岷山红车轴草资源调查与生物活性研究
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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.
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
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