含量。样品采用硝酸-高氯酸(4：1,v/v),在电热板上程序升温消解后测定。结果表明,药材中Na、Mg、K、Cu、Zn和Ca元素广泛地存在于深裂竹根七药材中,而As、Cr、Mo和Pb元素含量均低于限量。该方法简单、快速,可以作为深裂竹根七中Na、Mg、K、Cu、Zn、Ca、As、Cr、Mo和Pb的元素测定方法。使用UPLC法,测定了深裂竹根七中芦丁、木犀草素、槲皮素和白桦脂酸的含量。
采用纸片法和试管逐级稀释法,对深裂竹根七中的挥发油、总皂苷、总多酚和总多糖进行了抗菌活性的考察。以卡那霉素和氟康唑为阳性对照,通过对抑菌圈大小的测定,最低抑菌浓度(MIC)和最低杀菌浓度(MBC)的测定,系统地评价了深裂竹根七中四种提取物抗菌活性。在4种微生物实验中,结果总多酚显较强的抗菌活性,其他提取物未显示明显的抗菌活性。
采用两种经典的抗氧化活性实验方法(DPPH法和FRAP法)进行深裂竹根七4种提取物的抗氧化活性评价。以2,6-二叔丁基-4-甲基苯酚(BHT)为阳性对照,结果深裂竹根七中挥发油、总皂苷、总多酚和总多糖都具有一定的抗氧化活性,其抗氧化活性强弱顺序为：挥发油<多糖<皂苷<多酚
本研究采用HPLC-Q-TOF-MS、GC-MS、UV和FAAS等多种分析方法和手段,对深裂竹根七中的化学成分进行了定性和定量研究,考察了深裂竹根七的抗氧化和抗菌活性,对深裂竹根七药材的药效物质基础研究、质量控制和药理作用进行了初步地探索和研究。
Disporopsis pernyi (Hua) Diels which belongs to Disporopsis genus of Liliaceae family, mainly grows in south Asia up to an altitude of500-2500m. Disporopsis pernyi (Hua) Diels has been widely used in folk medicine for the treatment of abnormal sweating, chronic cough, women postpartum weakness and irregular menstrual etc. The chemical constituents, extraction process, the quality control, antimicrobial and antioxidant ability of Disporopsis pernyi (Hua) Diels were investigated in this study.
Nine chemical constituents were isolated from70%ethanol extraction of the root of Disporopsis pernyi (Hua) Diels, by using silica gel column chromatography, Sephadex-LH20column chromatography. The structures of following7compounds (Stearic acid, Palmitic acid, Rutin, Luteolin, Betulin, Salicylic acid, Glucose) were fully elucidated by chemical, physical and spectroscopic methods (MS, UV, IR and NMR). Among them, Stearic acid, Palmitic acid and Betulin were isolated from Disporopsis pernyi (Hua) Diels for the first time.
High performance liquid chromatography quadrupole of flight time mass spectrometry (HPLC-Q-TOF-MS) was applied to study the phenols components of Disporopsis pernyi (Hua) Diels. The analysis was performed on a Diamonsil C18column using gradient elution with the mobile phase consisting of0.1%formic acid in acetonitrile and water (pH2.4) and the flow rate was1.0mL·min-1. The detection was performed under anion pattern detection mode. Standards, references and other methods were used to identify the5phenols compounds of Disporopsis pernyi (Hua) Diels. Gas chromatography tandem mass spectrometry (GC-MS) method was developed to study the volatile compounds of Disporopsis pernyi (Hua) Diels. The essential oil analysis was carried out on SHIMADZU QP5050A series instrument coupled with HP-5MS capillary column (30m×0.25mm; film thickness0.25μm). Twelve volatile compositions of Disporopsis pernyi were identified for the first time.
The content of10elements were determined by Flame Atomic Absorption Spectrometry (FAAS). The samples needed to be digested in nitric acid-perchloric acid (4:1, v/v) on the heating pad before measured. The result shows that Na, Mg, K, Cu, Zn and Ca are widely existed in Disporopsis pernyi (Hua) Diels, while the content of As, Cr, Mo and Pb are all lower than limit content. This method was found to be simple, rapid, accurate and good for the determination of Na, Mg, K, Cu, Zn, Ca, As, Cr, Mo and Pb in Disporopsis pernyi.
Polyphenols were quantified by ultra-performance liquid chromatography (UPLC). A total of4polyphenolic compounds found in extract were identified and presented:rutin, luteolin, quercetin, betulinic acid.
Disk diffusion method and gradually tube dilution method were used to investigate the antibacterial ability of essential oil, total saponins, total polyphenols and total polysaccharide. Through the determing the size of inhibition zone and MIC, the antibacterial ability of essential oil, total saponins, total polyphenols and total polysaccharide of Disporopsis pernyi (Hua) Diels was evaluated systematically. The antimicrobial activity of the four extracts was evaluated against4microorganisms. The total polyphenols of Disporopsis pernyi showed antimicrobial activities comparable with standard antibiotics, while the other extracts did not showed antimicrobial activity.
The antioxidant potential was evaluated using following in vitro methods:FRAP and DPPH radical. The antioxidant activity of samples was compared with the activity of BHT. Results showed that all extracts possessed concentration-dependent antioxidant activity. The antioxidant activities were in descending order:BHT> total polyphenols> total saponins> total polysaccharide> essential oil.
To indicates the differences between composition compounds of essential oil, in vitro antimicrobial and antioxidant activity of Disporopsis pernyi and those of its adulterants(Polygonatum odoratum, Panax japonicus, Recineckea carnea and Tupistra chinensis). Although these herbs have similarities in composition, there were remarkable differences between them. The methanol extract of Tupistra chinensis showed antimicrobial activities against the tested microorganisms in the disc diffusion test. The antioxidant potential was evaluated using following in vitro methods:Ferric reducing antioxidant power (FRAP) and2,2-diphenyl-l-picrylhydrazil (DPPH) radical. Results showed that all extracts possessed concentration-dependent antioxidant activity. The abilities of antioxidant activities were in descending order:BHT>Tupistra chinensis>Recineckea carnea> Panax japonicus>Polygonatum odoratum>Disporopsis pernyi.
In conclusion, the constituents of the Disporopsis pernyi (Hua) Diels were investigated by HPLC-Q-TOF-MS, GC-MS, FAAS, UV and various analytical techniques. The the antioxidant and antimicrobial activities of Disporopsis pernyi and those of its adulterants were determined. This study provided useful information for research of chemical constituents study, quality control and biological activity of Disporopsis pernyi (Hua) Diels.
引文
[1]王莹.中药的应用与发展前景[J].中国卫生产业,2011,8：113.
[2]高振广.中药新药研究现状与方法对策[J].山东医学高等专科学校学报,2011,33：474-476.
[3]严铸云,李羿.中药品质研究现状与展望[J].成都医学院学报,2011,4：299-302.
[4]肖培根.新编中药志[M].北京：化学工业出版社.2001.6
[5]钱信忠.中国本草彩色图鉴[M].北京：人民卫生出版社.1982-1987。
[6]王冠元,聂凤提.深裂竹根七化学成分研究[J].中成药,2010,32(11)：1936-1938.
[7]黄泰康.现代本草纲目[M].北京：中国医药科技出版社.2000.3
[8]Yang Qingxiang, Xu Min, Zhang Yinjun, et al. Steroidal saponins from Disporopsis pernyi [J].Helv Chim Acta,2004,87(5):1248-1253.
[9]张素英.深裂竹根七根化学成分的研究田].贵州：贵州大学；2006
[10]王冠元,聂凤禔.深裂竹根七化学成分研究[J].中成药,2010,32(11)：1936-1938
[11]刘剑平,侯梅,易成.羟基喜树碱抑制ECV304细胞的实验研究[J].实用癌症杂志,2002,17(6)：580-582.
[12]蒙麦侠,杨智锋,贺浪冲.竹根七中具有血管生成抑制作用有效成分的筛选[J].中国中医药信息杂志,2008,15(1)：46-47
[13]中国科学院西北植物研究所.秦岭植物志1(1)[M].北京：科学出版社,1976：337-338.
[14]张志英.陕西中药名录[M].西安：陕西省科学技术出版社,1989：76.
[15]程虎印.太白山“七药’及其植物资源研究[J].陕西中医,1990,11(8)：371-372.
[16]张元,胡一冰,杜江,等.吉祥草总皂苷对实验性Freund's完全佐剂大鼠影响的实验研究[J].中药药理与临床,2010,26(5)：52-53.
[17]张元,杜江,许建阳,等.吉祥草总皂苷溶血、止咳、化痰、抗炎作用的研究[J].武警医学,2006,17(4)：282-284.
[18]谭苹,姚丽芳,彭承秀.40种秦岭“七药”最低抑菌浓度的测试[J].时珍国医国药,2001,12(6)：484-485.
[19]梁启超,李荣辉,刘爽.降低中药中重金属含量方法的研究进展[J].微量元素与健康研究,2012,29(2)：48-50.
[20]卢立明,宋少江,武勇,等.哈士蟆卵油化学成分研究(Ⅱ)[J].沈阳药科大学学报,2002,19(1)：25-26.
[21]刘桂艳,马双成.深绿山龙眼种子化学成分研究(Ⅰ)[J].中草药,2005,36(6)：814.
[22]陈凤凰,唐文明.核桃树皮的化学成分分析及活性研究[J].天然产物研究与开发,2008,20：16-18.
[23]姚巍,林文艳,周长新.蒙古蒲公英化学成分研究[J].中国中药杂志,2007,32(10)：926-929.
[24]马养民,王佩.文冠果叶化学成分的研究[J].中成药,2010,32(10)：1750-1753.
[25]刘明韬,韩志超,吴立军,等.龙胆的化学成分研究[J].沈阳药科大学学报,2005,22(2)：103-104.
[26]柏玲,齐艳明,张文治.还阳参化学成分研究[J].齐齐哈尔大学学报,2012,28(2)：22-24.
[27]Carrasco-Pancorbo A., C. Neusuβ, M. Pelzing, A. Segura-Carretero, A. Fernandez-Gutierrez. CE- and HPLC-TOF-MS for the characterization of phenolic compounds in olive oil[J]. Electrophoresis.2007,28:806-821.
[28]Ouni Y, Taamalli A, Gomez-Caravaca AM, Segura-Carretero A, Fernandez-Gutierrez A, Zarrouk M. Food Chem.2011,127:1263-1267.
[29]Fernandez EH, Pancorbo AC, Gutierrez AF. J. Agric. Food Chem.2011,59:2155-2167.
[30]Simirgiotis, M. J., Schmeda-Hirschmann, G., J. Food. Compos. Anal.2010,23:545-553.
[31]郑国华,陶君彦,王永忠.草木樨药材香豆素的含量测定[J].中国医院药学杂志2008,28(18)：1569-1572.
[32]刘莉,龙娉,姚建忠.HPLC法测定三种常见菊花中木樨草素的含量[J].药学实践杂志,2009,27(4)：289-293.
[33]SkrbieBD. Unified retention concept-statistical treatment of Kova'ts retention index[J]. Journal of Chromatography A,1997 (764):257-264.
[34]徐铸德,赵如松,张晓东.由气相色谱程序升温数据计算恒温保留指数[J].色谱,1994,12(3)：164-165.
[35]Zouari S, Ketata M, Boudhrioua N, Ammar E, Allium roseum L. Volatile compounds profile and antioxydant activity for chemotype discrimination-Case study of the wild plant of Sfax (Tunisia)[J]. Industrial Crops and Products,2013,41:172-178.
[36]Zhao CX, Liang YZ, Fang HZ, Li XN. Temperature-programmed retention indices for gas chromatography-mass spectroscopy analysis of plant essential oils[J]. Journal of Chromatography A,2005,1096:76-85.
[37]Pino JA, Queris O. Analysis of volatile compounds of mango wine[J]. Food Chemistry, 2011,125:1141-1146.
[38]Haoui IE, Derriche R, Madani L, Oukali Z. Analysis of the chemical composition of essential oil from AlgerianInula viscosa (L.) Aiton[J]. Arabian Journal of Chemistry,2011,25: 184-192.
[39]Radulovic N, Dordevic N, Stojanovic-Radic Z. Volatiles of the Balkan endemic Daucus guttatus ssp. zahariadiiand cultivated and wild-growing D. carota-A comparison study[J]. Food Chemistry,2011,125:35-43.
[40]Kovats E.. Gas chromatographische charakterisierung organischer verbindungen teil 1 retentions indices aliphatischer halogenide, alkohole, aldehyde und ketone (Characterization of organic compounds by gas chromatography. Part 1. Retention indices of aliphatic halides, alcohols, aldehydes and ketones)[J]. Helvetica Chimica Acta.1958,41:1915-1932.
[41]马亚兵,王海刚,高海青,等.中药微量元素与其药理的关系研究[J].’首都医药,2009,(22)：42-43.
[42]吴兆熹,胡克菲.马威中药炮制前后功效变化与微量元素关系浅析[J].微量元素与健康研究,2003,20(1)：35-36.
[43]陈丽坤,李绍华.诃子不同炮制品功效与微量元素的关系[J].微量元素与健康研究,2002,19(1)：36.
[44]祁俊生,徐辉碧,周井炎,等.解表植物类中药中微量元素与功效关系[J].计算机与应用化学,2003,20(4)：449-452.
[45]李增烈,廖宁逊.微量元素功效大,过量危害却不小[J].家庭用药,2004,(9)：40.
[46]范思敏,刘凤权.中药自然铜功效本草考证[J].黑龙江中医药,2000,(4)：54-55.
[47]黄文耀,邓琴,沈更新,等.保健食品中三价铬的测定方法[J].公共卫生与预防医学,2006,17(3)：66-67.
[48]黄作明,黄殉.微量元素与人体健康[J].微量元素与健康研究,2010,27(6)：58-62.
[49]白爱梅,李跃,范中学.砷对人体健康的危害[J].微量元素与健康研究,2007,24(1)：61-62.
[50]刘怀燕,潘晓洁,吴茜,等.铅中毒对儿童健康的危害.云南医药,2010,31(3)：356-359.
[51]WM/T2-2004,药用植物及制剂外经贸绿色行业标准[S].
[52]Olowoyo, J.O., Okedeyi, O.O., Mkolo, N.M.,Lion, G.N., Mdakane, S.T.R.; Uptake and translocation of heavy metals by medicinal plants growing around a waste dump site in Pretoria, South Africa[J]. South African Journal of Botany; 2012,78:116-121.
[53]Fallah, A. A., Saei-Dehkordi, S. S., Nematollahi, A., Jafari, T.; Comparative study of heavy metal and trace element accumulation in edible tissues of farmed and wild rainbow trout (Oncorhynchus mykiss) using ICP-OES technique[J]. Microchemical Journal,2011,98: 275-279.
[54]Altundag, H., Tuzen, M.; Comparison of dry, wet and microwave digestion methods for the multi element determination in some dried fruit samples by ICP-OES[J]. Food and Chemical Toxicology,2011,49:2800-2807.
[55]Naozuka, J., Vieira, E.C., Nascimento, A.N., Oliveira, P.V.; Elemental analysis of nuts and seeds by axially viewed ICP-OES[J]. Food Chemistry,2011,124:1667-1672.
[56]Larranaga,C.S., Blasco, I.N. Optimization of a slurry dispersion method for minerals and trace elements analysis in infant formulae by ICP OES and FAAS[J]. Food Chemistry,2009, 115:1048-1055.
[57]Sousa R.A., Baccan N., Cadore, S. Determination of elemental content in solid sweeteners by slurry sampling and ICP-OES[J]. Food Chemistry,2011,124:1264-1267.
[58]Karbasi, M.H., Jahanparast, B., Shamsipur, M., Hassan, J. Simultaneous trace multielement determination by ICP-OES after solid phase extraction with modified octadecyl silica gel; Journal of Hazardous Materials,2009,170:151-155.
[59]Jallath,J.S., Rodriguez, A.C., Casillas, J.H., Bustillos, L.T. Arsenic and heavy metals in native plants at tailings impoundments in Queretaro, Mexico[J]. Physics and Chemistry of the Earth,2012,37-39:10-17.
[60]Singleton V.L., J.A. Rossi. Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents[J]. American Journal of Enology and Viticulture.1965, 16:144-158.
[61]许瑞波,刘玮炜,马卫兴,等.山楂叶多糖的提取工艺研究[J].食品科技,2008,10：149.
[62]Duraipandiyan V, Ignacimuthu S. Antibacterial and antifungal activity of Flindersine isolated from the traditional medicinal plant, Toddalia asiatica (L.) Lam[J]. Journal of Ethnopharmacology 2009; 123:494-498.
[63]Chang HC, Huang GJ, Agrawal DC, Kuo CL, Wu CR, Tsay HS. Antioxidant activities and polyphenol contents of six folk medicinal ferns used as "Gusuibu" [J]. Bot Stud 2007; 48: 397-406.
[64]Giri L, Dhyani P, Rawat S, Bhatt ID, Nandi SK, Rawal RS. In vitro production of phenolic compounds and antioxidant activity in callus suspension cultures of Habenaria edgeworthii: A rare Himalayan medicinal orchid[J]. Ind Crop Prod 2012; 39:1-6.
[65]薛小娟,陈晓青,李钰.紫外分光光度法测定知母中总皂苷的含量[J].药物分析杂志,2006,26(11)：1659-1662
[66]兰霞,王洪新.比色法测定甘草中总皂苷的含量[J].时珍国医国药,2007,18(4)：886-887
[67]解红霞,薛培凤,张宏桂,等.可见分光光度法测定玉簪花中总皂苷的含量[J].北京中医药大学学报,2009,32(9)：624-625,645
[68]昝丽霞,钟小龙,孙文基.高效液相色谱法及分光光度法测定黄姜中总皂苷的含量[J].药物分析杂志,2008,28(1)：111-114.
[69]徐洋,严铭铭,付颖超,等.马齿苋中总皂苷提取工艺的优选[J].时珍国医国药,2010,21(12)：3104-3105.
[70]Jacobo-Velazquez D.A., G.B. Martinez-Hernandez, C.R.S. Del, C.M. Cao, L. Cisneros-Zevallos. Plants as biofactories:physiological role of reactive oxygen species on the accumulation of phenolic antioxidants in carrot tissue under wounding and hyperoxia stress[J]. J Agric Food Chem.2011,59:6583-6593.
[71]Nijveldt R.J., E. van Nood, D.E. van Hoorn, P.G. Boelens, K. van Norren, and P.A. van Leeuwen.2001. Flavonoids:a review of probable mechanisms of action and potential applications[J]. Am J Clin Nutr.74:418-425.
[72]JB Lowe,JD Marth. A genetic approach to mammalian glycan function[J]. Annual Review of Biochemistry,2003,72 (1):643-691.
[73]龚苏晓,高展,张铁军,等.微波辅助提取黄芪多糖及含量测定[J].时珍国医国药,2008,19(4)：784-786.
[74]王莉,陈正行,张萧艳.微波辅助提取米糠多糖的工艺[J].江苏大学学报,2007,28(3)：193-196.
[75]Y. Chen, M. Y. Xie, X. F. Gong. Microwave-assisted extraction used for the isolation of total triterpenoid saponins from Ganoderma atrum[J]. Journal of Food Engineering,2007,81: 162-170.
[76]陈文强,邓百万,刘开辉,等.猪苓多糖超声提取工艺条件优化[J].食品与生物技术学报,2008,27(4)：53-58.
[77]方积年,丁侃.天然药物-多糖的主要生物活性及分离纯化方法[J].中国天然药物,2007,5(5)：338-347.
[78]XB Yang, XD Gao, F. Han, et al. Purification,characterization and enzymatic degradation of YCP, a polysaccharide from marine filamentous fungus Phoma herbarum YS4108[J]. Biochimie,2005,87 (8):747-754.
[79]杨云,田润涛,苗明三,等.大枣多糖活性炭脱色工艺研究[J].河南中医学院学报,2004,19(1)：35-36.
[80]Kiho T, A Yamane, J Hui, et al. Hypoglycemic activity of a polysaccharide from the cultural mycelium of Cordy ceps sinensis and its effect on glucosemetabolism in mouse liver [J]. Biological and Pharmaceutical Bulletin,1996,19:294-296.
[81]刘成梅,付佳明,涂宗财,等.百合多糖降血糖功能研究[J].食品科学,2002,23(6)113-114.
[82]陈蔚,刘芳,愈茂华,等.黄芪多糖对NOD小鼠Ⅰ型糖尿病的预防作用[J].复旦大学学报(医学版),2001,28(1)：57-60.
[83]李磊,谢明勇,易醒.青钱柳多糖降血糖作用研究[J].中药材,2002,25(1)：39-41.
[84]Konno C, Murkamai M., Oshima Y, et al. Isolation and hypoglycemic activity of pnaxans Q, R, S, T and U, glycnas of Panxa ginseng roots[J]. Ethnopharmacology,1985,14 (1):69-74.
[85]Hiroshi Hikino, et al. Methaniems of hypoglycemic activity of ganoderan B:A glycan of Ganoderma lucidum fruit bodies[J]. Planta Medica,1989,55,423.
[86]Saenz MT, Tornos MP, Alvarez A, Fernandez MA, Garcia MD. Antibacterial activity of essential oils of Pimenta racemosa var.terebinthina and Pimenta racemosa var. grisea[J]. Fitoterapia 2004; 75:599-602.
[87]Ansorena D, Gimeno O, Bello IAJ. Analysis of volatile compounds by GC-MS of a dry fermented sausage:chorizo de Pamplona[J]. Food Research International 2001; 34:67-75
[88]Mandal S, Mallick N, Mitra A. Salicylic acid-induced resistance to Fusarium oxysporum f. sp.lycopersici in tomato[J]. Plant Physiology and Biochemistry 2009; 47:642-649
[89]Lou ZX, Wang HX, Rao SQ, Sun JT, Ma CY, Li J.p-Coumaric acid kills bacteria through dual damage mechanisms[J]. Food Control 2012; 25:550-554
[90]Dziri S, Hassen I, Fatnassi S, Mrabet Y, Casabianca H, Hanchi B, Hosni K. Phenolic constituents, antioxidant and antimicrobial activities of rosy garlic (Allium roseum var. odoratissimum) [J]. Journal of Functional Foods 2012; 4:423-432.
[91]Cai Y., Q. Luo, M. Sun, and H. Corke. Antioxidant activity and phenolic compounds of 112 traditional Chinese medicinal plants associated with anticancer. Life Sci[J].2004. 74:2157-2184.
[92]Zheng W., and S.Y. Wang. Antioxidant activity and phenolic compounds in selected herbs[J]. J Agr Food Chem.2001,49:5165-5170.
[2]高振广.中药新药研究现状与方法对策[J].山东医学高等专科学校学报,2011,33：474-476.
[3]严铸云,李羿.中药品质研究现状与展望[J].成都医学院学报,2011,4：299-302.
[4]肖培根.新编中药志[M].北京：化学工业出版社.2001.6
[5]钱信忠.中国本草彩色图鉴[M].北京：人民卫生出版社.1982-1987。
[6]王冠元,聂凤提.深裂竹根七化学成分研究[J].中成药,2010,32(11)：1936-1938.
[7]黄泰康.现代本草纲目[M].北京：中国医药科技出版社.2000.3
[8]Yang Qingxiang, Xu Min, Zhang Yinjun, et al. Steroidal saponins from Disporopsis pernyi [J].Helv Chim Acta,2004,87(5):1248-1253.
[9]张素英.深裂竹根七根化学成分的研究田].贵州：贵州大学；2006
[10]王冠元,聂凤禔.深裂竹根七化学成分研究[J].中成药,2010,32(11)：1936-1938
[11]刘剑平,侯梅,易成.羟基喜树碱抑制ECV304细胞的实验研究[J].实用癌症杂志,2002,17(6)：580-582.
[12]蒙麦侠,杨智锋,贺浪冲.竹根七中具有血管生成抑制作用有效成分的筛选[J].中国中医药信息杂志,2008,15(1)：46-47
[13]中国科学院西北植物研究所.秦岭植物志1(1)[M].北京：科学出版社,1976：337-338.
[14]张志英.陕西中药名录[M].西安：陕西省科学技术出版社,1989：76.
[15]程虎印.太白山“七药’及其植物资源研究[J].陕西中医,1990,11(8)：371-372.
[16]张元,胡一冰,杜江,等.吉祥草总皂苷对实验性Freund's完全佐剂大鼠影响的实验研究[J].中药药理与临床,2010,26(5)：52-53.
[17]张元,杜江,许建阳,等.吉祥草总皂苷溶血、止咳、化痰、抗炎作用的研究[J].武警医学,2006,17(4)：282-284.
[18]谭苹,姚丽芳,彭承秀.40种秦岭“七药”最低抑菌浓度的测试[J].时珍国医国药,2001,12(6)：484-485.
[19]梁启超,李荣辉,刘爽.降低中药中重金属含量方法的研究进展[J].微量元素与健康研究,2012,29(2)：48-50.
[20]卢立明,宋少江,武勇,等.哈士蟆卵油化学成分研究(Ⅱ)[J].沈阳药科大学学报,2002,19(1)：25-26.
[21]刘桂艳,马双成.深绿山龙眼种子化学成分研究(Ⅰ)[J].中草药,2005,36(6)：814.
[22]陈凤凰,唐文明.核桃树皮的化学成分分析及活性研究[J].天然产物研究与开发,2008,20：16-18.
[23]姚巍,林文艳,周长新.蒙古蒲公英化学成分研究[J].中国中药杂志,2007,32(10)：926-929.
[24]马养民,王佩.文冠果叶化学成分的研究[J].中成药,2010,32(10)：1750-1753.
[25]刘明韬,韩志超,吴立军,等.龙胆的化学成分研究[J].沈阳药科大学学报,2005,22(2)：103-104.
[26]柏玲,齐艳明,张文治.还阳参化学成分研究[J].齐齐哈尔大学学报,2012,28(2)：22-24.
[27]Carrasco-Pancorbo A., C. Neusuβ, M. Pelzing, A. Segura-Carretero, A. Fernandez-Gutierrez. CE- and HPLC-TOF-MS for the characterization of phenolic compounds in olive oil[J]. Electrophoresis.2007,28:806-821.
[28]Ouni Y, Taamalli A, Gomez-Caravaca AM, Segura-Carretero A, Fernandez-Gutierrez A, Zarrouk M. Food Chem.2011,127:1263-1267.
[29]Fernandez EH, Pancorbo AC, Gutierrez AF. J. Agric. Food Chem.2011,59:2155-2167.
[30]Simirgiotis, M. J., Schmeda-Hirschmann, G., J. Food. Compos. Anal.2010,23:545-553.
[31]郑国华,陶君彦,王永忠.草木樨药材香豆素的含量测定[J].中国医院药学杂志2008,28(18)：1569-1572.
[32]刘莉,龙娉,姚建忠.HPLC法测定三种常见菊花中木樨草素的含量[J].药学实践杂志,2009,27(4)：289-293.
[33]SkrbieBD. Unified retention concept-statistical treatment of Kova'ts retention index[J]. Journal of Chromatography A,1997 (764):257-264.
[34]徐铸德,赵如松,张晓东.由气相色谱程序升温数据计算恒温保留指数[J].色谱,1994,12(3)：164-165.
[35]Zouari S, Ketata M, Boudhrioua N, Ammar E, Allium roseum L. Volatile compounds profile and antioxydant activity for chemotype discrimination-Case study of the wild plant of Sfax (Tunisia)[J]. Industrial Crops and Products,2013,41:172-178.
[36]Zhao CX, Liang YZ, Fang HZ, Li XN. Temperature-programmed retention indices for gas chromatography-mass spectroscopy analysis of plant essential oils[J]. Journal of Chromatography A,2005,1096:76-85.
[37]Pino JA, Queris O. Analysis of volatile compounds of mango wine[J]. Food Chemistry, 2011,125:1141-1146.
[38]Haoui IE, Derriche R, Madani L, Oukali Z. Analysis of the chemical composition of essential oil from AlgerianInula viscosa (L.) Aiton[J]. Arabian Journal of Chemistry,2011,25: 184-192.
[39]Radulovic N, Dordevic N, Stojanovic-Radic Z. Volatiles of the Balkan endemic Daucus guttatus ssp. zahariadiiand cultivated and wild-growing D. carota-A comparison study[J]. Food Chemistry,2011,125:35-43.
[40]Kovats E.. Gas chromatographische charakterisierung organischer verbindungen teil 1 retentions indices aliphatischer halogenide, alkohole, aldehyde und ketone (Characterization of organic compounds by gas chromatography. Part 1. Retention indices of aliphatic halides, alcohols, aldehydes and ketones)[J]. Helvetica Chimica Acta.1958,41:1915-1932.
[41]马亚兵,王海刚,高海青,等.中药微量元素与其药理的关系研究[J].’首都医药,2009,(22)：42-43.
[42]吴兆熹,胡克菲.马威中药炮制前后功效变化与微量元素关系浅析[J].微量元素与健康研究,2003,20(1)：35-36.
[43]陈丽坤,李绍华.诃子不同炮制品功效与微量元素的关系[J].微量元素与健康研究,2002,19(1)：36.
[44]祁俊生,徐辉碧,周井炎,等.解表植物类中药中微量元素与功效关系[J].计算机与应用化学,2003,20(4)：449-452.
[45]李增烈,廖宁逊.微量元素功效大,过量危害却不小[J].家庭用药,2004,(9)：40.
[46]范思敏,刘凤权.中药自然铜功效本草考证[J].黑龙江中医药,2000,(4)：54-55.
[47]黄文耀,邓琴,沈更新,等.保健食品中三价铬的测定方法[J].公共卫生与预防医学,2006,17(3)：66-67.
[48]黄作明,黄殉.微量元素与人体健康[J].微量元素与健康研究,2010,27(6)：58-62.
[49]白爱梅,李跃,范中学.砷对人体健康的危害[J].微量元素与健康研究,2007,24(1)：61-62.
[50]刘怀燕,潘晓洁,吴茜,等.铅中毒对儿童健康的危害.云南医药,2010,31(3)：356-359.
[51]WM/T2-2004,药用植物及制剂外经贸绿色行业标准[S].
[52]Olowoyo, J.O., Okedeyi, O.O., Mkolo, N.M.,Lion, G.N., Mdakane, S.T.R.; Uptake and translocation of heavy metals by medicinal plants growing around a waste dump site in Pretoria, South Africa[J]. South African Journal of Botany; 2012,78:116-121.
[53]Fallah, A. A., Saei-Dehkordi, S. S., Nematollahi, A., Jafari, T.; Comparative study of heavy metal and trace element accumulation in edible tissues of farmed and wild rainbow trout (Oncorhynchus mykiss) using ICP-OES technique[J]. Microchemical Journal,2011,98: 275-279.
[54]Altundag, H., Tuzen, M.; Comparison of dry, wet and microwave digestion methods for the multi element determination in some dried fruit samples by ICP-OES[J]. Food and Chemical Toxicology,2011,49:2800-2807.
[55]Naozuka, J., Vieira, E.C., Nascimento, A.N., Oliveira, P.V.; Elemental analysis of nuts and seeds by axially viewed ICP-OES[J]. Food Chemistry,2011,124:1667-1672.
[56]Larranaga,C.S., Blasco, I.N. Optimization of a slurry dispersion method for minerals and trace elements analysis in infant formulae by ICP OES and FAAS[J]. Food Chemistry,2009, 115:1048-1055.
[57]Sousa R.A., Baccan N., Cadore, S. Determination of elemental content in solid sweeteners by slurry sampling and ICP-OES[J]. Food Chemistry,2011,124:1264-1267.
[58]Karbasi, M.H., Jahanparast, B., Shamsipur, M., Hassan, J. Simultaneous trace multielement determination by ICP-OES after solid phase extraction with modified octadecyl silica gel; Journal of Hazardous Materials,2009,170:151-155.
[59]Jallath,J.S., Rodriguez, A.C., Casillas, J.H., Bustillos, L.T. Arsenic and heavy metals in native plants at tailings impoundments in Queretaro, Mexico[J]. Physics and Chemistry of the Earth,2012,37-39:10-17.
[60]Singleton V.L., J.A. Rossi. Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents[J]. American Journal of Enology and Viticulture.1965, 16:144-158.
[61]许瑞波,刘玮炜,马卫兴,等.山楂叶多糖的提取工艺研究[J].食品科技,2008,10：149.
[62]Duraipandiyan V, Ignacimuthu S. Antibacterial and antifungal activity of Flindersine isolated from the traditional medicinal plant, Toddalia asiatica (L.) Lam[J]. Journal of Ethnopharmacology 2009; 123:494-498.
[63]Chang HC, Huang GJ, Agrawal DC, Kuo CL, Wu CR, Tsay HS. Antioxidant activities and polyphenol contents of six folk medicinal ferns used as "Gusuibu" [J]. Bot Stud 2007; 48: 397-406.
[64]Giri L, Dhyani P, Rawat S, Bhatt ID, Nandi SK, Rawal RS. In vitro production of phenolic compounds and antioxidant activity in callus suspension cultures of Habenaria edgeworthii: A rare Himalayan medicinal orchid[J]. Ind Crop Prod 2012; 39:1-6.
[65]薛小娟,陈晓青,李钰.紫外分光光度法测定知母中总皂苷的含量[J].药物分析杂志,2006,26(11)：1659-1662
[66]兰霞,王洪新.比色法测定甘草中总皂苷的含量[J].时珍国医国药,2007,18(4)：886-887
[67]解红霞,薛培凤,张宏桂,等.可见分光光度法测定玉簪花中总皂苷的含量[J].北京中医药大学学报,2009,32(9)：624-625,645
[68]昝丽霞,钟小龙,孙文基.高效液相色谱法及分光光度法测定黄姜中总皂苷的含量[J].药物分析杂志,2008,28(1)：111-114.
[69]徐洋,严铭铭,付颖超,等.马齿苋中总皂苷提取工艺的优选[J].时珍国医国药,2010,21(12)：3104-3105.
[70]Jacobo-Velazquez D.A., G.B. Martinez-Hernandez, C.R.S. Del, C.M. Cao, L. Cisneros-Zevallos. Plants as biofactories:physiological role of reactive oxygen species on the accumulation of phenolic antioxidants in carrot tissue under wounding and hyperoxia stress[J]. J Agric Food Chem.2011,59:6583-6593.
[71]Nijveldt R.J., E. van Nood, D.E. van Hoorn, P.G. Boelens, K. van Norren, and P.A. van Leeuwen.2001. Flavonoids:a review of probable mechanisms of action and potential applications[J]. Am J Clin Nutr.74:418-425.
[72]JB Lowe,JD Marth. A genetic approach to mammalian glycan function[J]. Annual Review of Biochemistry,2003,72 (1):643-691.
[73]龚苏晓,高展,张铁军,等.微波辅助提取黄芪多糖及含量测定[J].时珍国医国药,2008,19(4)：784-786.
[74]王莉,陈正行,张萧艳.微波辅助提取米糠多糖的工艺[J].江苏大学学报,2007,28(3)：193-196.
[75]Y. Chen, M. Y. Xie, X. F. Gong. Microwave-assisted extraction used for the isolation of total triterpenoid saponins from Ganoderma atrum[J]. Journal of Food Engineering,2007,81: 162-170.
[76]陈文强,邓百万,刘开辉,等.猪苓多糖超声提取工艺条件优化[J].食品与生物技术学报,2008,27(4)：53-58.
[77]方积年,丁侃.天然药物-多糖的主要生物活性及分离纯化方法[J].中国天然药物,2007,5(5)：338-347.
[78]XB Yang, XD Gao, F. Han, et al. Purification,characterization and enzymatic degradation of YCP, a polysaccharide from marine filamentous fungus Phoma herbarum YS4108[J]. Biochimie,2005,87 (8):747-754.
[79]杨云,田润涛,苗明三,等.大枣多糖活性炭脱色工艺研究[J].河南中医学院学报,2004,19(1)：35-36.
[80]Kiho T, A Yamane, J Hui, et al. Hypoglycemic activity of a polysaccharide from the cultural mycelium of Cordy ceps sinensis and its effect on glucosemetabolism in mouse liver [J]. Biological and Pharmaceutical Bulletin,1996,19:294-296.
[81]刘成梅,付佳明,涂宗财,等.百合多糖降血糖功能研究[J].食品科学,2002,23(6)113-114.
[82]陈蔚,刘芳,愈茂华,等.黄芪多糖对NOD小鼠Ⅰ型糖尿病的预防作用[J].复旦大学学报(医学版),2001,28(1)：57-60.
[83]李磊,谢明勇,易醒.青钱柳多糖降血糖作用研究[J].中药材,2002,25(1)：39-41.
[84]Konno C, Murkamai M., Oshima Y, et al. Isolation and hypoglycemic activity of pnaxans Q, R, S, T and U, glycnas of Panxa ginseng roots[J]. Ethnopharmacology,1985,14 (1):69-74.
[85]Hiroshi Hikino, et al. Methaniems of hypoglycemic activity of ganoderan B:A glycan of Ganoderma lucidum fruit bodies[J]. Planta Medica,1989,55,423.
[86]Saenz MT, Tornos MP, Alvarez A, Fernandez MA, Garcia MD. Antibacterial activity of essential oils of Pimenta racemosa var.terebinthina and Pimenta racemosa var. grisea[J]. Fitoterapia 2004; 75:599-602.
[87]Ansorena D, Gimeno O, Bello IAJ. Analysis of volatile compounds by GC-MS of a dry fermented sausage:chorizo de Pamplona[J]. Food Research International 2001; 34:67-75
[88]Mandal S, Mallick N, Mitra A. Salicylic acid-induced resistance to Fusarium oxysporum f. sp.lycopersici in tomato[J]. Plant Physiology and Biochemistry 2009; 47:642-649
[89]Lou ZX, Wang HX, Rao SQ, Sun JT, Ma CY, Li J.p-Coumaric acid kills bacteria through dual damage mechanisms[J]. Food Control 2012; 25:550-554
[90]Dziri S, Hassen I, Fatnassi S, Mrabet Y, Casabianca H, Hanchi B, Hosni K. Phenolic constituents, antioxidant and antimicrobial activities of rosy garlic (Allium roseum var. odoratissimum) [J]. Journal of Functional Foods 2012; 4:423-432.
[91]Cai Y., Q. Luo, M. Sun, and H. Corke. Antioxidant activity and phenolic compounds of 112 traditional Chinese medicinal plants associated with anticancer. Life Sci[J].2004. 74:2157-2184.
[92]Zheng W., and S.Y. Wang. Antioxidant activity and phenolic compounds in selected herbs[J]. J Agr Food Chem.2001,49:5165-5170.