| Zhiling Capsules are capsules made from the mycelium of Mortierella SP isolated from Cordyceps larvae through artificial culture and fermentation.As an important substitute for wild Cordyceps,Zhiling Capsule has the effect of nourishing the lungs and kidneys.It is mostly used for cough and swelling caused by the deficiency of both lung and kidney.It can also be used for the adjuvant treatment of various nephropathy,chronic bronchial asthma,chronic hepatitis and tumors.There are many similar fermented cordyceps mycelium products,and Zhiling capsules are not exclusive varieties.In addition,its standards are still included in the drug standards of the Ministry of Health(WS3-B-2312-97).The examination items include character,routine examination of capsule,two physical and chemical identification,one thin layer identification of adenosine and content determination of adenosine and mannitol.The quality standard is relatively extensive.In order to improve its market competitiveness,this topic through the experiment to carry out a comprehensive study on the improvement of quality standard.The original standard was included in the 1997 Ministry of Health Drug Standards Chinese Medicine Prescriptions Volume 12.This subject first investigated the properties,which were consistent with the original standard,and were brown granules;then,the water content,the difference in filling amount,and the disintegration time limit were investigated,and the results all met the requirements of the capsule under the pharmacopoeia standard.In the identification study,on the basis of the original standard,this subject first reviewed the two physical and chemical identifications of mannitol and free amino acids,which showed good specificity and reproducibility.The sample powder was fixed by the smear method with chloral hydrate test solution,and the microscopic identification of the light yellow sub-prismatic cells and cordyceps mycelia in the 15 batches of Zhiling capsules was completed.Secondly,in the thin-layer chromatography identification,the thin-layer identification method of adenosine in the original standard was improved,and pure water was used to extract and the expansion conditions were changed to chloroform-ethyl acetate-isopropanol-water-concentrated ammonia test solution(8:2:6:0.3:0.15)and disodium hydrogen phosphate silica gel GF254 thin-layer plate,15 batches of samples and the reference substance adenosine and uridine showed the same color fluorescent spots at 254nm.In addition,the experiment added the thin-layer identification of three free amino acids of leucine,valine,and alanine.It is achieved by using n-butanol-water-glacial acetic acid(4:1:1)as the developing agent,continuous vertical development twice,spraying ninhydrin test solution,and heating at 105℃ for color development.Finally,in the identification of ergosterol,a sample solution was prepared by ultrasonic extraction with methanol,and spotted on a thin-layer silica gel G plate,with petroleum ether(60～90℃)-ethyl acetate-formic acid(5:1:0.1)as the development After spraying with 10%sulfuric acid ethanol solution,heating at 105℃ until the spots are clearly colored.As a result,all the batches showed the same color spots in the relative position of the control under the sunlight and 365nm.However,the thin-layer identification study of mannitols could not be completed due to unclear spots.In the assay study,the original standard contained only adenosine and mannitol.On this basis,this experiment added two nucleosides,guanosine and uridine,and ergosterol.High Performance Liquid Chromatography(HPLC)was used to determine the content of cordycepin and mannitol.As far as adenosine is concerned,by optimizing extraction conditions and chromatographic conditions,the content of adenosine in 15 batches of Zhiling capsules has been increased from the original standard not less than 0.25mg/capsule to 0.65～0.98mg/capsule.Then the guanosine content was determined at 256nm by degreasing with petroleum ether,ultrasonic extraction with 5%methanol aqueous solution,methanol-triethylamine solution as the mobile phase,and the final content range was 0.10～0.55mg/capsule.Secondly,the extraction methon of uridine is the same as guanosine,but the mobile phase is changed to a potassium dihydrogen phosphate aqueous solution-methanol system,and the detection wavelength is 254nm.As a result,the content of uridine in the 15 batches of Zhiling capsules ranges from 0.06 to 0.48 mg/capsule.There is no research on ergosterol in the original standard.The experiment was performed by ultrasonic extraction with methanol,and the detection was completed at 283nm with pure methanol as the mobile phase,and good results of 0.38～0.95mg/capsule were obtained in 15 batches of Zhiling capsules.The original standard titration method was used for the determination of the content of mannitol.In this experiment,sugar column and differential detector were tried to be used to determine the content of the water bath samples with ultra-pure water as the mobile phase at the column temperature of 80℃ and the test cell temperature of 55℃.The results were repeated several times in the later period of the methodology investigation and the results were not good.At last,the HPLC method was abandoned and titration method was used to complete the determination of mannitol.In the new titration method,this subject simplified the original reflux for 2 hours into ultrasonic extraction for 30 minutes.As a result,the content was also increased from no less than 18mg/capsule to 18.40～26.23mg/capsule.At the same time,this subject also conducted an exploratory study on the content of another important nucleoside substance-cordycepin.The results showed that cordycepin showed extremely low chromatographic peaks under 5 different extraction conditions,so we gave up the research on its content.Traditional Chinese medicine fingerprinting technology has become a highly internationally recognized quality control technology for traditional Chinese medicines.In the 2020 edition of the Chinese Pharmacopoeia,many traditional Chinese medicines and proprietary Chinese medicine preparations have been added to with fingerprint as a new means of quality control.But it has not been used in fermented cordyceps mycelium products and Cordyceps sinensis.This subject creatively carried out the fingerprint study and similarity comparison of 14 batches of Zhiling capsules,and the results showed that the similarity was not less than 0.854,and there were 7 chromatographic peaks in total.The three main chromatographic peaks of adenosine,uridine and guanosine were identified and confirmed,and the results of precision,repeatability,stability and chromatographic adaptability were good.In summary,the quality standards established in this experiment are more comprehensive,systematic,and reproducible,which can better reflect the quality standards of Zhiling Capsules and increase its market competitiveness. |
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