| Dendrobium is the second largest genus in the orchid,there are 76 species discovered in China,and nearly 40 kinds are used of medicine.Dendrobium was set out in the Eastern Han Chinese "Shen Nong Ben Cao Jing" firstly.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 showed that they have a variety of active anti-tumor,immunomodulatory,anti-oxidation,lowering blood glucose and other activities.Dendrobioum huoshanense and Dendrobium officinale were recorded in books on Chinese materia medica,covering its' exact name,origin,plant morphology,which are the two most valuable medicinal Dendrobium speciations.Dendrobium huoshanense is confined to distribute in Huoshan County and neighboring areas.Dendrobium officinale and Dendrobium moniliforme would pretend to be Dendrobium huoshanense frequently in the local area of huoshan.Dendrobium loddigesii would pretend to be Dendrobium huoshanense constantly in Guangdong area.Now the wild resources of Dendrobium have been depleted and the species are at risk of extinction along with unlimited excavation for a long time.Recently some progress have been made on the tissue culture and artificial cultivation of Dendrobium huoshanense merely in Huoshan County and neighboring areas,but the demand still exceeds supply and the price is quite expensive.Under the tall requirement,a wide variety of counterfeit goods of Dendrobium huoshanense turned up.On account of the lack of authentic sample sources,there are not yet credible and complete on quality control for Dendrobioum huoshanense now.On the basis of the district records,the population of Dendrobium officinale are mainly distributed in Guangdong,Guangxi,Zhejiang,Fujian,Yunnan,Jiangxi and so on.Currently a larger scale of the tissue culture and artificial cultivation have developed in all the main origin of Dendrobium officinale,but the demand exceeds supply and the prices are relatively expensive.Our previous research discover that the characteristic spectrum of Dendrobium officinale from Guangxi and Yunnan provenance is similar,but the differences and correlation of the characteristic spectrum of Dendrobium officinale coming from mainly nationwide origins is not yet clear.The germplasm samples of Dendrobium were provided by the domestic manufacturing enterprises and are identified correctly.Dendrobium huoshanense was studied qualitatively and quantitatively by characteristic fingerprint technology combined with content determination methods.What is more,there were comparative studies between Dendrobium huoshanense and Dendrobium officinale,Dendrobium moniliforme,Dendrobium loddigesii by HPLC characteristic spectrum analysis method,which can provide the methodology basis for quality control of Dendrobium huoshanense.In addition,we also study and contrast the discrepancy and correlation of the HPLC characteristic spectrum on the stems and leaves of Dendrobium officinale from different provenance source.Objective:1.To establish HPLC characteristic spectrum of flavonoids and the pre-column derivation HPLC characteristic spectrum of polysaccharide from Dendrobium huoshanense;to establish the content determination method of Schaftoside,Isoschaftoside,D-Mannose and D-Glucose from Dendrobium huoshanense by HPLC.to compare the differences of the characteristic spectrum and the related content of Dendrobium huoshanense and Dendrobium officinale,Dendrobium moniliforme,Dendrobium loddigesii,which can provide the basic methods for quality control and identification of Dendrobium huoshanense.2.To establish HPLC characteristic spectrum of Dendrobium officinale and compare the generality and discrepancy of Dendrobium officinale from different provenance source,which provide a reference to discriminate provincial characteristics.To establish the characteristic spectrum of flavonoids of the leaves of Dendrobium offcinale from three main provenance source by HPLC and study the feasibility of identifying different provenance source quickly.Methods:1.The characteristic spectrum analysis method combined with content determination by HPLC was used to improve the level of quality control of Dendrobium huoshanense and Dendrobium officinale.The characteristic spectrum mainly includes the screening of chromatographic conditions,the methodological evaluation,the alignment of characteristic peaks,and 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 compare the different breed and different provenance source of Dendrobium.2.The characteristic spectrum of flavonoids from Dendrobium huoshanense was established.HPLC was employed with Kromasil 100-5 C18 column,the acetonitrile-phosphoric acid solution((p=0.1%)(gradient elution)was used as a mobile phase,the detection was seted at 340 nm with flow rate and column temperature being of 1 mL/min and 35?,respectively.3.The polysaccharide pre-column derivation HPLC characteristic spectrum of Dendrobioum huoshanense was established.Dendrobium polysaccharide samples were hydrolyzed with Hydrochloric Acid and derivated by 1-Phenyl-3-methyl-5-pyrazolone(PMP).HPLC was employed with Kromasil 100-5 C18 column,the acetonitrile-0.02 mol/L ammonium acetate(pH=6.7)solution(gradient elution)was used as a mobile phase,the detection was seted at 250 nm with flow rate and column temperature being of 1 mL/min and 30 ?,respectively.4.HPLC-MS method was used to identify Schaftoside and Isoschaftoside from Dendrobium huoshanense referring to chromatographic retention time and UV absorption spectral.What is more,the HPLC method for the determination of Schaftoside and Isoschaftoside from Dendrobium huoshanense was established.HPLC was employed with Zorbax SB-Aq column,the acetonitrile-phosphoric acid solution((p=0.1%)(gradient elution)was employed as a mobile phase,detection wavelength was 340 nm,column temperature was 30 ?,and flow rate was 1.0 mL/min.5.The pre-column derivation HPLC method for the determination of D-Mannose and D-Glucose from Dendrobium huoshanense was established.Dendrobium polysaccharide samples were hydrolyzed with Hydrochloric Acid and derivated by 1-Phenyl-3-methyl-5-pyrazolone(PMP).HPLC was employed with Zorbax Eclipse XDB-C18 column,the acetonitrile-0.02 mol/L ammonium acetate solution(gradient elution)was used as a mobile phase,the detection was seted at 250 nm with flow rate and column temperature being of 1 mL/min and 30 ?,respectively.6.HPLC characteristic spectrum of Dendrobium officinale was established.HPLC was employed with Kromasil 100-5 C18 column,the acetonitrile-formic acid solution(?=0.2%)(gradient elution)was employed as a mobile phase,detection wavelength was 270 nm,column temperature was 35 ?,and flow rate was 1.0 mL/min.The different provenance sources of Dendrobium officinale were classified on the basis of the analyzation of characteristic spectrum of the samples and the same points and discrepancy of the characteristic peaks.7.The characteristic spectrum of flavonoids from the leaves of Dendrobium officinale by HPLC was established.HPLC was employed with Kromasil 100-5 C18 column,the methanol-water(gradient elution)was used as a mobile phase,detection wavelength was 340 nm,column temperature was 25 ?,and flow velocity was set as 0.8 mL/min.to study the feasibility of identifying different provenance sources of Dendrobium officinale quickly by the analyzation of characteristic spectrum of the samples and the similarities and differences of the characteristic peaks.Results:1.The HPLC characteristic spectrum analysis method of flavonoids from Dendrobium huoshanense is feasible and practical.(1)24 common peaks were separated from 10 batches of manual cultivation Dendrobium huoshanense.The similarities of 10 batches were 0.918?0.990.(2)Preliminary studies found most peaks of a batch of wild Dendrobium huoshanense were consistent with the cultivar of Dendrobium huoshanense,but the counter peak area ratios were quite different,the similarity between a batch of wild Dendrobium huoshanense and common pattern of 10 batches of the cultivar samples was 0.839.(3)The HPLC characteristic spectrum of Dendrobium huoshanense,Dendrobium officinale,Dendrobium moniliforme and Dendrobium loddigesii were quite large.By UV spectra and retention time counterpoint,one flavonoid peaks were marked in the six batches of Dendrobium officinale,which was peak t1,corresponding to the peak 2 of Dendrobium huoshanense.Six batches of Dendrobium officinale do not contain Schaftoside and Isoschaftoside and there were some differences for the numbers of flavonoid peaks of the six batches of Dendrobium officinale.Dendrobium moniliforme had mainly two larger peaks,and had not corresponding peaks to Dendrobium huoshanense;There were fewer flavonoid peaks in the three batches of Dendrobium loddigesii which were also not containing Schaftoside and Isoschaftoside.The characteristic spectrum of Dendrobium huoshanense to be a mutual mode control,the similarities of 6 batches of Dendrobium officinale,3 batches of Dendrobium moniliforme and 3 batches of Dendrobium loddigesii were 0.502?0.613,0.387-0.391,0.269?0.333.The studies provide the methodology basis for the identification between Dendrobium huoshanense and its adulterant.2.The polysaccharide pre-column derivation HPLC characteristic spectrum of Dendrobium huoshanense,Dendrobium officinale,Dendrobium moniliforme,Dendrobium loddigesii had certain similarity and differences.(1)Six common peaks were separated in 10 batches of Dendrobium huoshanense,mainly composed of D-Mannose and D-Glucose,which the peak area percentage was 71.81%and 27.19%,respectively,containing traces of D-Galacturonic acid,D-Galactose,D-Xylose and D-Arabia sugar,which the peak area percentage was 0.12%,0.42%,0.19%,0.26%,respectively.The similarities of 10 batches were 0.994?1.000.(2)Six common peaks were separated in 3 batches of Dendrobium officinale,3 batches of Dendrobium loddigesii and 3 batches of Dendrobium moniliforme,respectively,which were similar to Dendrobioum huoshanense,mainly composed of D-Mannose and D-Glucose.The peak area percentage of D-Mannose and D-Glucose were 78.56%and 20.42%,46.08%and 52.05%,78.12%and 19.26%,respectively.There were traces of D-Galacturonic acid,D-Galactose,D-Xylose and D-Arabia sugar,which the peak area percentage was less than 0.5%?0.8%?1.2%,respectively.(3)There were certain differences of the ratio of peak area of D-Mannose to D-Glucose(Am/Ag)in the four kinds of Dendrobioum.The ratio of Am/Ag in Dendrobium huoshanense,Dendrobium officinale and Dendrobium moniliforme were 2.0?3.7,3.2?5.0,3.2?6.3,respectively;while the ratio of Am/Ag in Dendrobium loddigesii was 0.7?0.8,lower 1.0,which was different relatively from other three Dendrobium.These results showed that there were not significant difference of Am/Ag with Dendrobium huoshanense and Dendrobium officinale,Dendrobium moniliforme,so the specialization of Am/Ag of Dendrobium officinale in the Pharmacopoeia of Peoples Republic of China was not very good.(4)The polysaccharide pre-column derivation HPLC characteristic spectrum of 10 batches of Dendrobium huoshanense to be a mutual mode control,the similarities of common pattern of 3 batches of Dendrobium officinale and 3 batches of Dendrobium moniliforme were 0.993,0.992,which indicated the ratio of polysaccharide component of the three kinds of Dendrobioum were similar relatively;but the similarity of Dendrobium loddigesii was 0.889 merely.3.Two Flavone C-glycosides were identified from Dendrobium huoshanense,which are Schaftoside znd Isoschaftoside.The regression equation of schaftoside and isoschaftoside were Y = 10.289X + 1.869,Y=8.958X+4.116,which the linear ranges were 5.84?70.08 ?g/mL,r=0.9999,9.64?115.68 ?g/mL,r=0.9999,respectively.The content of Schaftoside and Isoschaftoside from 10 batches of Dendrobium huoshanense were 0.0297?0.1515,0.0122?0.0866 mg/g,respectively.The content of Schaftoside and Isoschaftoside from one batche of wild Dendrobium huoshanense were 0.1463,0.0794 mg/g,respectively.4.The content of D-mannose and D-glucose(Cm and Cg)of 10 batches were 16.08%?29.47%,6.64%?16.08%,respectively;the range of the sum of Cm and Cg content in ten batches of Dendrobium huoshanense was 25.73%?44.37%.The Cm and Cg content were 21.11%?27.04%,4.94%?10.05%in the three batches of Dendrobium ofcinale,respectively,and the range of Cm+g content was 26.04%?37.09%.The Cm and Cg content were 7.50%?9.62%,11.20%?13.97%in three batches of Dendrobium loddigesii,respectively,and the range of Cm+g content was 20.40%?21.76%.The Cm and Cg content were 10.15%?10.71%,3.88%?4.24%in the three batches of Dendrobium moniliforme,respectively,and the range of Cm+g content was 14.03%?14.71%.The results showed that the Cm and Cg content of Dendrobium huoshanense and Dendrobium officinale were similar,the Cm content of Dendrobium loddigesii and Dendrobium moniliforme was all significantly lower.In addition,the range of the ratio of Cg between Dendrobium moniliforme and other three Dendrobium was 1/3?1/2 merely.5.The HPLC method for establishment of fingerprint analysis of Dendrobium officinale was developed and seted up.It was found that the HPLC characteristic spectrum of Dendrobium officinale from different provenance source had certain differences.(1)The provenance sources of Dendrobium officinale were divided primarily according to the common features and differences of HPLC characteristic spectrum of Dendrobium officinale from different habitat.The study come up with concept of provenance source include Danxia landform,Tiepilan and native species from Zhejiang.(2)The main component of three provenance source is basically consistent,but the variability exists in ratio of peak areas and numbers of characteristic spectrum.37 common peaks were separated in 10 batches from Danxia landform and the similarities were 0.907?0.972,which peak 23 had strongest UV absorption.41 common peaks were separated in 5 batches from Zhejiang landform and the similarities were 0.817?0.933.37 common peaks were separated in 5 batches from Tiepilan landform and the similarities were 0.893?0.954,which peak 25 had strongest UV absorption.(3)The characteristic spectrum of Dendrobium officinale of the provenance sources from Danxia landform to be a mutual mode control,the similarity of the common pattern of the specimens from Tiepilan landform and from Zhejiang landform were 0.700 and 0.855;The characteristic spectrum of Dendrobium officinale of the provenance sources from Zhejiang landform to be a mutual mode control,the similarity of the common pattern of the specimens from Danxia landform and from Tiepilan landform were 0.855 and 0.681,indicating that similarities of HPLC characteristic spectrum of Dendrobium officinale from different provenance sources have certain differences.6.The HPLC characteristic spectrum of the leaves of Dendrobioum huoshanense from three main provenance source were established and different provenance source of the leaves of Dendrobium officinale could be identified quickly by the analyzation of the characteristic spectrum of the samples.(1)The HPLC characteristic spectrum of Dendrobium officinale from different provenance sources were quite large.14 mutual peaks were separated in leaves of Dendrobium officinale from the three provenance sources and two characteristic common peaks were identified,which were Isoschaftoside and Schaftoside.12 common peaks were separated in 10 batches from Danxia landform and the similarities were 0.880?0.996.13 common peaks were separated in 6 batches from Zhejiang landform and the similarities were 0.774?0.981.9 common peaks were separated in 7 batches from Tiepilan landform and the similarities were 0.922?0.974,which the peak area of peak 10 and peak 11 were larger relatively.(2)The characteristic spectrum of the leaves of Dendrobium officinale whose provenance sources from Danxia landform to be a mutual mode control,the similarities of common pattern of Danxia landform with 6 batches from Zhejiang and 7 batches from Tiepilan were 0.395,0.061,respectively.The similarities of common pattern of Tiepilan landform with 6 batches from Zhejiang was 0.370,indicating that the variability exists in component and ratio of peak areas of component flock and numbers of characteristic spectrum.Conclusion:1.The analysis means of HPLC characteristic spectrum of Dendrobium huoshanense was accurate and reliable,has a good repetitiveness,and the HPLC characteristic spectrum between Dendrobium huoshanense and Dendrobium officinale,Dendrobium moniliforme,Dendrobium loddigesii were distinctly different,which provided the methodology basis for identification and quality control for Dendrobium huoshanense.2.The polysaccharide pre-column derivation HPLC characteristic spectrum of Dendrobium huoshanense,Dendrobium officinale,Dendrobium moniliforme,Dendrobium loddigesii have certain similarity and differences and their similarities are relatively high.But Dendrobium loddigesii can be identified by the relatively low ratio of peak area of mannose to glucose among the four Dendrobioum samples.3.Two Flavone C-glycosides are identified from Dendrobioum huoshanense,which are Isoschaftoside,Schaftoside.The HPLC means for the determination of Isoschaftoside and Schaftoside are feasible,providing effective content components as the indicators for Dendrobioum huoshanense.4.The HPLC means for the determination of D-Mannose and D-Glucose are feasible.The content of D-Mannose and D-Glucose between Dendrobium huoshanense and Dendrobium officinale are similar and relatively higher,but significantly lower of Dendrobium moniliforme and Dendrobium loddigesii.The sum of mannose and glucose content had a large proportion in the composition of the total polysaccharide,which could basically reflect the content of total polysaccharides.5.The main component of three provenance source is basically consistent,which shows stability of internal quality of Dendrobium officinale,but the variability exists in ratio of peak areas and numbers of characteristic spectrum,which provides a reference to discriminate provincial characteristics from Danxia landform,Tiepilan and native species from Zhejiang.6.The HPLC characteristic spectrum of flavonoids on leaves of Dendrobium officinale from different provenance source have certain differences.By means of the amount of characteristic peaks,the ratio of peaks and similarity evaluation,different provenance sources of Dendrobium officinale can be identified quickly. |
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