| Dendrobium is the second largest genus in the orchid,there are 76 species in China,and nearly 40 kinds are used of medicine.Now dendrobium officinale is the most valuable representative.Dendrobium firstly was set out in the Eastern Han Chinese "Shen Nong Ben Cao Jing".It have been widely used in the treatment of weakness after febrile diseases,thirsty,fire excess from yin deficiency,asthenia-syndrome and other diseases.Modern pharmacological experiments have proved that they have a variety of active anti-tumor,immunomodulatory,anti-oxidation,lowering blood glucose and other activities.Due to the improved living standards in recent years,traditional medicinal and health care function of Dendrobium are got attention,and Dendrobium is surged in demand.Fengdou comes from the plants which are short,the stems are stout and fleshy,the texture is soft and rich in mucus ingredient of dendrobium stem.And the market has reached more than 20 species of Fengdou.Fengdou is easy to save,has become a natural green products enjoys a high reputation both at home and abroad.In recent years,wild Dendrobium resources have been seriously damaged.Dendrobium artificial cultivation industry is still in its infancy,rare Dendrobium market is in short supply.As Dendrobium variety,lookalike,quality varies,prices vary greatly,but mostly different Dendrobium variety of complex components with similar,the existing quality standards Dendrobium is difficult to effectively control the quality of different Dendrobium.Dendrobium Fengdou conceptual confusion,breed confusion,counterfeiting,shoddy phenomenon is still very serious,and the quality problem of the precious of Dendrobium officinale and devonianum are particularly prominent.Such as devonianum often pretend to be Dendrobium officinale,and aphyllum often adulterated devonianum,etc.Therefore,to establish a reliable,perfect quality control standard for Dendrobium quality has very important significance.Traditional Chinese medicine fingerprint technology is recognized as the most effective method of traditional Chinese medicine quality control at home and abroad.This study choose Dendrobium officinale,devonianum and aphyllum as the research object,using the traditional Chinese medicine fingerprint technology,comparativing study the three kinds of Dendrobium of high performance liquid chromatography(HPLC)characteristic spectrum,to provide methods for its quality control level.In order to make the authenticity,advantages and disadvantages,adulteration judgment,effectively control the quality of Dendrobium.Objective1.To establish HPLC characteristic spectrum analysis method of fresh Dendrobium officinale and study out the main features of thecharacteristic peak;to investigate the correlation of specific chromatogram between stems and leaves;to explore the applications in the optimal selection tissue culture seedling;to explore regularity of HPLC characteristic spectrum in different growing season;to provide the basis of the methods to optimization the resources,the best harvest and quality control.2.To establish the HPLC characteristic spectrum analysis method Fengdou of Dendrobium officinale,devonianum and aphyllum;to provide the basic methods for quality control and identification.3.To establish the HPLC characteristic spectrum of amino acid pre-column derivatization of Dendrobium officinale,devonianum and aphyllum,to compare the similarities and differences of HPLC characteristic spectrum of amino acid of the three Fengdous.Methods1.Use the HPLC characteristic spectrum analysis method of fresh Dendrobium officinale to analysis 10 batches of samples.specific chromatogram similarity evaluation system software writed by the State Pharmacopoeia Commission Specific chromatogram similarity evaluation system software(2004A version)was adopted to generate a total of mode;and use the same method to analysis and comparison of the same batch of fresh Dendrobium stem and leaf samples,the same germplasm samples of different growth fresh Dendrobium,Dendrobium Stem with fresh tissue culture samples correlation characteristic spectrum.2.Establishment Dendrobium officinale,devonianum and aphyllum of HPLC characteristic spectrum analysis method,analysising 10 batches of samples,generating a total of three kinds of Dendrobium characteristic spectrum mode.comparing the three characteristic spectrum Dendrobium commonalities and differences,and the similarity evaluation.3.The HPLC characteristic spectrum of amino acid column derivatization of Dendrobium officinale,devonianum and aphyllum was established and correlations of them were researched with HPLC characteristic spectrum technology,analysising 10 batches of samples,generating a total of three kinds of Dendrobium amino acid characteristic spectrum mode,comparing the three characteristic spectrum Dendrobium commonalities and differences,and the similarity evaluation.Results1.The establishment of fresh Dendrobium HPLC characteristic spectrum analysis method is feasible and practical.(1)Fresh Dendrobium officinale were marked out of a total of 35 common peaks,10 batches of samples' similarty were 0.900?0.956,fresh Dendrobium officinale characteristic peaks can be divided into four categories,which peaks 17 to Peak 27 were Dendrobium officinales' exclusive features common peaks.(2)Four batches of fresh Dendrobium officinale stem and leaves were analysed,the stems were marked out a total of 33?35 peaks,leaves were marked out of a total of 17 peaks;the similarity between the four batches of stems were 0.914?0.964,and the leaves were 0.916-0.988,the same batch of stems and leaves were 0.802?0.921,suggesting that the characteristic components of fresh Dendrobium officinale stem and leaf have some relevance.(3)The different growing season of the same germplasm(6,9,12month)of fresh Dendrobium officinale samples have the similar HPLC characteristic spectrum,the similarity were 0.941?0.971.(4)Study were found that Dendrobium Tissue Culture of different germplasm have the similar characteristics common peaks with fresh Dendrobium officinale.2.The HPLC characteristic spectrum analysis method Fengdou of Dendrobium officinale,devonianum and aphyllum were established.(1)Fengdou of Dendrobium officinale,devonianum and aphyllum each were marked out a total of 36 peaks,27 peaks and 42 peaks;the similarity of 10 batches of samples each were 0.898?0.969,0.880?0.977 and 0.901?0.961.(2)The characteristic spectrum of Dendrobium officinale to be a mutual mode control,the similarity of 10 batches of devonianum and aphyllum were 0.654?0.784and 0.159?0.261;while devonianum to be a mutual mode control,the similarity of 10 batches of officinale and aphyllum were 0.364?0.495 and 0,096?0.242;indicating that similarity of HPLC characteristic spectrum of Dendrobium officinale,devonianum and aphyllum were very low.(3)Combination with the three Dendrobium Fengdou in the retention period the characteristic peaks of spectrum UV analysis.In the retention time of 60?90 min,the biggest difference of the characteristic spectrum of Dendrobium officinale between Dendrobium devonianum.the number of characteristic peaks,peak shape and the peak ratio were different,and Dendrobium devonianum have some typical characteristic peaks of flavonoids,and Dendrobium officinale's peak area is not large but more specific set of characteristics near at 270 nm absorbtion wavelength.And the biggest difference of Dendrobium aphyllum was in the retention time of 25?35 min with significantly large proportion of 3?4 absorption peak group,which could identificated of three Fengdou of Dendrobium officinale,Dendrobium devonianum and Dendrobium aphyllum.3.The analysis method of HPLC characteristic spectrum of amino acid precolumn derivatization of Dendrobium officinale,devonianum and aphyllum was established,and compared the three characteristic spectrum Dendrobium commonalities and differences.(1)The HPLC characteristic spectrum of amino acid precolumn derivatization of Dendrobium officinale,devonianum and aphyllum each were marked out a total of 20?21 peaks,and marked out 15?16 peaks of beneficial to human body amino acids,the similarity of 10 batches of samples each were 0.920?0.999,0.866?0.992 and 0.927?0.998.The similarity of same Dendrobium is relatively high.(2)The characteristic spectrum of amino acid pre-column derivatization of Dendrobium officinale to be a mutual mode control,the similarity of 10 batches of devonianum and aphyllum were 0.693?0.950 and 0.434?0.713;while devonianum to be a mutual mode control,the similarity of 10 batches of officinale and aphyllum were 0.758?0.848 and 0.773?0.948.indicating that similarity of HPLC characteristic spectrum of amino acid pre-column derivatization of Dendrobium,devonianum and aphyllum has certain differences.(3)Most characteristic amino acid peaks area of officinale are much larger than the devonianum and aphyllum,peak a is the strongest peak of officinale,accounting for 30.8%of total peaks area;Although the strongest peak of devonianum is peak a too,but its only 17.7%of total peaks area,while peak 1(aspartic acid)is the strongest peak of aphyllum.accounting for 14.4%of total peaks area.In the retention time of 17 min,the arginine peak(peak 5)of devonianum and aphyllum are both bigger than officinale.In the retention time of 20?22 min.officinale has a group of characteristic peaks(peak b.Peak 7 Peak 8)are relatively uniform,while the Peak 7 of devonianum and aphyllum are very small.And the peak e of aphyllum is far greater than officinale and devonianum.Conclusion1.The analysis method of HPLC characteristic spectrum of Dendrobium officinale accurate and reliable.has a good stability,provided the methodology basis for quality control for Dendrobium.(1)Studies have shown that HPLC characteristic spectrum of Dendrobium officinale stems and leaves has relevance,and provided a reference for the development and application of Dendrobium officinale leaves;and the peak area of flavonoids in leaves were larger than stems,through the the characteristic spectrum of leaves also can identification the resources.(2)The different growth period of fresh Dendrobium officinale haved the similar HPLC characteristic spectrum,prompted that Dendrobium officinale has distinct genetic characteristics.(3)Study found that tissue culture of different germplasm have the similar characteristics common peaks with Dendrobium officinale,suggesting that analysis of HPLC characteristic spectrum of the original stem or tissue culture can preferred the better germplasm.2.The HPLC characteristic spectrum of Dendrobium officinale,devonianum and aphyllum were quite large.Through UV spectrum of characteristic peaks can be identified the three Dendrobium,and provided a method for quality control of the three Dendrobium,and also provided a reference for other Dendrobium.3.The characteristic spectrum of amino acid precolumn derivatization of Dendrobium officinale,devonianum and aphyllum have certain similarity and differences.By the number of characteristic peaks,the ratio of peaks and similarity evaluation can provide a reference for the identification of three Dendrobium. |
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