| In this study, the nucleoside composition and content in Hirsutella sinensis mycelia, the anamorph of aweto, were analyzed and the RP-HPLC and SEC-HPLC fingerprint analysis methods for small and big molecule respectively were also studied.RP-HPLE/ESI-MS results showed that the H. sinensis water extract contained cytidine, uridine, inosine, guanosine, adenosine, but didn't contain codycepin. RP-HPLC analysis further showed that the contents of these nucleosides were: cytidine 565.452±3.157, uridine 1004.056±2.418, inosine 40.957±0.400, guanosine 905.858±7.073, adenosine 748.621±4.915 (μg/g). The regression equations were: A=1.491E-005C-0.766,y=1.365E-005x-0.533, y=9.159E-006x-0.034, y=9.118E-006x-0.128, y=1.032E-005x -0.161, y=9.788E-006x - 0.038, respectively(x: the concentration, y: the peak-area). The recoveries were 87.20±3.52%, 97.46±0.80%, 98.06±1.04%, 87.54±1.02%, 76.92±1.58% and 100.50±1.42%, respectively. Extraction method study revealed that hot-water extraction was better than 50% methanol-water hot and ultrasonic extraction. The proportion of the six nucleosides were 17.3︰30.8︰1.3︰27.7︰22.9︰0.0 in H. sinensis, which was similar to that in C. sinensis. By calculating the"cosine value of vectorial angle", their similarity was 0.89. The similarities of RP-HPLC profiles of six Cordyceps or its products were calculated with a"The fingerprint similarity calculating software from National Pharmacopoeia Committee". The result indicated that the similarity between aweto and H. sinensis was the highest (0.799). The similarities between aweto and C. sinensis CS1, Jinshuibao capsules, mycelia and fruiting bodies of C. militaris were 0.718,0.680,0.737 and 0.504, respectively. Therefore, the nucleoside content and the RP-HPLC fingerprint analysis could be markers and quality control method for H. sinensis and its related products.In order to establish a method to quantitatively evaluate the similarity of big molecule substances, the polysaccharide extracts from Ganoderma genus (5 species, 23 strains in total) were analyzed by using size exclusive chromatography HPLC (SEC-HPLC). The result revealed that by using"cosine value of vectorial angle"to process all time point signal data as variables in the effective separation scope (10-23min), the similarity could be properly calculated. Base on similarity, the clustering relation of the Ganoderma genus could be recurred with"Between-group linkage"method, and the accuracy was 87%. By applying this method, the big molecule substances in H. sinensis, C. sinensis CS1, Jinshuibao capsule, mycelia and fruiting bodies of C. militaris were analyzed, and compared to that of aweto. The results showed that the molecular weight distribution of C. sinensis and H. sinensis was very close (similarity 0.966), higher than that in between aweto and C. sinensis CS1, Jinshuibao capsules, mycelia of C. militaris, except that in between fruiting bodies of C. militaris (the similarities 0.966,0.843,0.805,0.675 and 0.978 respectively). The results were coincident with that of RP-HPLC analysis. Further studies on different H. sinensis extracts from which were cultured with ten kinds of medium and harvested at 1, 3, 6 months showed a relatively high similarities. It suggested that the culture conditions had inapparent effect on SEC-HPLC chromatogram shifting. It could also be used as an analytical method for quality control of Cordyceps and related products.This study provides methods and data base for quality control for the anamorph of aweto, and provided new methods and ideas for big molecule fingerprint analysis for medicinal mushroom extracts. |
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