Study On The Extraction And Separation Of Lignans And HPLC Fingerprint Of Schisandrae Chinensis Fructus

Schisandrae Chinensis Fructus is the dried ripe fruit of the Magnoliaceae plantSchisandra (Schisandra chinensis (Turcz.) Baill.). Schisandrae Chinensis Fructus has beencommonly used as a traditional Chinese medicine for a thousand years. It is used to treatdiseases such as cough, nocturnal spermatorrhea, enuresis, frequent urination, spontaneoussweating, night sweats, heat diabetes, heart palpitations, insomnia.It is reported to containlignans, volatile oil, polysaccharide and other chemical composition.Modern medicineresearch has demonstrated that most of the biological activites and pharmacological effectsof it can be attributed to its lignan constituents, particularly the C18-dibenzocyclooctadiene-type lignans, which can lower the glutamate-pyruvate transaminase (SGPT) level in serum,and show anti-hepatitis,antioxidative, antitumor, anti-HIV, calcium antagonism,and animmuno-modulating activities.Chromatographic fingerprint analysis borrowed the concept of fingerprint identificationfrom forensic. Chromatographic fingerprint analysis uses modern analytical techniques tocharacterize and describe the chemical information of traditional Chinese medicine in theway of graphic (image).It emphasizes the similarity of the groups of the same medicine, theuniqueness in species groups rather than the absolute uniqueness of individual. Theidentification of Chinese herbal medicines based on the chemical composition of plantmetabolism,it is different between individuals because of the different environment for thegrowth,meanwhile, there are bound to groups shared similarities between individualsbecause of the the hereditary of biological metabolism. This kind of graph (image) withspecies uniqueness and individual similarity has the significance of fingerprint. The mostbasic property of chromatographic fingerprint analysis is that the similarity is reflected in theintegrity and ambiguity.This research project is established based on the requirements of the project of NationalKey New Drug Creation.Using Schisandrae Chinensis Fructus as the object of study, three aspect of research areincluded in this dissertation1. The optimum extraction conditions were studied and filtered ①Two solvent extraction were studied: reflux extraction and ultrasonic extraction.②The xtraction conditions were filtered:Solvent multiples, solvent concentration, extractiontimes and extraction time were selected as four factors affecting the extraction rate of lignansfrom Schisandrae Chinensis Fructus. The optimum extraction conditions were determinedafter L9(34) orthogonal experiment:ultrasonic extracted three times with12times of95%ethanol,25min each time. The optimum extraction conditions were verified by experiments,the average content of total lignans is18.2316mg/g.2. Purification and identification of lignans from Schisandrae Chinensis FructusThe optimum conditions were used to extract the lignans from Schisandrae ChinensisFructus, the conventional method, column chromatography was used to the separation ofcrude extract,7components were isolated; Two compounds were isolated and purified bysilica gel column chromatography and recrystallization.The identification results of TLC,HPLC and1H-NMR confirmed that compound I and II were Schisandrin and γ-Schisandrin.3. Study on the HPLC fingerprint of Schisandrae Chinensis Fructus①The best analytical conditions were established based on the number,height,shape ofpeaks,separation and analysis time and so on. The conditions of HPLC analysis:detector:SPD-20AV UV detector; Detection wavelength:254nm; Chromatography column:AgilentZORBAX300SB-C18(4.6×250mm,5μm) chromatography column; Column temperature:27°C; Mobile phase:methanol-water gradient elution; Flow rate:0.5mL/min; Injectionvolume:10μL.The preparation methods of solution is:The powder of samples wereweighed up precisely and placed in a100mL Erlenmeyer flask，and were ultrasonicextracted2times with15times of methanol,25min each time.The extract was Ultrasounddissolved with methanol and dilute to50mL,5mL was removed to25mL volumetric flaskand constant volume with methanol,0.45μm microporous membrane was used to filtrate.②Methodological study confirmed the applicability of the HPLC conditions.13samples from different origins were analysed under the conditions. The Hierarchical clusteranalysis gived by SPSS and the analysis results gived by chromatographic fingerprintsimilarity evaluation system get uniform results,and11kinds of samples were used toestablish the representative HPLC fingerprint.12Common peaks were calibrated and5components were determined compared with the HPLC chromatogram of referencestandard(Peak No.2： Schisandrin，Peak No.5：Schisandrol B, Peak No.6： SchisantherinA, Peak No.9：Deoxyschizandrin，Peak No.11：Schisandrin B). The analysis results gived by chromatographic fingerprint similarity evaluation systemindicated the similarity between samples and representative HPLC fingerprint are over0.9,so the HPLC fingerprint can be used to distinguish between different Schisandrae ChinensisFructus and be used as reference of quality assessment of Schisandrae Chinensis Fructus: itcan be considered to the same medicinal materials if the similarity between the sample andthe representative HPLC fingerprint is over0.90.