Study On The Comprehensive Quality Control Mode And Preliminary Activity Of Ganoderma Lucidum

Ganoderma lucidum is a well-known edible mushroom and traditional Chinese medicine（TCM）,it contains a variety of physiological activity,such as anti-tumor,anti-aging,anti-oxidation,hypoglycemic,regulating immunity activity.Currently,the species of Ganoderma lucidum on the market were complex.It is not scientific and comprehensive to evaluate the quality of Ganoderma lucidum only by determining the total content of triterpenoids and polysaccharides.In this project,the Ganoderma lucidum triterpenoids and amino acids as research object were studied systematically from the aspects of extraction,content determination and fingerprint by various advanced technology of quality control.（1）Five extraction methods of Ganoderma lucidum triterpenoids were compared,including heating reflux extraction,ultrasonic extraction,microwave extraction,ultrasonic-assisted enzymatic extraction and microwave-assisted enzymatic extraction.Response surface methodology（RSM）with Box-Behnken design（BBD）method was successfully applied to optimize the factors of the ultrasonic extraction.The maximum yield of triterpenoids reached 0.7338+0.0150%,under the condition of 87%ethanol,liquid-to-solid ratio 28:1mL/g and extraction time 36 min.（2）Ten triterpenoid compounds were determined by multi-index quantitative,including lucideric acid C,lucideric acid LM₁,ganoderic acid G,lucideric acid B,ganoderic acid A,lucideric acid A,ganoderenic acid D,ganoderic acid D,lucideric acid D and ganoderic acid F.On this basis,the ganoderic acid A was took as reference,and the relative correction factors between ganoderic acid A and other nine components were calculated.A quantitative analysis of multi-components by single marker（QAMS）was established.In addition,a new pre-column fluorescence labelling method with HPLC-FLD was developed for determination of total triterpene acids in Ganoderma lucidum by using4-Bromomethyl-7-methoxycoumain as derivative reagent.The total content of triterpene acids was 0.18%in Ganoderma lucidum karst,and0.19%in Ganoderma sinense.This method had strong specificity,which was a powerful supplement to the quality control of Ganoderma lucidum.（3）Based on HPLC-LTQ-Orbitrap-MSⁿ and literature dates,24Ganoderma lucidum triterpenoids were identified.The common mode of HPLC fingerprint（including 11 characteristic peaks:lucideric acid LM₁,ganoderic acid G,ganoderic acid B,lucideric acid E,ganoderenic acid A,ganoderic acid A,lucideric acid A,ganoderenic acid D,ganoderic acid D,lucideric acid D and ganoderic acid F）and TLC fingerprint（including 9characteristic peaks）were generated by Chempattern^TM software.By cluster analysis,the origins of Ganoderma lucidum from different sources were distinguished quickly.Ganoderma lucidum and Ganoderma sinense were distinguished by using principal component analysis.The fingerprint method combined with the stoichiometry could be applied to identify the species and origins of Ganoderma lucidum.（4）Amino acids were extracted from the Ganoderma lucidum by using an improved hydrochloric acid-hydrolysis method.The optimal preparation conditions were as follows:liquid-to-solid ratio 20:1mL/g,extraction time 9.5h and hydrochloric acid concentration 7.0mol/L.Under this optimal condition,the extraction yield of amino acids was4.78±0.21%.Then,the crude extract was subjected to activated charcoal decoloration and ethanol precipitation to give the final product and the content of amino acids was 17.78%.The composition of amino acids in Ganoderma lucidum was analyzed by HPLC with PITC pre-column derivation method.The Ganoderma lucidum mainly contained 17 amino acids in them of which 8 were human essential amino acids.Analyzing the composition of amino acids in Ganoderma lucidum could also be effective in controlling the nutritional quality of Ganoderma lucidum.（5）Three major components in Ganoderma lucidum（triterpenoids polysaccharides and amino acids）were extracted and activity analyzed respectively.The results showed that Ganoderma lucidum triterpenoids exhibited highα-glucosidase inhibitory with IC₅₀ value of 301.63μg/mL,and the IC₅₀ value of positive control（Acarbose）was 1801.01μg/mL.For antioxidant activity,the Ganoderma lucidum triterpenoids displayed a certain capacity of scavenging DPPH free radicals as a slow antioxidant.The IC₅₀ value of triterpenoids was 229.03μg/mL,and the IC₅₀ value of positive control（VC）was 18.20μg/mL.This project provided technical guidance for the establishment of a comprehensive and systematic method of quality control on Ganoderma lucidum.It also offered scientific basis for the research and production of Ganoderma lucidum health care products and pharmaceuticals.