| Antrodia camphorata is aphyllophorales,porous,thin hole bacteria genera.Antrodia effective part is sporophore Antrodia,which parasite on the Taiwan camphor tree.It isthe perfect hangover cure,has the good activi ty of protecting liver and anti-tumor[1].Modern researches showed that the main physiological active substances are polysaccharide,triterpene,antrodia Antrocampin,adenosine,superoxide dismutase(SOD)and small molecule protein(including immune proteins)[3].Triterpene component is the main active ingredient of antitumor.Due to Antrodia is almost artificial cultivation products on the market at present,the wild Antrodia is scarce and expensive.The main cultivation mode for Antrodia is basswood,sol id agar and liquid fermentation.Different ways will lead to differences in the quality of medicinal materials.Therefore,this study was performed for total tri terpenoid on effective part of the preparation of extraction and purification process and HPLC fingerprint of Antrodia herbs.Objective:To establish HPLC fingerprint of Antrodia medicinal material,in order to provide standard reference for identification of antrodia medicinal activities and cultivations:By using ultraviolet-visible spectrophotometry,establish of Antrodia mthod and condition in determination of Antrodia camphorata total triterpenoids(ACTT);In total triterpenoid extraction rate as the index,used single factor combined with central composite design-response surface methodology to investigate different extraction methods,different extraction solvents,solvent amount of times,extraction temperature,extraction timeextraction times methods for Antrodia camphorata total triterpenoids extraction yield,the influence of optimum process conditions of total triterpene extract.At the same time using macroporous resin of antrodia antitumor activity component concentration and purification,provide the scientific basis for the further development and utilization of Antrodia.Methods:1.The factors of fingerprints for the chromatographic were investigated,including the mobile phase,the mobile phase gradient,separation of different columns,column temperature,flow rate,detection wavelength and acquisition time.According to the investigation,the best conditions for chromatographic fingerprint was determined,and then methodology study was carried out.Finally,10 different batches of sample was determined under optimum chromatographic conditions.The HPLC fingerprints for effective parts of Antrodia camphorate herbs was established.2.Establishment of the measurement method of total triterpenoid:using ultraviolet spectrophotometric method for determination with Antrodia camphorata total triterpenoid content as one of the indicators.Determine the chromogenic system;Using single factor experiment,to inspect the dosage of chromogenic agent,the water bath temperature,the water bath time,ice water bath time on measurement of total triterpenoid content,determine the best method of content determination.3.Total triterpenoid extraction technology research:in the total triterpenoid extraction rate as the index,using the single factor experiment to investige the way of total triterpene extract,ethanol concentration,solvent amount,extraction temperature,extraction time and extraction times on the effects of total triterpenes extraction rate,determine the optimal extraction process and validation.4.Total.triterpenoid purification technology research:do static adsorption and desorption test,determine the resin type,dynamic test on the sample investigation,sample weightconcentration on sample,elution solvent,elution volume and other factors,which affects the purity of total triterpenes.Results:1.Set up the HPLC fingerprint of Antrodia effective materials,its chromatographic conditions:Kromasil C18 reverse phase chromatography column(C18 250 mm*4.6 mm,5 ?m);gradient elution of acetonitrile-0.1%phosphate system;column temperature of 30?;the flow rate of was 1.0mLˇmin-1;detection wavelength of 254nm;Sample volume was 10 mu L;the acquisition time of 115 min.2.Antrodia total triterpenoid content determination method is established,and its content determination conditions as follows:the vanillin-glacial acetic acid-perchlorate as chromogenic system,detection wavelength of 548 nm,chromogenic condition is:0.4mL dosage of 5%vanillin-glacial acetic acid,0.8 mL dosage of perchlorate,water bath temperature for 60?,water bath time was 20 min,ice water bath was 5 min.3.Total triterpenoid Antrodia extraction technology was optimized:extraction methods for the extraction of reflux method,the dosage of solvent was 70%ethanol,ethanol was 20 bv,water bath temperature was 70 ?,60 min bath time,extraction 2 times.Extraction yield of Antrodia total triterpenoid is up to 11.79%.4.Establish the preparation of high purity total triterpenoid purification process.5.The total triterpenoid purification process was optimized:the macroporous resin model was HPD100,the amount of the sample(g)was 1 g of the crude drug,the eluent was 60%ethanol,the elution volume was 5BV,the total triterpenoid purity was above to 50%.Conclusion:1.To establish HPLC fingerprint of Antrodia medicinal materials,reflect the characteristics of Antrodia medicinal material composition,can solve the problem its quality identification defects,control the better quality of medicinal materials and suggest the direction for the artificial cultivation.2.Set up of Antrodia total triterpenoid content measurement method which is simple,operational,stable,repeatable,accurate.At the same time the star design-response surface method is adopted to optimize the extraction technology for total triterpenes,determine the best extraction technological conditions,can provide the reference for the follow-up quality standards of medicinal and theirs development.3.Using macroporous adsorption resin to purify Antrodia total triterpenoid.The method is simple and practical.It can provide a reference for the late preparation. |
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