Gentiana Germplasm Research

Radix Gentianae Macrophyllae, a traditional Chinese medicine, has been frequently used to dispel rheumatism and ease pain. There are four species of Gentiana (G. macrophylla, G. straminea, G. dahurica and G. crassicaulis) recorded as the original plants in the Chinese Pharmacopoeia. Besides, G. officinalis and G. siphonantha,two other Gentiana species which are mainly planted in Gansu and Hebei, Sichuan and Qinghai respectively, are often used as the substitutes. In this study, the main morphological variations were observed and recorded on the basis of extensive collection of germplasm resources. The DNA fingerprints of the collective germplasms were established by AFLP.Six species of Gentiana including G. macrophylla,G. straminea, G. dahurica and G. crassicaulis, G. officinalis and G. siphonantha were compared in morphological features, chemical composition, analgesic and inflammatory activities, which would provide the scientific information for the further rational utilization of Gentiana resources. The main results showed as follows:1.Abundant morphological variations were observed and recorded in G. macrophylla, G. straminea, G. crassicaulis and G. dahurica.On this basis,89 germplasms were collected according to the main morphological characteristics.2. The seeds of the six Gentiana species were compared and the seed surface was observed by scanning electron microscopy. Obvious differences were observed among the six species containing the exterior color, aspect ratio,1000-grain weight, hilum size and the striation length.3.Totally,1068 bands containing 1067 polymorphic bands were generated by AFLP from the 89 Gentiana germplasms using the seven primer combinations. The average number of bands amplified by each primer pair was 152.27 and all the 89 germplasms shared one common band, viz. E+ACT/M+CAA-385.5.4. Seven primer combinations generated 159,220,219 and 154 specific bands in the germplasms of G. macrophylla,G. straminea, G. dahurica and G. crassicaulis, respectively. AFLP fingerprint identification efficiency was 97.5%,95.2%,100% and 100%, respectively.5. The cluster analysis was carried out using the non-weighted group average method (UPGMA) based on DICE similarity coefficient. The result showed that the clustering of the four Gentiana species listed in the Chinese Pharmacopoeia was basically consistent with the morphological classification. The cluster analysis based on the average similarity coefficient among the six different species revealed that the genetic relationship was the closest between G. officinalis and G. dahurica, and G. crassicaulis was distant from other 5 species.6. In the 40 germplasms of G. macrophylla, the contents of gentiopicroside in the germplasms from Fengxian were higher than those from other areas. The correlation analysis between the main biological characteristics and the content of gentiopicroside showed that the content of gentiopicroside in roots had a very significant correlation with the shape of leaves and the density of flowers. The germplasms with narrower leaves or denser inflorescences had the higher content of gentiopicroside. Moreover, the content of gentiopicroside had significant correlation with the color of stems and calyx. The deeper was the color, the higher content of gentiopicroside.7. Four iridoid and secoiridoid glycosides including loganic acid, swertiamarin, gentiopicroside and sweroside were simultaneously detected in the six Gentiana species by HPLC. Contents of the four marker compounds varied among different samples even those from the same species. The contents of gentiopicroside in G. siphonantha and G. officinalis were 9.09% and 9.63% respectively, which were higher than the average content (7.72%) of the 19 batches of samples. The HPLC fingerprint similarity indexes was between 0.996 and 0.994, in which the similarity of both G. siphonantha and G. officinalis were higher than 0.991, the average similarity index. Among the four Gentiana species listed in the Chinese Pharmacopoeia, the contents of the four iridoid and secoiridoid glycosides were relatively higher in G. macrophylla and G. crassicaulis, while the contents in G. dahurica were the lowest. The contents of the four iridoid and secoiridoid glycosides in G. crassicaulis at different harvest time were determined. The content of gentiopicroside in July was the highest for both three years old and two years old roots.8. The total ion chromatograms of fatty acids in the roots of six Gentiana species showed high similarity. The relative percentage content of the three main fatty acids (palmitic acid, oleic acid and linoleic acid) accounted for 84.29-96.46% in the six species. Though the relative percentage content of each fatty acid differed in the six species, no significant difference was found by MANOVA analysis.9. Macro and trace elements were analyzed by FAAS. Though the contents of four macro elements including Ca, K, Na and Mg differed in the roots of the six Gentiana species, the contents of the four trace elements containing Fe, Zn, Mn and Cu displayed high consistency, viz. Fe>Zn>Mn>Cu. The contents of Ca, Mg, K, Zn and Mn in G. macrophylla in Shaanxi were all higher than those in other three Gentiana species listed in the Chinese Pharmacopoeia.10. The results of analgesic and inflammatory activities showed that G crassicaulis had the best analgesic effect for the lasting pain stimulus caused by intraperitoneal injection of acetic acid; G. dahurica had the best effect for pain relief caused by thermal stimulation; G. macrophylla had the best anti-inflammatory effect for the inflammation induced by xylene; G. straminea showed the highest inhibition rate on the cotton ball-induced chronic inflammation. As indicated by MANOVA analysis, no significant difference in the analgesic and inflammatory activities was found among the six Gentiana species. The results supported from the perspective of pharmacological effect that G. siphonantha and G. officinalis could be used as the alternate original plants for Radix Gentianae Macrophyllae.