Study On Extraction And Separation Of Scopoletin Of Mulberry Leaves, Content Analysis And Antibacterial Activity In Vitro

China has a long history of planting mulberry and rearing silkworm.According to traditional medicine,each part of Morus can basically be used as medicines.Dynasties of Chinese Pharmacopoeias all have a record of medicinal efficacy of leaves,Ramulus Mori,Cortex Mori and Sorosis.Scopoletin is one kind of coumarin compounds,which exists in Scopoletin of solanaceae plants,Belladonna,umbelliferous plants,dahurian angelica and ect.It has effects onAntifungal,inhibiting decay of trees.In addition,there was activities for inhibiting monoamine oxidase,diminishing in flammation,expelling wind to relieving pain,Eliminating Phlegm,Spasmolysis, inhibiting tumor,Anti-Nasopharyngeal Carcinoma 9KB cells and ect.It also be found in mulberry which is important to study on the extraction and separation,screening of contents and Bioactivity of Scopoletin from mulberry.1.Extraction of ScopoletinWe intend to take newly-bred Artificial Triploid Mulberry Variety Jialing 20 which comes from Biotechnology College,Southwest University for our research materials.We take methods of ethanol extracting,then extraction,Crystallization,direct-reverse chromategraphy HPLC ect.to separate.We take Silica,polyamide,Macroporous Resin ect.for separation materials to extracte,separate and purify Scopoletin.Compare TLC with Standard,identify by Rf,we obtain the pure of Scopoletin.We isolate Scopoletin from leaves of Jialing 20 weighing 8kg,which weighing 23mg.2.Content Determination of Scopoletin from different Mulberry VarietiesThe Content of Scopoletin from7 Species,27 Varieties which are now widely planted in production is exactly determined by HPLC.The comparative results is(1)These is a big difference among Morus,that the content of Scopoletin is the highest in the leaves of Huosang(164.2375μg/g) while that is the lowest in the leaves of Dahongpi(25.1μg/g).(2)The content of Scopoletin is also different in different seasons.The result indicates that the content of Scopoletin in the leaves in winter is higher than that in summer,and the content of Scopoletin in the leaves of Huosang in summer is 164.2375μg/g while the content of Scopoletin is the highest in the leaves of Huosang in winter,which is up to 294.05μg/g,which provides the basis for the utilization of the leaves in winter.(3)The content of Scopoletin is also different in different parts.We compare the content of Scopoletin from different parts of mulberry and find that content of Scopoletin in branches(152.15μg/g) from Huosang and Sha 2 is lower than leaves(219.275μg/g),which shows that in the same variety the content of Scopoletin from leaves is higer than that from branches.We also compare the content of Scopoletin from the the leaves of broussonetia papyrifera,which has a near relationship with mulberry.The result indicates that the content of Scopoletin from most varieties of mulberry is higer than that from broussonetia papyrifera,while that is proximately between small parts varieties of mulberry and roussonetia papyrifera,which indicates that the content of Scopoletin has great difference among moraceae plants.That is the content of Scopoletin from most varieties of Morus is higer than that from broussonetia,which provides the experimental basis for the utilization and extraction of Morus.3.The research on antibacterial activity of Scopoletin in vitroThe activity of Scopoletin in vitro against bacteria and fungi is initially determined with the method of agar diffusion and agar dilution.Two methods are consistent.The results showed that Scopoletin inhibits Pseudomonas aeruginosa and Bacillus subtilis to certain extent,while Escherichia coli and Staphylococcus aureus are not significantly inhibited.The minimum inhibition concentration of Scopoletin to Pseudomonas aeruginosa is 62.5μg/mL to 125μg/mL,while to Bacillus subtilis is 125μg/mL to 250μg/mL.These is a big difference between different inhibition concentration and with the increase in concentration of scopolamine,inhibition also increases.Scopoletin also inhibits Candida to certain extent,and to a direct proportion relationship between the concentration gradient,and there is the relationship between the time.The optimum inhibition concentration is 500μg/mL to 1000μg/mL and the best inhibition time is 48 hours.