摘要
红车轴草(Trifolium pratense L.)隶属于豆科(Leguminosae)车轴草属多年生草本植物,又名红三叶、红花苜蓿和三叶草等,原产欧洲、西亚,现广泛分布于世界各地。红车轴草不仅是优良的饲用牧草,而且在许多国家已经成为人们的传统食品和调味品。我国甘肃岷县于1944年引种栽培岷山红车轴草(Trifolium pratense cv.Minshan),现已大面积种植。该县位于甘南、陇中黄土高原及陇南山地接壤区,属于典型的高寒阴湿区地域,在此地发展以红车轴草为主的草地畜牧业具有优越的天然条件。本文基于提高我国红车轴草的药用价值和饲用价值,系统研究了岷山红车轴草的营养成分和所含异黄酮类包括大豆甙元、染料木素、芒柄花素和鹰嘴豆芽素A的分布和含量,并对其提取物的抑菌作用和抗氧化活性进行了初步研究,为进一步开发利用红车轴草提供资料。主要实验结果如下:
1、岷山红车轴草的生物学性状特征调查
据实地调查:岷山红车轴草根系发达,茎直立或上升,多分枝,高15-30cm;掌状复叶,具3枚小叶;头状花序、顶生,花冠红色或紫红色:荚果小,通常具1粒种子;花期8-9月,果期8-9月。岷山红车轴草种植当年一般可以刈割利用1次,种子成熟极少;第二年后每年可刈割2-3次,可连续刈割利用5-7年。岷山红车轴草株高60-110cm,鲜草产量为45-67.5 t/hm~2,最高可达90t/hm~2,鲜干比为6-7:1茎叶比接近1:1,生长第两年种子产量达150-375kg/hm~2。
2、草体营养成分测定
我国甘肃岷山红车轴草营养成分测定结果表明:全草中粗蛋白质、粗脂肪、粗灰分、粗纤维等的含量分别为21.76%、3.25%、10.42%、28.78%。叶、茎、花中钾含量分别为1.89%,2.62%,2.14%;叶、茎、花中钙含量分别为5.67%,4.88%,5.20%。
3、岷山红车轴草种子脂溶性成分分析
岷山红车轴草种子脂溶性成分中不饱和脂肪酸含量占优势,其中单不饱和脂肪酸(Z)-6-十八烯酸占14.60%,多不饱和脂肪酸亚油酸占29.64%。最终共鉴定出6种脂肪酸,主要包括:3种饱和脂肪酸及其酯类,占脂肪酸总含量的9.64%,分别为棕榈酸、硬脂酸、三十烷酸,其中棕榈酸占5.91%,硬脂酸占3.24%,三十烷酸占0.49%;3种不饱和脂肪酸包括(Z)-6-十八烯酸、亚油酸和草酸葵基—丙基酯,占脂肪酸总含量的44.41%,其中单不饱和脂肪酸(Z)-6-十八烯酸甲酯占14.60%,多不饱和脂肪酸亚油酸占29.64%,草酸葵基—丙基酯占0.17%。
4、草体不同部位异黄酮类物质含量测定
用高效液相色谱法对岷山红车轴草不同部位(花、茎、叶)中四种主要异黄酮类物质(大豆甙元、染科木素、芒柄花素和鹰嘴豆芽素A)的含量进行测定,并与白车轴草的异黄酮含量进行对比。实验结果表明,按干物质计算,红车轴草叶片中4种异黄酮成分的百分含量分别为:大豆甙元0.019%,染枓木素0.024%,芒柄花素0.113%以及鹰嘴豆芽素A为0.082%:而茎和花中相关异黄酮类成分含量相对较低,仅为叶片中含量的1/5左右。红车轴草中异黄酮成分含量以芒柄花素利鹰嘴豆芽素A为主,约占4种异黄酮物质总量的99.8%。经比较表明:岷山红车轴草中异黄酮类物质的含量远高于白车轴草以及大豆中的含量,是白车轴草种同种物质含量的2-4倍。
5、草体提取物的抑菌作用研究
据抑菌实验测定:岷山红车轴草的甲醇提取物(MET)对金黄色葡萄球菌、白色葡萄球菌、大肠杆菌、枯草芽孢杆菌、巨大芽孢杆菌等有抑菌作用;岷山红车轴草石油醚-乙酸乙酯-甲醇提取物(PEM)对金黄色葡萄球菌、白色葡萄球菌、大肠杆菌、枯草芽孢杆菌、巨大芽孢杆菌、凝结芽孢杆菌等有抑菌作用。但两种提取物对不同菌种的抑菌活性有所差异,对常见菌群金黄色葡萄球和大肠杆菌等都有明显抑菌作用。
6、草体及种子提取物的抗氧化活性研究
抗氧化活性分析表明,岷山红车轴草全草PEM和MET对DPPH·自由基清除率均低于阳性对照BHT,但亦表现出较强的抗氧化活性,具有一定的开发价值。红车轴草种子脂溶性成分对DPPH·自由基具有一定的清除作用,其半数抑制浓度(EC_(50))为17.97mg/mL,但是显著低于阳性对照BHT;在β-胡萝卜素漂白体系中,0.04mg/mL利0.08mg/mL浓度的的岷山红车轴草种子脂溶性提取物抗氧化速率分别为29.9%利32.1%,对亚油酸的氧化只有一定的抑制作用。
Trifolium pratense L.,also called red clover,meadow clover,purple clover or cow clover,is a perennial herb and attributes to Trifolium of Leguminosae.It originally came from Europe and West Asia,and distributed in most parts of the world.T.pratense is widely used as forage plants,food and spices in many countries.T.pratense cv.Minshan was introduced in Minxian County in 1944 and cultivated in a large area.Minxian County located in the border area of Gannan,the Loess Plateau and Longnan mountainous.It is a typically alpine humid geographical area and has superior natural conditions to the development of T.pratense.In order to achieve full development and use of T. pratense cv.Minshan,this paper mainly studied the nutritional constituents,the distribution and contents of isoflavones(daidzein,genistein,formononetin and biochanin A) from different parts of T. pratense cv.Minshan.The antimicrobial effects and antioxidant activities of the extracts from different parts of T.pratense cv.Minshan were also determined.The main results were as follows:
1.Observation of morphological traits
T.pratense cv.Minshan has strong roots,upright or ascending stems,more branches,with a height of 15-30 cm;palmate compound leaf,a three lobular;capitulum,red or purple corolla;small pod, usually more than one seed;florescence and fructification from August to September.T.pratense cv. Minshan was mowed and used once a year.The mature seeds are very few in the first year.In the second year,it can be mowed two or three times,and continued to mow for 5 to 7 years.T.pratense cv. Minshan has a height of 60~110 cm,and the production of fresh forage is 45~67.5 t/hm~2.The highest production can achieve to 90 t/hm~2.The ratio of fresh and dry measure is 6 to 7:1,the ratio of stems and leaves is close to 1:1,and the seed yield can reach to 150~375 kg/hm~2 in the coming year.
2.Nutrients constituents of T.pratense cv.Minshan
According to the nutritional constituents,the contents of crude protein,crude fat,crude ash,crude fiber,microelement and amino acid of T.pratense cv.Minshan were measured.The results showed that the contents of crude protein,crude fat,crude ash and crude fiber of T.pratense cv.Minshan were 21.76%,3.25%,10.42%,28.78%,respectively;the contents of potassium in the leaf,stem and flower were 1.89%,2.62%and 2.14%,respectively;the contents of calcium in the leaf,stem and flower were 5.67%,4.88%and 5.20%,respectively.
3.Fat-soluble components in seeds of T.pratense cv.Minshan
The fat-soluble components in seeds of T.pratense cv.Minshan were preponderant unsaturated fatty acid,which including 6-octadecenoic acid,methyl ester(14.60%),and 9,12-octadecadienoic acid (Z,Z)- methyl ester(29.64%).Six kinds of fatty acids are identified,including three kinds of saturated fatty acids and esters which account for 9.64%of the total fatty acid contents,the three saturated fatty acids and esters were hexadecanoic acid,methyl ester(5.91%),octadecanoic acid,methyl ester(3.24%), and triacontanoic acid,methyl ester(0.49%).The three kinds of unsaturated fatty acids were 6-octadecenoic acid,methyl ester(14.60%),9,12-octadecadienoic acid(Z,Z)-methyl ester(29.64%) and oxalic acid,decyl propyl ester(0.17%) which account for 44.41%of the total fatty acid contents.
4.Contents of isoflavone in different parts of T.pratense cv.Minshan
HPLC was used to separate and determine four isoflavones,including biochanin A,formononetin, genistein and daidzein,from different parts of T.pratense cv.Minshan.The contents of daidzein, genistein,formononetin and biochanin A of leaves were 0.019%,0.024%,0.113%and 0.082%, respectively.The contents of isoflavones of stems and flower are lower,and only account 1/5 those of the leaves.Formononetin and biochanin A were the main percentage of isoflavone ingredients,which is about 99.8%of the four kinds of isoflavones.Isoflavone contents of red clover were much higher and about two to four times than that of the white trefoil and the soybean.
5.Antimicrobial effect of the whole grass extract of T.pratense cv.Minshan
The extracts of the whole grass of T.pratense cv.Minshan were subjected to a screening for their possible antimicrobial activities.The methanol(MET) extract was found to inhibit the growth of bacteria tested,including Staphylococcus aureus,Staphylococcus epidermidis,Escherichia coli, Bacillus subtilis and Bacilus megaterium.The petroleum ether-ethyl acetate-methanol(PEM) extract has the antimicrobial effects on Staphylococcus aureus,Staphylococcus epidermidis,Escherichia coli, Bacillus subtilis,Bacilus megaterium and Bacillus coagulans.We can draw the conclusion that both extracts have the obvious antimicrobial activities for common bacteria,such as Staphylococcus aureus, Escherichia coli and so on.
6.Antioxidant activities of whole grass and seed extracts of T.pratense cv.Minshan
In DPPH·free radical-scavenging assays,MET and PEM extracts of T.pratense cv.Minshan showed strong radical scavenging activities,but they were lower than the positive control,BHT.The 50%inhibitory concentration(EC_(50)) of the fat-soluble components from seeds of T.pratense cv. Minshan was 17.97 mg/mL,which is significantly lower than the positive control,BHT.In theβ-carotene/linoleic acid system,oxidation of the linoleic acid was effectively inhibited by the fat-soluble extract with 29.9%and 32.1%of inhibition at 0.04 and 0.08 mg/mL,respectively.
引文
[1]陈默君,贾慎修主编.中国饲用植物[M].北京:中国农业出版社,2002:654-655.
[2]Frame J.The potential of tetrapliod red clover and its role in United kingdom.Journal of the British Grassland Society.1976,31:139-152.
[3]Lancashire J A,The distribution and use of forage legumes in New Zealand.In:Burns J C,et al.Forage Legumes for Energy-Efficient Animal Proceeding of a-Trilateral Workshop Hold in N.Z.1984:5-23.
[4]Burns,J C Environmental and management limitation of legume-based forage system in the Northern United States.In:Burns J C,et al.Forage Legumes for Energy-Efficient Animal Proceeding of a-Trilateral Workshop Hold in N.Z.1984:56-71.
[5]樊江文.红车轴草的研究和利用[J].草业科学,1994,11(5):10-11.
[6]苏加楷.中国牧草新品种选育的回顾与展望[J].草原与草坪,2001,95(4):3-9.
[7]刘岩,刘顺航.红车轴草的研究进展[J].中草药,2007,38(5):5-8.
[8]王志明,岳民勤,杜文化.集饲用和药用价值于一体的牧草新秀--岷山红车轴草[J].草业科学,2005,22(4):33-35.
[9]张文淑.红车轴草[J].农业养殖技术,2002,15:27.
[10]Davies D R,Winters A L,Lemans D K.The effect of inoculants treatment of alternative crop forages on silage quality and in vitro rumen function.Sweden:Silage Conf,Uppsala.1999:131-132.
[11]Linda McGraw.Red clover silage boasts benefits over alfalfa silage.Agricultural Research.2001,49(7):7.
[12]Amy Spillman.Nutrient boost for alfalfa silage.Agricultural Research.2003,51(12):20.
[13]Hunt Ⅳ.Potential production of red clover varieties in South-West Scotland.J.of the British Grassland Society.1975,3:209-216.
[14]江月兰,何毅,杨学兰.红车轴草品种比较试验[J].草业科学,1997,10:39-41.
[15]林成招.大豆异黄酮植物雌激素对大鼠乳腺癌细胞和小肠上皮细胞生长的影响及其机理研究[D]南京农业大学,2004.
[16]甘肃省畜牧厅.甘肃省种草区划[M].北京:中国农业科技出版社,1991,141-142.
[17]马强.红车轴草化学成分的研究[J].中国药学杂志,2005,40(14):1057-1059.
[18]陈寒青,金征宇等.红车轴草不同部位中异黄酮含量的测定[J].食品科学,2004,25(6):150-153.
[19]胡冬华,梁久来.红车轴草中异黄酮成分提取工艺优化和方法学研究[J].吉林中医药,2007,27(12):61。
[20]边清泉.红车轴草提取物中异黄酮含量的高效液相色谱分析法的研究[J].绵阳师范学院学报,2003,22(2):52-55.
[21]KROYER G T.Red clover extract as antioxidant active and functional food ingredient.Innovative Food Science and Emerging Technologies.2004,5(1):101-105.
[22]陈学福,史高峰.超声波提取红车轴草异黄酮工艺的研究[J].安徽农业科学,2006,34(24):6426-6427.
[23]边清泉,杨振萍.红车轴草提取物中异黄酮定量方法的研究[J].中国中药杂志.2003,28(11):1044-1047.
[24]陈封政,李书华.红车轴草中总异黄酮含量测定[J].乐山师范学院学报,2005,20(5):49-50.
[25]Wong E.Detection and estnnahon of oestrogemc conshhients in red clover.J.Sci.FdAgric.1962,13:304-308.
[26]Wong E.Isoflavone contents of red and subterranean clovers.J.Sci.FdAgric.1963,14:376-379.
[27]Nilssoe A.The quantitative detennieatioe of biochaiue A in red clover samples by means of an isotope dilution method.Arkiv for Kemi Band 21nr.1963,10:87-91.
[28]BeckA B.The oestrogeeic isollavoees of subterranean clover.Aust.J.Agric.Res.1964,15:223-230.
[29]Golden A F,Tones R.A routine method for predicting the fonnoeoeetie content of red clover.J.Sci.Fd Agric.1978,29:925-929.
[30]Patron J J,Collies W J,Stern W R.Quantitative analysis of the isollavoee phyto-oestrogees geeisteie,fonnoeoeetie and biochaeie A in subterranean clover leaves by high-performance liquid chromatography.J.of Chromatography.1982,247:366-368.
[31]Nicollier G F,Thompsoe A C.Separation and quaetitatioe of estrogeeic isollavoees from clovers by high-performance liquid chromatography.J.of Chromatography.1982,249:399-402.
[32]Dziedzic S Z,Dick J.Analysis of isollavoees ⅲ Beegalgram by high-performance liquid chromatography.Journal of Chromatography.1982,234:497-499.
[33]Kitada Y,Mizobuclu M,Ueda Y.Analysis of isollavoees in Puerariae radii by high-performance liquid chromatography with amperometric detection.J.of Chromatography. 1985, 347:438-442.
[34] Dorr K K, Guest D I. Rapid, sensitive high-performance liquid chromatographic assay for isollavoeoids from cowpea(Vigea ueguicμLata). J. of Chromatography. 1987,387:536-540.
[35] Jones A E, Price K R, Femvick G R. Development and application of a high-performance liquid chromatographic method for the analysis of phytoestrogees.J. Sci FoodAgric. 1989, 46:357-364.
[36] Fraeke A A, Caster L J, Cerna C M. Qiiaetilicatioe of phytoestrogees ie legiunes by HPLC. J. Agric. Food Chem. 1994, 42:1905-1913.
[37] Vetter J. Isollavoees ie different parts of common Trifolium species J. Agric. Food Chem. 1995, 43:106-108.
[38] Klejdus B, Vitamvasova D, Kubae V. Reversed-phase high-performance liquid chromatographic determination of isollavoees in plant materials after isolation by solid-phase eltractioe. J. of Chromatography A. 1999, 839:261-263.
[39] Hutabarat L S, Greeelield H, MμLhollaed M. Quantitative detennieatioe of isollavoees and coumestrol ie soybean by cohumi liquid chromatography. J. of Chromatography. 2000, 886:55-63.
[40] Rijke E D, Zafra-Gomez A, Ariese F. Detennieatioe of isollavoee glucoside maloeatesie Trifolium prateese L. (redclover) eltracts: quaetilicatioe and stability shidies.Journal of chromatography A. 2001, 932:55-64.
[41] Krem L, Ueterrieder, Ruprechter R. Quaetilicatioe of isollavoees ie red clover by high-performance liquid chromatography. Journal of Chromatography B. 2002,777:123-128.
[42] Wu Q L, Wang M F, Simoe J E. Detennieatioe of isollavoees i.e. red clover and related species by high-performance liquid chromatography combined with ultraviolet and mass spectrometric detection. Journal of Chromatography A. 2003,1016:195-209.
[43] Mazur W Fotsis Wahala K, et al. Isotope Dilution Gas Chromatographic-Mass Spectrometric Method for the detennieatioe of isollavoeoids, comnestrol, and ligeaes in food samples. Analytical Biochemistry. 1996, 233:169-180.
[44] Wang C Y, Huang H Y, Kuo K L, et al. Analysis of Puerariae radii and its medicinal preparations by capillary electroplioresis. Journal of Clmomatograpliy A. 1998.802:225-231.
[45] Lapcik O, Hainpl R, Al-Maliarik N, et al. A novel radioiimnimoassay for daidzein. Steroids.1997,62:315-320.
[46]Lapcik O,Hill M,Hampl R,et al.Identification of isollavonoids ⅲ beer.Steroids.1998,63:14-20.
[47]Wang W Q.Radioiimnunoassay determination of fonnononetin in marine plasma and mannnary glandμlar tissue.Proc Soc Elp Biol Med.1998,217:281-287.
[48]Lapcik O,Hampl R,Hill M,et al.Radioiimnunoassay of Free Geiustein in Human Senun.J.Steroid Biocliem.Molec Biol.1998,64:261-268.
[49]Lapcik O,Hill M,Ceniy Ⅰ,et al.Inununoanalysis of isollavonoids ⅲ Pisum sativiun and Vigna radiata.Plant Science.1999,148:111-119.
[50]Bemetau-Pelissero C,Arnal-Sclmebelen B,Lainothe Ⅴ et al.ELISA as a new method to measure genistein and daidzein in food and human Iluids.Food Chemistry.2003,82:645-658.
[51]O so ski AL,Kennelly E J1,Phytoestrogens:a review of the p resent state of research,Phytother Res.2003,17:8452-8691.
[52]江苏新医学院.中药大辞典[M].上海:上海科学技术出版社.1977:134-133.
[53]Chen HQ,Jin ZY,Tao GJ,et al.Determ ination of isoflavones in different parts of Trifolium pratense by high performance liquid chromatography.Food S ci.2004,25(6):1502-1531.
[54]Mark Eric G.Method of preparing and using compo sitionsextracted from vegetable matter for the treatment of cancer.US:6900240[P],20052-52311.
[55]Atkinson C,Oosthuizen W,Scollen S.Modest protective effects of isoflavones from a red clover derived dietary supplement.J N utr.2004,134(7):1759-1764
[56]Beck Ⅴ,Roh r U,Jungbauer A.Phytoestrogens derived from red clover:An alternative to estrogen rep lacement therapy Steroid Biochem Mol Bio.2005,94(5):499-518.
[57]尹春萍,樊龙昌等.红三叶总异黄酮对人乳腺癌MCF-7细胞增殖和凋亡的影响[J].中草药,2007,38(2):232-234.
[58]Adlerereutz H,Mazur W1.Phytoestrogens and western diseases Ann Med.1997,29(1):95-120.
[59]De Rijke E,Zafra Gomez A,Ariese F.Determination of isoflavone gluco side malonates in Trifolium pratense L.(red clover) extracts:quantification and stability studiesl J Chromatogr A.2001,932(1):55-60.
[60]A dlerereutz H,Goldin B R,Go rbach S L,et al.Soybean phytoestragen intake and cancer riskl J Nutr.1995,125:757-770.
[61]Liu J,Burdette JE,Xu H,et al.Evaluation of estrogenic activity of plant extracts for the potential treatment of menopausal symptom s.J.Agric Food Chem.2001,49(5):2472-2479
[62]张荣庆,韩正康.芒柄花素槲皮素对奶牛初乳中4种酶活力的影响[J].南京农业大学学报,1992,15(3):131-132.
[63]Ingram D,Sanders K,KdybabaM,et al.Case control study of phytoestrogens and brest cancer.Lancet.1997.350(9083):990-994.
[64]De Rijke E,Zafra2GomezA,A riese F,et al.Determination of isoflavone gluco side malonates in Trifolium pratense L.(red clover ) extracts:quantification and stability studies.J.Chromatogr A.2001,932(1):60-64.
[65]Peter H M,vande Weijer,Barentsen R.Isoflavones from red clover(Promensilor)significantly reduce menopausal hot flushsymptoms compared with placebo.Maturitas.2002,42(3):187-193.
[66]陈学福,史高峰.红三叶异黄酮的研究进展[J].中草药,2006,(02).309-312.
[67]江苏新医学院.中药大辞典[M].上海:上海科学技术出版社,1975:10-12.
[68]黄胜阳,屠鹏飞.红车轴草异黄酮化合物的分离鉴定[J].北京大学学报(自然科学版),2004,40(4):544-549.
[69]Burdette J E,Liu J,Lantvit D,et al.Trifolium pretense(redclover) exhibits estrogenic effects in vivo inovariectomized Sprague-dawleyrats.J Nutr.2002,132(1):27-30.
[70]杨丽珍,周俊芬等.红车轴草异黄酮对老年雌性大鼠性激素水平及SOD活性、MDA含量的影响[J].中国中医基础医学杂志,2007,13(7):513-514.
[71]Rice L,SamediV,Medrano T A,et al.M echanism s of the growth inhibitory effects of the isoflavonoid biochanin A on LNCaP cells and xenorafls.Prostate.2002,52(3):201-212.
[72]Beck Ⅴ,Unterrieder E,Krenn L,et al.Comparison of hor-monalactivity(estrogen,androgen and progestin) of standardized plant extracts for large scale use in hormone replacement therapy.J.Steroid Biochem Mol Biol.2003,84(2-3):259-268.
[73]安春志,刘莉,刑学锋.中药指纹图谱的现状和研究进展[J].第一军医大学分校学报,2005,28(12):197-200.
[74]陶巧凤.几种色谱技术在中药质量控制中的应用进展[J].中国现代应用药学杂志,1999,16(5):12-14.
[75]Da Porto C.,Calligaris S.,Celotti E.,et al.Antiratical properties of commercial cognacs assessed by the DPPH· test.J.Agrc.Food Chem.2000,48:4241-4245.
[76]Dapkevicius,A.,Venskutonis,R.,Van Beek,T.A.,& Linssen,P.H.(1998).Antioxidant activity of extracts obtained by different isolation procedures from some aromatic herbs grown in Lithuania.J.of the Science of Food and AgricuLture.77:140-146.
[77]王汝富,李新媛等.岷山红三叶及其产业化发展思路[J],草原与草坪,2007,1:69-72.
[78]王林嵩.生物化学实验技术[M].北京:科学出版社,2007,9:57-61.
[79]虞大红,吴海霞.实验化学[M].化学工业出版社,2007,5:23-24.
[80]陈毓荃.生物化学实验方法和技术[M],科学出版社,2002:18-22.
[81]章银良.食品检验教程[J].化学工业出版社,2006,8:231-234.
[82]段金秀,张向东,惠文,曹致中.盐酸浸提法和消煮法测定牧草中钾钙含量的比较研究[J].甘肃农业大学学报,2006,4:34-37.
[83]严付华,陈波等.高效液相色谱法荧光检测红车轴草提取物中的苯并(α)芘[J].中国中药杂志,2007,32(10):935-937.
[84]陈寒青.红车轴草异黄酮的分离、纯化、结构鉴定及生理功能研究[D].江南大学,2005:23-33.
[85]刘宁芳,徐庆国等.不同三叶草品种的农艺性状与营养特性比较[J].作物研究,2007,2:128-130.
[86]王晶,周禾.红三叶与紫花苜蓿蛋白质利用率的比较[J].草业科学,2005,23(1):52-55
[87]陈寒青,金征宇.我国不同产地红车轴草异黄酮含量的测定[A].天然产物研究与开发.2007,19:631-634.
[88]常平,张颍等.红车轴草提取物中异黄酮成分的分析[J].分析检测食品科学.2007,28(09):449-452.
[89]Chen H Q,Jin Z Y,Tao G J,et al 1 Determination of isoflavones in different parts of Trifolium pratense by high performance liquid chromatography.Food Sci.2004,25(6):150-153.
[90]李松;黎国兰;王洪福;边清泉。火焰原子吸收光谱法测定红车轴草及其提取物中铁和锰[J].理化检验--化学分册2007年第43卷1:21-23.
[91]徐颖.高效液相色谱法检测大豆异黄酮含量[J].粮油食品科技,2006,6(14):33-35.
[92]刘逢芹,董其亭,李宏建.野葛根、藤及其提取物中相关异黄酮成分的含量测定[J].中国中药杂志,2007,7(32):1487-1489.
[93]陈封政,李书华,王熊清.红车轴草最佳采收期的研究[J].时珍国医国药2007,18(4):847-848.
[94]胡喜兰,韩照祥,陶莹.DPPH·法测定17种植物的抗氧化活性[J].食品科技,2006,10:264-268.
[95]谷利伟,谷文英,过世东.新型生长调节剂--异黄酮类植物雌激素[J].饲料添加剂,2000,21(12):26-28.
[96]郑小江,向东山,唐巧玉.武陵地区主要冬季牧草营养品质的比较与分析[J].安徽农业科学,2007,25(6):150-153.
[97]Editorial Board of China Herbal,State A dministration of Traditional Chinese Medicine,China Herbal,Shanghai:Shanghai Scientific and Technical Publishers.1999:372-390
[98]江苏新医学院.中药大辞典[M].上海:上海科学技术出版社.1975,1:21-27.
[99]Freeman C L.Factors affecting scale of enter prise.Producing herbage seed.PalmerstonNorth:New Zealand Grassland Association Inc.1985:37-40.
[100]Pedson G A.White clover and other perennial clovers.IA:Iowa State University Press.1995:226-236
[101]曹志东,陈学通,马先锋.岷山红车轴草改良退化山地草甸效果研究[J].草业科学,2004,8:52-55.
[102]Dornstader E,Jisa E,Unterrieder Ⅰ,et al.Estrogenic activity of two stander dizedred clover extracts(Menollavon) intended for large scale use in hormone replacement therapy.J Steroid Biochem Mol Biol.2001,78(1):67-68.
[103]王志明,岳民勤,杜文华.岷县草地资源可持续发展面临的问题及治理对策[J].草原与草坪,2005,(1):18-19.
[104]甘肃省草原总站.甘肃草地资源[M].兰州:甘肃科学技术出版社,1999.10.
[105]何世炜,常生华,武得礼.饲用山羊豆引种研究初报[J].草业科学,2003,20(11):28-29.
[106]李玲,何桂霞,裴刚.红车轴草中异黄酮成分含量测定方法的研究[J].江西中医学院学报,2004,16(6):45-46.
[107]索志新,斯建勇,沈连钢.红车轴草的化学成分研究[J].中草药,2007,1:8-10.
[108]辽宁省林业土地研究所.东北草木植物志[M].第五卷.北京:科学出版社,1976.
[109]郑杰,刘艳平.红车轴草提取物对胃癌BGC-823细胞凋亡的影响[J].生命科学研究,2006,10(2):178-182.
[110]Dornstauder E,Jisa E,Unterrieder Ⅰ,et al,Estrogenic activity of two standerdized red clover extracts(Meno llavon) intended for large scale use in hormone replacement therapy.J.Steroid Biochem Mol Biol.2001,78(1):68-70.
[111]Rice Lamedi Ⅴ,Medrano T A,et al,Mechanisms of the grow thinhibitory effects of the isoflavonoid biochanin A on LNCaP cells and xeno rafts.Prostate.2002,52(3):201-212.
[112]Aziz Turkoglu,Mehmet Emin Duru,Nazime Mercan,Ibrahim Kivrak,Kudret Gezer,Antioxidant and antimicrobial activities of Laetiporus suphureus(BuL.) Murrill,Food Chemistry.2007,101:267-273.
[113]Dornstauder E,Jisa E,Unterrieder Ⅰ,et al.Estrogenic activity of two standerdize dried clover extracts(Menollavon) intended for large scale use in hormone rep lacement therapy.J Steroid Biochem Mol Biol.2001,78(1):71-75.
[114]徐桂花,沈益新,刘信宝,周建国.红三叶与白三叶春季植物雌激素含量和种类的差异[J].中国草地学报,2006,(06):39-42.
[115]吴彩霞,王倩嵘等.SPME/GC/MS法分析白车轴草花挥发性化学成分[J].河南大学学报(医学版),2006,25(3):17-20.
[116]马强,雷海民等.红车轴草挥发油成分的GC-MS分析[J].中草药,2005,36(6):828-829.
[117]黄锋,林黎林,胡娟娟.黄花倒水莲抗氧化活性研究[J].中国天然药物,2006,4(4):291-294.
[118]蔡双莲,李敏.多不饱和脂肪酸的研究进展[J].生命科学研究,2003,7(4):289-304.
[119]唐传核,徐建祥,彭志英.脂肪酸营养与功能的最新研究[J].中国油脂,2000,25(6):20-23.
[120]郑子新,张荣欣.构成脂肪的脂肪酸和必须脂肪酸(营养与健康卷)[M].四川:四川人民出版社.1999,(6):50-51.
[121]张媛,王喆之.荔枝种子脂溶性成分的GC-MS分析.食品科技,2007,28(4):267-268.
[122]马强.红车轴草和川续断的化学成分及质量研究[D].大连理工大学,2005.
[123]Nagmani M,Cao H A,Lu L-J W.Potency of different is of lavones on growth inhibition of end ometrial can cercellsin culture.J.Soe Gynecol Invest.1998,51,Supplement:112A.
[124]Yanagiharak,Itoa,Toge T,et al.Antiproliferative effects of isoflavoneson human cancer cell lines established from the gastroint estinaltract.Cancer Research.1993, 53:5815-5821.
[125]Lyn Cookbd,Stottman H L,Yany,et al.The effects of phytoestrogens on human pancreatic tumor cells sinvitro.Cancer Letters.1999,142:111-119.
[126]Fung M C,Szetoyy,Leung K N,et al.Effects of biochanin A on the growth and differentiation of myeloid leukemiawehi-3B(JCS)cells.LifeSciences.1997,61(2):105-115.
[127]Nestel P L,Pomeroy S,Kay S,et al.Isoflavones from red clover improve systemic arterial Compliance but not plasma lipids inmenopausal women.Journal of Clinical Endocrinology& Metabolism.1999,84:895-898.
[128]Akiyama T,IshidA J,Nakagawa S,et al.Genistein,a specific inhibitor of tyrosine-specific protein kinases.Journal of Biological Chemistry.1987,262(12):5592-5595.
[129]Adler Creutz H.Phytoestrogens and breast cancer.J.of steroid biochemistry &MolecμLar Biology.2003,83:113-118.
[130]Le Bail J C,Champavier Y,Chmlia A J,et al.Effects of phytoestrogensonaromatase,3-β and 17-β-hydroxysteroiddehydro-genase acticities and human breast cancer cells.Life Sciences.2000,66(14):1281-1291.
[131]孙健,耿彤,潘勤,肖峰.红车轴草异黄酮的研究进展[J].国外医药(植物药分册),2007,(04):27-30.
[132]谷文英,余飞,陈莹,储东生.不同生长时期红三叶异黄酮含量变化的研究[J]草原与草坪,2006,(03).61-64.
[133]王妍琪,单安山.异黄酮类化合物的营养作用及其应用[J].中国饲料,2003,18:18-20.
[134]李崇明,向阳,翟莉,曾庆恢.红车轴草中总异黄酮提取精制工艺研究[J].中草药,2004,(10).1135-1136.
[135]彭向雷,王蕊,王悦等.大豆异黄酮研究进展(J).中国食品卫生杂志,1998,3:38-41.
[136]王国杰,韩正康.植物异黄酮研究新进展[J].天然产物研究与开发,1994,6(3):81-83.
[137]钟瑞敏,张振明,王羽梅.杨梅叶芳香精油的成分鉴定及抗氧化活性[J].华南理工大学学报,2006,34(3):49-53.
[138]黄锋,林黎琳,杜冠华等.尾叶远志抗氧化活性研究[J].天然产物研究与开发,2006,18:717-720.
[139]武雪芬,郑超瓶,,李桂兰.金银花叶提取物的抗氧化作用研究[J].河南化工,1999,10:10-11.
[140]马先锋.红三叶引种试验研究初报[J].四川草原,2006,127(6):10-11.
[141]岳民勤,杜文华等.岷山红三叶示范推广及产业化开发进程[J].草业科学,20047,24(5):34-36.
[142]陈寒青,金征宇.红车轴草异黄酮的组成及主要生理功能的研究进展[J].食品与发酵工业,2004,30(11):70-76.
[143]朱宇旌,张勇,王纯刚等.红车轴草黄酮抗氧化性研究[J].食品科技技,2006(4):78-81.
[144]谢明杰,陆敏,邹翠霞.大豆异黄酮的抑菌作用[J].大豆科学,2004,01-105.
[145]曾虹燕,周朴华等.红车轴草有效成分的研究进展[J].中草药,2001,02:54-57.
[146]He Xiao-guo,Lin Lang-ze.Analysis of flavonoids from red Clover by liquid chromatography-electrospray mass spectrometry.J Chromatogr A,1996,755:127-132.
[147]金汝城,赵国磊,李贵文,逄艳.微波辅助提取红车轴草异黄酮的工艺研究[J].食品科学,2007,(09).165-168.
[148]Ranich T,Bhathena S J,Ⅴ elasquezM T1 Protective effects of dietary phytoestrogens in chronic renal disease.J.RenN utr.2001,11(4):1832-1931.
[149]ShiSF,YuCQ1 Progress in research on phytoestrogens andtheir effect targets.J.Chin Integr Med.2005,3(5):4082-4101.
[150]Grahan Edmund K,A lan James H.CardivascuLar and bonetreatment using isoflavones.U S:20040072765,20042042091.
[151]Grahan Edmund K1 Methods of cholesterol reduction using isoflaovens.U S:20040170713.20042092021.
[152]Grahan Edmund K.Dietary supplements comp rising soyhypocotyls containing at least one isoflvaone.U S:7045155.20062052161.
[153]Mark Eric G.Method of preparing and using composition sextracted from vegetable matter for the treatment of cancer.U S:6900240.20052052311.