Study On Isolation, Purification, Identification Of Isoflavones In Trifolium Pratense And Its Physiological Functions

Trifoliun pratense L. (Leguminosae), known as red clover, meadow clover, purple clover,cow clover, a perennial plant, grows in many parts of the world. It is also widely distributed inChina. Trifolium pratense ( T. pratense) is one of the most important forage plants, which hasbeen used by the Oriental and the European cultures, and more recently also by the Americans,as a medicinal herb for the treatment of eczema and psoriasis. T. pratense contains protein,amino acids, sugars, vitamins and so on. But in recent years, considerable research hasshowed that T. pratense is one of the richest sources of isoflavones and more attention hasbeen paid to T. pratense isoflavone. Isoflavones based on the structural similarity to theestrogens 17β-estradiol have been found to exert potential health benefits in age-related andhormone dependent diseases, including cancer, menopausal symptoms, cardiovascular diseaseand osteoporosis. But recent years in China, no attention is paid to T. pratense isoflavone,which proved a limited research work carried out in this area.In this work, systematic studies were carried out on the extraction technology, isolation,purification, identification, quantification method and types of T. pratense isoflavones. Theapplication of isoflavones in LPS-induced in vitro model of Parkinson's disease and the effectof different supplemental form and level of isoflavones on the performance andphysiochemical indexes of laying hens during late period of the laying cylce were alsoinvestigated. In addition, the contents of isoflavones, protein and amino acids in differentparts of T. pratense were determined.The contents of protein and amino acids in different parts of T. pratense were measured.The results showed that the contents of crude protein in the leaf, flower and stem of T.pratense were 21.57%, 18.46%, and 8.87%, respectively. The contents of total amino acids inthe leaf, flower and stem were 17.73%, 13.71%, and 6.44%, respectively.HPLC was used to separate and determine four isoflavones, including biochanin A,formononetin, genistein and daidzein, from dried leaf, flower and stem of T. pratense. Therewere different distributions of the tested isoflavone compounds among the different plantparts. Contents, based on dry matter, of the total four isoflavones compounds in the leaf, stemand flower of T. pratense were 0.856%, 0.403% and 0.258%, respectively, among which, thehighest contents was found in the leaf and the lowest in the flower, Biochanin A andformononetin were two kinds of predominant isoflavones in T. pratense. The content ofbiochanin A or formononetin was the highest in the leaf and the lowest in the flower. Contentsof genistein and daidzein in different parts were low.The types of T. pratense isoflavones were analyzed by HPLC and UV. The resultsshowed that there were about twenty types of isoflavones in T. pratense.Based on the UV scanning spectra, three-wavelength UV spectrophotometry wasestablished to determine the content of total isoflavones in T. pratense. This method couldeliminate the interference of pigments and ethanol-soluble protein in the ethanol extract of T.pratense. It was thought to be a simple, fast and accurate method.The extraction technology of isoflavones in T. pratense was studied by single factor andorthogonal experiment. The optimal condition for T. pratense isoflavones was established asfollows: 80% ethanol as solvent, ratio of material to liquid was 1∶15 (w/v), reflux extractionat 80℃ for three times and one hour for each time, the extraction rate of isoflavones in T.pratense was as high as 91.90%.For isolation of isoflavone, the adsorption and desorption performance of 14 types ofmacroporous adsorbent resins were studied and compared. AB-8 resin was selected for itshigh adsorption and desorption capacity. The effect of concentration, pH and flow rate of theinfusion on the adsorption of AB-8 resin were studied with dynamic adsorption experiment.The extract solution at a concentration of 0.79-1.11mg/mL, pH of 4.24, flow rate of 2BV/hwere the optimal condition. When using 4BV 80% ethanol as eluent in 2BV/h speed,desorption rate of isoflavones could reach up to 93.72% and elution peak was satisfactory.Eight isoflavone compounds were isolated and purified from T. pratense by columnchromatography of silica gel, polyamide and Sephadex LH-20 and their chemical structureswere identified by UV, ESI/MS, 1H-NMR, 13C-NMR and DEPT-NMR. They were irilone,formononetin, pratensein, daidzein, calycosin, genistein, biochanin A and ononin.The application of seven isoflavone aglycones (biochanin A, formononetin, genistein,daidzein, pratensein, calycosin and irilone) isolated from T. pratense in LPS-induced in vitromodel of Parkinson's disease was studied for the first time. The results showed that differentisoflavone attenuated LPS-induced decrease in DA uptake and the number of DA neurons,inhibited LPS-induced microglia activation and the generation of proinflammatory factorsincluding TNF-α, NO and superoxide in primary mesencephalic neuron-glia cultures. Inaddition, in primary microglia-enriched cultures, different isoflavone inhibited the productionof proinflammatory factors (TNF-α, NO and Superoxide ). The action of seven isoflavoneswere in the following order: genistein > biochanin A > pratensein > daidzein > calycosin >formononetin > irilone. In a conclusion, isoflavones from T. pratense played a role in theprotection of dopaminergic neurons through the inhibition of microglia activation and thegeneration of proinflammatory factors.The effect of different supplemental form and level of isoflavones from T. pratense onthe performance and physiochemical indexes of laying hens during late period of the layingcylce were also investigated. 450 55-week-old ISA brown laying hens were randomly dividedinto nine groups. Group one was the control, Group two to group five were formononetin-treated groups, with formononetin supplemented to the basal diet at the level of 5, 10, 20,40mg/kg, respectively, group six to group nine were isoflavone mixture-treated groups, withisoflavone mixture supplemented to the basal diet at the level of 5, 10, 20, 40mg/kg,respectively. Each treatment consisted of five replications of 5 cages (two birds per cage). Theduration was 9 weeks. The results showed that egg production was increased significantlycompared to the control when dietary supplemental level increased from 0 to 20mg/kg forformononetin and from 0 to 5mg/kg for isoflavone mixture, but different levels offormononetin or isoflavone mixture had no effect on feed/egg ratio, average egg size andaverage daily feed intake. The percentages of soft and broken eggs were markedly decreasedwhen the diet was supplemented with formononetin or isoflavone mixture at the level of5mg/kg. When formononetin or isoflavone mixture was added to the diet, the levels of T3, T4,Insulin and E2 in serum were increased significantly. The activities of CAT, GSH-Px and SODin serum and liver were increased significantly when formononetin was added to the diet atthe level of 20mg/kg or isoflavone mixture at the level of 5mg/kg, but there was no differenceamong different added levels. When the diet was supplemented with 20mg/kg formononetinor 10mg/kg isoflavone mixture, the concentrations of triglyceride and T-cholesterol in serumwere decreased significantly. Different levels of formononetin or isoflavone mixture in thediet had no effect on the activity of AKP and phosphorus level in serum, but the level ofcalcium in serum was increased significantly when isoflavone mixture was added to the dietat the level of 10mg/kg.