| Lingzhi is the Chinese name given to the Ganoderma family of mushrooms, which was considered the most valuable medicine in ancient China and was believed to bringlongevity. Today, Lingzhi is still widely revered as a valuable health supplement and herbal medicine worldwide, as studies into the medicinal and nutritional values of Lingzhi revealed that it does indeed contain certain bioactive ingredients that might be beneficial for the prevention and treatment of a variety of ailments, including important diseases such as hypertension, diabetes, hepatitis, cancers, and AIDS.Following the successful cultivation of Ganoderma, mushroom fruiting bodies, mycelia and related products have been widely used as functional foods and medicine. However, identification and classification data applicable to Lingzhi is wholly inadequate, and the present position relating to the taxonomy of Ganoderma species cultivated in China is chaotic. Uncertainty over species identity may have resulted in the misinterpretation and/or different explanations of experimental data relating to Lingzhi, thereby creating a major problem for research and quality control of Lingzhi species cultivated in China. Chromatographic fingerprint analysis provides a more rational approach to the authentication and quality assessment of traditional Chinese medicine (TCM), and many TCM materials have been classified in recent years based on this methodology. Although HPLC has been used previously in Lingzhi classification, chromatographic fingerprinting and related data analyses have not been applied.There are six parts in this dissertation. Chemical identification of Lingzhi species cultivated in China using HPLC fingerprints, studies on similarity evaluation of chromatographic fingerprint for edible fungi, studies on pattern classification, fingerprint analysis of Lingzhi mycelia cultured for different time, uses of fingerprint analysis in Lingzhi cultivation and study on correlation between characteristic of Lingzhi HPLC fingerprint and pharmacodynamics were discussed in this dissertation.1 Chemical identification of Lingzhi species cultivated in China using HPLC fingerprinting Sample preparation: Powdered sample (1 g) was transferred into a 50 mL volumetricflask, made up to volume with 95% analytical alcohol, and the mixture was shaken forapproximately 5 min and then left to stand for 14 h. Suspensions were filtered throughanalytical filter paper and 10 mL of filtrate was evaporated to dryness at 60℃undervacuum. Dried extract was re-dissolved in 1.5 mL methanol.HPLC conditions: Sampleswere separated on a reversed-phase column (YMC-Pack ODS-AQ, 5μm,250 mm×4.6 mmi.d.YMC,Japan).The mobile phase consisted of 1% (v/v) acetic acid in Milli-Q water (A)and methanol (B) using an isocratic program of 52% (A) and 48% (B). The flow rate was1.0 mL/min and the temperature of the column was maintained at 30℃.The detectionwavelength was set at 250 run, and the total recording time was 90 min. The 105 strainswere divided into 20 groups, the similarities of the samples in the same group were allhigher than 0.96. The standard fingerprints of group A and group B were constructed.2 Studies on similarity evaluation of chromatographic fingerprint for edible fungiA similarity calculation software named Dminer is developed cooperated with Demen. A new matching algorithm is put forwarded based on sequence subsection and similarity seaking was used in similarity calculating.3 Studies on pattern classificationThe 105 strains were divided into 20 groups using Hierachicar Cluster Analysis of SPSS 13.0. Two discriminant functions were got using Discriminant Analysis, they are Canonical Discriminant Function and Fisher's Discriminant Function.Canonical Discriminant Function:Y1=-23.448 + 24.895X1 + 14.394X6-20.210X8-8.528X20+ 5.995X24 + 6.902X32 Y2=-9.938 - 16.254X1 + 23.608X6+ 8.213X8- 10.460X20 +2.146X24 + 2.686X32Y3=-6.562 - 2.331X1 + 5.499X6+ 1.582X8+ 3.101X20-1.823X24+0.533X32Y4=-0.169-1.768X1 + 2.676X6-2.539X8+1.062X20-0.403X24+1.141X32Discriminant standard:Y1<0 and Y2<0 and Y3>0 and Y4>0 : the first group Y1>0 and Y2>0 and Y3<0 and Y4<0 : the second group Y1>0 and Y2<0 and Y3>0 and Y4=0 : the third group Y1<0 and Y2<0 and Y3<0 and Y4>0 : the fourth group Y1<0 and Y2<0 and Y3<0 and Y4<0 : the fifth group Fisher's Discriminant Function:Y1=-65.602- 192.393X1+ 197.155X6+ 114.916X8 - 37.872X20-10.227X24+11.469X32 Y2=-3088.592 + 1185.282X1 + 1854.772X6-1135.129X8 - 940.602X20 + 485.312X24 + 581.760X32 Y3=-12443.598 + 4237.206X1 + 1740.703X6 - 3327.746X8 - 1062.902X20 + 870.498X24 + 1017.906X32 Y4=-4.167 + 37.605X1 + 50.804X6 - 35.973X8 - 25.317X20 + 13.763X24 + 16.992X32 Y5=-1.609Discriminant standard: the sample belong to the group of the biggest function value.4 Development of HPLC fingerprints of mycelia cultivated for different time and different conditionThe extracelluar triterpenoids at different static culture time and different shaking culture time showed low inhibition effect to L1210,the inhibition ratios all below 45%, on the HPLC fingerprints the peaks appeared before 40 min.The fingerprints showed that static culture could be good for the synthesis of triterpenoids and those peaks appeared between 60min and 75min. The inhibition ratio of G. lucidum 156 was higher than 90% at 148 h of static culture condition and the inhibition ratio of G. lucidum 16 was higher than 90% at 268 h of static culture condition.The similarity between the HPLC fingerprints of G. lucidum 156's fruiting bodies and mycelia cultured for 70 days is 0.90113. The inhibition ratio of the extracts of G.lucidum 156 fruiting bodies to L1210 is 71% and that of the extracts of G. lucidum 156's mycelia cultured for 70 days is 90%. This result showed that suitable culture condition can get mycelia with higher inhibition effect to L1210.5 Uses of fingerprint analysis in Lingzhi cultivationBy comparing the HPLC chromatographies of samples of different earthed time, different harvesting time and different culture methods we found that the triterpenoids of these samples changed a lot and the difference of triterpenoids contents of these samples changed from 20% to 70%. The triterpenoids contents of samples cultivated with Sawdust Medium were higher than these cultivated with logs. The triterpenoids contents of samples harvested before autumn were higher than these harvested at autumn period. The triterpenoids contents of samples earthed for 1 m were higher than these earthed for 1.5 m. The similarities of HPLC fingerprints of different earthed time ,different harvesting time and different culture methods were all higher than 0.98. Four parts of fruiting bodies were used for tissue isolation and the isolated strainswere cultured on four media. The similarities between chromatograms of these sampleswere calculated. The results are as follows: under the same culture condition,thesimilarities of HPLC fingerprints of the strains isolated from different parts were all higherthan 0.99. Meanwhile, the similarities of HPLC fingerprints of the strains isolated from thesame part were all higher than 0.96 in different media. The effects of different isolationtissues and media to the HPLC chromatograms of the fruiting bodies were not distinctive.The triterpenoid content of strain 18 cultured on medium C is the highest among the testedfruiting bodies. The best isolated part is the upper layer of pileus and medium C is the bestmedium. The triterpenoid composition in the fruiting bodies of Ganoderma lucidum ismainly decided by their genetic factors; the growth condition will affect the content oftriterpenoid compound and this effect can be showed by different peak-area ratios of thechromatogram.6 Study on correlation between characteristic of Lingzhi fingerprint and pharmacodynamicsAt the premise of the acquired characteristic of fingerprint and pharmacodynamic data, grey relational analysis was selected to study the relationship between represented by fingerprints and the inhibition effect of Lingzhi to K562. The results showed that the inhibition effects are highly related with the constituents of the retention time tR31.397,tR 25.058,tR25.980,tR26.681,tR27.772,tR29.431,tR32.225,tR15.739,tR22.523,tR16.829, tR20.131 min. According to the relational grade, the sequence(from small to big) of contRibution to inhibition effect is as followes,tR11.329,tR37.709,tR38.680,tR12.918, tR 10.699, tR 35.692, tR 14.689,tR 34.573, tR 41.242, tR 13.375, tR 44.671, tR 28.590, tR 14.012, tR 12.279, tR31.397, tR25.058, tR25.980, tR27.772, tR 26.681, tR 29.431, tR 32.225, tR 15.739, tR22.523, tR 16.829, tR20.131.
|
PDF Full Text Request