Minshan Trifolium Pratense Resources Survey And Biological Activity

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 traitsT.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/hm².The highest production can achieve to 90 t/hm².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/hm² in the coming year.2.Nutrients constituents of T.pratense cv.MinshanAccording 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.MinshanThe 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.MinshanHPLC 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.MinshanThe 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.MinshanIn 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(EC₅₀) 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.