| Sika deer antler anti-osteoporosis capsule(hereinafter referred to as the antler capsule)composed of four herbs of sika deer antler,pueraria,malaytea scurfpea fruit,cucumber seeds,In order to control its quality,16 samples of drug samples and 4 negative control samples were used as test materials.From the two aspects of qualitative identification and quantitative analysis,the pilose antler capsule was analyzed and evaluated.And the psoralen and psoralen were used as the index components to establish the fingerprint of the antler capsule,to further improve and perfect the quality standards of antler capsules for the actual production and clinical application to provide a scientific basis.Qualitative identificationIn this study,according to the provisions of the 2015 regulations of "Chinese Pharmacopoeia",and comprehensive and relevant literature of various kinds of identification methods,he sample processing method,TLC conditions,colorimetric conditions and sample volume were optimized,so as to select the most suitable method for the identification of.The identification method used in this experiment has the characteristics of strong,simple and accurate.Results showed that he stripes and spots of the chromatograms of sika deer antler,radix puerariae,malaytea scurfpea fruit,and cucumber seed were clear.And the control sample has the same color spots in the corresponding position,negative control without interference,can be included in the quality standards of antler capsules.According to the "Chinese medicine injection fingerprints of the technical requirements(temporary)" in the relevant provisions of psoralen and psoralen as an index to establish the velvet capsule HPLC fingerprints.Using Agilent-C18(5m,4.6x250mm)chromatographic column;methanol(A):water(B)gradient elution;detection wavelength of 318 nm;column temperature:25℃;injection volume:10μL as chromatographic conditions of fingerprint of 16 samples and 4 negative control samples fingerprint detection,ethodology test has proved that the method is stable accurate and reliable.Using the fingerprint similarity evaluation system of Chinese medicine(2012),the fingerprint of each sample was analyzed and the similarity was evaluated,results showed that the similarity of 1-16 samples were above 0.8.Quantitative analysisThe content of psoralen,isopsoralen and puerarin were detected by high performance liquid chromatography(HPLC);the content of collagen was detected by bicinchoninic acid method(BCA);the contents of polysaccharides and phosphorus were determined by UV spectrophotometry;amino acids were deteremined by amino acid automatic analyzer;and the content of calcium,zinc and iron by atomic absorption detection.The detection of psoralen and isopsoralen: Antler capsule methanol ultrasonic extraction 30min;methanol-water gradient elution;detection wavelength 240 nm.The linear relationship of psoralen and isopsoralen were good in the range of 5 ~ 25μg / mL;the repeatability of the method was good and the RSD were 1.15% and 0.76%;the sample recovery rate were99.7%and 100.7%.Determination of psoralen and isopsoralen content in the antler capsules were not lower than 0.207mg/g,0.209mg/g.The detection of puerarin: Antler capsules 30% ethanol ultrasonic extract 30 min;mobile phase: methanol-water(25:75);the detection wavelength of 250 nm.The linear relationship of puerarin was good in the range of 0.96~4.8μg/mL;the repeatability of the method was good and the RSD was 2.01%;the sample recovery rate was 98.4%.Determination of Puerarin content in the antler capsules was not lower than 38.57μg/g.The detection of collagen: Extraction of the collagen in antler capsules by trypsin hydrolysis,the glucose standard solution,the sample solution,the negative reference substance solution and the BCA working solution were pointd into the 96-well plate,the absorbance at 562 nm was measured by a microplate reader.The linear relationship of collagen was good in the range of 0.5 ~ 10μg / mL.Determination of collagen content in the antler capsules not less than 29.73%.The detection of polysaccharides: The samples were extracted with HCl solution in boiling water for 60 min,neutralization with NaOH solution and add the DNS,boiling water bath for 5 min,spectrophotometer determination of 503 nm absorbance,it is concluded that the content of total sugar.The samples were extracted by 85% ethanol,and the ethanol was recovered and adds the DNS,boiling water bath 5min,the absorbance of 520 nm was measured by spectrophotometer to obtain the content of reducing sugar.Polysaccharide = total sugar-reducing sugar.The linear relationship of total sugar and reducing sugar were good in the range of 0.02~0.08mg/mL.Determination of polysaccharide content in the antler capsule is not less than 0.88 mg/g.The detection of amino acids: The samples were added with hydrochloric acid and hydrolyzed at 110 ° C for 22 h.Instrument condition:ion exchange column 4.6mm × 60 mm,3μm sulfonic acid cationic resin;column temperature 57 ℃;reaction temperature 135 ℃;separation buffer flow rate: 0.40 mL / min;ninhydrin reaction solution flow rate: 0.35 mL / min;wavelength 570,440 nm,analysis time 30 min.Determine the amount of amino acids in the antler capsule shall not be less than 31.53%,the total essential amino acid is not less than 10.88%.The detection of inorganic elements: After digestion with perchloric acid and nitric acid.The digestive fluid was measured by atomic absorption spectrophotometer,the linear relationship of the three elements of calcium,zinc and iron were in good range with 4~20μg/mL、0.5~2μg/mL、2~8μg/mL.The absorbance at 660 nm was measured by ultraviolet spectrophotometer,the content of phosphorus was measured,the linear relationship of phosphorus content was good in the range of 5 ~ 50μg / mL.Determination of the antler capsule calcium,zinc,iron,phosphorus content of four elements shall not be less than 32.33%,6.39%,0.12mg/g,0.62 mg/g.The quantitative analysis method has the advantages of precision,stability and good reproducibility,which can be used for the quality control of the components of the sika deer antler anti-osteoporosis capsule. |
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