Quality Research Of Dendrobium Loddigesii Based On The HPLC Characteristic Spectrum Technology And The Comparison With Dendrobioum Huoshanens

Dendrobium is originally recorded in Sheng Nong’s herbal classic,which is the traditional valuable Chinese traditional medicine.It have been widely used in the treatment of weakness after febrile diseases,thirsty,fire excess from yin deficiency,asthenia-syndrome and other diseases.The traditional precious Dendrobium include Dendrobioum huoshanens,Dendrobium officinale,Dendrobium nobile and Dendrobium fimbriatum,but with different kinds of the changes of the cultivated Dendrobium,contains source and its name also had changed.There are more than 50 species used as medicine at present.The records of the sources of Dendrobium were the plants of Dendrobium in the 1963 edition of "Chinese pharmacopoeia",while the record of the sources of Dendrobium loddigesii,Dendrobium fimbriatum,Dendrobium chrysanthum,Dendrobium officinale and Dendrobium nobile in the 1977,1985,1990,1995,2000 editions of "Chinese pharmacopoeia",and the record of the sources of Dendrobium nobile,Dendrobium officinale and Dendrobium fimbriatum and its allied species in 2005 edition of "Chinese pharmacopoeia".But the rerords of sourses of Dendrobium were Dendrobium nobile,Dendrobium chrysotoxum,Dendrobium fimbriatum and its allied species of the same genera in the 2010 and 2015 editions of "Chinese pharmacopoeia",with Dendrobium officinale single listed seperatelly,which shows that the varieties of Dendrobium is widely applied and part of standard is not completely.Dendrobium loddigesii is also named as Dendrobium loddigesii includes "Mei Hua Shi Hu,Fen Hua Shi Hu,Xiao Huang Cao and Xiao Huan Cao",and was recorded as one of the sources of Dendrobium name as "Mei Hua Shi Hus" in "Zhong Hua Ben Cao".Dendrobium loddigesii is mainly distribute in Guizhou,Yunnan,Guangxi and Guangdong.Dendrobium loddigesii mainly contain bibenzyls such as gigantol,alkaloids such as shihunine and shihnidine,and polysaccharide.Modem pharmacological studies have found that polysaccharide component is the main active ingredient of Dendrobium,which has the pharmacology effect of promoting immune and antitumor and so on.At present,the quality study of Dendrobium loddigesii is few,only a small amount of thin layer chromatography(TLC),the content of total polysaccharides,which could not perfectly control its quality.Because of the similar appearance between Dendrobium loddigesii Fengdou and Dendrobium huoshanense Fengdou.Dendrobium loddigesii Fengdou is usually pretended as Dendrobium huoshanense Fengdou.Therefore,it is necessary for the distinction difference between two kinds of Dendrobium.This study intends to establish a system of quality control method to provides the basis for quality control for Dendrobium loddigesii,and provide reference for identification of Dendrobium huoshanense.Objective:1.To establish HPLC characteristic spectrum of medium polarity part and the pre-column derivation HPLC characteristic spectrum of polysaccharide from Dendrobium loddigesii;combine with the content determination of gigantol,D-Mannose and D-Glucose and TLC for the quality standard of Dendrobium loddigesii,comparing with Dendrobium huoshanense to provide reference for identification of Dendrobium huoshanense.2.To establish HPLC characteristic spectrum of the water solution of Dendrobium loddigesii and to analysis the correlation between Dendrobium loddigesii and its water solution,evaluating the effect of extract by decoction.Methods:1.The characteristic spectrum analysis method combined with content determination by HPLC was used to improve the level of quality control of Dendrobium loddigesii 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 wrote by the State Pharmacopoeia Commission Specific chromatogram similarity evaluation system software(2004A version)was adopted to generate a total of mode by averaging method to analysis the similarity.2.The characteristic spectrum from Dendrobium loddigesii and its water solution was established.HPLC was employed with Kromasil 100-5 C18 column,the acetonitrile-phosphoric acid solution(cp=0.2%)(gradient elution)was used as a mobile phase,the detection was seted at 280 nm with flow rate and column temperature being of 0.8 mL/min and 35 ℃,respectively.3.The polysaccharide pre-column derivation HPLC characteristic spectrum of Dendrobium loddigesii 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.The pre-column derivation HPLC method for the determination of D-Mannose and D-Glucose from Dendrobiunm loddigesii 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 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.5.The HPLC method for the determination of gigantol from Dendrobium loddigesii was established with Kromasil 100-5 C18 column,acetonitrile-water(36:64)was used as a mobile phase,detection wavelength was 210 nm,column temperature was 30 ℃,and flow rate was 0.5mL/min.6.The thin-Layer Chromatography was adopted to identify alkaloids,bibenzyls and flavonoids from Dendrobium loddigesii.7.Compare with Dendrobium huoshanense by the same method as Dendrobium loddigesii.Results:1.The HPLC characteristic spectrum analysis method of medium polarity part from Dendrobium loddigesii was established.(1)22 common peaks were separated from 17 batches from Guangxi,Guangdong,Yunnan and Guizhou of Dendrobium loddigesii and two common peaks are identified,which were naringenin and gigantol.After getting rid of the batch with great different,the similarity of whom is only 0.596,the similarities of 16 batches were 0.854～0.969.(2)16 common peaks among the HPLC characteristic spectrum of Dendrobium loddigesii were separated from 10 batches of the HPLC characteristic spectrum of water solution of Dendrobium loddigesii.The similarities of 10 batches were 0.909～0.968,with high similarities,and the similarities of every batch sample between Dendrobium loddigesii and its water solution of Dendrobium loddigesi were 0.815～0.910,which promptd conventional water decoction can basically keep the main characteristic omponent of Dendrobium loddigesii.(3)The HPLC characteristic spectrum of 3 batches of Dendrobium huoshanense and Dendrobium loddigesii were quite large.There were hardly same common peak except peak 14(naringenin).The characteristic spectrum of Dendrobium loddigesii to be a mutual mode control,the similarities of 3 batches of Dendrobium huoshanense were only 0.173～0.339.2.D-Mannose and D-Glucose were the main content of polysaccharide pre-column derivation HPLC characteristic spectrum from Dendrobium loddigesii,with a great difference with Dendrobium huoshanense.(1)Six common peaks were separated in 10 batches of Dendrobium loddigesii,mainly composed of D-Glucose and D-Mannose and which the peak area percentage were 42.95%and 53.99%,respectively,containing traces of D-Galacturonic acid,D-Galactose,D-Xylose and D-Arabia sugar,which the peak area percentage were 0.50%,1.12%,0.66%,0.77%,respectively.The similarities of 10 batches were 0.934～1.000.(2)Six common peaks were also separated in 3 batches of Dendrobioum huoshanense,which were similar to Dendrobium loddigesii,mainly composed of D-Mannose and D-Glucose.The peak area percentage of D-Mannose and D-Glucose were 76.99%and 21.73%.There were traces of D-Galacturonic acid,D-Galactose,D-Xylose and D-Arabia sugar,which the peak area percentage were 0.14%,0.58%,0.21%and 0.35%respectively.(3)There was difference in the polysaccharide pre-column derivation HPLC characteristic spectrum between Dendrobioum huoshanense and Dendrobium loddigesii,by which to be a control mode,the similarities of 3 batches of Dendrobioum huoshanense were merely 0.811～0.828,with low similarities.3.The method for content determination of D-mannose and D-glucose was established.The regression equation of D-mannose was A,/As =0.9561X+0.0027,r=0.9999.The linear ranges of whom were 0.010～1.0 μg.And the regression equation of D-glucose was Ag/As=0.9145X-0.0022,r=0.9999,the linear ranges of whom were 0.040～0.80 μg.The content of D-mannose and D-glucose(Cm,and Cg)of 7 batches were 5.07%～9.42%,8.43%～13.71%,respectively;the range of the sum of Cm and Cg(Cm+g)in 3 batches of Dendrobium loddigesiiwas 13.50%～22.61%.The contents of Cm and Cg were 18.44%～19.85%,5.30%～10.72%in the three batches of Dendrobioum huoshanense,respectively,and the range of Cm+g,was 25.15%～30.02%.The results showed that the Cm+g of Dendrobioum huoshanense is higher than Dendrobium loddigesii,with the Cg similar,the Cm in Dendrobium huoshanense were about 1～4 times as high as in Dendrobium loddigesii.The ratio of peak areas of D-mannose and D-glucose in Dendrobium loddigesii and Dendrobium huoshanense were 0.56～0.88 and 1.96～4.07 respectly,with obvious difference.The range of the ratio of peak areas of D-mannose and D-glucose was diffenent with Dendrobium offcinale in "Chinese pharmacopoeia",which is 2.4～8.0.4.The method for content determination of gigantol was established.The regression equation of gigantol was Y=16841 X + 2.4747,r=0.9999,which the linear ranges were 0.00802～0.1203 μg,r=0.9999.The content of gigantol from 10 batches of Dendrobium loddigesii were 0.0739～0.171mg/g.In the same condition,the peak of gigantol in Dendrobioum huoshanense is obviously small,which could not be found.5.The method for TLC of Dendrobium loddigesii was of specificity,with great different with other species of Dendrobium.(1)Identification of alkaloids:The mobile phase system was chloroform-methanol(10:0.8)and color developing agent was bismuth potassium iodide.Neither Dendrobium loddigesii nor Dendrobium huoshanense was identified the spot of dendrobine.There was a main spot from Dendrobium loddigesii while Dendrobium huo,shanense was not identified.(2)Identification of bibenzyls:The mobile phase system was petroleum ether(60～90℃)-ethyl acetate(6:4)and color developing agent was 10%sulfuric acid ethanol.There were two main spots,one of which was gigantol from Dendrobium loddigesii while Dendrobium huoshanense was identified only one with lighter colour.(3)Identification of flavonoids:The mobile phase system was methylbenzene-Methanol-butanone(6:1.5:2)and color developing agent was alchlor.Dendrobium loddigesii,and Dendrobium huoshanense were both identified the spot of naringenin(365nm).The spots from Dendrobium loddigesii identified were more than Dendrobium huoshanense and the colour of Dendrobium loddigesii was more obvious.Conclusion:1.The analysis means of HPLC characteristic spectrum polysaccharide pre-column,content determination of gigantol,D-Mannose,D-Glucose and TLC of Dendrobium loddigesii were accurate and reliable,with a good repetitiveness.The HPLC characteristic spectrum and TLC shows an obvious difference between Dendrobium loddigesii and Dendrobium huoshanense,which provides effective basis of quality control of Dendrobium loddigesii and identification of Dendrobium huoshanense.2.The water decoction of Dendrobium loddigesii can basically keep the main characteristic component of its herbal,which provides certain reference basis for the marble material base of Dendrobium loddigesii.3.The content determination of D-Mannose and D-Glucose are high in Dendrobium loddigesii,which is appropriate for the indicators of quantify.4.There is great difference in the HPLC characteristic spectrum,contents of D-Mannose,ratio of peak areas of D-mannose and D-glucose and TLC with Dendrobium loddigesii and Dendrobium huoshanense,which provides certain reference of distinguishing Dendrobium huoshanense and Dendrobium loddigesii.