Study On The Technology Of Lyophilized Excipients Of Lycium Barbarum L. From Black Fruit Based On The Evaluation Of Antioxidant Activity And Chemical Composition

1.Objective Lycium ruthenicum Murr(Lrm)is a traditional Tibetan medicine,which can be used to treat heart disease and gynecological diseases.Nowadays,Lrm is widely used as health care products because of its well-known antioxidant.Lrm is rich in anthocyanins,resulting in a dark purple ripe fruit.Anthocyanins are susceptible to high temperature,so the traditional drying method cannot fully save its effective ingredient.This study is to use the freeze-dried shaping preparation technology to prepare the Lrm lyophilized shaping preparation.In order to maximize the protection of anthocyanins,we performed the preparation selection and the optimization from three aspects:the Appearance character index,the Chemical index and the Biological activity index.Thus,our study may provide theoretical and technical support for the application of freeze-dried shaping preparation technology in other traditional Chinese medicine containing thermal instable components.2.Methods Based on the biological activity of Lrm,this research studied the antioxidant effects by using the H2O2-induced injury in H9c2 cells model and selected simple and repeatable evaluation indexes of bioactivity.Moreover,the detection methods of total anthocyanins and polysaccharides were established.The selection of formula was carried out by single factor and Experimental design with four evaluation indexes(the preparation solution viscosity,appearance character,H9c2 apoptosis rate and total anthocyanin content).After selecting the best prescription,the freeze-dried curve was optimized with the principle of appearance character,microstructure as evaluation index,energy saving and environmental protection.After determining the optimum freeze-dried route,the packaging of the preparation was screened by the appearance character,water content and total anthocyanin content under accelerated conditions.Three batches were prepared according to the best process to preliminarily evaluate the procedure.The differences of appearance character,microstructure,disintegration time,water content and weight were evaluated.At the same time,the biological activity index and the active ingredient of Lrm Freeze-dried shaping tablets,the fresh Lrm and the dry Lrm were compared.3.Results(1)The optimal concentration of the H2O2 was 400 ?mol/L,and the treatment time was 6 h.The MTT assay showed that Lrm could enhance the H9c2 cell viability,the drug concentration used was 1.00 mg/mL,and the drug delivery time was 8 h.Next,the drug delivery model was determined by 1 mg/mL Lrm solution pretreated h9c2 for 2 h before adding 400 ?mol/L H2O2 for 6h.Finally,the cells were collected for Tunel assay,Realtime-PCR and western-blotting.Eventually,the H9C2 apoptosis rate was selected as the biological activity index of preparation process selection.The results of these tests indicated that Lrm had antioxidant effect and could protect the cells from oxidative stress damage.(2)This study established methods to detect the total anthocyanins by pH-Differential Method and the polysaccharide by Phenol-Sulfuric Acid Method.The best formula was mannitol 3.2%,Pullulan 3%,and Lrm powder 100mg/tablets evaluated by appearance index,preparation solution viscosity,anthocyanin content and apoptosis rate.Low energy consumption and good shape of the freeze-drying curve includes-20?,-12.5?,-5?,10 ?,35? five temperature sector,and the total time is 985 min.The best packaging is double aluminum bubble cover.(3)The 3 batches prepared according to the final process are homogeneous in appearance,orderly in microstructure,less than 15s in disintegration time,and water content is 4.3%.The weight difference also meets the requirement.The TUNEL assay results showed that there was no significant difference between freeze-dried Lrm and fresh Lrm in antioxidant activity(P>0.05),and the total anthocyanin content of one freeze-dried Lrm tablet was equal to 1.1 fresh Lrm.4.ConclusionThe freeze-dried shaping preparation of the Lrm is in accordance with the preparation in appearance,bioactivity and chemical composition.The total anthocyanin content is higher than that of the traditional dried Lrm,suggesting that the freeze-dried shaping preparation technology has important significance in the preservation of thermal instability components.