Study On Optimization Of Extraction And Fermentation Conditions Of Flavonoids From Polygonum Hydropiper And Daphniphyllum Calycinum

Polygonum hydropiper Linn.(Polygonaceae, Polygonum) was acrid in taste and warm in nature, with the effect of invigorating spleen to eliminate dampness, dispersing wind and eliminating stagnation, eliminating stasis and relieving pain, killing parasites to relieve itshing. It was clinically applied for the treatment of dysenter, gastroenteritis, diarrhea, rheumatic joint pain, bruises and swelling pain, dysfunctional uterine bleeding. It also could be used for snake bites, eczema and other symptoms. Daphniphyllum calycinum Bench(Daphniphyllaceae, Daphniphyllum) was bitter, astringent in taste and flat in nature, toxic. It possessed the functions of heat-clearing and detoxicating, expelling wind and activating blood, reducing swelling and alleviating pain. It was clinically used for the treatment of rheumatic pain, sores, traumatic fractures and snake bites.The application of composite prescription composed of P. hydropoper L. & D. calycinum Bench. has a long history for the treatment of gastrointestinal disease. It was proved that flavonoids were bioactive components in P. hydropoper & D. calycinum. In this paper, optimization of extraction technology of total flavonoids from P. hydropoper & D. calycinum was conducted. Then, qualitative and quantitative analytical method of composite prescription were established. Finally, microbial fermentation conditions of composite prescription were optimized with flavonoids content as index. It aimed at obtaining cheap, efficient, zero emission of traditional Chinese medicine fermentation products. The contents of this paper was as followed:1. Different extraction methods(including hot water extraction, ultrasonic extraction, ethanol is heated to reflux, soxhlet extraction) were compared with total flavonoids content as index. It was found that ultrasonic extraction method was optimal. Orthogonal design was applied for extraction technology optimization of flavonoids. The optimum extraction conditions of P. hydropoper were 60% ethanol concentration, solid-liquid ratio 1:30, extraction time was 50 min, ultrasonic extraction temperature is 80℃, under these conditions, total flavonoids was 53.3mg / g. The optimal extraction process parameters of D. calycinum were ethanol concentration of 60%, solid-liquid ratio 1:50, extraction time 40 min, extracted twice, under these conditions, total flavonoids was 35.3mg / g.2. Physicochemical Properties and assaying of P. hydropoper L. and D.calycinum Bench.were identified by the methods of HPLC, UV, and TLC. HPLC method regard rutin, Hyperoside, quercetin glycosides, quercetin, isorhamnetin, kaempferol as reference substance. The standard curve regression equation of rutin:y=20785x-1885.8, R2=0.9998. hyperoside: y=19860x+5368.9, R2=0.9997. quercetin glycosides: y=16884x+6745, R2=0.9998. quercetin: y=32354x+1850, R2=0.9998. isorhamnetin: y=38458x-8908, R2=0.9999. kaempferol:y=39924x-4444.3, R2=0.9999. UV-visible spectrophotometry method take rutin as reference substance, the regression equation y=12.003x+0.0037 was obtained which correlation coefficient was R2=0.9999. TLC regard rutin, hyperoside as reference substance,spotted on polyamide TLC plate, sodium dodecyl sulfate: n-butanol: hexane: water: formic acid(0.27: 0.63: 0.10 : 3: 0.2) as the agent, to 3% Al Cl3 as chromogenic reagent. Qualitative identification test was shown that samples and reference substance had the same color fluorescent spots at corresponding position on TLC plate.3. The fermentation effects of different strains including Bacillus subtilis, Bacillus licheniformis, Aspergillus niger, Trichoderma, Rhizopus oligosporus, white rot fun gi, Red Aspergillus were compared. Bacillus subtilis was chosen as optimal fermentation strains. Single factor experiments were conducted to investigate the influence of initial p H, temperature, humidity, fermentation time, amount of bacteria, amount of medium and water on fermentation. We determined the optimum fermentation conditions were as followed: medium initial p H 7.0, fermentation temperature 28℃, fermentation humidity 80%, ratio of solid/ liquid Fengliao : medium: water = 1: 1: 0, amount of bacteria was 33μL/g.4. Studies showed that bacillus subtilis fermentation maple smartweed can increase to a certain extent flavonoids ingredients dissolution, make the body more easily absorbed. after fermentation quercetin, kaempferol, isorhamnetin has different degrees of increase.