Study On Preparation And Properties Of Limonene Liposomes

Limonene,whose scientific name is 1-methyl-4-?1-methyl vinyl?cyclohexene,is an important monocyclic monoterpene.It widely exists in citrus,vegetables and other plants,especially in the essential oils of citrus fruits.For example,the content of sweet orange peel essential oil is as high as 90%⁹5%.It is reported that citrus processing produces more than 800 thousand tons of citrus peel every year,which is the main raw material of D-limonene.Studies have shown that limonene has many physiological functions such as antibacterial,anti-oxidation,anti-cancer,anti-cough and anti-asthma and it has been widely used in the fields of food,spices,medicine and chemical industry.However,limonene is difficult for further preserve and use because limonene is easily to be oxidized and volatilized,and is hardly soluble in water.Therefore,this feature limits its application and promotion in food,medicine and health products.Liposome,as a new type of microcapsule carrier,has hydrophilic and lipophilic properties,which can improve the stability and bioavailability of the core material and it has been widely used.In this study,pre-experiments screen and determine the ethanol injection method for the preparation of limonene liposomes.By single factor and Box-Behnken response surface test,with encapsulation efficiency as the main evaluation index,the formulation and preparation process of limonene liposome were optimized,and the optimal preparation conditions of limonene liposomes were determined.A number of physical and chemical properties of limonene liposomes prepared under optimal conditions were studied,including the encapsulation efficiency,average particle size,polydispersity index,Zeta potential,morphology and so on.The experi-ments investigated the effects of temperature,light,pH,oxidizing and reducing agents on the storage stability of limonene liposomes.The stability and release of limonene liposomes were studied by vitro release and simulated gastrointestinal fluid.The main results are as follows:1.In this experiment,the content of limonene was detected by gas chroma-tography,and the linear relationship between the content of limonene and the peak area was established.The determination of limonene by gas chromatography was performed at an initial temperature of 40?for 1 min,with a temperature of 15?/min to 160?,a pause of 1 min,an inlet temperature of 250?,and the flow rate is 1.2mL/min.Without splitting,the injection volume was 0.1?L;use the external standard autosampler to inject directly.The standard curve of limonene was plotted as Y=46000X+74860,R²=0.9990,and the linear correlation was good.2.Limonene lipsomes were prepared by ethanol injection method with limonene as the material.The effect of preparation of main influence factors in the process includes the addition of soybean lecithin,cholesterol and limonene,the temperature and pH of phosphate buffer solution,magnetic stirrer speed etc.One-factor-at-a-time design was used to investigate the effects on encapsulation efficiency.On the basis of single-factor tests,three main process parameters that influence encapsulation eff-iciency including the cholesterol content,the limonene content and the temperature of phosphate buffer solution were optimized by response surface analysis based on a three-variable,three-level Box-Behnken experimental design.The optimal conditions for preparing limonene liposomes were determined as the amount of cholesterol is 8.8mg,limonene was added to 12.7 mg,and the temperature of PBS is 51?.Under these conditions,the actual encapsulation efficiency of limonene liposomes was?67.44±0.58?%,which showed a relative error of 0.4%when compared with the predictived value.3.The physicochemical indexes of limonene liposomes prepared under the above optimum conditions were tested.These indexes were as follows:the encap-sulation efficiency was?67.44±0.58?%,the average particle size was?165.4±2.08?nm,PDI was0.185±0.011,and the Zeta potential was?-16.23±0.569?mV.The prepared limonene liposomes were basically spherical or ellipsoidal by transmission electron microscopy,and a few of them were irregular,with no obvious adhesion and coalescence.Overall,liposomes showed good embedding effect on limonene.4.The vitro release properties of limonene liposomes were investigated and the release curves in vitro were draw.The dialysis membrane method was used,and the limonene phosphate buffer solution?0.5%Tween-80?was used as the control group to study the vitro release of limonene liposomes.The results showed that limonene solution could rapidly spread to the dissolution medium and achieve equilibrium in a relatively short time,while limonene liposomes showed sustained release,whose release rate was relatively slower,and the release rate of limonene was?42.92±1.78?%during 24hours.According to the fitting curve of dynamic model,the first order dynamic release model was obtained.The equation was Mt=45.64(1-e^-0.183t),and it has a good fit.5.During digestion experiment,artificial gastrointestinal fluid environment was investigated the release of limonene liposomes.The results showed that limonene liposomes in simulated gastric fluied was relatively stable,the initial release rate was relatively larger during 0¹ hour,and then the release rate decreased.The cumulative release rate in 3 hours was?11.78±1.15?%,indicating that limonene liposomes can be more stable in gastric fluid.But in the experiments of simulated intestinal fluid,the liposomes of limonene had a larger increase in particle size and a faster digestibility.At the end of 3 hours,the release rate reached?36.08±0.74?%,and no burst release phenomenon was found during the process.6.The stability of limonene liposomes was evaluated.With temperature,light,pH,and the redox agent as the influence factor,the physical and chemical stability of limonene liposomes on during a certain storage time was studied.The results showed that both temperature and light had a certain impact on the limonene liposomes within28 storage days,and the effect of temperature on the stability of liposomes was greater than that of light.The results indicated the stability of liposomes stored at?4±2??under dark showed best,whose indexes were relatively stable.Compared with the control group,adding 0.5%of reducing agent had a certain protective effect on limonene liposomes.Compared with pH 5.5 and pH 6.5,limonene liposomes diluted with pH 7.5 buffer solution have better stability.When 0.5%oxidant agent was added to the liposomes,the encapsulation efficiency and particle size of liposomes all have more significant changes.While adding 0.5%reducing agent,the change of liposome was smaller.This indicated that reducing agent had a certain protective effect on the limonene liposomes,which was more stable.