| Dendrobium officinale is a kind of rare medicinal herbs,which grows in the south of China.That is what is called "ginseng is in the north,as dendrobe in the south”.It belongs to dendrobium species,which is one kind of perennial herbaceous plants and can be harvested from November to next March.The stems can be used to make medicine.It can strengthen stomach and promote fluid production,nourish Yin and clear heat.For injury of fluid due to heat disease,dry mouth polydipsia,insufficiency of stomach yin,eat less retching,after the illness and Hot,bone-steaming,head dark unknown,bones and muscles impotent soft,etc..As people increasingly pay attention to health,dendrobium officinale becomes a new favor health in the area of health care market.Meanwhile,it's rapid development also brings series problems on cultivated varieties,cultivation area environment and technology,product development and quality assurance,etc.,which all restrict the sustainable development of industry.Our previous studies found that it has obvious differences between Dendrobium leaves and stems of tiepilan species(Yunnan,Guangxi),Danxia species(Guangdong,Fujian,Zhejiang,Jiangxi),Zhejiang native species(Zhe jiang),Because can't buy the corresponding reference substance reference standard,influenced the determination of characteristic flavonoid content of Dendrobium?Therefore,this experiment aims to Separat the flavonoids constituents from Dendrobium leaves of Danxia provenance,to measure the different flavone contents in five different producing area,which are Yunnan,Zhejiang,Guangdong,Guangxi and Fujian.By means of separate and identify its biological activities,the study aims to provide a certain reference for the identification and evaluation to the quality of medicinal materials.Objective1?This paper takes the leaves of dendrobium officinale in Danxia as raw material to extract,separate,and purify flavonoid monomeric compound,which lays a good foundation for discussing pharmacologic action of the leaves of dendrobium officinale,expanding medicinal parts of dendrobium officinale,and making full use of resources.2?The measurement method of multiple flavonoids compound content of stems of dendrobium officinale in different regions shall be established by PR-HPLC.3?Through content determination,the difference of dendrobium officinale from different origin can be examined,in order to provides a method for the identification of origin of dendrobium officinale,and some reference for improving quality standards of it.4?External antioxidant experiments shall be used to explore the physiological activity of isolated anti-oxidation monomer compound.And provide experimental evidence for screening flavonoids natural antioxidant.Methods1?General flavone of leaves of dendrobium officinale in Danxia shall be extracted with 70%,and different polarities extraction parts of crude extractof leaves of dendrobium officinale shall be sorted with organic solvent extraction method(petroleum ether,ethyl acetate,water saturation n-butyl alcohol).2?Extract of n-butyl alcohol part shall be isolated and purified by using macroporous resin AB-8,Sephadex LH-20,and ODS column chromatography.3?The flavonoids compounds can be obtained through isolation and purifying with the method of physical and chemical properties and modern instruments(ESI-MS,1H-NMR,1C-NMR).4?Taking the isolated compound as reference substance,the content of stems of dendrobium officinale in Guangdong,Guangxi,Zhejiang,Yunnan,and Fujian shall be measured with RP-HPLC method.Chromatographic conditions:Agilent ZORBAX SB-Aq(250×4.6mm,5 ?m)was adopted,the mobile phase was acetonitrile(A)-0.2%formic acid solution(B)with gradient elution at a flow rate of 1.0 mL · min-1,the gradient elution program was:0?15 min,12%?12%A;15?80min,12%?15%A,the detection wavelength was 340 nm,the column temperature was 30?;the injection volume was 20?L.5?Scavenging activity of isolated monomeric compound which is purified from leaves of dendrobium officinale in Danxia to DPPH and ABTS free radical,and reducing capacity of isolated monomeric compound to copper shall be determined respectively through three different antioxidant system.Results1?Three compounds including Vitexin-4"-0-glucoside,vitexin-2"0-pyranxyloside,and rutin gained by separation and purification are obtained from dendrobium officinale at the first time.2?By the analysis of chromatographic conditions,the separation of Vitexin-4"-0-glucosid?vitexin-2"-0-pyranxyloside and rutin which are contained in different areas' Dendrobium can meet the equirements of HPLC determination.The regression equation of Vitexin-4"-0-glucosid was Y ?2463713X-46232.5,r = 0.9997,which the linear range was 23.2?116.0 ug/mL.The regression equation of vitexin-2"-0-pyranxyloside was Y= 2355633X-24328.6,r=0.9997,which the linear range was 13.65?68.25 ug/mL.The regression equation of rutin was Y=1720543X-12895.2,r=0.9995,which the linear range was 77.4?387.0 ug/mL.The average sample recovery rate(n=6)were 99.58%,99.64%,99.39%,RSD was 0.88%,1.46%and 0.78%.3?Content determination results:dendrobium officinale in Danxia,Yunnan,Zhejiang,and Guangxi all contain Vitexin-4"-0-glucoside and vitexin-2"-0-pyranxyloside,but that of Danxia and Yuannan contain rutin.The content of Vitexin-4"-0-glucoside are:Yunnan Provenance 50-70ug/g,Zhejiang provenances 20-40ug/g,Guangxi provenance 10-30ug/g,Danxia Provenance 15-35ug/g.The content of vitexin-2"-0-pyranxyloside are:Yunnan Provenance about 30ug/g,Zhejiang provenances 10-30ug/g,Guangxi provenance about 15ug/g,Danxia Provenance 10-30ug/g.The content of rutin are:Yunnan Provenance 30-115ug/g,Danxia Provenance 75-340ug/g,Differences between the same provenance is relatively large,Still need to enlarge sample size for content determination.4?DPPH.Radical-scavenging Assay.:When the concentration of Vitexin-4"-0-glucoside is 1.0mg/mL,the inhibition was 48.82%,which similar to 0.lmg/mL BHT effect.It' s show that vitexin-4"-0-glucoside have some inhibition for DPPH.Radical-scavenging,but the effect is not strong.5?ABTS+ radical-scavenging Assay:Vitexin-4"-0-glucoside and BHT have a similar effect in the 0.5mg/ml,the inhibition was 86%,indicating that Vitexin-4"-0-glucoside has a strong cleaning effect on ABTS+ radical scavenging.6?Cu2+-reducing Power Assay:When the concentration of Vitexin-4"-0-glucoside is 1.Omg/mL,the Relative reducing power was 48.82%,while the relative reducing power of lmg/mLBHT up to 100%,indicating that vitexin-4"-0-glucosidase has a weak effect on Cu2+-reducing.7.The antioxidant experiment results of Vitexin-4"-0-glucoside:capacity of ABTS+ radical-scavenging>capacity of DPPH · Radical-scavenging>capacity of Cu2+-reducing.Conclusions1?There are differences of Vitexin-4"-0-glucoside,vitexin-2"-0-pyranxyloside,rutin contained in dendrobium officinale in different areas,but the origin of dendrobium officinale can be tested by the method of the content determination of RP-HPLC.It is recommended to Vitexin-4"-0-glucoside as Dendrobium common components for determination of the content of different areas' Dendrobium.2?It is calculated that the causes of flavonoids contained in different dendrobium officinale of different areas may be affected by climate and cultivation methods.In order to guarantee the quality,it is recommended that the standardized planting are needed.3?The measure of flavonoids RP-HPLC in dendrobium officinale is simple,accurate,reliable,and repeatable which provides the methods for the distinction of the origin and quality control of dendrobium officinale.4?Vitexin-4"-0-glucoside can offer antioxidant benefits,so it can be further confirmed and explored its antioxidation mechanism. |
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