Study On Procyanidins From Tartary Buckwheat Husk And Its Prevention Dental Caries And Antioxination Activity

Buckwheat are extensively cultivated in China and its starch, protein and the composition, structure and biological activity of flavonoids were studied commonly, but Procyanidins in buckwheat (the BWPC) and the prevention of dental caries and anti-oxidative effect studies have not been reported in the related literature. For this reason, this study was designed to research the inhibition of BWPC on growth and metabolism of Streptococcus mutans besides the extraction and identification of the BWPC. Through analyzing on the changes of the Streptococcus mutans such as growth situation,acid production and the ability to scavenge Superoxide anion radical, we can not only get the conclusion of the efficiency of BWPC on dental caries and lay the foundation for the further study of the effect of BWPC on prevention of oral diseases, but also provide experimental evidence and ideas for the value of enhancing the deep processing of tartary buckwheat.The main results are as follows:1. Study on the content determination, extraction and purification of procyanidins in tartary buckwheat husk.1.1 The content of procyanidins in tartary buckwheat husk is the highest, the seed is next and the buckwheat rice is the lowest, so procyanidins from buckwheat husk was extracted as activity polyphenols.1.2 The extraction yield of procyanidins from tartary buckwheat husk was compared using different solvents, such as water, ethanol/water, acetone/water, methyl / water, the results showed the yield of acetone/water is the best, the alcohol/water system's is next, follows is the methyl alcohol/ water system and the water is the worst. When acetone/water is 60% (v/v), we can get the highest extraction rate. Using the ethanol/water system as the eluent, When the concentration of ethyl alcohol surpassed 70% ,it can get the maximum extraction taking 77% of the maximum extraction rate of acetone. Considering the friendliness of environment and saves of cost, the 70% ethyl alcohol/water was chose as the eluent to extract procyanidins from tartary buckwheat husk.1.3 Using the 70% ethyl alcohol/water system to extract BWPC, extraction temperature and time affected the extraction yield significantly, and the effect of ratio of material to liquid is not significant. The three factor's relations about extraction ratio is: extraction temperature > extraction time > the material and fluid ratio. Ridge analysis indicated that the more excellent extraction parameters are: extraction temperature 71.8℃, extraction time 118.9 min, and 1:16.9 of ratio of material. The absorptance of extract can reach 0.674 (546nm) .the predictive value was 0.709, the yield of extraction achieved 95% of the theoretical prediction. The result surpassed the most superior result in the center combination test, and increased 5.8%.1.4 Comparing 5 kinds of resins on the static adsorption and desorption ability of the BWPC, the most efficient condition of the ADS-17 resin on the dynamic adsorption analysis of BWPC was studied. The result indicated that the ADS-17 resin was the ideal adsorbent, the ADS-17 resin achieved balance needing 5 h in the solution. The best condition of dynamic adsorption analysis is using the methyl alcohol/water (70/30, V/V) as the eluent, the rate of flow is 3 mL/min, sample solution pH6.3, the diameter height ratios of Chromatographic analysis column was 1 : 6, The purity of the last product achieved 52%.2. Qualitative analysis of BWPC2.1 Anthocyanidin reactionUsing the method of butyl alcohol - hydrochloric acid to qualitative analyze the purified product, the typical characteristic reaction of the product is decomposing to red anthocyanidin under the condition of acidity and the heating.2.2 Ultra-violet spectrograph analysis of BWPCUltraviolet spectrum of the product was consistented with typical spectral characteristic of procyanidins.2.3 Mass spectrum analysis of BWPCThe ESI-MS/MS analysis indicated that BWPC consist of the monomers (catechin or epicatechin), the dimers, the trimers, the tetramers, pentamers of BWPC, and their molecule-ion (M-H) - m/z respectively are 289, 577, 865, 1153, 1441, its corresponding molecular weights are 290, 578, 866, 1154, 1442. Through MS/MS analysis, the main fragment ions were corresponded to possibly ion produce of molecular weights 290, 578, 866, 1154,1442 procyanidins and the monomers. 2.4 HPLC analysis of BWPCThe result of RP-HPLC analysis indicated that: the compound was divided into 6 compounds which had the typical fiavanols ultraviolet characteristic absorption. Comparing to the procyanidins of B1, B2, B3 and C, EC, the EGCG standard sample chromatograph peak retention time, the compound did not include the above 6 materials.3. The effect of BWPC on dental caries3.1 The result of BWPC on streptococcus mutans growth indicated: MIC of BWPC on streptococcus mutans is 8mg/mL, the lowest sterilization concentration (MBC) is 16mg/mL. The result indicated that BWPC can prevent dental caries through effecting streptococcus mutans growth.3.2 The result of BWPC on streptococcus mutans producing acid indicated that: In designates of the concentration scope (0.5-4mg/mL), producing acid ability of streptococcus mutans was powerfully inhibitted by BWPC, the negative control which did not add BWPC had the extremely significance difference (p<0.01), the value ofΔpH along with the BWPC concentration increasing reduced gradually. The results indicated that BWPC can prevent dental caries through effecting streptococcus mutans acid production.3.3 The result of chemiluminescence indicated that: BWPC had the scavenging O₂^-·free radical ability. In 200～1000mg/L concentration scope, it had a good scavening effect , along with sample density's increasing, the elimination of the free radical ability enhanced. The results indicated that BWPC can prevent dental caries through scavenging O₂^-·.4. The study of BWPC antioxidant and antibacterial4.1 Anti-lipin oxidizing ability result showed that BWPC had the anti-oxidation function, and it had a good concentration effect relations, the concentration effect equation was Y=0.0006x+0.1759, its IE50 was 0.54mg/mL. Taking the bamboo leaves flavanone, the sorghum original anthocyanidin and BHT as the comparison, it indicated that bamboo leaves flavanone's in lipin system IE50=5.084mg/mL, IE50 bamboo leaves flavanone/IE₅₀ BWPC = 9.49, it explained that BWPC in lipin system's antioxidation ability were by far more stronger than bamboo leaves flavanone. But when in same level experimental condition (0.3mg/mL), its antioxidation ability was the same as sorghum proanthocyanidin quite, but obvious weaker than BHT. 4.2 The result of BWPC the total antioxidation experiment by FRAP method indicated: The BWPC total antioxidation ability in the scope of 40mg/L～200 mg/L had concentration effect relations , and its concentration effect relations were: Y=0.0085x-0.0305, R2=0.9985, its antioxidation value was 8.5mmolFe2+ /g BWPC, which was 2.7 times than bamboo leaves fiavanone .4.3 In the experiment, along with concentration (0.5%～2.0%) increasing, the bacteriostasis circle's diameter increased, the bacteriostasis experimental result indicated that BWPC can suppress backwoods coli's growth effectively.