Analysis Of Phenolic Compounds Of Propolis From Different Geographical Origins And Its Inhibition Of Streptococcus Mutan

Recently, the application of propolis has gained much attention, as it has a variety of biological activities such as anti-oxidant, anti-microbial, anti-inflammatory and anti-tumor, all of which are closely linked to its complex chemical ingredients. Therefore, it is of great value to make a research into the chemical constituents and biological activities of propolis. As is known to all, China spreads across a large area of lands, covering the temperate, subtropical and tropical zones. And the properties of propolis changes with the region as well. As a result, the relationship between the conditions of environment and regions and the properties of propolis has become the hot spot in the research of propolis.In this article, we make an analysis of the phenolic compounds from the ethyl acetate extraction of propolis (EaEP) by the application of High performance liquid chromatography-Electrospray Ionization-mass spectrometry/mass spectrometry (HPLC-ESI-MS/MS) and make a quantitative analysis of the seven kinds of flavonoid compounds in EaEP as well. We also make a research to explore the properties of the growth, acidogenetic and adherance of Streptococci mutans(S.mutans) in diffrent regions, thus buiding a link between the geography positions and the anti-caries activities of propolis. In addition, we have explored the mechanism of the bacteriostatic effects of phenolic compounds derived from propolis on S.mutans by the observation of the morphology of S.mutans which has added EaEP before with the use of SEM and TEM.The main results and contents are as follows:1. Extraction of phenolic compounds in propolis:By comparison the adherence inhibition rate of S. mutans in the wall of test tube between ethyl acetate extraction of propolis,95%ethanol extraction of propolis and70%ethanol extraction of propolis, we found that EaEP has the highest adherence inhibition rate of S. mutans in the wall of test tube. It’s higher than95%ethanol extraction of propolis and70%ethanol extraction of propolis. It’s also higher than the control of NaF. It explained that the anti-caries activity of EaEP is better than the other two solvent extraction. So ethyl acetate was the extraction solvent of propolis active ingredients in this research. Separation of propolis phenolic compounds:we established the HPLC-DAD separation conditions of phenolic compounds in EaEP. Mobile phase A was0.3%formic acid, mobile phase B was acetonitrile, the detection wavelength was280nm, using a Thermo Hypersil GOLD C18(250mmx4.6mm,5.0μm) column at a column temperature30℃, flow rate1mL/min conditions to separate the propolis phenolic compounds in gradient elution way, the substances can basically achieve the baseline separation. The relative standard deviations of methodological study which includes precision, stability, repeatability are controlled within5%, the lower limit of detection, recoveries in the range of93.40%～103.69%, and the relative standard deviations are less than2%, indicating the HPLC-DAD analysis of scientific and reliable.2. Identification of propolis phenolic compounds:Use HPLC-ESI-MS/MS method to isolate and identify EaEP. By analyzing the UV spectral information corresponding to the mass of the parent ion and secondary fragment ions of collected peaks of EaEP after HPLC separation, and combining with the known reference flavonoids, we identified26kinds of phenols including7kinds of phenolic acids,19kinds of flavonoids. Among them, quercetin, apigenin, kaempferol, isorhamnetin, chrysin, pinocembrin and galangin were identified by comparing the relative retention time, UV, and other identification of the fragment ions whit the reference, luteolin, pinobanksin、7-methoxychrysin、 pinobanksin-3-O-propionate、5-methoxygalangin、5-methoxy pinobanksin-3-O-acetate、5-methoxy pinobanksin、7-methoxyquercetin. sakuranetin、pinobanksin-3-O-acetate、 pinobanksin-3-O-butanoate、prenyl caffeate、pinobanksin-3-O-pentanoate、3,4-dimethoxycinnamic acid、caffeic acid、benzyl caffeate、phenethyl caffeate、benzyl p-coumarate、cinnamyl caffeate were identified by analyzing MS fragmentation pathway and combineing with the results of previous studies. The HPLC-ESI-MS/MS analysis method established in this study was accurate and reliable. It could be achieved on the purposes of propolis phenols qualitative, quantitative identification. By comparing China diagram of the total peak areas of EaEP liquid chromatography and7kinds of typical flavones content in12origin (from warm temperate, temperate, tropical, northern subtropical, southern subtropical, temperate plateau), we found that7typical flavonoid content and total peak area of EaEP in Hubei Yichang (Northern Subtropics), Shanxi Taiyuan (warm temperate zone), Zhejiang Hangzhou (Northern Subtropics) are higher than other GEOGRAPHICAL ORIGINS.3.Determining the MIC values of EaEP against S. mutans, the results showed that7kinds of typical flavonoids content of EaEP and MIC values were negatively correlated significantly (P=0.004<0.01), the total peak area of EaEP and MIC values were negative correlated significantly (P=0.004<0.01), which explaned the fact that the higher content of7typical flavonoid in EaEP, the higher content of polyphenols, the stronger inhibitory effect on the growth of S.mutans, the stronger the anti-caries activity of EaEP; determining EaEP to suppress acid production of S.mutans, the results showed that the total content of7typical flavonoids in EaEP and ΔpH values were positive correlated significant (P=0.005<0.01), the total peak area of EaEP and ΔpH value were positive correlated significant (P=0.006<0.01), it illustrated that the higher the7typical flavonoids content in EaEP, the higher the content of polyphenols, the inhibition of acid production was stronger, that is, the propolis of this origin has stronger anti-caries activity; determining the IC50, the result showed that total content of7flavonoids in EaEP, total peak area and IC50values were not correlated, indicating that phenolic compounds in EaEP and the total content of7kinds of flavonoids were of no relevance to adhesion in the glass wall.4.By scanning electron microscopy and transmission electron microscopy, we found that contrast to adding no EaEP to S.mutans, the surface of the bacterial cell became rough, sticky substance produced, the shape of the bacterial cell became irregular, cell wall was damaged, the cell membrane permeability changed, cytoplasm leaked, also bright white arc-shaped material appeared near the cell wall of some bacteria; division of S.mutans also have been greatly affected after adding EaEP, the cell membrane of dividing cell was not clear, the two daughter cells have different sizes and during the course of dividing the cell wall of the two daughter cells was destroyed, cytoplasm leaked, indicating that propolis destroy S.mutans cell structure and cell division, and also explained the anti-caries activity of propolis to some extent.