Studies On Isolation And Identification Of Two Flavonol Glycosides From Fujian Shuixian Tea And Their Inhibition Of Oral Pathogenic Bacteria

Fujian Shuixian Tea is popular in all parts of Fujian and Guangdong,and is exported to Southeast Asian countries and regions.It has the functions of refreshing teeth,helping digestion and lowering cholesterol.Fujian Shuixian Tea contains a variety of active ingredients,mainly flavonoids.In this study,two new flavonoid tetraglycosides from Fujian Shuixian tea were isolated and identified.The contents of the two substances in different seasons and varieties were compared,and the antibacterial effects with the mechanism of the two substances were studied.The main findings are as follows:1?The two compounds were separated and purified using AB-8 macroporous resin by gradient elution at a flow rate of 1.5 BV/h.80%ethanol elution fraction was collected and spin-dried to obtain semi-preparative samples.The conditions of semi-preparative HPLC separation was as follows:mobile phase;0.1%aqueous formic acid-0.1%formic acid acetonitrile by gradient elution,low rate:10 mL/min,injection volume:0.5 mL.After two preparations,the purity of the two flavonol glycosides reached 98.77%and 97.08%,respectively,named F1 and F2.Structural identification is performed using modern techniques including mass spectrometry and nuclear magnetic resonance spectroscopy.F1 and F2 were Quercetin 3-0-[(E)-p-coumaroyl-(1?2)][a-L-arabinopyranosyl-(1?3)][?-D-glucopyranosyl-(1?3)-a-L-rhamnopyranosyl(1?6)]-?-D-glucopyranoside and kaempferol 3-0-[(E)-p-coumaroyl-(1?2)][?-L-arabinopyranosyl-(1?3)][?-D-glucopyranosyl-(l?3)-a-L-rhamnopyranosyl(1?6)]-?-D-glucopyranoside,respectively.2?The content of F1 and F2 in Shuixian tea from different producing areas and different seasons was determined by UPLC method for analyzing flavonoids in Shuixian tea.The results showed that,among the Shuixian teas in the four producing areas of Jianye,Wuyi,Zhangping and Weinan,the F1 content of Jianou Shuixian was the highest(1.3 mg/g),and the F2 content of Wuyi Narcissus was the highest(0.85 mg/g);The F1 content in Shuixian tea from four producing areas was higher than F2.The F1 content in Shuixian tea increased with the growth period,reaching a maximum of 1.25 mg/g in early Winter.While the F2 content fluctuated around 0.5 mg/g in the whole year.3.The inhibitory effects of F1 and F2 on Porphyromonas gingivalis and Fusobacterium nucleatum were determined by a two-fold dilution method.The minimum inhibitory concentration was in the range of 0.12-0.24 mM,indicating that F1 and F2 have strong inhibition effects on oral pathogenic bacteria.The inhibition ability of F1 and F2 on the biofilm of pathogenic bacteria was evaluated by crystal violet staining.The results showed that F1 and F2 could reduce the formation of bacterial biofilm in a dose-dependent manner,and the biofilm under high concentration(0.24 mM)was inhibited 70%;It was found that F1 and F2 had strong iron carrier activity in the range of 0.0075-0.06 mM by calculating the iron chelation rate,which was greater than 50%at 0.06 mM.The results showed that F1 and F2 could reduce the occurrence of oral diseases greatly by inhibiting bacterial growth and biofilm formation with chelating iron ions.