Preparation And Evaluation Of Oregano Essential Oil Loaded Nanostructured Lipid Carrier

Oregano essential oil（OEO）is one of the important active ingredients of Origanum Vulgare Linn.It has a variety of medicinal properties,including antioxidant,antibacterial,antifungal,anti-inflammatory properties.It also has the pharmacological activities of regulating blood glucose and cardiovascular disease,anti-tumor,anti-virus and other pharmacological activities.At present,there are more studies on the pharmacological activity of this essential oil,and less research on its preparation field.Based on this,this study prepared oregano essential oil loaded nanostructured lipid carriers（OEO-NLC）,and established the multi-component content determination method by gas chromatography（GC）.carried out prescription process optimization,freeze-drying process research,preparation quality evaluation and activity evaluation.The problems of insoluble and strong volatility of oregano essential oil were effectively solved,and its application quality was improved to a certain extent,providing more basis for its industrial application1.Literature analysis and research ideasBased on the nanocolloid delivery system,the composition and structural characteristics of essential oils loaded nanostructured lipid carriers（EOs-NLCs）were introduced.Nanostructured lipid carriers（NLCs）had an impact on the physical and chemical properties,stability and active ingredients of essential oils.Explained how to overcome the shortcomings of essential oils and protect essential oils from adverse environmental influences,so as to improve the stability,bioavailability and safety of essential oil,and realize the slow and controlled release of active components.This paper summarized the development and current situation of oregano essential oil loaded nanostructured lipid carrier in the fields of pharmacy,food,cosmetics and skin care products,and summarized Oregano essential oil and The research progress of its preparations was expected to lay the foundation for the research of oregano essential oil loaded nanostructured lipid carrier.2.Establishment of a method for the determination of multi-component content of oregano essential oil loaded nanostructured lipid carrierA method for the determination of multi-component content of oregano essential oil loaded nanostructured lipid carrier was established by gas chromatography,and to investigate the content of carvacrol,p-cymene and thymol in OEO-NLC.The results showed that p-cymene,thymol,carvacrol and the internal standard n-undecane were separated well,and the blank nanostructured lipid carrier had no interference to the content determination of the sample and had good specificity.The linear ranges of p-cymene,thymol and carvacrol were 1.976～126.464μg·m L^-1,17.888-1144.832μg·m L^-1and 8.926～571.290μg·m L^-1,respectively.The precision test results showed that the relative standard deviation（RSD）values of p-cymene,thymol and carvacrol were 0.95%,0.21%and 0.32%,respectively,which indicated that the precision of the instrument was good.The results of reproducibility test showed that the RSD values of p-cymene,thymol and carvacrol were 1.62%,1.75%and 1.61%,respectively,indicating that the method had good reproducibility.The results of the stability test showed that the RSD values of p-cymene,thymol,and carvesol were 1.84%,0.52%,and 0.60%,respectively,indicating that the OEO-NLC test product was stable within48 hours;The sample recovery rates are all between 95%and 105%.The average contents of the three components were 0.1294mg/m L,2.1974mg/m L and 1.1023mg/m L,respectively,and the RSD values were 1.71%,0.99%and 1.11%,respectively,indicating that the preparation process was stable and the method was feasible.3.Preparation of oregano essential oil loaded nanostructured lipid carrierUsing preicirol ATO-5 as solid lipid,decanoyl/octanoyl-glycerides as liquid lipid,poloxamer 188 and egg yolk lecithin as composite emulsifiers,oregano essential oil loaded nanostructured lipid carrier was prepared by melt emulsification ultrasonic dispersion method.Through the single factor test,the lipid dosage,emulsifier ratio,and emulsifier dosage were selected as box Behnken response surface factors,and the particle size and three index components of p-cymene,thymol and carvacrol were used as evaluation index to optimize the formulation.The results showed that the optimal formulation of oregano essential oil loaded nanostructured lipid carrier was as follows:preicirol ATO-5 0.14g,decanoyl/octanoyl-glycerides 0.06g,egg yolk lecithin0.1g,poloxamer 188 0.3g,oregano essential oil 0.2 g,the melting temperature was73°C,stirring at 1000r/min speed for 25min,75%amplitude ultrasonic dispersion for15min（ultrasound 5S,interval 7S）,cooling and solidification for 30min to obtain OEO-NLC.In the study of OEO-NLC freeze-drying process,10%sucrose was finally selected as the lyophilized protective agent of OEO-NLC,and the quality of the freeze-dried powder was the best.4.Quality evaluation of oregano essential oil loaded nanostructured lipid carrierThe OEO-NLC prepared by the optimal formulation process was light yellow opalescence under natural light,clear and transparent.Transmission electron microscopy（TEM）results showed that the nanoparticles were spherical and evenly distributed,the average particle size was 45.00±0.48nm,the particle size distribution was 0.230±0.007,and the Zeta potential was-29.4±0.56mv;the encapsulation efficiency of p-cymene,thymol,and carveol were 89.53%92.33%and 91.76%respectively,and the drug loadings were 0.42%,6.42%,and 3.33%,respectively.FTIR and DSC results showed that OEO has been successfully embedded in nanostructured lipid carriers;XRD results showed that the perfect crystal structure of glyceryl distearate in OEO-NLC has changed,forming a new disordered phase,which once again showed that the preparation of OEO-NLC was successful;the in vitro release results showed that OEO-NLC had a sustained release effect;The results of storage stability showed that OEO-NLC should be stored at 4℃in light protection compared with 25℃.5.Preliminary evaluation of antibacterial activity of oregano essential oil and its nanostructured lipid carrierA preliminary study on the in vitro antibacterial activity of oregano essential oil and its nanostructured lipid carrier was carried out by using the double dilution method of micro broth.MIC and MBC of oregano essential oil and its nanostructured lipid carrier againsted two kinds of gram positive bacteria（Enterococcus faecalis,Listeria monocytogenes）,two kinds of gram negative bacteria（Shigella dysenteriae,Pseudomonas aeruginosa）and Candida albicans were determined.The MIC results showed that within 24 hours,the antibacterial activity of oregano essential oil and its nanostructured lipid carrier was equivalent to that of the four bacteria,while the antibacterial activity of oregano essential oil nanostructured lipid carrier against Candida albicans was stronger than that of oregano essential oil.The results of MBC showed that the oregano essential oil and its nanostructured lipid carrier had similar bactericidal effect,with the best bactericidal effect against Shigella dysenteriae,followed by the other three bacteria,and the worst bactericidal effect against Candida albicans.