CN107496490A - One kind extraction glycoside substance method - Google Patents

One kind extraction glycoside substance method Download PDF

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CN107496490A
CN107496490A CN201710893479.XA CN201710893479A CN107496490A CN 107496490 A CN107496490 A CN 107496490A CN 201710893479 A CN201710893479 A CN 201710893479A CN 107496490 A CN107496490 A CN 107496490A
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extract
barrenwort
glycoside
medicine
epimedium
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CN107496490B (en
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杨桂芹
王志标
刘霞
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Anhui Jin Yuan Pharmaceutical Co Ltd
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Anhui Jin Yuan Pharmaceutical Co Ltd
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Abstract

One kind is disclosed from plant extract glycoside substance method, and the composition comprising Shorthorned Epimedium P.E and gadol extract, the extract have especially good antiphlogistic effects compared with general extraction mixture.The composition can be used for preparing the medicine, health products or drink that can strengthen immune function of human body, radiation proof and anti-inflammatory.

Description

One kind extraction glycoside substance method
Technical field
The present invention relates to extractive technique field, more particularly to extracts glycoside thing from barrenwort using multistage column chromatography for separation Matter method, and it is related to the composition comprising Shorthorned Epimedium P.E and rhodiola root derivative.
Background technology
Barrenwort is traditional traditional tonic medicine in China, is initially recorded in《Sheng Nong's herbal classic》On, it has tonifying kidney and strengthening yang, by force Muscle reinforcing and bone strengthening, the effect of remove rheumatism.Epimedium herb is the dried leaf of Berberidaceae plant barrenwort, and existing pharmacopeia is included shared The kinds such as Epimedium sagittatum, E. Pubescens and korean epimedium herb are more.Barrenwort has the work(of kidney-replenishing, strengthening the bones and muscles, wind-damp dispelling Effect.Icariin is one of monomer composition, and having improves cardiovascular system, regulation endocrine, strengthen immunity, sex hormone sample Deng effect, while also there is positive therapeutic action in antitumor, anti-liver poison etc..
Modern pharmacological research shows that barrenwort can increase cardiovascular and cerebrovascular CBF, promotes hematopoiesis function, immunologic function and bone Metabolism, there is anti-aging, antitumor and other effects.
Recent researches find, the monomeric substance such as glycoside material, Flavonoid substances and polysaccharose substance in barrenwort, Improve blood vessel inner skin cell function and process barrenwort Chinese medicine better than tradition especially, but their separation and Extraction has very big difficulty.
CN101264120B discloses a kind of barrenwort flavonol glycosides medicine materical crude slice, is made up of barrenwort effective part extract, Icariin content is 10-20%wt, and icariside I I contents are 2-5%wt, general flavone content 40-60%wt, total yellow Ketone include icariside I I, newly determine towards leaves of pulse plants glycosides 1, towards the leaves of pulse plants K, icariin, towards leaves of pulse plants glycosides I, towards leaves of pulse plants glycosides third, towards leaves of pulse plants glycosides A, newly towards the leaves of pulse plants Glycosides 2 and Hyperoside, the preparation method of barrenwort flavonol glycosides medicine materical crude slice include：Weigh the korean epimedium herb overground part after crushing Point, with ethanol water refluxing extraction；Extract solution filtering, be concentrated under reduced pressure, then diluted with ethanol water after merging, after standing from The heart, upper Diaion-HP20 resin columns, with ethanol water gradient elution, collect 60-70%v ethanol waters elution liquid, decompression Paste is concentrated into, crushed after being dried, obtains barrenwort flavonol glycosides medicine materical crude slice.
CN101843629A discloses the new application of icariin and the epimedium flavone containing icariin, and in particular to The extract prepare be used for treat, prevent, mitigate and/or alleviate the disease relevant with nervous system myelin disease damage and/or Purposes in the medicine of illness, or preparing the purposes in being used to mitigate myelinoclasis and/or promote the medicine of myelin reparation.
CN104711300A discloses a kind of preparation method of icariine, and this method is included Shorthorned Epimedium P.E in fruit Enzyme digestion reaction is carried out in the presence of glue enzyme, obtains icariine, it is preferable that described pectase include polygalacturonase and Arabinofuranosidase.
CN101933550A discloses the electuary containing barrenwort, and the electuary is by barrenwort, ground coffee, cordyceps sinensis or pupa Cordyceps sinensis is formed, and the percentage by weight of each material is as follows：Barrenwort and cordyceps sinensis or Cordyceps militaris sum account for 2-99%, ground coffee 1- 98%, or barrenwort 1-95%, cordyceps sinensis or Cordyceps militaris 1-96%, ground coffee 1-98%.
CN104171175A discloses a kind of barrenwort tea, and it is prepared in following manner：A, collection is selected：Every year Spring gathers Epimedium brevicornum leaves, chooses debris and old leaf；B, finish：Finished 1-3 minutes at 160 DEG C~200 DEG C；C, rub：Will Epimedium brevicornum leaves after fixing are rubbed, and are rubbed into bar；D, fried dry：It will rub to Epimedium brevicornum leaves in stripes at 130 DEG C -90 DEG C Carry out fried dry, first through 130 DEG C of fryings of high temperature, fry to it is half-dried when gradually descend temperature regulating gradually to descend temperature adjustment again through frying to 110 DEG C Degree is to 90 DEG C, untill fried dry.
CN101607976A discloses a kind of preparation method of icariin, takes epimedium herb, with 30-90% methanol Or alcohol reflux extraction, recycling design, it is condensed into medicinal extract；Extracted repeatedly with petroleum ether, chloroform, ethyl acetate and n-butanol successively To near colourless, recovery n-butanol obtains coarse extract to dry；Then silica gel column chromatography separation and polyamide column chromatography point are carried out successively From producing icariin highly finished product, purity >=98%.
CN104688795A discloses a kind of method that general flavone is prepared by barrenwort, and this method comprises the following steps：a. Barrenwort cured leaf is extracted by ethanol solution first, obtains extract solution；B. by the extract solution that step a is obtained with macropore tree Fat post adsorbs, and then collects last time ethanol eluate with water and the macroporous resin column described in ethanol elution successively；C. will step The eluent that rapid b is obtained is dried, and obtains described general flavone.
CN101669980B discloses a kind of method that separating icariin and general flavone are extracted from barrenwort, including with Lower step：First extracted using 20-40% ethanol as solvent counter current, extract solution centrifuges, and separating liquid is respectively with microfiltration membranes except heavy Filtering and Ultra filtration membrane, then with nanofiltration membrane treatment, obtain trapped fluid；This trapped fluid is dried, below 60 DEG C of drying temperature, dried Thing moisture content≤5.0%, dried object are epimedium active constituent enriched substance, and wherein Icariin content reaches more than 60%, excessive Sheep leaves of pulse plants general flavone content reaches more than 80%, UF membrane each unit icariin, the epimedium flavone rate of transform >=98%.
CN101899077A discloses the extracting method of icariside I in Korean epimedium leaves, and its step and condition are such as Under：A. the volumetric concentration that Korean epimedium leaves use is 15 times of its quality is 70% alcohol reflux 4 hours, filters out ethanol extraction Liquid, the volumetric concentration that the dregs of a decoction to flow back use is 10 times of korean epimedium herb leaf quality is extracted 2 hours for 70% alcohol reflux, filter Go out ethanol extract, merge filtrate twice；B. filtrate is depressurized at 50 DEG C and steams solvent, obtains extract, contained in extract Moisture be not more than the 5% of its quality；Obtained by c. using in the absolute ethyl alcohol dissolving step b of 2 times of korean epimedium herb leaf quality Extract, concentrated hydrochloric acid is added, korean epimedium herb leaf quality gram: the volume mL of concentrated hydrochloric acid is 1:0.05~0.15, in 50 DEG C of water-baths Heat 15-25 hours, left at room temperature over night, filtering；D filtrates are concentrated under reduced pressure into the 20% of its volume at 50 DEG C, and it is excessive to add Korea The distilled water of 2 times of sheep leaves of pulse plants leaf quality separates out precipitation, is deposited at 50 DEG C and is washed with the ethanol solution that volumetric concentration is 20%, is washed Being deposited at 50 DEG C afterwards is dried in vacuo, and obtains icariside I powder.
CN105998140A discloses a kind of rhodiola root composition, and it is made up of following component：Rhodiola root 1.5-6g, thorn five Add 3-27g, Radix Angelicae Sinensis 2-12g.
CN102487979B discloses a kind of Rhodiola rosea micro-powder lotus seed paste, by weight percentage form include lotus seeds 50-70%, Momordica grosvenori 5-15%, vegetable oil 10-20%, Radix Rhodiolae micropowder 7-15%；The Radix Rhodiolae micropowder is rhodiola root ultramicro grinding Particle diameter made of machine is 0.1-10 μm of micro mist.
CN103012512A discloses a kind of isolation and purification method of rhodioloside in natural rhodiola root, takes natural rhodiola root Cut into slices for raw material, obtain Aqueous extracts through ultrasonic wave water extraction, low temperature alcohol precipitation and after being concentrated under reduced pressure, then peeled off through solvent Removal of impurities, solvent extraction and back extraction the stage obtain the rhodioloside aqueous solution of high-purity, most afterwards through being concentrated under reduced pressure, absolute ethyl alcohol knot Crystalline substance, separate and be dried to obtain rhodioloside product.
CN101804156A discloses the preparation technology and antifatigue effect of compound rhodiola root extractive, and it is by rhodiola root 20g-30g, sealwort 20g-30g, fruit of Chinese magnoliavine 10g-20g, barrenwort 10g-20g, jujube 3g-5g, what 8-10 times of coarse powder addition was measured After water immersion 4h-6h, 100 DEG C of heating extractions, then ultrasonic 1h-1.5h, temperature is 30 DEG C, power 80W, then with gauze mistake Filter, filter residue is each once with 6 times of amounts and 4 times of amount water repetition above steps respectively again, merges filtrate three times, vacuum drying, adds people Join three alcohol type saponin(e 0.5g-1g, obtain compound rhodiola root extractive.
" barrenwort Study on extraction ", Chen Luoyi etc., Chinese Journal of Modern Applied Pharmacy, 2000 (s1):12-14, research are excessive Sheep leaves of pulse plants extraction process, wherein using Icarrin as Testing index, from L9 (3~4) orthogonal test table, knot Fruit shows that decocting method is close with alcohol extracting method extraction effect, and barrenwort uses decocting method, and simple process, cost is low, and extraction efficiency is high, It is comparatively ideal production technology.
From above-mentioned prior art, remain in the prior art in number of drawbacks, barrenwort extraction of the prior art Thing is actually the mixture of many kinds of substance, wherein including flavones ingredient, more than ten kind leaves of pulse plants glycosides, the leaves of pulse plants time glycosides, a variety of glucosides, wooden fat Element, alkaloid and polysaccharide, maltol, the vertical glycosides of sand, Quercetin, icariine A, Epimedium acuminatium glycosides, anhydroicartin, isoamyl Alkene kaempferol, ginkegetin, Isoginkgetin, bilobetin, epimidin A, Hyperoside, also containing certain Volatile oil, brown eleostearic acid, stearic acid, oleic acid, the leukotrienes of amount.In these materials, effective component is uneven, or even some components Drug effect is relatively low, relatively low so as to not only result in integral medicinal effect, and because some poorly efficient compositions can influence high-drug-effect composition on the contrary The performance of energy.In addition, there exists a further problem in that, Shorthorned Epimedium P.E and gadol extract are mixed although having in the prior art The report of compound formulation is prepared, but is generally all to mix two kinds of extracts containing complicated ingredient, not only effect is poor, and Synergy is also poor.This area needs a kind of efficient barrenwort and gadol extract and preparation method thereof.
The content of the invention
To solve above-mentioned technical problem present in prior art simultaneously, sugar is extracted from barrenwort the invention provides one kind Glycoside substance method, the glycoside material have of a relatively high pharmacological activity compared with other compositions in extract, special It is not to improve immunity and anti-inflammatory efficacy.In addition, the composition comprising Shorthorned Epimedium P.E and gadol extract is additionally provided, Acted synergistically by the compounding of the two, it is possible to increase comprehensive pharmacological activity.Therefore, the invention provides following technical scheme.
In one aspect of the invention, there is provided one kind uses multistage column from plant extract glycoside substance method, this method Chromatography.
Preferably, wherein the plant is barrenwort.
Preferably, in the multistage column chromatography for separation, first order column chromatography uses reverse phase silica gel post.
Most preferably, the extraction and isolated glycoside material are the compound shown in lower formula (I)：
Research shows that compared with general flavone compound such as icariine A and B, it has the glucoside compound Wider bioactivity, such as anti-osteoporosis disease, androgen, anti-inflammatory and antitumaous effect.Therefore, the application makees the compound For Objective extraction compound.
For the present invention, particularly preferably, the compound shown in the formula (I) can be made by following method, the party Method comprises the following steps：
(1) barrenwort herbal medicine powder is extracted (preferably with MeOH (preferably 3 × 6L, each soak time are about 20h) at room temperature 1.18kg), the extract of merging is evaporated under reduced pressure to obtain brown residue (162.5g) i.e. methanol extract liquid；
(2) reverse phase silica gel post (such as RP-18 silicagel columns) is utilized, first by MeOH-H2O mixed solutions are to the remnants Thing carries out gradient elution, and to carry out pillar layer separation, preferable elution step is 0:1 (1L), 1:9 (1L), 2:8 (1L), 3:7 (1L), 4:6 (1L), 5:5 (2L), 6:4 (2L), 7:3 (1L), 1:0 (2L), then using acetone (2L), 10 is obtained successively and is washed De- component, it is designated as eluting component A-J；
(3) elution fraction B (such as 6.5g) is subjected to chromatographic isolation on silicagel column (preferably 60cm × 5cm), used CHCl3-MeOH-H2O mixed solutions carry out gradient elution, and (gradient is preferably 20:1:0 (1L), 10:1:0 (2L), 30:8:1 (2L) and 15:6:1 (2.5L)), 4 elution components are obtained, are designated as elution fraction B1-B4；
(4) elution fraction B2 (such as 300.2mg) is used into MeOH-H2O(1:1) in Sephadex LH-20 (preferably 100cm × 3cm) on separate, obtain 7 time elution fractions, be designated as elution fraction B2.1-B2.7；
(5) it is by silica gel CC posts (preferably 80cm × 2.5cm), eluent by elution fraction B2.6 (such as 95.0mg) EtOAc-CHCl3-MeOH-H2O(16:8:3:1), eluent is concentrated under reduced pressure into dry, obtains pale yellow powder (such as 6.1mg), Compound as shown in formula (I).
Analyzed through HPLC, the purity of compound shown in formula (I) is 99.7% in extract.Through1HNMR and mass spectral analysis, its It is consistent with the known compound through sign described in CAS.Positive ion mode ESI-MS shows that molecular ion peak appears in m/z 667([M+Na]+) and 1311 ([2M+Na]+)。IR(KBr):3423,2940,1660,1605,1512,1450。1In HNMR, in δ (H) the A2B2 coupled systems that 7.88 (d, J=8.6, H-C (2 ', 6 ')) and 7.11 (d, J=8.6, H-C (3 ', 5 ')) occur are shown The presence of para-orientating group in B rings；δ (H) 6.72 (d, J=10.0, H-C (1 "), 5.68 (d, J=10.0, H-C (2 "), The serial hydrogen atom signal that 2.14 (s, Me (4 ")) and 1.45 (s, Me (5 ")) occur shows γ be present, γ-dimethyl chromene ring.
Experiment finds that it is extremely difficult to want separation to obtain compound or extract shown in the formula (I) of high-purity, wherein A major reason for causing to be difficult to obtain high-purity compound is the interference of albumen in extraction process, in addition, some low liters Polysaccharide is also the impurity of more difficult separation.The applicant obtains the formula of extreme high purity by above-mentioned 5 grades of column chromatography for separation first (I) compound shown in.There is close level and matching relationship, it sets selection and order non-between 5 grades of piece-rate systems Chang Guanjian.
Certainly, it will be appreciated by those of skill in the art that other elution fractions may also be used for obtaining it is other valuable Material rather than whole discard.
In another aspect of this invention, there is provided a kind of composition comprising Shorthorned Epimedium P.E and gadol extract, The Shorthorned Epimedium P.E is the glycoside material obtained according to the above method, is preferably compound shown in (I).Can also be at this Saussurea involucrata extract, wolfberry fruit extract or other extracts with required pharmacological activity are added in composition.
Preferably, the purity of the glycoside material is more than 99.0wt%.It is highly preferred that the purity of the glycoside material More than 99.2wt%.Most preferably, the purity of the glycoside material is more than 99.7wt%.
The gadol extract is preferably rhodioside.Rhodioside can take the conventional method of this area to be carried Take.It is further preferred that the gadol extract is modified gadol extract, most preferably, the gadol extract is logical Cross to the compound shown in the lower formula (II) of rhodioside modification acquisition or (III)：
Compared with general natural nucleoside material, formula (II) or immunological regulation of the compound to normal mouse shown in (III) Effect becomes apparent from, and due to electron withdraw group Cl and F introducing, enhances it and removes the ability of free radical, can dramatically increase mouse Special antibody secreting cell number, delayed allergy intensity can be strengthened with 50mg/ (kgd) dosage, also showed in addition Go out the mixed lymphocyte reaction (MLP) to special-shaped mouse and the phagocytic function of macrophage, and can reduce to a certain extent white The activity of cytokine, show it is a kind of up-and-coming immunomodulator both it.
Material with structure formula (II)~(III) can synthesize by the following method：MCM- is added in reaction vessel 22 molecular sieves alternatively property catalyst, then adds chloroform solvent, then by natural salidroside and the acetyl of chloro or fluoro Chlorine (i.e. acylating agent) is with 1:1~1.3 mol ratio is added in reaction dissolvent (natural salidroside and chloro or the acetyl of fluoro Chlorine adds preferably in the form of chloroformic solution), reaction temperature is 20-30 DEG C, reaction time 10-30min, and separation and drying are The compound can be obtained.This method has the advantages of high income, impurity are few.It is particularly due to the shape-selective work of MCM-22 molecular sieves With the generation of side reaction can be significantly reduced.
In another aspect of this invention, there is provided the purposes of the composition, it, which is used to prepare, can strengthen human immunity work( Medicine, health products or the drink of energy.
Preferably, the medicine is Chinese traditional compound medicine.Preferably, the composition is used to prepare oral capsule, electuary Deng.The health products include oral-cavity article such as chewable tablets or lozenge.
In a preferred embodiment of the present invention, in the Chinese traditional compound medicine, Shorthorned Epimedium P.E and rhodiola root The weight ratio of derivative is 1:20-20:1.When adding Saussurea involucrata extract, the weight ratio of Saussurea involucrata extract and Shorthorned Epimedium P.E For 1:10-1:20.Such composition has the effect of especially good anti-inflammatory, strengthen immunity and/or radiation proof.
Brief description of the drawings
Fig. 1 is the compound according to the embodiment of the present application 11HNMR analysis diagrams；
Fig. 2 is the suppression curve figure to PTP1B according to the compound of the embodiment of the present application 1.
Embodiment
Embodiment 1：The extraction of compound shown in formula (I)
Using compound shown in following operation extraction formula (I)：(1) MeOH (3 × 6L, each soak time are used at room temperature It is about 20h) extraction barrenwort herbal medicine powder (1.18kg, raw material are purchased from Tonghua, Jilin Province city Jin Chang towns), the extract of merging is subtracted Pressure evaporation obtains brown residue (162.5g) i.e. methanol extract liquid；(2) RP-18 reverse phase silica gel posts are utilized, first by MeOH- H2O mixed solutions carry out gradient elution to the residue, to carry out pillar layer separation, elution step 0:1 (1L), 1:9 (1L), 2:8 (1L), 3:7 (1L), 4:6 (1L), 5:5 (2L), 6:4 (2L), 7:3 (1L), 1:0 (2L), then using acetone (2L), 10 elution fractions are obtained successively, be designated as eluting component A-J；(3) by 6.5g elution fractions B silicagel column (60cm × Chromatographic isolation is carried out on 5cm), using using CHCl3-MeOH-H2O mixed solutions carry out gradient elution (gradient 20:1:0 (1L), 10:1:0 (2L), 30:8:1 (2L) and 15:6:1 (2.5L)), 4 elution components are obtained, are designated as elution fraction B1-B4； (4) by 300.2mg elution fraction B2 MeOH-H2O(1:1) separated on Sephadex LH-20 (100cm × 3cm), obtain 7 Individual secondary elution fraction, is designated as elution fraction B2.1-B2.7；(5) by 95.0mg elution fractions B2.6 by silica gel CC posts (80cm × 2.5cm), eluent EtOAc-CHCl3-MeOH-H2O(16:8:3:1), eluent is concentrated under reduced pressure into dry, it is light obtains 6.1mg Compound shown in yellow powder, as formula (I).
Embodiment 2：Compound shown in formula (I) suppresses PTP 1B (PTP1B) and alpha-glucosidase Ability.Specific rejection ability is as shown in table 1 below：
Table 1：Compound shown in formula (I) suppresses the ability of PTP 1B (PTP1B) and alpha-glucosidase
In the test, malol and acarbose are used as positive reference substance.Method of testing is according to standard method with micro Method is carried out, and is specifically：Body series reaction μ L, the PTP1B substrates p-NPP of cumulative volume 100 is dissolved in buffer solution (50mmolL- 1HEPES pH value 7.3,100mmolL-1NaCl, 0.1%BSA, 1mmolL-1DTT in), pNPP reactions are final concentration of 5mmol·L-1, extract dissolves by DMSO, takes the μ L of enzyme liquid 0.5~1, is diluted by 250 μ L PBSs and is fully mixed, takes The μ L of enzyme liquid 160 diluted add to 96 orifice plates, add the μ L of DMSO (extract solvent) 1, soft mixing, at the same by PTP1B with Extract adds 35mmolL after adding 96 orifice plates incubation at room temperature 5min-1The μ L of p-NPP 20,37 DEG C of lucifuges react 30min, 0.5mol·L-1Sodium hydroxide terminating reaction, ELIASA reads absorbance (A) value at wavelength 405nm, according to normalized form meter Calculate corresponding half-inhibition concentration (IC50) value；Also make suppression curve of the compound to PTP1B simultaneously (referring to Fig. 2).
Embodiment 3：The manufacture of capsule comprising Shorthorned Epimedium P.E and gadol extract
The capsule manufacture method of Shorthorned Epimedium P.E and gadol extract comprising embodiment 1, wherein the rhodiola root Extract is compound shown in formula (II), and the weight ratio of Shorthorned Epimedium P.E and rhodiola root derivative is 5:1, each capsule contains 0.1g contents.
Embodiment 4：Antiinflammatory action of the capsule of testing example 3 to mice auricle swelling
Kunming mice 40 is taken, body weight 150-220g, male and female half and half, is randomly divided into 5 groups, i.e. model group, conventional extract Capsules group (0.08gkg-1, control group, wherein Shorthorned Epimedium P.E according to described in CN101264120B method obtain, its It is remaining same as Example 3), the Capsules group (0.08gkg of embodiment 3-1), daily gastric infusion 1 time, continuous 4d, in last dose 30min afterwards, every mouse right ear exterior feature two sides are applied the μ L of dimethylbenzene 20, cause mice auricle swelling model, compared with left ear, Cervical vertebra is taken off after 20min and puts to death mouse, two ears are cut along auricle baseline, with diameter 6mm card punch respectively in left and right ear antimere Circular auricle is laid, precise weighing, using its difference as inflammatory swelling degree (mg), and calculates swelling inhibiting rate.
Swelling inhibiting rate=(model group swelling-administration group swelling)/model group swelling × 100%
Compared with model group mice ear degree, the compound capsule group of embodiment 3 is compared with compareing each dosage group of Capsules group The inhibitory action of mice ear caused by paraxylene has significant difference, shows to prompt FUFANG XUELIAN JIAONANG to mitigate diformazan Benzene causes mice auricle swelling degree, has certain antiinflammatory action, see the table below 2：
Table 2：Compound capsule paraxylene causes the influence of mice auricle swelling
Group Dosage/gkg-1 Swelling/mg Inhibiting rate/%
Model group - 8.55±1.22 -
Embodiment 3 0.08 4.01±0.92 53.10%
Control group 0.08 5.96±1.23 30.29%
By above example clearly it can be seen from the present invention Shorthorned Epimedium P.E to PTP1B) and alpha-glucosidase With good inhibitory action, and relative to extract of the prior art (i.e. crude extract, the mixture of Multiple components) Compare, there are obvious more preferable antiphlogistic effects.
This written description discloses the present invention, including optimal mode using example, and also enables those skilled in the art Manufacture and using the present invention.The present invention can patentable scope be defined by the claims, and this area skill can be included Other examples that art personnel expect.If this other examples have the structural elements of the not literal language different from claims Element, or if this other examples include equivalent structure element of the literal language without substantial differences with claims, Then this other examples are intended to be within the scope of claims.In the case where inconsistent degree will not be caused, by reference to It will be incorporated herein in place of all references referred to herein.

Claims (10)

1. one kind uses multistage column chromatography for separation from plant extract glycoside substance method, this method.
2. according to the method for claim 1, wherein the plant is barrenwort.
3. method according to claim 1 or 2, wherein in the multistage column chromatography for separation, first order column chromatography uses Reverse phase silica gel post.
4. a kind of composition comprising Shorthorned Epimedium P.E and gadol extract, the Shorthorned Epimedium P.E is will according to right The glycoside material for asking the method any one of 1-3 to obtain.
5. composition according to claim 4, wherein the purity of the glycoside material is more than 99.0wt%.
6. composition according to claim 5, wherein the purity of the glycoside material is more than 99.2wt%.
7. composition according to claim 6, wherein the purity of the glycoside material is more than 99.7wt%.
8. the purposes of the composition according to any one of claim 4-7, it, which is used to prepare, can strengthen immune function of human body Medicine, health products or drink.
9. purposes according to claim 8, wherein the medicine is Chinese traditional compound medicine.
10. purposes according to claim 9, wherein in the Chinese traditional compound medicine, Shorthorned Epimedium P.E and rhodiola root spread out The weight ratio of biology is 1:20-20:1.
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