| (?)(called Spang rgyan dkar pol), namely Alpine gentian herb, is a staple Tibetan medicine which is used commonly. It is clinically used to cure virus disease, a variety of fever and laryngitis. It is the monarch drug in the product of fifteen flavors gentian flower pill and twenty five flavors gentian flower pill and other classical Tibetan medicines, and it has a certain effectiveness. However there are some problems about this medicine,such as, its complex sources, classification confusion, inconsistencies of species contained in modern Tibetan medicine books and varieties of national, local standards. Moreover,the method of quality control is simple, the internal quality evaluation criteria is lacking, and there is not enough scientific basis to support its usage in clinical. To solve these problems, a combination method of literature analysis, field survey, crude drug research, chemical composition analysis, and efficacy evaluation of this traditional Tibetan medicine was used. Species systematization and quality evaluation of mainstream varieties of Tibetan medicine Spang rgyan dkar pol has been conducted, and the following results have been obtained.1The literatures of alpine gentian herb have been carded systematically, providing primary data for its clinical usage and reasonable development.We first found that the Tibetan medicine Spang rgyan dkar pol was first recorded in Somaratsa, and corrected the wrong statement in Tibetan medicine monographs Chinese Materia Medica Tibetan Volume and other literatures that it was first recorded in rGyud-bzhi. In variety of "Bangjiangabao" used in Tibetan herbal medicine and related literatures has been arranged and the problems of varieties of merging, naming objections in these literatures has been analyzed. Eventually,we found that the species of the white gentian plants recorded in these literatures were up to7kinds of groups, one variant. We figured out the effect, indications and usage of" Spang rgyan dkar pol" in Indian medicine and Tibetan medicine,and we concluded more than70classic Tibetan medicine prescriptions that containing the alpine gentian herb. There are2/3of the Formulas are used for the treatment of respiratory diseases clinically.2Commodity and utilization status of Alpine gentian herb has been investigated deeply, and the mainstream varieties and usage of their goods has been clarified.According to the investigation, coupled with literature research, we concluded that Gentiana szechenyii Kanitz was closest to the description recorded in ancient herbal literature. In addition, G. algida Pall."Stemless" plants called " Spang rgyan dkar pol qiong wa (florets)" were the species recorded in the standards. The former, extensively used in Tibetan medicine factories, is widely circulated in the medicine market. The latter and pine gentian "stemmed" plants, G. purdomii Marq. are considered as alpine gentian herb in Tibetan medicine, and they are mainly used by local Tibetan hospitals and Tibetan doctors. G. stipitata Edgew is used as alpine gentian herb in Aba Sichuan, Changdu Tibet, and parts of Qinghai, and it is also recorded in some Tibetan medicine books.The plant morphology of G. striata Maxim, G. yunnanensis, G. sinoornata Balf.f. has a great difference with the description in Tibetan Herbal, and they are not commodity or are not used by hospitals, Tibetan doctors working in Villages and towns, even they are not local common varieties.The description of Gentiana nubigena in Tibetan Herbal is different with the description recorded in "Flora of China", perhaps mistaken. No herbs commercial medicinal materials circulations are found for the above varieties.In addition, it was found that there were Gentiana straminea Maxim, Gentiana filistyla mixed in the commercial medicinal materials of alpine gentian herb; and white gentian was replaced by blue flower gentian in some Tibetan medicine factories.3Resources investigation for original plants of alpine gentian herb and the medical plant of gentiana in Tibetan areas of Sichuan has been Conducted to provide a reference for its protection of resources, rational development and utilization. To cooperate with the fourth national chinese medicine resources survey, we performed the investigation on original plants of alpine gentian herb and gentiana medical plant in Tibetan areas of Sichuan. The results showed that a total of28kinds of gentiana medicinal plants, in which, there were5kinds (6types) of original plants involves white gentian (G. szechenyii Kanitz, G. algida pall."stemless" plants, G. algida pall."stemmed" plants, G. purdomii Marq., G. stipitata Edgew., G. striata Maxim.), as well as G. crassicaulis Duthie, G. straminea Maxim., G. veitchiorum Hemsl., G. sino-ornata Balf, etc. which have the important medicinal value. With a wide distribution, rich in resources, it has a good prospect for the development and utilization.4The quality research of mainstream species of Alpine gentian herb4.1The morphological and histological study on5kinds (6types) of Alpine gentian varieties herb has been conducted for the first time, and the similarities and differences of its original plants, medicinal properties and microscopic characteristics has been identified, providing the reference for its identification of species.On the original plant morphology, we conduct the identification mainly from the thickness of the roots, the growing way of stem and leaves, the size and shape of flower, corolla color and the presence or absence of spots and so on.On medicinal materials characters, we conduct the identification mainly from the shape of corolla and calyx, the shape of leaf and the way of growing, calyx and leaf edges and so on.On microscopic characteristics, we conduct the identification mainly from the flowering branch central stem’s cross-section endodermis obvious or not, the ratio of cortex and radius, the inner surface of corolla, medicinal powder catheter type and type of calcium oxalate crystals and so on.SEM observation results of5kinds(6types) of white gentian pollen and seed coat showed that the surface ornamentation of pollen and seed coat’s Grade II surface ornamentation were the important basis for identification of varieties.4.2We established the thin-layer chromatography identification method of gentiopicroside, swertiamarin in white gentian as well as the thin-layer chromatography identification method of homoorientin and isovitexin, and performed thin-layer chromatography of white gentian having different origins, and compared these white gentian with G. filistyla (G. veitchiorum Hemsl, G. sin-ornata Balf.f), G. straminea Maxim (counterfeit). We identified their similarities and differences, providing the reference basis for the analysis of the differences of their chemical composition.The separation effect of the established TLC method was well, with many clear chromatographic spots which had a moderate Rf values. By comparing TLC identification for5kinds (6types) of white gentian, G. filistyla, G. straminea Maxim, the results showed that at same position of Control sample of gentiopicroside, swertiamarin glycoside in the Sample chromatography of G. algida pall."stemless" plants, G. algida pall."Stemmed" plants, G. purdomii Marq., G. veitchiorum Hemsl, G. sinoornata Balf.f. and G. straminea Maxim, there were chromatographic significant fluorescence quenching spots, while G. szechenyii Kanitz, G. stipitata Edgew. did not have this kind of spot at same position. The number, size and shade of chromatography spots of different varieties were different. The TLC results of homoorientin and isovitexin, except G. striata Maxim., G. straminea Maxim., the chromatography for the rest varieties of Control sample, there were significantly yellow-green fluorescent spots at the same position of homoorientin and isovitexin, but the number, size and shade of chromatography spots of different varieties were different. These results suggested that the chemical compositions in different varieties were different.4.3Identification study on different white gentian, Gentiana filistyla and adulterants has been conducted by Using Fourier transform infrared (FTIR), and the characteristic infrared spectroscopy for identifying the difference between different varieties has been established, and result showed that second derivative infrared spectroscopy could distinguish the difference between similar varieties.4.4The HPLC fingerprint identification method of Alpine gentian herb has been established for the first time. We utilized the principal component analysis and cluster analysis to conduct comparative analysis in total62batches of of Alpine gentian herb, G. filistyla and adulterants samples. The results showed that different varieties or different parts of same variety and different storage time had a large difference in the peak number and peak area in HPLC fingerprint identification. The similarity of G.szechenyii Kanitz and G. stipitata Edgew was high. There was no significantly difference between stem G. algida Pall and stemless G. algida pall, but it was similar to G. purdomii Marq. Through statistical analysis, G. szechenyii Kanitz and G. stipitata Edgew were clustered together; stem G. algida pall, stemless G. algida pall and G. purdomii Marq were clustered into one class. G. filistyla were clustered into one class which was quite close to G. algida pall and G. purdomii Marq, while quite far from G. szechenyii Kanitz and G. stipitata Edgew. Moreover, the aboveground and underground part of G. szechenyii Kanitz, G. stipitata Edgew and the G. urnula was a little bit far, revealing that the differences of the chemical composition was remarkable and the distance of the same variety having different storage time was quite distant.4.5In accordance with the method in first Chinese Pharmacopoeia (Appendix Ⅸ H First Act) and (Appendix Ⅸ K), measurement in terms of water, total ash and acid insoluble ash of in total43batches of Alpine gentian herb samples has been conducted. The results showed that, the highest of moisture content was10%, the lowest was6%, and the average was7.82%; the highest of total ash content was7.1%, the lowest was3.5%and the average was5.4%; the highest of acid insoluble ash content was3.8%, the lowest was0.6%,and the average was1.71%. This provided a reference for the development of limited standards.4.6We established the method for determination of the extract from Alpine gentian herb, using70%ethanol as solvent, method for the determination of hot dip. According to the determination of43different batches samples for Alpine gentian herb, the maximum value of the extractive content is38.25%, the minimum value was21.98%and the average was29.73%; while the G. Stipitata Edgew had a higher extract content.4.7The HPLC method for simultaneous determination of gentiopicroside, Swertiamarin, homoorientin and isovitexin in white gentian has been established for the first time. Determination for43different groups white gentian samples having different origins and different storage time has been conducted, the results showed that the content of isovitexin in the G. szechenyii Kanitz, G. stipitata Edgew. was higher, followed by iso-orientin, while gentiopicroside, swertiamarin were not detected. G. algida Pall. The content of gentiopicroside in G. purdomii Marq. was higher, followed by iso-orientin. The4kinds of chemical composition of G. striata Maxim. were low. The4chemical compositions in the sample having long storage time were generally low.5Coupled with Tibetan medicine efficacy and clinical usage of Tibetan medicine Spang rgyan dkar pol, we conducted pharmacodynamic comparative study on G. szechenyii Kanitz, G. algida Pall, G. stipitata Edgew, G. veitchiorum Hemsl and Tibetan Medicine (fifteen flavors Bangjian pill) for the first time, providing a scientific basis for its clinical application.Antibacterial experiments in vivo, Tibetan medicine (fifteen flavor gentian flower pill) showed a good antibacterial effect. For antibacterial experiments in female mice, G. stipitata Edgew and G. veitchiorum Hemsl showed good antibacterial effect. A large number of colloidal precipitate in aqueous extract was observed. It was presumed that this was associated with these substances. Further study should be conducted to understand it. Overall, the antibacterial effect:Tibetan medicine> G. stipitata Edgew> G. szechenyii Kanitz> G. algida Pall> G. veitchiorum Hemsl.The experiments for the effects on mice chronic bronchitis showed that Tibetan medicine (fifteen flavor gentian flower pill), G. algida Pall and G. veitchiorum Hemsl had a better therapeutic effect on chronic bronchitis, and it might be associated with their effects of anti-inflammatory and antioxidant, whereas G. szechenyii Kanitz and G. stipitata Edgew had no signoficant effects for the mice chronic bronchitis.6The study conducted in this paper, provided a reference for determining the source of varieties and establishing the standards of quality standards of Tibetan medicine Spang rgyan dkar pol, and the study also laid the foundation for sustainable development and utilization of its resources.The study found that the current commodity circulation of the mainstream was G. szechenyii Kanitz which is the closest to the description recorded in ancient books of herbal medicine. There are a small number of commodities medicines for G. stipitata Edgew. G. algida Pall and G. purdomii Marq are being used by some Tibetan Hospital and Tibetan doctors. G. striata Maxim is only recorded in the Tibetan Medicine Annals. The results of quality evaluation showed that G. szechenyii Kanitz was quite similar to G. stipitata Edgew, while G. algida Pall and G. purdomii Marq were similar. Stemless G. algida Pall plants and stem G. algida Pall plants had no significant difference. G. striata Maxim was quite different with the others. Synthesized the results of study in this paper, coupled with the using habits and resources status; it is recommended that the using varieties for alpine Gentian Herb were G. szechenyii Kanitz, G. stipitata Edgew, G. algida Pall and G.purdomii Marq. |
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