Extraction And Separation Of Total Flavonoids From Fenugreek Seeds And Their Biological Activities

Trigonella foenum-graecum L,belonging to the family Leguminosae,is an annual plant mainly distributed in China,Eastern Mediterranean,Iran Plateau and Himalayan Region.It has been widely used as vegetable and flavouring substance.Recent pharmacological studies have indicated that fenugreek has various bioactivities such as hypoglycemic,antilipemic,antioxidant,antineoplastic and antibacterial.Phytochemical studies on fenugreek have revealed the presence of steroidal saponins,flavonoids,triterpenoids,carbohydrate and alkaloids.Steroidal saponins and flavonoids are the main components with healthy function.At present,researches of fenugreek are focused on extraction process of active components and separation of steroidal saponins,there are few studies on flavonoids from fenugreek seed and pharmacological activities.This paper explored a systematical study on extraction process,purification and biological assessment of the chemical constituents from fenugreek seed.Moreover,the antioxidant and antibacterial ability of total flavonoids were studied.A multiple responses surface methodology（MRSM）was applied to optimize the extraction of fenugreek seed.The effects of factors along with the correlation between all responses（flavonoids content,DPPH assay,OH^-assay）were studied.Through single factor and BBD experiment design,the optimal process of extraction of total flavonoids from fenugreek seeds were obtained:ethanol concentration of 72%,material to solvent ratio of 35 m L/g,ultrasonic time of 41 min,ultrasonic power of 500 W.By investigating the effects of various factors on the dynamic adsorption and desorption of flavonoids in AB-8 macroporous resins,and the optimum process of dynamic adsorption and desorption was obtained:volume of uptake of 4BV（100ml）,concentration of uptake solution of 0.5mg/ml,sample solution p H of 5.0,and eluting speed of 4BV/h used with 20%,40%ethanol concentration as the favourable strippant.Under such circumstances,the purity of total flavonoids in the fractions collected is the highest,and the purity of flavonoids TFC₁（61.88%）and TFC₂（71.86%）with different purity were obtained by purification,which was more 12 times higher than that of total flavonoids from crude extracts.LC-HRMS analysis showed that there were 7 kinds of flavonoids with known structure in two components.Radical-scavenging and antioxidant activities were evaluated by DPPH,OH^-,T-AOC assay,in all antioxidant studies,TFC₁and TFC₂showed strong antioxidant activity.In addition,antibacterial activities were evaluated by CFU,MIC,LIVE/DEAD assay,although the two compounds had a certain ability to antibacterial,the effect was not ideal.It has been reported that metal organic skeleton（MOFs）materials can be loaded with drugs to enhance the biological activity of raw materials.In this paper,by prepared metal-organic skeleton（Fe-100）was used as carrier to loaded on TFC_2,and were characterized and activities were studied.The results of this experiment are as follows:release rate of TFC-Fe under acidic conditions is higher than that under neutral and alkaline conditions.Antioxidant capacity of TFC-Fe was higher than Fe-100,but slightly lower TFC₂.The antibacterial test screened out the target bacteria of drug-resistant E.coil and S.aureus.MIC values were 11.71±0.31ug/ml and 24.58±1.04 ug/ml.CFU,LIVE/DEAD tests showed that TFC-Fe had good antibacterial activity,and scanning electron microscopy test further proved that TFC-Fe could inhibit bacterial growth by destroying bacterial integrity.