Researching On The Extraction,the Separation And The Activity Of Sennoside From Senna

Senna leaf is a kind of Chinese herbal medicine used as antibacterial,purgative and hemostatic which has a good application prospect.The constituents of Senna pod are similar to those of senna leaf.Therefore,this article aims to optimize the extraction and purification process of sennoside A and B from Senna pods and determine its bacteriostasis effect.In order to make full use of Senna resources,expand the medicinal parts and provide certain reference for the development and utilization of sennoside A and B of Senna pods.The main research contents and conclusions were as follows:1.Determination of content detection method of sennoside A and B and investigation the quality and stability of raw materialA simple and rapid method for the content detection of sennoside A and B by HPLC was confirmed.C18 column?250 mm×4.6 mm,5?m?was used,with acetonitrile-0.4%phosphoric acid water?20:80?as the mobile phase,340nm as detection wavelength,and column temperature was 25?.We found that sennoside A and B in Senna pod was higher than that in Senna leaf,and the content distribution in different parts was as follows:pod shell>blade?leaf petiole>fruit petiole>seed through the research on the quality of Senna leaf and Senna pod.The accelerated test of raw material showed that Senna pod should be sealed and stored in low temperature,dry and dark places,of which the validity period was one year.2.Optimization of extraction process of sennoside A and B from Senna podThe total extraction amount of sennoside A and B as the index,the single factor experiment combined with the orthogonal test was used to optimize the extract parameters while optimum pH for storage of extraction solution was investigated.The optimal extraction process parameters were obtained as follows:0.5%NaHCO₃solution,ratio of material to liquid 1:15,temperature 70?,extraction for 2 times and each time for 2 h.Under these conditions,the extraction rate could reach 94.84%.The results of the classical constant temperature test showed that the optimal pH for storage of Senna pods extract solution at room temperature was 7.5 with the recommended storage time was no more than 24 h.3.Optimizing the purification process of sennoside A and B by macroporous resin and investigating the stability of the extractFive kinds of macroporous resins DM130,AB-8,LX-17,HPD417 and ADS-F8,were screened by static absorption test and the dynamic column chromatographic parameters were optimized.The results showed that AB-8 resin had the best absorption and desorption performance.The optimum column chromatographic parameters were as follows:resin saturated absorption capacity was 0.21g·g^-1?equivalent as crude drug?,diameter-height ratio was 1:8,sample flow rate was 1BV·h^-1,elution flow rate was 2 BV·h^-1,impurities were removed by 3 BV water,target components were eluted with 3 BV 30%ethanol.The eluate was concentrated to dryness under reduced pressure at 70?and 0.08 MPa vacuum to obtain an extract with a total content of sennoside A and B of 26.05%with the yield of 7.18%,the enrichment recovery of 90.24%.The results of high temperature and accelerated test of sennoside A and B extracts showed that it should be sealed and stored in low temperature,dry and dark places,of which the validity period was one year.4.Commissioning of pilot-sacle process of the extracts of sennoside A and BAfter weighing 3 batches of 40 kg dry Senna pods,pilot production of sennoside A and B extracts was executed according to the optimized extraction and purification process.The ratio of material to liquid was adjusted to 1:10,the flow rate of the sample was to 2 BV·h^-1.As a result,the extract of sennoside A and B with a content of 26.15%was obtained,and the yield was 7.28%,the recovery rate was83.05%.5.Preparation of high purity sennoside A and BThe 26.65%extract was used as the raw material for silica gel column chromatography,and ethyl acetate-n-propanol-water?4:4:3?was used as the eluent.The sennoside A bright yellow needle crystals with a content of 97.39%can be obtained by one time column separation,and the sennoside B yellow needle crystals with a content of 95.42%can be obtained by the second time column separation.6.Preliminarily exploring the antibacterial activity of Senna podThe antibacterial effect of Escherichia coli,Streptococcus agalactiae,Staphylococcus aureus and Pseudomonas aeruginosa were determined.The results showed that the alkaline extract solution,aqueous extract solution,alcohol extract solution,alkali extract,sennoside A,B extract,water extract of senna pod and sennoside A,sennoside B had no antibacterial effect on the 4 kinds of bacteria.The ethanol extract had a certain antibacterial effect against Staphylococcus aureus and Pseudomonas aeruginosa,and the MIC of it for both bacteria were 25 mg?mL^-1,the MBC were 25 mg?mL^-1,while had no antibacterial effect on Escherichia coli and Streptococcus agalactiae.It was indicated that Senna pod contains antibacterial components which were easily soluble in alcohol,but sennoside A and sennoside B were not its antibacterial active ingredients.