| Objective:According to the deficiency of liver and kidney,the liver depression and qi stagna and the deficiency of spleen and lung is easy to produce phlegm-dampness,and then accumulate blood stasis toxin leads to Alzheimer’s disease（AD）.So soothing the liver and regulating qi,invigorating spleen and stomach,clearing lung and eliminating phlegm are one of the keys to the treatment of AD.In this study,we propose a multi-target syndrome sequential therapy of“in the early stage,Eucommia ulmoides leaves replenish liver and kidney,strong muscles and bones and run through the whole process;in the middle stage,Aurantii Flos soothing the liver and regulating qi,removing phlegm and blood stasis;in the later stage,Gardeniae Flos clearing away heat and purging fire and detoxifying”.To develop the individual case experience of ancient dialectical treatment into a group integration program of sequential treatment combined with the strategic needs of"healthy aging"to develop dietary intervention series products for hypofunction in the elderly,Eucommia ulmoides leaves combined with Aurantii Flos and Gardeniae Flos was obtained by traditional decoction process to construct Fufang Duzhongye Jiaonang to meet the whole treatment needs of AD.In order to develop and utilize the resources of Eucommia ulmoides leaves,the pharmacodynamic material basis of Eucommia ulmoides leaves were preliminarily studied,the fingerprint and content determination method of index and characteristic components of Eucommia ulmoides leaves from different habitats were established,the harvest period and drying method of Eucommia ulmoides leaves were determined,and the pharmacognostic study was carried out,which provided a scientific basis for the development and quality control of effective parts and APIs of Eucommia ulmoides leaves.On the basis of completing the research on the APIs and capsules of Aurantii Flos and Gardeniae Flos,this paper mainly carried out the research on the preparation technology,quality standard and stability of the APIs and capsules of Compound Eucommia ulmoides leaves,providing a theoretical basis for the formulation of their preparation standards,So as to promote the innovative new drugs of Jiangxi genuine and characteristic medicinal materials,Eucommia ulmoides leaves,Aurantii Flos and Gardeniae Flos preparations to enter the clinical application faster.Method:（1）A disease model of Heterogeneous and Multifactorial Alzheimer’s disease（H/MAD）caused by aging,ischemia cardiovascular and cerebrovascular diseases and Aβprotein deposition was established.The pharmacological efficacy of Eucommia ulmoides leaves in the treatment of H/MAD was confirmed by the determination and observation of learning and memory,spatial exploration behavioral changes,changes in serum physiological and biochemical parameters in H/MAD model rats before and after administration.Spectroscopy was employed to analyze and structural identify the compounds and standards produced from the separation and purification of Eucommia ulmoides leaves using macroporous resin,silica gel,ODS-C18,preparative high performance liquid chromatography and other techniques.（2）HPLC was employed to evaluate and analyze the similarity of finger prints of 20 batches of Eucommia ulmoides leaves from various habitats,and t he optimal origin of the leaves was determined through component content and multivariate statistical analysis techniques such as cluster analysis（CA）.A mu lti-component method was established to determine the optimum harvest time a nd drying method of Eucommia ulmoides leaves,and pharmacognostics study o f Eucommia ulmoides leaves was carried out by character identification,micros copic identification and physicochemical identification.（3）Orthogonal design was used to determine the optimum extraction process of compound Eucommia ulmoides leaves by taking the contents of chlorogenic acid,geniposide and total flavonoids as indicators,and taking into account the extract yield.On this basis,according to the enrichment process of single herb,determined the optimal enrichment preparation process of compound Eucommia ulmoides leaves API,and the API was prepared by pilot test.The influence factor test and accelerated long-term test were carried out for the APIs of prepared compound Eucommia leaves,Eucommia leaves,Aurantii Flos and Gardeniae Flos,and the changes of the pharmacological and chemical properties of the four raw materials with time under different storage conditions and the stability study were investigated.（4）With the angle of repose as the evaluation index,the effects of the number of comminuted mesh,the type of excipients and the amount of excipients on the flowability of the compound Eucommia ulmoides leaves API were investigated,and the type of capsule shell and the amount of sample were investigated through the bulk density,finally determined the preparation process of the capsule.The compound Eucommia ulmoides leaves capsule was tested according to the capsule test items.The content of Fufang Duzhongye capsules was determined by HPLC with quercetin-3-O-sambubioside,rutin,isoquercitrin,Nicotiflorin,Astragalin in Eucommia ulmoides leaves,Narirutin,naringin,hesperidin,neohesperidin in Aurantii Flos,geniposide in Gardeniae Flos and chlorogenic acid and catechin in common components of the three herbs as index components.Investigated the stability of compound Eucommia ulmoides leaves capsules for 6 months accelerated and 9 months long-term,and established the quality standard of compound Eucommia ulmoides leaf capsules.Results:（1）The experimental results showed that Eucommia ulmoides leaf extract could shorten the latency period of H/MAD model rats,increase the number of stage passes,prolong the residence time in the fourth quadrant,increase the content of T-AOC in brain,decrease the content of TCh E,decrease the content of MDA in the serum of H/MAD model rats,and increase the content of CAT,SOD,and GSH-PX.Eucommia ulmoides leaf extract could exert antioxidant damage to slow down the aging of the body,and reducing neuronal injury and apoptosis.32 compounds were identified,including 1 sesquiterpene macrocolumn alkanes,13 lignans,7 simple phenylpropanoids,4 iridoids,5 flavonoids,1 phenolenes,and 1 monoterpenoid.（2）The similarity of HPLC fingerprints of 20 batches of Eucommia ulmoides leaves from different habitats was 0.883-0.998.A total of 19 common peaks were calibrated in the fingerprints,and the structures of 9 chromatographic peaks were identified by using reference materials.CA and PCA analysis methods could broadly divide 20 batches of Eucommia ulmoides leaves from different habitats into 4categories.OPLS-DA analysis results showed that 20 batches of Eucommia ulmoides leaves from different habitats had certain differences,and peaks 5,7,11 and 12 with VIP value>1 were the landmark components affecting the differences of Eucommia ulmoides leaves.The content of 20 batches of Eucommia ulmoides leaves from different habitats was determined,the contents of total iridoid terpene,chlorogenic acid and total flavonoids were taken as indexes.The results were consistent with multiple statistical analysis,indicating that except for two batches of Eucommia ulmoides leaves from Zhangjiajie boundary,Hunan Province,the content of Eucommia ulmoides leaves from Nanchang Xinjian District（school medicine planting garden）was the highest.（3）The comprehensive scoring results showed that the optimal decoction and extraction process of compound Eucommia ulmoides leaves was decoction thrid in 30minutes with the ratio of material to liquid 1:10.Combined with and referring to the early enrichment process of Aurantii Flos and Gardeniae Flos,pilot scale-up tests were performed to prepare compound Eucommia ulmoides leaves APIs;the influencing factor tests of APIs showed that the characteristics of the four APIs did not change significantly under high temperature and light conditions,and they were easy to absorb moisture lumps,deepen the color,and decrease the component content under high humidity conditions.The stability test of the drug substance showed that the properties,TLC identification,moisture content and content of the four apis did not change significantly under accelerated long-term conditions,indicating good stability.（4）The investigation results of angle of repose and bulk density show that:the smaller the number of crushing mesh,the smaller the angle of repose,the better the fluidity,and the greater the bulk density.Finally,the preparation process of capsules was determined as 65 mesh compound Eucommia ulmoides leaves APIs:dextrin:1%silicon dioxide=155:142:3 fully mixed and filled into No.1 capsules.The quality standard of Fufang Duzhongye Jiaonang was established:hard capsule,the content was dark gray,the TLC identification spots were consistent with the control sample,the disintegration time limit,water content,filling difference,heavy metal and microbial detection were in line with the requirements,and the content of the index components did not change significantly,all in line with the relevant provisions of the capsule.The stability test results showed that:the characteristics,TLC identification,water content,disintegration time and content of the capsule did not change significantly,which was in line with the relevant regulations of capsule preparation,indicating that the capsule had good stability and was suitable for industrial production.Conclusion:The HPLC fingerprint and multi-component content determination method of Eucommia ulmoides leaves established in this study are simple and reliable,which can be used to identify different sources of Eucommia ulmoides leaves,and provide basis for the selection and quality evaluation of the best place of origin,drying method and harvest time of Eucommia ulmoides leaves.The preparation process of Fufang Duzhongye Jiaonang was determined,including the preparation method of the API,the type of excipients and the dosage of excipients,and the finished product of the capsule was prepared in accordance with the specifications of the pilot test,which confirmed the good stability of the compound Eucommia ulmoides leaves capsule,and provided a theoretical basis for industrial production and quality control. |
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