Study On The Identification And Content Determination Of Ganoderma Lucidum

Objective:The aim was to study the feasibility of using DNA barcoding to distinguish Ganoderma lucidum,based on internal transcribed spacer sequences,as well as specific PCR identification,and to establish specific HPLC methods for determination of ergosterol,uridine,adenosine and ganoderic acid A,respectively.It can be proposed to provide scientific basis for improving the quality control of Ganoderma lucidum,and simultaneously,to compare the contents of Ganoderma lucidum collected from different areas.Method:Firstly,collected 12 batches of Ganoderma lucidum and tested the characters,moisture,total ash,extract content and microscopic identification according to the“Chinese Pharmacopoeia”provisions.Secondly,a specific PCR identification method was developed.Thirdly,HPLC methods for determination of content were developed.1.The method for determination of ergosterol's content.The separation was performed on an Welch Ultimate LP-C₁₈8 column,?4.6×150mm,5?m?with methanol as the mobile phase at a flow rate of 1mL/min.DAD,with the 283nm detection wavelength,the column temperature of 26?and the injection volume of 10?l.Meanwhile,examined methodology.2.The method for determination of the content of uridine and adenosine.The separation was performed on an Agilent ZORBAX Eclipse XDB-C₁₈8 column?4.6×150mm,5?m?,methanol?A?-0.01mol/L potassium dihydrogen phosphate solution?B?as the mobile phase with a gradient elution at a flow rate of 0.8mL/min.The methanol ratio:5%?0min??5%?10min??13%?15min??13%?25min?.DAD,with the260nm detection wavelength and the column temperature of 26?.The injection volume:3?l reference substance and 10?l sample.Meanwhile,methodology was examined.3.The method for determination of ganoderic acid A's content.The separation was performed on an Welch Xtimate^TM-C₁₈8 column?4.6mm×250mm,5?m?,with the mobile phase of acetonitrile-0.01%acetic acid solution?13:87?at a flow rate of 1mL/min.The column temperature of 30?and the injection volume of 10?L.ELSD,the temperature of the drift tube was set at 105?,and the flow rate of nebulizing gas at2.8L/min.Meanwhile,methodology was examined.Results:The traits,moisture,total ash,extracts,and microscopic identification of Ganoderma lucidum were all in accordance with the provisions of the“Chinese Pharmacopoeia”.As well as the specific PCR identification method can clearly and accurately appear target bands.The regression equation of ergosterol was Y=1531.1X+3.358?r=1?.It had a good linear relationship within the range of 0.0832¹.664?g,and the average recovery rate was 98.68%?RSD=1.9%?.The regression equation of uridine and adenosine were Y=7.5404X-10.409?r=0.9998?and Y=27.845X-9.2962?r=0.9999?.There displayed good relationship within the range of 2.825⁹0.4?g/mL and 0.985⁹8.5?g/mL,respectively.The average recovery rate of uridine was 97.22%?RSD=0.56%?,and adenosine's was 97.71%?RSD=0.60%?.The regression equation of ganoderic acid A was Y=1.765X+5.5352?r=0.9998?.The logarithm of the peak area was in good linear relationship with the logarithm of the injection volume,within the injection volume ranged from 0.8928 to 5.3568?g.The average recovery rate was 95.81%?RSD=0.8%?.Conclusion:The specific PCR method was quick and effective.And the three HPLC determination methods were simple,accurate and reproducible,which can provide a reference method for the quality control of Ganoderma lucidum.The quality of Ganoderma lucidum,collected from different regions,were uneven.The content of the three kinds of compounds varied greatly.So it was important to its quality issues.