Separation And Purification Of Phloretin From Apple Tree Branches And Design And Evaluation On Its Drug Delivery System

China has a wide area of apple cultivation and abundant resources.However,a large number of apple branches are produced every year due to apple tree pruning and other reasons,which are mostly abandoned,resulting in resource waste and environmental pollution.Dihydrochalcone compounds were abundant in apple tree branches,among which the content of phloridzin and phloretin were higher,and phloretin is the aglycone of phloridzin which has important pharmacological activities.The current studies on phloretin mainly focus on its pharmacological activity,and there are few studies on the extraction,purification and improvement of water solubility of phloretin.The research on the extraction and purification of phloretin is mainly based on the traditional extraction and separation technology,which has a long production cycle and it is difficult to expand production,the most important is the low yield and poor quality of phloretin.Phloretin has poor water solubility,stability and bioavailability,but like most flavonoids,which has good lipid solubility.Therefore,we used an efficient and environmentally friendly method to extract phloretin from apple tree branches,and the extract was isolated and purified to obtain high purity phloretin.Finally,the bioavailability of phloretin was improved by using three natural carrier materials.The results of the study are as following.1.Tween-80 as a surfactant,cellulase and pectinase as complex enzyme,phloretin was extracted from apple tree branches by surfactant micellar assisted enzyme method,The total extraction rate of phlorizin and phloretin were taken as the indicators,the conversion of phlorizin into phloretin in the pretreatment solution according to the hydrolysis reaction mechanism was the conversion amount of phloretin.The optimum extraction conditions as follows:the extraction temperature is 55?,the pH value is 4.5,the enzymatic hydrolysis time is 3.5 hours,the enzyme concentration is 2.5%,the ratio of pectinase to cellulase is 3:1,the amount of surfactant is 5%,the solid-liquid ratio is 1:11.Under the above conditions,the total extraction amount of phloretin was 25.18 mg/g,and the extraction amount of phloretin was 7.26 mg/g.On the basis of surfactant micellar assisted enzymatic extraction of phloretin from apple branches,ultrasonic microwave assisted extraction of phloretin was carried out,the optimal conditions were as follows:the ratio of solid to liquid was 1:20,the surfactant content was 6.0%,the microwave power was 400 W,and the microwave irradiation time was 6min.Under these conditions,the total extraction amount of the root cortex element was 28.21 mg/g,and the extraction amount of the root cortex element was 10.51 mg/g.Therefore,pretreatment with surfactant micellar assisted enzyme,and then extraction of apple branches by ultrasonic-microwave synergistic method can increase the extraction rate of phloretin.Ethanol was used as the solvent to extract apple branch,and the total extraction rate of phloretin and the extraction rate of phloretin were 22.59 mg/g and 0.33mg/g,respectively.2.Phlorizin and phloretin was separated from extract by surfactant micellar turbidity point and their yields were 85.4%and 84.9%.Dry solid powder can be obtained by washing the upper phase liquid with chloroform.The content of phlorizin and phloretin in the solid powder is 8.9%and 3.2%respectively.The solid powder treated with ethanol,dilute hydrochloric acid and ethyl acetate was used to obtain 59.63%phloretin.With dimethyl sulfoxide as the solvent,water as the reverse solvent,using the solvent precipitation method to purification of phloretin,the best purification condition is:the volume of solvent and the solvent ratio was 1:10,deposition time was 5 min,deposition temperature was 28?,the concentration of the raw phloretin was 46 mg/mL,purity and yield of phloretin were 98.11%and 86.74%respectively.The purified samples were determined by HPLC,FTIR,DSC and LC-MS,and the purified samples were identified as phloretin.3.The phloretin is loaded with porous starch,and the effects of different factors on the drug loading and encapsulation rate of porous starch were investigated,the optimal conditions were determined as follows:adsorption time was 30 min,concentration of phloretin was 150 mg/mL,ratio of rhizocellin to porous starch was 1:3 Under this condition,the drug loading of porous starch loaded phloretin was 12.8%,and the encapsulation rate was 44.4%.The morphology of rhizoctin,porous starch and porous starch loaded with phloretin was characterized by scanning electron microscopy.The morphology of porous starch loaded phloretin was basically the same as that of porous starch.The adsorption medium and mechanical stirring had no effect on the morphology and porosity of porous powder.The porous starch loaded phloretin,the pores were filled with phloretin and the specific surface area decreased obviously.The results of FTIR,X-ray diffraction and differential thermal scanning showed that the crystallinity of phloretin decreased obviously after being loaded with porous starch.In the thermogravimetric curve,the retention rate of porous starch loaded phloretin was between that of phloretin and porous starch.According to the retention rate of each substance,the drug load of porous starch loaded phloretin was calculated to be 13.7%,which was basically consistent with that measured by HPLC.4.The inclusion complex of phloretin was prepared by mixing phloretin with hydroxypropyl-?-cyclodextrin in ethanol medium at a material ratio of 1:1.Cellulose membrane with water content of 83.4%was prepared by static culture and freezing and thawing treatment.The aqueous solution of the inclusion compound was adsorbed by bacterial cellulose.The inclusion compound supported by bacterial cellulose was freeze-dried,and the drug load was 2.52%,the encapsulation rate was 57.2%.SEM,FTIR,X-ray diffraction,differential thermal scanning and thermogravimetric analysis showed that the inclusion complex was formed by phloretin and hydroxypropyl-?-cyclodextrin at a mass of 1:1.The bacterial cellulose membranes are tightly packed with filaments and the freeze-dried cellulose membrane adsorbed the inclusion solution showed a more porous network structure.The inclusion compound of hydroxypropyl-?-cyclodextrin was successfully adsorbed by bacterial cellulose membrane.According to the retention rate of each substance in the thermogravimetric curve,the calculated drug load of bacterial cellulose was 2.43%,which was basically consistent with the results determined by HPLC.5.The saturated solubility of phloretin,porous starch loaded phloretin,inclusion compound,bacterial cellulose loaded inclusion compound in artificial gastric juice,pH 4.5 acetic acid-sodium acetate buffer and artificial intestinal fluid was determined,the saturated solubility in the medium of artificial gastric juice were 29.07?g/mL,40.43?g/mL,49.14mg/mL and 48.87mg/mL,which were 28.93?g/mL,44.29?g/mL,59.81mg/mL and 60.12mg/mL in acetic acid-sodium acetate buffer medium and 61.40?g/mL,81.90?g/mL,64.09mg/mL and 65.58mg/mL in artificial intestinal fluid medium.The saturated solubility of porous starch loaded phloretin,inclusion compound and bacterial cellulose loaded inclusion compound were higher than that of the raw phloretin in the three medium.At 12h,the cumulative release rate of porous starch loaded phloretin,inclusion compound and bacterial cellulose loaded inclusion compound was 12.7 times,17.4 times and 9.5 times of the raw phloretin in the medium of artificial gastric juice,which was 10.4 times,16.2 times and 8.7 times higher than that of the raw phloretin in the pH 4.5 acetic acid-sodium acetate buffer medium,and 7.8 times,7.3 times and 5.9 times of the raw phloretin in the medium of the artificial intestinal fluid respectively.The results showed that the solubility and dissolution rate of phloretin could be improved by several carriers.The stability of phloretin,porous starch loaded phloretin,inclusion compound,bacterial cellulose loaded inclusion compound in artificial gastric juice,pH 4.5 acetic acid-sodium acetate buffer and artificial intestinal fluid was determined.Phloretin and its delivery system has good stability in the artificial gastric juice and pH 4.5 acetic acid-sodium acetate buffer,phloretin is prone to degradation in the artificial intestinal juice,porous starch,hydroxypropyl-?-cyclodextrin and bacterial cellulose loaded phloretin can alleviate the degradation of phloretin and improve the stability of it,and the effect of bacterial cellulose on the degradation rate of phloretin was stronger than that of porous starch which was stronger than hydroxypropyl-?-cyclodextrin.The above results showed that the three carriers could improve the solubility of phloretin and enhance the stability of it.The results of lipid oxidation resistance,hydroxyl radical scavenging ability and reducing power indicated that phloretin had good antioxidant activity in vitro,and the antioxidant capacity of the phloretin loaded with carriers was better than that of the raw phloretin6.The studies of the blood concentration in mice shown that the bioavailability of porous starch loaded phloretin,inclusion compound and bacterial cellulose loaded inclusion compound were 1.89,2.39 and 4.56 times of that of the raw phloretin,respectively.The bioavailability of phloretin increased with the increase of solubility of it.Compared with the other two carriers,the bacterial cellulose membrane expanded after absorbing water,and the slow release drug reduced the degradation rate of phloretin in gastrointestinal tract,which made it have the highest bioavailability of phloretin.The higher the release rate,the faster the degradation rate may be in the gastrointestinal tract.Therefore,the improvement of the bioavailability of phloretin does not absolutely depend on the increase of its solubility.The histological distribution of phloretin showed that the maximum concentration of phloretin in the heart,liver,spleen,lung and brain organs of rats in the phloretin and inclusion compound group appeared 1h after administration.After 2h of administration,the concentration of phloretin in heart,liver,spleen,lung and brain organs reached the maximum that treated with porous starch loaded phloretin and bacterial cellulose group loaded inclusion compound.The maximum concentration of phloretin in the viscera of rats in the group of porous starch loaded phloretin,inclusion compound and bacterial cellulose loaded inclusion compoundwas higher than that in the raw phloretin group,which were 1.20,1.21 and 1.34 times of the raw phloretin in the heart,1.58?1.88?1.94 times in liver,1.32,1.76 and 1.75 times in lung,1.63,1.89 and 1.86 times in spleen,3.00?3.90 and 5.10 times in brain.The concentration of phloretin in the kidney of rats in the raw phloretin group,porous starch loaded phloretin group,inclusion compound and bacterial cellulose inclusion compound group reached the maximum at 4h,6h,4h and 6h after administration,which were 1.46,1.59 and 1.83 times of the raw phloretin,respectively.