Study On The Separation And Purification Of Antibacterial Components From Sapindus Mukurossi And Its Application

The pericarp of Sapindus mukurossi contains a large number of oleanolic acid saponins,which have been proved to have strong surfactivity and biological activities such as antimicrobial,anti-inflammatory,anti-oxidant and antitumor activities.The ancient medical literature records Sapindus mukurossi has skin clean and oil control effect,therefore,with the yield of saponins and the minimum inhibitory concentration?MIC?against Propionibacterium acnes ATCC 6919?P.acnes ATCC 6919?as evaluation indicators,the preliminary purification effect of the compound extractant was investigated,based on the pericarp of Sapindus mukurossi.To understand the basic information for the resource of Sapindus mukurossi,the bacteriostasis active components of the extract were investigated,and the mechanism of its bacteriostasis was preliminaryly explored.In addition,face mask products were developed based on the extracts from Sapindus mukorossi pericarp,so as to expand the field of exploitation and utilization of Sapindus mukurossi.The results were shown as follows.With the yield,purity of saponins and the MIC against P.acnes ATCC 6919 as evaluation indicators,this paper investigated the effects of three solvents and four kinds of extraction technologies on the extraction of saponins.The results showed that the three solvents had significant differences in the extracting effect of Sapindus saponins?P<0.05?.Among them,the highest yield of water extracted saponins was 56.84%.Ethanol was the second and ethyl acetate was the worst.Instead,the purity of saponins obtained by ethyl acetate extraction was the highest,which was 83.24%.On the basis of water extraction,the purity of saponins was increased with 4.29%by adding Aspergillus oryzae fermentation and ethyl acetate extraction?83.24%?87.53%?,but the yield of saponins was reduced to 4.59%.The bacteriostasis test showed that the obtained ethyl acetate fraction after water extraction and fermentation?SSEW?has the best inhibitory effect against P.acnes ATCC 6919,and MIC was 0.50 mg/m L.The optimum conditions for purifying saponins by compound extractants were studied to improve the yield of saponins and reduce the MIC of extract against P.acnes ATCC 6919.The optimum extraction conditions were as follows:the binary compound system containing ethyl acetate and 20%isoamyl alcohol was used as extractant,the ratio of material?the fermented condensed saponin solution of Sapindus pericarp?SSFW??to solution was 1:2 at a frequency of 70 times/min,and the extraction was performed 3 times for 4 min each time.Under this optimal reaction condition,this paper obtained the product called EAWF.The saponin yield of EAWF was 7.83%,with an increase of 3.24%over SSEW,and the MIC for P.acnes ATCC 6919 decreased from 0.50 mg/m L to 0.25 mg/m L.It showed that above extraction conditions were of practical significance to increase the yield and bacteriostasis of saponins.Besides,this article used a semi-preparative C₁₈ column to further purify EAWF.According to peak intensities,seven components was collected and their antibacterial effects were compared.Results showed that the antibacterial substances mainly concentrated in the chromatographic peaks with retention time from 47-53 min,of which the component with the retention time of 48.38 min?F48?had the best inhibitory effect against P.acnes ATCC 6919,and the MIC was only 0.12 mg/mL.The composition of the component with the best bacteriostasis effect was further analyzed by HPLC/MS.It was concluded that this component contained four types of oleanolic acid saponins,namely Mukurozi-saponin E1(Relative molecular mass??Mr?=924?,Sapindoside L?Mr=924?,Mukurozi-saponin G?Mr=966?,and Sapindoside M?Mr=966?.The antimicrobial mechanism of EAWF against P.acnes ATCC 6919 was investigated.The antibacterial test showed that EAWF had a concentration-dependent effect on the antibacterial rate.When the concentration of EAWF was 2.00 mg/m L?8MIC?,the antibacterial rate reached 100%.Besides,this paper found that content of 260 nm absorbance and alkaline phosphatase?AKP?,the concentration of K⁺and Mg²⁺in the supernatant of P.acnes ATCC 6919 had increased after treated by EAWF.And the destruction of P.acnes ATCC 6919 was positively correlated with EAWF concentration.Further,the observations with transmission election microscope?TEM?showed that EAWF caused the cell wall bec thinning obviously,cytoplasm was concentrated and produced a cavity,cell membrane was separated from cell wall.Therefore,studies on the increasing content of 260 nm absorbance and AKP,the concentration of K⁺and Mg²⁺in the supernatant of P.acnes ATCC 6919,and observations with TEM revealed that EAWF could influence the structure of the cell wall and membrane,and led to the increment of cell permeability,further caused the leakage of intracellular nucleic acid and AKP.SSFW and F48 were added to two kinds of basic mask formulas,and the preliminary study was conducted on the application of Sapindus mukurossi in face mask.The four kinds of mask developed in this paper possessed good color,uniform texture,suitable drying time,and good moisturizing effect.Furthermore,two kinds of mask added with F48 had better antibacterial properties than the masks with SSFW,and the 20%F48-paste mask had the best inhibitory effects on the three kinds of pathogenic bacteria?P.acnes ATCC 6919,Escherichia coli ATCC 8099,Staphylococcus aureus ATCC 6538?from acne,with inhibition rate being54.49%,32.11%and 33.91%,respectively.In addition,it confirmed that the inhibition of tyrosinase by two kinds of mask added with F48 was stronger than those with SSFW,and the20%F48-washed mask had the best inhibitory effects on tyrosinase,with inhibition rate of58.55%.To sum up,the results of this study would provide a theoretical basis for the further development and utilization of Sapindus mukurossi.