Preparation And Characterizon Of Perilla Frutescens Essential Oil Microcapsules And Its Antibacterial Activity

Perilla frutescens is a labiatae plant with extremely high medicinal and nutritional value,which is the dry leaf(or with twigs)of Perilla frutescens(L.)Britt.It was first recorded in Tao hongjing’s "Mingyi Bielu" and listed in "the catalogue of medicine and food homologous articles" published by the Ministry of Health.Traditional Chinese medicine(TCM)believes that Perilla frutescens tastes spicy,warm in nature,distributes to lung,and spleen channels,which has the functions of relieving cold,promoting qi and stomach.Volatile oil is one of the main active components of Perilla frutescens.According to the literature,perilla frutescens essential oil(PFEO)has significant antibacterial,anti-inflammatory and antioxidant effects,but it has the problems of volatile,oxidative degradation,unstable,poorly water-soluble and low oral bioavailability,which seriously limits its clinical efficacy.Therefore,this paper prepared PFEO into microcapsules by using microencapsulation technology,in order to improve the stability and bioavailability of the volatile oil,and finally facilitate the application and improve the curative effect.Based on this,this paper mainly studied PFEO extraction process optimization,component analysis,thermal stability,preparation and characterization of PFEO microcapsules,and antibacterial activity,and prepared a safe and effective,stable quality of PFEO microcapsule,in order to facilitate the industrial application of volatile oil formulations.1.Study on extraction process optimization,component analysis and thermal stability of the PFEO.(1)Extraction of volatile oil by steam distillation,on the basis of single factor experiment,the extraction time,immersion time and the ratio of liquid to materialwere taken as independent variables.and the yield of PFEO was used as the response value.Box-behnken response surface method was used to optimize the extraction process of PFEO.The results showed that the P value of the model was less than0.0001,indicating that the model was significant and could analyze and predict the extraction process parameters of PFEO;The effect size of the three factors on the oil yield was: extraction time > immersion time > the ratio of liquid to material;The best extraction process parameters of PFEO were: extraction time 3.2 h,immersion time1.4 h,the ratio of liquid to material 10: 1(m L/g),and the optimal predicted oil yield was 0.8599%,and the measured value was 0.8454%.It showed that the method was reasonable and feasible,which can provide reference for the selection of extraction parameters of steam distillation.(2)The chemical constituents of PFEO were investigated by GC-MS.The results showed that 11 compounds of the volatile oil were identified,the main components were perillaldehyde(73.59%),d-terpene diene(8.63%),caryophyllene(6.81%)of PFEO.And the method for the determination of Perillaldehyde in PFEO was studied.The results showed that the method was suitable for the component analysis of PFEO.(3)The thermal stability of PFEO under different heating temperatures and heating times were studied.The content of perillaldehyde in PFEO was determined by HPLC.The results showed that when the PFEO was heated to 24 hours at 25℃,40℃,60℃,80℃ and 100℃,the corresponding retention rates of Perillaldehyde were 95.76%,93%,88%,45.36% and 1.42%,respectively.It could be seen that the PFEO is sensitive to temperature,and when it reached a certain temperature,its active component Perillaldehyde degraded quickly,which indicated that it was greatly affected by temperature and time.2.Study on preparation process of PFEO microcapsules.(1)Preparation of PFEO microcapsules by spray-drying method to improve the stability and bioavailability of essential oil.Arabic gum and gelatin were selected as capsule material and emulsifier,PFEO was used as core material,and PEG6000 was used as plasticizer and anti-adhesive agent.According to the principle of Box-Behnken central composite design and the results of single-factor investigation,ratio of core material and capsule material,amount of solid,and temperature of inlet air,which have a greater effect on the microcapsules,were selected as independent variables.The comprehensive score of the encapsulation rate,drug loading and yield of microcapsules was taken as the response value.The optimum preparation process of PFEO microcapsules were obtained through response surface analysis method.The results showed that the P value of the model was 0.0005 less than 0.01,indicating that the model was significant and could be used to analyze and predict the preparation process of PFEO microcapsules;The effect size of the three factors on the comprehensive score was: amount of solid > temperature of inlet air > ratio of core material and capsule material;The best preparation process parameters of PFEO microcapsules were: ratio of core material and capsule material 0.3,amount of solid24%,temperature of inlet air 153℃,and the optimal predicted comprehensive score was 55.52%,and the measured value was 55.19%.This method provided a basis for the preparation of PFEO microcapsules by spray drying.(2)The method for HPLC analysis of perillaldehyde in PFEO microcapsules was established.The systematic applicability,linear relationship,precision,repeatability,stability,and sample recovery of the determination of perillaldehyde in PFEO microcapsules were investigated.The results were as following: The results of systematic applicability showed that the number of theoretical plates and separation degree of perillaldehyde were more than 8000 and 1.5,respectively.The blank microcapsule had no interference to the determination of perillaldehyde in PFEO microcapsules,which all met the requirements of pharmacopoeia;perillaldehyde showed good linearity in the concentration range of 2.58～154.8μg/m L;the RSD value of precision experiment of perillaldehyde was 0.23%,indicating that the instrument was good;the RSD value of repeatability experiment of perillaldehyde was 1.17%,indicating that the method was good;the stability experiment results showed that the RSD value of perillaldehyde was 0.09%,indicating that the sample was basically stable within 24 hours;The sample recovery experiment results showed that the recovery rates of perillaldehyde were 95%～105% and the RSD value of perillaldehyde was 1.75%,indicating that the recovery rate of perillaldehyde wasgood and the method was feasible.The content of perillaldehyde in PFEO microcapsules was obtained by reference substance.The results showed that the average content of perillaldehyde was 190.58 mg/g and the RSD value of perillaldehyde was 0.01%,which suggested that the content difference was small and the preparation process was stable.3.Quality evaluation of PFEO microcapsules.(1)The physicochemical properties of PFEO microcapsules were measured,including scanning electron microscopy(SEM),particle size and particle size distribution,encapsulation efficiency and drug loading,Fourier infrared spectroscopy(FTIR),X-ray diffraction(XRD),thermogravimetric analysis(TG),differential scanning calorimetry(DSC),in vitro dissolution and GC-MS analysis of the microcapsules.The results were as following: SEM results showed that the surface of the microcapsules was dense,smooth and round;the particle size of the microcapsules appeared uniform distribution,with the average particle size of 3.562μm;encapsulation efficiency and drug loading of the microcapsules were 77.00% and18.46%,respectively;FTIR results showed that most characteristic peaks of PFEO remained unchanged when the blank microcapsule contained PFEO,which indicated that the PFEO was successfully embedded into the microcapsules,and the chemical stability of the PFEO was good after encapsulation;XRD results showed that all samples had amorphous structures;results of TG and DSC showed that the thermal stability of the PFEO microcapsules was significantly higher than that of the PFEO;dissolution results in vitro showed that the cumulative release rate of microcapsules reached 75.63% after 12 hours,indicating that the microcapsules have good sustained-release properties and conform to the Higuchi diffusion model;GC-MS analysis results of the microcapsules showed that chemical composition of the PFEO had no significant change before and after the microencapsulation,but its relative content increased and decreased.(2)The effects of high temperature and high humidity(60℃,75%)on the stability of PFEO microcapsule were studied.The results showed that the relative contents of PFEO and PFEO microcapsules were 62.07% and 80.46%,respectively,when placedat 60℃ for 10 days.Compared with PFEO,it showed that the microcapsule was more stable to heat;When the relative humidity was 75% for 10 days,the relative contents of PFEO and PFEO microcapsules were 48.56% and 81.16% respectively,indicating that the moisture stability of the microcapsule was significantly higher than that of PFEO.(3)The antibacterial activity of PFEO microcapsules was studied.The antibacterial activity of PFEO microcapsules was determined by agar drilling method.The results showed that the PFEO microcapsules had long-lasting antibacterial activity against Staphylococcus aureus,Escherichia coli,Bacillus subtilis and Pseudomonas aeruginosa.Meanwhile,the minimal inhibitory concentration(MIC)and minimum bactericidal concentration(MBC)of PFEO microcapsules were determined.The results showed that the MIC and MBC of the microcapsules against Staphylococcus aureus,Escherichia coli and Bacillus subtilis were 0.16 mg/m L and 0.33 mg/m L,respectively,and the MIC and MBC of the microcapsules against Pseudomonas aeruginosa were 0.33 mg/m L and 0.66 mg/m L,respectively.