| Objective: Shihu,originally contained in the Shen Nong Ben Cao Jing,in which D.huoshanense has been regarded as genuine since the ancient times and was known as the soft gold in Chinese herbal medicines.(1)In view of the multitudinous sources and greatly uneven quality of Dendrobium species,the evolution and changes of the germplasm and quality evaluation of Shihu in ancient and modern times were studied,to provide the herbalism basis for the quality evaluation of Dendrobium.(2)In view of the phenomenon that there are numerous adulterants of D.huoshanense currently,it is necessary to provide a method for rapid identification of D.huoshanense and its relative species.(3)To study the relationship between the distribution and structure of alkaloids and coumarin compounds in the stems of D.huoshanense and its related species.Methods: Through the investigation of the ancient Chinese Materia Medica,the germplasm,the genuine producing area,the processing methods,the usage and quality evaluation of Shihu were studied.By continuous measurement of the length,diameter,number of vascular bundles of the internode and the transverse section observation of the stems of D.huoshanense and its most confusing adulterant D.officinale,D.moniliforme and D.henanense,their macroscopic and microscopic characteristics were compared.The paraffin section method was used to compare the microstructure of the transverse section in stems of 19 Dendrobium species.Based on the study of the microstructure,the regularities of distribution of alkaloids and coumarin compounds in the stems of D.huoshanense and its related species were determined by histochemical localization and Raman spectroscopy.Results:(1)The textual research was conducted on the evolution and changes of the quality evaluation of Shihu.The germplasm of Shihu had a broad and narrow sense since ancient times,the broad Shihu referred to the genus Dendrobium and its relative genera,the narrow one sometimes referred to the Dendrobium plants which growing in the rocks,sometimes referred in particular to one or several Dendrobium plants.Since the Qing Dynasty,D.huoshanense has been regarded as a genuine regional drug.Shihu of different germplasm and different processing methods had different emphases on its clinical application.The commercial specification as Fengdou might start from the Qing Dynasty,and it has become a mainstream form of dendrobium commodity up to now.There were many sources of Dendrobium medicinal materials,and the quality evaluation of Shihu in traditional Chinese medicine deemed that the heavier,sticky and slag free ones had the better quality.(2)A method of rapid identification of D.huoshanense and its related species was established.In the identification of the macroscopic characteristics,there were significant differences among D.huoshanense,D.henanense,D.officinale,D.moniliforme.According to the total length,the total number of nodes,the length and diameter of each internode and its changing trend of stems,D.huoshanense and its related species were able to distinguish.The most important difference was that the diameter of second internode and third internode had the biggest change trend in D.huoshanense.In the identification of the microscopic characteristics,there was a certain change rule in the vascular bundles numbers of each internode in stems of D.huoshanense and its related species.The vascular bundles numbers of D.huoshanense and D.moniliforme began to gradually reduce from the base,while that of D.henanense and D.officinale present a trend of first increased and then decreased.In the number of vascular bundles,D.officinale had the maximum number,then D.moniliforme,and D.huoshanense and D.henanense had the less number.In addition,there were some differences in the thickening degree of epidermic cells,number of fibers and the existence of siliceous blocks in the stem of D.huoshanense and its related species.(3)The identification of stems of 19 Dendrobium species was developed by their microstructure.There were some similarities of the transverse section of stems of the 19 species,all belong to the typical structure of the stem from the monocotyledon.However,there are some differences in the details of the organization among different species.It is mainly manifested in the thickness of cuticle,the thickening degree of epidermal cells,the existence of subcutaneous cells,the types of vascular bundles,the type and distribution of calcium oxalate crystals,the existence of siliceous blocks and etc.(4)The histochemical study of D.huoshanense and its related species was carried out.The results indicated that,the modified bismuth potassium iodide,iodide-iodine potassium and picric acid had the higher consistency on the detection response of alkaloids in D.huoshanense,D.henanense,D.officinale and D.moniliforme.The alkaloids in stems of these four plants were mainly found in some parenchyma cells and vascular bundles.And the contents of D.moniliforme and D.henanense were higher than that of D.huoshanense and D.officinale.According to the rare difference between the fluorescence reaction before and after the dissolution of coumarin by ethanol elution of D.huoshanense,D.henanense,D.officinale and D.moniliforme,it showed that the contents of coumarin were lower or scarce in those 4 Dendrobium species.The Raman spectrum of D.huoshanense and its related species can be divided into two types.After comparing with the standard Raman spectra library,it was suggested that there were 7-diethylamino-4-methyl coumarin,cellulose and starch in these samples.Taking the signal intensity at 1156 cm-1 and 1522 cm-1 of 7-diethylamino-4-methyl coumarin as the Raman spectrum of D.huoshanense,it showed that the coumarin compounds were mainly distributed in the cell wall around the vascular bundles and parenchyma cells near epidermal.Conclusions:(1)The textual research showed that Shihu was divided into sweet and bitter taste with different clinical application in ancient herbal books.Those records about the quality evaluation of Shihu in traditional Chinese medicine deemed that the heavier,sticky and slag free ones had the better quality,which is consistent with D.huoshanense,to reveal the herbalism basic for its representative of genuine regional drug.(2)The macroscopic characteristics can be able to identify D.huoshanense,D.officinale and D.moniliforme,but not to D.henanense.However,the macroscopic characteristics combined with the microscopic characteristics can be better for the identification of D.huoshanense,D.henanense,D.officinale and D.moniliforme.The processing way for ―Longtou fengwei‖ of D.huoshanense revealed the scientific connotation of viewpoint about assessing quality by distinguishing the features of traditional Chinese medicinal materials for Shihu,which also reflected the ancients‘ protection consciousness of genuine regional drug.(3)The alkaloids in the stems of D.huoshanense and its related species were mainly distributed in the parenchyma cells and vascular bundles.Innovation points:(1)Based on the textual research in the ancient materia medica,the quality evaluation of Shihu considered that the heavier,sticky,sweet and slag free ones had the better quality,which revealed the herbalism basis for the geoherbalism of D.huoshanense,and the clinical application of sweet and bitter taste of Shihu had its own emphasis.(2)On the basic of method of combining macroscopic characteristics with microscopic characteristics,D.huoshanense and its related species were effectively and rapidly identified,and the scientific connotation of ―Longtou Fengwei‖ of D.huoshanense was revealed.(3)The distributions of alkaloids and cumarins in D.huoshanense and its related species were preliminarily ascertained,which laid a foundation for its quality evaluation and resource utilization. |
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