| Dendrobium officinale Kimura et Migo.is originally recorded in the "Shen Nong's herbal classic",which is the China's rare traditional Chinese medicine.With the effect of benefiting stomach,nourishing yin and clearing heat,it is mainly used to treat the symptoms of febrile stomachache,retching,lung heat cough,lumbar acid soft embolism and so on.Modern pharmacology pointed that the D.officinale has anti-tumor,antioxidant,hypoglycemic,improve immunity and other physiological activity.Since the low reproduction rate in natural environment,coupled with uncontrolled picking,D.officinale was once on the verge of exhaustion.At present,artificial cultivation of D.Officinale are more and more mature,there has formed a large scale planting in the major producing areas(mainly distributes in Zhejiang,Guangdong,Guangxi,Fujian,Yunnan,Jiangxi and other provinces).Different provenances,added up weith different geography,climate and other factors,making the D.Officinale 's composition and content are so different.After analyzed the HPLC characteristic chromatogram of D.officinale in different areas,and according to the difference and regularity of characteristic spectrum,our team put forward the sources concept named "Danxia landform","Zhejiang local" and"Tiepilan".The study showed that the flavonoids were the main difference between different provenances.On this basis,our team identified New Zealand Vitexin ?,New Zealand I,Apigenin-6-C-a-L-rham-no-8-C-?-D-glucoseside,Apigenin-6-C-?-D-glucose-8-C-?-L-rhamnoside and rutin from D.officinale leaf after purification,separation and extraction.In addition,by using the electrospray mass spectrometry,New Zealand Vitexin ?,New Zealand Vitexin I,schaftoside,isoschaftoside,Apigenin-6-C-a-L-rhamno-se-8-C-?-D-gluco-seside,Apigenin-6-C-?-D-glucose-8-C-a-L-rhamnoside,rutin were identified and isolated from D.officinale.Therefore,in order to provides the basis of parting D.officinale sources,different provenances and wild D.officinale were analyzed by HPLC characteristic chromatogram combined with the content determination methods,aiming at the problems that most of flavonoids are isomers and difficult separation.In addition,the method would be used to compare the difference between D.officinale and its confusion varieties D.Paxt.Objective:1.To optimize the HPLC characteristic spectrum of flavonoids from D.Officinale for providing the reference for the division of provenance regions.To explore the difference of HPLC characteristic between wild imitation samples and common greenhouse cultivars,and to explore the differences of flavonoids in D.candidum and D.Paxt.2.To establish a method to determine the content of 5 flavonoid glycosides(New Zealand Vitexin II,New Zealand Vitexin I,schaftoside,Apigenin-6-C-a-L-rhamn-ose-8-C-?-D-glucoside,rutin),2 flavonoids(isoschaftoside and Apigenin-6-C-?-D-glucose-8-C-a-L-rhamnoside)by QAMS and mannose and glucose of D.officinale,which would be used to analyzed the difference in the content of different sources.Methods:1.HPLC characteristic spectrum was employed with Kromasil 100-5 C18 column,the acetonitrile methanol tetrahydrofuran(10:22:5)-0.05%phosphoric acid(gradient elution)was used as a mobile phase,the detection was set at 340nm.By the UV spectroscopy,retention time and reference control,the flavonoids were identified.And through the method of Ward system clustering analysis,the characteristic peak area and the numbers were used to classify provenances of D.officinale.On the basis of the clustering analysis,similarity evaluation would be done by the State Pharmacopoeia Commission of traditional medicine fingerprint similarity evaluation software system software(version 2004 A)2.Owing to the situation that individual component reference is difficult to obtain,the methods that the HPLC characteristic used,combined with the QAMS,was used to determine the content of 7 flavonoids of different provenances of D.officinale.3.To optimize the pre-column derivatization HPLC method to determine the content of mannose and glucose.Results:1.It was feasible to establish the HPLC characteristic analysis method of flavonoids in D.officinale.And the separation of each characteristic peak reached the requirement of content determination(1)13 common peaks were separated from 30 batches of D.Officinale and 7 common peaks were identified,which were New Zealand Vitexin ?,New Zealand Vitexin I,schaftoside,isoschaftoside,Apigenin-6-C-a-L-rhamnose-8-C-?-D-glucoside,Apigenin-6-C-?-D-gluc-ose-8-C-a-L-rhamnoside and rutin.The samples were divided into 3 categories by cluster analysis method,8 peaks were marked from 5 batch of the class I,which was mainly from Guangdong,Shaoguan,Jiangxi,Jinggangshan,Fujian and other areas of Danxia landform;10 peaks were marked from 11 batches of class II,which was mainly from Guangxi and Yunnan two areas;11 peaks were marked from 14 batches of class ?,which was composed of every region in Zhejiang province.And the results of clustering was consistent with the previous classification of the team.(2)Apigenin-6-C-a-L-rhamnose-8-C-?-D-glucoside,Apigenin-6-C-?-D-glucose-8-C-a-L-rhamnoside is the exclusive ingredients in "Tiepilan"(class ?)and the similarity of 11 batches was 0.749?0.980;peak 10 and peak 12 to peak 13 were higher on peak area in "Zhejiang local"(class ?),the similarity of 14 batches was 0.714?0.904;Danxia landform had high peak area on rutin and the similarity of 5 batches was 0.955?0.985.The Danxia landform common pattern to be a mutual mode control,the similarity of the common pattern of the specimens from Zhejiang local and Tiepilan were 0.257,0.579 respectively,and the similarity between Zhejiang local and Tiepilan was 0.344,indicating that different provenances of D.Officinale on flavonoid glycosides had certain differences.(3)10 common peaks were separated from 5 wild samples and the similarity was 0.863?0.924.There were not significantly different between the wild and the greenhouse one on the main peak area of flavonoids,but the wild one had more abundant components.(4)9 common peaks were separated from 10 batches of samples D.Paxt.and 5 common peaks were identified,which were New Zealand Vitexin ?,New Zealand Vitexin I,schaftoside,isoschaftoside and rutin.And among them,the peak Z1 to peak Z3 were the inconsistent with D.Officinale.The similarity of 10 samples was 0.929?0.987.Compared with the D.Paxt.control map,the similarity of Tiepilan,Zhejiang local and Danxia landform were 0.206,0.413 and 0.550 respectively,suggesting that the flavonoid glycosides between the D.Paxt.And D.officinale were quiet different.2.It was feasible to establish a method to determine the content of 5 flavonoids and 2 flavonoids by QAMS.(1)Established two relative correction factors:the Isoschaftoside and Schaftoside(S1),and the Apigenin-6-C-a-L-rhamnose-8-C-?-D-glucoside and Apigenin-6-C-?-D-glucose-8-C-a-L-rhamnoside(S2),which were 1.009 and 1.004 respectively.(2)The regression equation of Zealand Vitexin ?,New Zealand Vitexin I,Schaftoside,Apigenin-6-C-a-L-rhamnose-8-C-?-D-glucoside and rutin were Y=1770.7X 11.105,Y=1889.5X 5.476,Y=2046.5X 14.891,Y=1719.5X-4.402 and Y=1202.7X 11.916 respectively,which the linear ranges were 0.00925?1.85 ?g,r=1.0000,0.00843?1.686?g,r=1.0000,0.00714?1.428?g,r=0.9999,0.1195?2.39 ?g,r=0.9999 and 0.0165?3.3 g,r=1.0000 respectively.(3)The content of Apigenin-6-C-?-L-rhamnose-8-C-?-D-glucoside and Apigenin-6-C-?-D-glucose-8-C-?-L-rhamnoside from 11 batches of Tiepilan were 0.0422?0.232 mg/g,0.0154?0.138 mg/g respectively,which were the specific ingredient of Tiepilan.And the content of Apigenin-6-C-a-L-rhamnose-8-C-(3-D-glucoside was probably 3 times than the Apigenin-6-C-?-D-glucose-8-C-a-L-rhamnoside in the same sample.While the content of New Zealand Vitexin ?,New Zealand Vitexin I,schaftoside,isoschaftoside of 5 batches of Danxia landform were 0.0206?0.1126mg/g?0.0100?0.1298mg/g?0.0023?0.0234mg/g? 0.0026?0.0268mg/g respectively,11 batches of Tiepilan were 0.0349?0.1113mg/g?0.0052?0.0563mg/g?0.0012?0.0123mg/g ? 0.0016?0.0133mg/g respectively,and 14 batches of Zhejiang local were 0.021?0.1055mg/g?0.009?0.0548mg/g?0.0055?0.0308mg/g and 0.0066-0.0416mg/g,which indicated that there have a intersection on the above components from the 3 classes.Rutin content in the Danxia landform is 0.2828?0.6068mg/g,is higher than the other.Zhejiang local took the second place,which except the two group were 0.1558mg/g and 0.2432mg/g,rutin content of 12 samples is 0.02?0.0972mg/g,the Tiepilan is the lowest(0.0083?0.106mg/g).(4)The content of New Zealand Vitexin ?,New Zealand Vitexin ?,schaftoside,isoschaftoside and rutin in 9 batches of D.Paxt.Were 0.0181?0.0426mg/g,0.0139?0.0491 mg/g,0.0077?0.0307 mg/g,0.0107?0.400 mg/g and 0.0369?0.1924 mg/g respectively,indicating that there was not significant difference between the D.Paxt.And D.Officinale on the content of this 5 components.3.There were some differences on the content of rhamnose and glucose in different provenances.(1)The content of D-mannose and D-glucose(Cm and Cg)of Danxia landform types were 17.43%?36.19%,4.24%?18.39%,respectively;the range of the sum of Cm and Cg content was 9.56%?32.68%,the area ratio(Am/Ag)between mannose and glucose peak is 2.59?12.36.The Cm and Cg content were 8.82%?24.44%,0.74%?9.09%in the Tiepilan,respectively,and the range of Cm+g content was 21.67%?54.58%,Am/Ag is 1.97?4.26.The Cm and Cg content were 10.42%?36.40%,1.38%?9.47%in Zhejiang species,respectively,and the range of Cm+g content was 12.67%?39.78%,Am/Ag is 1.40?9.29.The results indicated that there were great differences on the content and the area ratio of mannose and glucose in different provenances.It also indicated that there was no significant difference on the regularity in provenances,but the Tiepilan had the higher content than the others.(2)The Cm and Cg content were 15.59%?20.67%,5.30%?18.03%in 5 lots of wild samples,respectively,and the range of Cm+g content was 23.83%?33.62%,the Am/A was 0.90?3.63.It had higher content of Cg relatively,which showed some differences with the greenhouse one.Conclus ion:1.The analysis means of HPLC characteristic spectrum of D.Officinale is accurate and reliable,and it can reach the aim to distinguish different provenances and the confusion variety D.Paxt.quickly.2.The QAMS determination method of 7 flavonoids from D.Officinale is feasible,which provides a effective reference for the study of the quality of D.Officinale.3.There are great differences on the content of mannose and glucose in different provenances,but the regularity of them has not obvious.4.The imitating wild samples shows more abundant on the components of flavonoids and it has the higher content on polysaccharide. |
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