| Objective To analyze and determine the constant and trace elements,collagen protein and fatty acid composition in Gray of animal drugs.We believe these compositions in Gray promote the skin and bone repair and healing.The researches of Gray illuminate the pharmacodynamic components for the follow-up research on the skin and bone repair and healing.Methodexp.1 To determination of Gray Na,K,Ca,Mg,and P,Sr,Ba,Mn,Fe,Cu,Zn after the sample were processed by hydro digestion method(HNO3-HClO4),using inductively coupled plasma atomic emission spectrometry(ICP-AES),moreover, we analyzed the Ca's form in Gray.exp.2 Based on the hydroxyproline is the collagen specificity amino acids,we decomposed the collagen in samples into hydroxyproline after hydrolysis with hydrochloric acid,It can be derivatized with phenylisothiocyanat(PITC)for the determination,by HPLC(reversed-phase C18column A:0.1mol/L NaAc-CH3CN (94:6)(pH6.5);B:CH3CN-H2O(80:20,v/v)as mobile phase,detection at 254nm), so the collagen content can be determined by hydroxyproline.exp.3 Use the HIP solvent(low toxicity reagent which is composed of n-hexane/ isopropanol)extraction Gray's lipid composition,and then through the saponification, extraction and derivative byω-acetophenone of bromide,supplemented with catalytic reagents Triethanolamine.HPLC condition:MeOH- CH3CN-H2O(325:135:50)as the mobile phase,the C18 column separation,UV-VIS detector at 242 nm.We detecte the tetradecanoic acid,palmic acid,octadecane acid,oleic acid and linoleic acid.Resultsexp.1 We had determined 11 kind of constant elements and trace element simultaneously,which are Na,K,Ca,Mg,P,Sr,Ba,Mn,Fe,Cu and Zn.The minimum of Precision RSD is Na element(0.46%)and the maximum is Cu element (4.01%);the minimum of the average recovery rate of 11 kinds of trace elements and major elements in the six samples we had determined is Mg element(90.90%)and the maximum is Cu element(109.14%).The content of each element in Gray is:Na 7.880mg/g,K0.5071mg/g,Mg2.896mg/g,Sr0.3223mg/g,Ba0.1187mg/g,Mn 0.01864mg/g,Fe0.5908mg/g,Cu0.01543mg/g,Zn 0.1410mg/g,Ca214.9mg/g,P 83.28mg/g,which proved that Ca and P are highest,it is related to the bone structure of Gray which exists in form of Ca3(PO4)2.The content ratio Ca/P is 2 generally.According this experiment,the content ratio Ca/P is about 3.We can infer that there is free calcium in the test solution of Gray,and it had been proved,the average content is 3.838mg/g.exp.2 In the linearity range 0.02～0.20mg/mL the linear relationship is good(r=0.9999),the average recovery rate is 102.0%,RSD is 2.47%,the hydroxyproline content of 4 batch of Gray(n=3)is 14.13mg·g-1,14.20mg·g-1, 14.33mg·g-1,14.39mg·g-1respectively.According to the content of hydroxyproline in collagen are about 10%,and then we can calculate that the content of collagen in Gray is about 14.25%.exp.3 Simultaneously examines 5 kind of fatty acid,respectively for the tetradecanoic acid,palmic acid,octadecane acid,oleic acid and linoleic acid.We also have established these 5 fatty acid quota examination method,the tetradecanoic acid linearity range is 0.05～0.45μg,r is 0.9997,the linoleic acid linearity range is 0.10～0.90μL,r is 0.9995,the palmic acid linearity range is 0.10～0.90μg,r is 0.9994, the oleic acid linearity range is 0.10～0.90μL,r is 0.9993,the octadecane acid linearity range is 0.033～0.30μg,r is 0.9996;the average recovery rate respectively is 100.27%,98.74%,101.94%,99.36%,97.99%,method RSD respectively is 2.92%, 3.76%,1.88%,2.55%,1.44%;We have determined the 5 kind of fatty acids of 3 batch of Gray(n=3),its average content respectively be 0.0597μg/g,0.121μL/g, 0.112μg/g,0.116μL/g,0.0472μg/g.Conclusion This experiment indicates in Gray of the traditional Chinese medicine, the pharmacodynamic components that promote the skin and bone repair and healing compose complex and diverse.In the experiments which analyzed and determined these components,we first used the advance and rapid ICP-AES method to determine the constant element and trace elements in Gray.We first establish two methods,one is to analysis the collagen in Gray after PITC derivatization,and the other is to determine the fatty acid composition of Gray by the high-performance liquid chromatography.These two methods determined the content of collagen protein and fatty acid in Gray precisely.At present,the Chinese medicine researches in animals are not more extensive and in-depth study than in plants,Gray is one of them.We established three methods in this study,which provide standard of methods for rapid and effective analysis Gray in the further.These researches also illuminate the pharmacodynamic components for further study on promoting the skin and bone repair and healing of Gray. |
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