Construction And Evaluation Of Limonoids Nanostructure Lipid Carrier

Limonin compounds are a class of secondary metabolites of tetracyclic triterpenoids containing furan rings and highly oxidized.They are important bioactive substances in lemon seeds and have anti-cancer,anti-viral,anti-oxidation,insecticidal,bacteriostatic,anti-inflammatory and analgesic activities.In spite of this,due to the high extraction cost and low extraction efficiency of limonin compounds,at present,most processing enterprises will treat lemon residue and seeds as waste.In addition,limonin compounds are fat-soluble substances with poor water solubility and low utilization rate,which limits the application of limonin in food,medicine,agriculture and other fields.In order to effectively utilize limonin-like in lemon fruit and improve the application value of lemon fruit,this study firstly used reverse micelle method to extract limonin-like in lemon seeds,optimized the extraction process to determine the best extraction technology,and investigated the antibacterial activity of the extracted substance.Then,limonoids nanostructure lipid carrier(Lmd-NLC)were prepared by microemulsification method,and the optimal formulation was determined.The effects of nano lipid carrier technology on anticancer activity of limonin were investigated.In addition,the limonoids nanostructure lipid carrier were applied in lemon juice to prepare a healthy drink rich in limonids,which provides reference for the application of limonin-like in food field.The main results of this study are as follows:(1)Optimization of extraction process of limonoids by reverse micelles andfunctional analysis of antibacterial activityThe experiment of limonids was extracted into two key parts: preparation process of initial extract and reverse micelle extraction.The optimum conditions of the two parts were obtained through process optimization.In the initial stage,the main factors affecting extraction efficiency were the extraction time of 1.5 h,the liquid to material ratio of 30:1(m L/g),the HPMC addition amount of 0.5%,the material liquid p H at 6.5,and at this condition the extraction rate was(3.34±0.02)mg/g.P-B test and B-B test results showed that the optimal formulation of limonoids extraction were dioctadecyl dimethyl ammonium chloride(DODMAC)44.30 mmol/L,orthohexanol 13.86%,bromotetradnet 3.88%,and the average yield of limonoids under the corrected process conditions was(87.64±1.12)%.The bacteriostatic test showed that the MIC and MBC of limonoids acted on Penicillium digitatum were 1250 μg/m L and 2500 μg/m L,and the MIC and MBC of limonoids acted on Penicillium italicum were 1250 μg/m L and 5000 μg/m L,respectively.Furthermore,after the determination of AKP,it was found that the AKP activity gradually increased with the extension of action time and the increase of purity.(2)The construction of limonoids nanostructure lipid carrier and analysis of anticancer activityBy single factor test and response surface test,the optimal preparation process of Lmd-NLC was determined as following: liquid lipids accounted for58.60% of total lipids,surfactant accounted for 2.05 times of total lipids,Drugs accounted for 8.94% of the total lipids,and the encapsulated rate of limonids under the corrected process conditions was(94.81±1.2)%.The diameter of the prepared Lmd-NLC was normally distributed,the mean particle size was(127.6±1.5)nm,the polydispersion coefficient(PDI)was(0.233±0.012),and the average Zeta potential was(-14.36±0.34)m V.Cell tests showed that Limonoids and Lmd-NLC showed significant inhibitory activity on the proliferation of MCF-7 and A549 cancer cells,and this effect increased with drug concentration and process time.After 48 h treatment with the same drug concentration,Lmd-NLC showed significantly higher inhibitory activity against the proliferation of MCF-7 and A549 cancer cells than limonoids,showing obvious slow-release properties.(3)Application of Limonoids nanostructure lipid carrier in lemon compound juiceThrough single factor test and orthogonal test,the optimal formula of Composite juice was lemon juice 20.0%,honey 12.0%,white sugar 10.0% and pectin 0.15%.The combined sensory score of the composite lemon juice under this formulation condition was 92.2 ± 1.2.Comparing the contents of limonoids in lemon NFC juice and lemon complex juice,the contents of limonoids in complex juice were as high as 73.32μg/m L,which was much higher than 6.87μg/m L in NFC juice.Stored for 60 days in the same environment,the content of limonoids in NFC juice fluctuated by 17.9%,while the content of limonoids in complex juice fluctuated by only 2.3%,suggesting that the limonods degradation in fruit juice could be effectively alleviated by coating with nanotechnology.The release rate of Lmd-NLC in simulated gastric juice and intestinal juice was investigated.The release rate of nanoparticles was 38.7% within 2h in simulated gastric juice and complete release within 2h in simulated intestinal fluid.The results indicated that limonoids was mainly released in intestinal fluid,which could effectively improve its bioavailability.