CN104725450B - A kind of method that high-purity oleuropein is extracted from jasmine - Google Patents
A kind of method that high-purity oleuropein is extracted from jasmine Download PDFInfo
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Abstract
The present invention relates to a kind of method that high-purity oleuropein is extracted from jasmine, this method includes ethanol water extraction, macroporous resin column absorption, polyamide column absorption with preparing oleuropein.The purity that the method for the present invention prepares oleuropein reaches by weight 98.0~99.5%, and oleuropein extraction rate reached to 2~4%, the method for the present invention is stablized, and favorable reproducibility is simple and efficient, is very suitable for industrialized production.
Description
【Technical field】
The invention belongs to extract Technique of Chinese Medicine Efficacious Ingredient fields.More particularly it relates to one kind is from jasmine
The method for extracting high-purity oleuropein.
【Background technology】
Jasmine is Oleaceae jasminum plant jasmine (Jasminum officinale L.var.grandiflorum)
Dry flower also known as jasmine pin, know tender tea leaves flower, have soothing liver-qi stagnation, promoting qi circulation and relieving pain the effect of.China's its treatment among the people disappears
Change the illnesss such as bad, duodenal bulbar ulcer, chronic hepatitis, hepatic sclerosis.Jasmine is widely cultivated all over the world, and resource is very
It is abundant.Result of study shows that jasmine mainly contains iridoid glycosides, triterpene saponin and flavone compound, middle ring
The content of alkene ether terpene glycosides compound oleuropein is relatively high, and there are larger research and development to be worth.
Oleuropein molecular formula is C25H32O13, there are multiple biological activities, such as it is antiviral, antibacterial, anti-inflammatory, hypoglycemic,
It has a extensive future in sector applications such as medicine, health food, cosmetics.
At present, oleuropein is mainly from olive (Olea europaea L.), lilac (Syringa oblate
Lindl.), extract and obtain in the plants such as daphne lilac (Syringa microphylla Diels).Extracting method mainly has heat
Extraction, ultrasonic wave assisted extraction, microblogging assisted extraction etc..These methods are there are certain technological deficiency, such as energy consumption is big, consumption
Material is more, Extracting temperature height causes oleuropein easily to decompose.CN101955503A discloses a kind of from spending more jasmine (Jasminum
Polyanthum Franch.) in extraction oleuropein method：Jasmine is spent more into drying and is added to CO2Supercritical extract device
In, by the use of ethyl alcohol as entrainer, extract is obtained, is dissolved in water, is extracted 1~3 time with equivalent ethyl acetate, pipettes water phase, is led to
The absorption of AB-8 large pore resin absorption columns is crossed, is eluted with 35% ethanol water, 3~8 times of amount column volume eluents is collected, depressurizes back
It receives ethyl alcohol and concentrates, when refrigeration 12~36 is small, filtration, filter residue addition absolute ethyl alcohol recrystallization, separation crystallization, washing, drying obtain
It is more than 97% oleuropein to purity.This method does not provide the final recovery rate of oleuropein, and the supercritical CO used2Extraction
Take instrument investment cost high, operating condition is harsh.The present inventor exists《Chinese experimental pharmacology of traditional Chinese medical formulae magazine》6th phase (2013) report
The process conditions of oleuropein, the solid that is obtained using the technique in application XDA-16 types macroporous resin enrichment purifying jasmine
Oleuropein average mass fraction is only 32.82% in powder.Have not yet to see high-purity oleuropein scale manufacturing technique
Research report, research and develop new oleuropein preparation process, prepare with scale high-purity oleuropein, right from jasmine
Application and development in jasmine and the application and development for oleuropein, are respectively provided with important meaning.
The present invention is directed to the problem of oleuropein content being enriched in the prior art from jasmine is not high, provides one
The method that kind extracts high-purity oleuropein from jasmine.
【The content of the invention】
[technical problems to be solved]
The object of the present invention is to provide a kind of methods that high-purity oleuropein is extracted from jasmine.
[technical solution]
The present invention is achieved through the following technical solutions.
The present invention relates to a kind of methods that high-purity oleuropein is extracted from jasmine.
The step of this method, is as follows：
A, ethanol water extracts
Dry jasmine bud raw material is taken to impregnate 2~4 times under conditions of room temperature in ethanol water, every time 46~
50h, the extracting solution obtained every time are filtered, and filtrate merges, and are then concentrated under conditions of 46~50 DEG C of temperature, dry
Obtain jasmine extraction medicinal extract M1；
B, macroporous resin column is adsorbed
The jasmine extraction medicinal extract M that step A is obtained1It is dissolved with methanol aqueous solution, the solution allowed is big by pre-processing
Hole resin column is adsorbed, and then with distilled water flushing, then is eluted with 65~75% ethanol waters by weight, obtained second
Alcohol eluen E1Dry concentration, obtains pale yellow powder M under conditions of 42~48 DEG C of temperature2；
C, polyamide column adsorbs
Polyamide dress column by pretreatment, the pale yellow powder M that step B is obtained2It is dissolved with methanol aqueous solution, allows obtain
Solution adsorbed by pre-processing polyamide column, then with distilled water flushing, then with 75~85% ethanol water by weight
Solution elutes, obtained ethanol eluate E2Dry concentration, obtains off-white powder M under the conditions of 42~48 DEG C of temperature3；
D, oleuropein is prepared
The off-white powder M that step C is obtained3It is separated by silica gel column chromatography, with dry method mode loading, then with chloroform first
Alcohol mixed solvent elutes, and solvent is removed under reduced pressure in obtained eluent, obtains oleuropein crude product in methyl alcohol in 0~4 DEG C of temperature
Under conditions of crystallize, filter, it is dry, obtain the oleuropein.
A preferred embodiment of the invention, in step, jasmine bud and 55~65% second by weight
The weight ratio of alcohol solution is 1:10~14.
According to another preferred method of implementation of the present invention, in stepb, the macroreticular resin be selected from XDA-16,
The macroreticular resin of HPD-100, XDA-1 or HPD-400, D-101 types；The macroreticular resin pre-treatment step is as follows：Described
Macroreticular resin impregnates 22~26h using 90~95% ethanol waters by weight, wet method dress post, then with by weight 90~
95% ethanol water elutes, until efflux mixes not white muddiness with water, is then eluted with water to not having ethyl alcohol smell.
According to another preferred method of implementation of the present invention, in stepb, macroporous resin column is adsorbed with elution requirement such as
Under：Sample solution mass concentration is 0.06~0.10gmL of crude drug-1, column blade diameter length ratio 1:2~4, the weight ratio of applied sample amount is M1/ big
Hole resin=3:8~12,1.5~2.5BVh of adsorption flow rate-1, 6~10BV of washing distillation water consumption；Eluting solvent is with weight
65~75% ethanol waters are counted, dosage is 3.5~4.5BV, and elution flow rate is 1.5~2.5BVh-1。
According to another preferred method of implementation of the present invention, in step C, the granularity of the polyamide is 30~90
Mesh；
The polyamide prepolymer processing step is as follows：30~90 mesh polyamide are in 90~95% ethanol waters by weight
22~26h is impregnated, is then charged into chromatographic column, 1.8~2.2mol/L sodium hydrate aqueous solutions is added in and impregnates 3.5~4.5h, connect
It and is washed with distilled water to neutrality；Then 3.5~4.5mol/L aqueous hydrochloric acid solutions are added in and impregnate 7~9h, then with distillation washing
It washs to neutrality；It is eventually adding 90~95% ethanol waters by weight and impregnates 9~11h, then be washed with distilled water to no alcohol taste.
According to another preferred method of implementation of the present invention, in step C, the adsorption conditions of polyamide column are as follows：Loading
The weight ratio of amount is M2/ polyamide=16.0-16.5mgg-1, sample solution mass concentration is 3.40~3.60mgmL-1, loading
Liquid pH is 5.8~6.2, column blade diameter length ratio 1:2~4, adsorption flow rate is 1.8~2.2BVh-1；Washing distillation water consumption 1.8~
2.2BV, eluting solvent are 75~85% ethanol water by weight, and dosage is 3.5~4.5BV, elution flow rate 1.8~
2.2BV·h-1。
According to another preferred method of implementation of the present invention, in step D, the silica gel column chromatography purification step is as follows：
Allow the off-white powder M that step C is obtained3The loading in a manner of dry method, applied sample amount M3:Silica gel=1:8~12, it is then 6 with weight ratio
~8:1 chloroform methanol mixed solvent elution, the eluent of Fractional Collections detect oleuropein content using HPLC, contain by described in
The eluent merging for by weight more than 90% is measured, solvent is recovered under reduced pressure, obtains oleuropein crude product, it exists in methyl alcohol again
It is crystallized, is filtered under conditions of 0~4 DEG C of temperature, it is dry, obtain the oleuropein.
According to another preferred method of implementation of the present invention, in step D, the off-white powder M that step C is obtained is allowed3It is logical
Sephadex column chromatography is crossed to be purified.
According to another preferred method of implementation of the present invention, the sephadex column chromatography purification step is as follows：Step C
Obtained off-white powder M3It is dissolved with ethanol water, then filtered, filtrate is by sephadex column, then with by weight
30% ethanol water elutes, and the eluent of Fractional Collections detects oleuropein content using HPLC, is with weight by the content
The eluent of gauge more than 90% merges, and solvent is recovered under reduced pressure, obtains oleuropein crude product, it is again in methyl alcohol in temperature 0~4
It is crystallized, is filtered under conditions of DEG C, it is dry, obtain the oleuropein.
The oleuropein product that the method is prepared according to the present invention, its purity for by weight 98.0~
99.5%.
The present invention is described in more detail below.
The present invention relates to a kind of methods that high-purity oleuropein is extracted from jasmine.
The step of this method, is as follows：
A, ethanol water extracts
Dry jasmine bud raw material is taken to impregnate 2~4 times under conditions of room temperature in ethanol water, every time 46~
50h, the extracting solution obtained every time are filtered, and filtrate merges, and are then concentrated under conditions of 46~50 DEG C of temperature, dry
Obtain jasmine extraction medicinal extract M1。
In this step, jasmine bud and the weight ratio of 55~65% ethanol waters by weight are 1:10~
14。
The jasmine bud raw material that the present invention uses is current market sales of product, such as purchased from Hui nationality's medicinal material
The product of trade center, the product are accredited as Oleaceae through Institute of Radiation Medicine of Military Medical Science Institute
(Oleaceous) dry flower of plant jasmine (Jasminum officinale L.var.grandiflorum).Using gas
Phase chromatographic process measures, and the water content of the dry flower is less than 2% in terms of jasmine bud raw material gross weight.
In the present invention, using oleuropein as reference substance, determined by ultraviolet spectrophotometry total iridoid glycoside is passed through
Content, it is evaluation index to calculate total iridoid glycoside yield (being calculated by medicinal material), using L9(34) orthogonal experiment method is to following water coolings
Leaching method, decocting cooking method, each factor of ethyl alcohol cold-maceration and ethanol refluxing process are investigated, and determine 4 kinds of extracting methods extractions
Condition is as follows：
Each method weighs 3 parts of jasmine medicinal materials respectively, and every part of 20g carries out parallel test.
Water cold-maceration：Dry jasmine medicinal material is taken, 12 times of amount waters are impregnated 3 times, each 72h, filtering, three at room temperature
Secondary filtrate merges, and Conventional concentration, drying obtain dry extract, weigh.
Ethanol refluxing process：Take dry jasmine medicinal material, 12 times of 60% ethanol water amounts of amount are heated to reflux 3 times, every time
Ethyl alcohol is recovered under reduced pressure in 2h, filtering, three times filtrate merging, and Conventional concentration is dry, obtains dry extract, weighs.
Decocting cooking method：Dry jasmine medicinal material is taken, 16 times of amount waters decoct 3 times, each 1h, filtering, and filtrate is closed three times
And Conventional concentration, drying, dry extract is obtained, is weighed.
Ethyl alcohol cold-maceration：Take dry jasmine medicinal material, 12 times of 60% ethanol water amounts of amount, in soaking at room temperature 3 times, every time
48h, filtering, filtrate merging three times, Conventional concentration, drying obtain dry extract, weigh.
Total iridoid glycoside content uses Conventional UV spectrophotometry in each extract.Weigh 4.0mg every time
Dry extract is placed in 100ml volumetric flasks, and methanol dilution is added to shake up to scale, the molten of every 1ml dry extracts containing 0.040mg is made
Liquid, using this solution as test liquid.Trap is measured under normal conditions at wavelength 232nm using ultraviolet specrophotometer,
The total iridoid glycoside concentration C of test liquid is calculated by standard curve1, total iridoid glycoside is calculated by following formula receive according to medicinal material
Rate, and variance analysis is carried out to result：
Total iridoid glycoside yield %=(C1/0.0400×MDry extract)/20.00×100
Result of the test shows that the total iridoid glycoside average recovery rate of 4 kinds of extracting methods is respectively：Water cold-maceration
20.67%, ethanol refluxing process 21.72%, decocting cooking method 17.33%, ethyl alcohol cold-maceration 23.26%, wherein ethyl alcohol cold-maceration
Average recovery rate highest, hence it is evident that better than other methods, and simple to equipment requirement, stablize feasible, therefore select ethyl alcohol cold-maceration
For the preparation method of jasmine flower extract.
B, macroporous resin column is adsorbed
The jasmine extraction medicinal extract M that step A is obtained1It is dissolved with methanol aqueous solution, the solution allowed is big by pre-processing
Hole resin column is adsorbed, and then with distilled water flushing, then is eluted with 65~75% ethanol waters by weight, obtained second
Alcohol eluen E1Dry concentration, obtains pale yellow powder M under conditions of 42~48 DEG C of temperature2。
The macroreticular resin that the present invention uses is selected from XDA-16, HPD-100, XDA-1 or HPD-400, the macropore tree of D-101 types
Fat.These resins are all current market sales of products, such as are sold by Xi'an Sunresin New Materials Co., Ltd.
XDA-16, XDA-1 type；HPD-100, HPD-400 type sold by Cangzhou Bon Adsorption Material Science and Technology Co., Ltd；By Tianjin
D-101 types of Xing Nanyun energy Polymer Technologies Co., Ltd sale etc..
The macroreticular resin is required for being pre-processed before use, and its object is to remove in resin synthesis process
The a small amount of oligomer for not completing polymerization usually contained, the substance and some soluble impurities that do not participate in polymerisation.In advance
Processing can not only improve resin stability, can also play the role of activated resin, improve its operating capacity.
The pre-treatment step for the macroreticular resin that the present invention uses is as follows：The macroreticular resin using by weight 90~
95% ethanol water impregnates 22~26h, wet method dress post, then is eluted with 90~95% ethanol waters by weight, until stream
Go out liquid and not white muddiness is mixed with water, be then eluted with water to there is no ethyl alcohol smell.
The present invention uses macroporous resin column processing jasmine extraction medicinal extract M1Solution condition is as follows：
Macroporous resin column adsorption conditions are as follows：
In the present invention, the jasmine extraction medicinal extract M that step A is obtained1Dissolved with methanol aqueous solution, acquired solution it is dense
It is 0.05~0.15g crude drugs that degree, which is usually every milliliter, it is therefore preferable to 0.06~0.10g.The excessive concentration of the solution is too low
All it is unfavorable, reason is that excessive concentration is easy to cause leakage, and concentration is too low can extend the production time, and raising is produced into
This.The concentration of the methanol aqueous solution is typically by volume 40~80%, and preferably by volume 50~70%.
The column blade diameter length ratio 1 of macroporous resin column:2~4.
It is 3 that applied sample amount extracts medicinal extract with the ratio between macroreticular resin in gram for jasmine in gram:8~12.
1.5~2.5BVh of adsorption flow rate-1, BV is bed volume, similarly hereinafter.
Distilled water flushing condition：6~10BV of washing distillation water consumption.
Elution requirement：Eluting solvent is 65~75% ethanol water, 3.5~4.5BV of eluting agent are washed by volume
1.5~2.5BVh of separation of flow speed-1。
The HPLC method assay methods of oleuropein content are described as follows：
I, the preparation of reference substance solution
Precision weighs 20.2mg oleuropein reference substances, puts in 50mL volumetric flasks, and addition methanol dilution to scale shakes up,
It is 404 μ gmL to obtain concentration-1Oleuropein reference substance solution.
II, chromatographic condition
The 1200 type high performance liquid chromatographs sold using Agilent companies of the U.S., the sale of Agilent companies of the U.S.
ZORBAX SB-C18 chromatographic columns (4.6mm × 250mm, 5 μm), flow velocity 1.0mLmin-1, 25 DEG C, Detection wavelength 232nm of column temperature,
Mobile phase is acetonitrile-water (by volume, 22: 78).Oleuropein reference substance solution chromatogram is shown in attached drawing 1.
III, linear relationship are investigated
Precision absorption 0.5,1,2,4,6mL oleuropein reference substance solutions, are diluted to 10mL, in above-mentioned chromatographic condition respectively
Lower sample detection.Using concentration as abscissa, peak area draws out regression curve for ordinate, is acquired following times by its regression curve
Return equation：
Y=486803X-3577560, r=0.9999,
Oleuropein is in 20.2~242.4 μ gmL-1In good linear relation.
M is determined using previously described HPLC methods2In oleuropein content, and calculated by following calculation formula
The oleuropein rate of recovery：
Oleuropein rate of recovery %=(M2Middle oleuropein quality/M1Middle oleuropein quality) × 100
Oleuropein mass fraction is calculated by following calculation formula：
Oleuropein mass fraction %=(M2Middle oleuropein quality/M2Quality) × 100
Oleuropein recovery rate is calculated by following calculation formula：
Oleuropein recovery rate %=(M2Middle oleuropein quality/quality of medicinal material) × 100
Result of the test shows that the oleuropein rate of recovery is more than 90.0%, and oleuropein mass fraction is more than 32.0%,
The recovery rate that oleuropein is calculated by medicinal material is more than 6.0%.
C, polyamide column adsorbs
Polyamide dress column by pretreatment, the pale yellow powder M that step B is obtained2It is dissolved with methanol aqueous solution, allows obtain
Solution adsorbed by pre-processing polyamide column, then with distilled water flushing, then with 75~85% ethanol water by weight
Solution elutes, obtained ethanol eluate E2Dry concentration, obtains off-white powder M under the conditions of 42~48 DEG C of temperature3。
In the present invention, the polyamide should be the polyamide for having adsorption and de-adsorption performance.The present invention uses
Polyamide be current market sales of product, such as by Taizhou City of Zhejiang Province road and bridge tetramethyl biochemistry plastic molding and processing plant, Shanghai source leaf give birth to
Object Science and Technology Ltd. or the polyamide of Sinopharm Chemical Reagent Co., Ltd.'s production, its granularity are 30~90 mesh.
The polyamide prepolymer processing step is as follows：30~90 mesh polyamide are in 90~95% ethanol waters by weight
22~26h is impregnated, the purpose of immersion polymerize in the unpolymerized starting monomer in polyamide production process is removed and low molecule
Object；Then polyamide is fitted into chromatographic column, adds in 1.8~2.2mol/L sodium hydrate aqueous solutions and impregnate 3.5~4.5h, then
Neutrality is washed with distilled water to, what the effect that sodium hydrate aqueous solution impregnates was to adsorb on removing polyamide functions relatively strong
Acid impurities；Then 3.5~4.5mol/L aqueous hydrochloric acid solutions are added in and impregnate 7~9h, are then washed with distilled water to neutrality, salt
The effect that aqueous acid impregnates is that is adsorbed on removing resin functions stronger alkaline impurities；It is eventually adding by weight
95% ethanol water impregnates 9~11h, then is washed with distilled water to no alcohol taste.
It is inquired into below to influencing polycaprolactam factor.
First, polyamide particle size influences research
I, Static Adsorption ability is investigated
9 parts of pretreatments, 3 kinds of each 0.5g of mesh number polyamide (each 3 repetitions) are weighed, are respectively placed in conical flask with stopper, respectively
Add in 750.4 μ g/mL M of 15mL mass concentrations2Solution (the 246.2 μ g/mL containing oleuropein), carries out Static Adsorption at room temperature
18h shakes once per 2h.Supernatant is pipetted, measures wherein oleuropein mass concentration, calculates polycaprolactam amount and absorption
Rate.The polyamide of different adsorption saturation degree is placed in conical flask with stopper, each 95% ethyl alcohol of 15mL that adds in carries out static state in room temperature
18h is eluted, is shaken once per 2h, isolated eluent.Then the oleuropein mass concentration of eluent is measured, calculates elution
Rate, and same group of processing is averaged.The result is shown in the following table 1.
Q=(C0- Cr)V1/W
D=(C0- Cr)/C0× 100%
E=2Ce V2/ Q × 100%
In formula：
Q adsorbances (mg/g), C0Initial mass concentration (mg/mL), CrAdsorb supernatant mass concentration (mg/mL), V1Sample
Liquor capacity (mL), W resin qualities (g), D adsorption rates (%), E eluting rates and V2Elute liquor capacity (mL)
Table 1：Influence (n=3) of the granularity to polyamide Static Adsorptive capacity
The investigation of II, dynamic adsorption capacity
9 parts of pretreatments, 3 kinds of each 0.5g of mesh number polyamide (each 3 repetitions) are weighed, precision weighing is packed into respectively with wet method
Internal diameter is in 1.2cm chromatographic columns.Respectively take 750.4 μ g/mL M of 15mL2(the 246.2 μ g/mL containing oleuropein) solution is with flow velocity
2BV·h-1Upper prop adsorbs, and collects efflux, measures the mass concentration of oleuropein, calculates polycaprolactam amount and adsorption rate.Respectively
Column with 30mL by volume 95% ethyl alcohol with flow velocity 2BVh-1Elution, measures oleuropein mass concentration in eluent, and calculating is washed
De- rate, result of the test are listed in Table 2 below.
Table 2：Influence (n=3) of the granularity to polyamide dynamic adsorption
Table 1 and table 2 the result shows that, polyamide granularity has certain influence to polyamide Static Adsorption and desorption performance, but poor
Not not less.In view of subsequent operation feasibility, it is 30-90 mesh polyamide to determine the granularity selected.
2nd, polyamide enrichment and purification method is studied
I, the influence of applied sample amount
It weighs 2g processing polyamide and is packed into chromatographic column, a certain amount of sample solution (750.4 μ g/mL of concentration) is passed through into polyamide
Column collects efflux, is collected once per 1/3BV (10mL 1BV), measures the oleuropein mass concentration of every part of efflux,
Mass concentration curve is shown in shown in Fig. 5.Should the result shows that, there is a small amount of oleuropein, optimal loading in the 14th part of efflux
It measures as 13/3BV, 43.3mL, i.e., optimal applied sample amount is 16.25mgg-1(M2/ polyamide)
The influence of II, sample solution pH
It weighs 8 parts of processed polyamide of every part of 2g, is packed into chromatographic column, (concentration is by 8 parts of sample solutions of different pH
3.50mg·mL-1, volume 10mL) and it is added separately in polyamide resin column, other steps are identical with A's.Measure oleuropein
Mass concentration calculates the adsorbance and adsorption rate of oleuropein according to previously described formula, and the results are shown in Table 3 for this.The knot
Fruit shows sample solution pH oleuropein adsorption rate highests at 6.0, therefore the Optimal pH of sample solution is 6.0.
Table 3：Influences of the sample solution pH to oleuropein adsorption rate
III, sample solution concentration, the orthogonal design of column blade diameter length ratio and adsorption flow rate
Using L9(34) orthogonal, using the oleuropein rate of recovery as evaluation index, to purifying process herb liquid quality
3 concentration, column blade diameter length ratio and adsorption flow rate conditions optimize screening.
Weigh 2g processing polyamide (bed volume 10mL), 10mL mass concentrations 3.50mgmL-1, pH 6.0 M2Sample
Liquid is added in polyamide resin column, first with 3BV distilled water flushings, is eluted with 95% ethanol waters of 5BV, other steps and A's
It is identical.The mass content of oleuropein in ethanol eluate is measured according to previously described method, olive is calculated by following formula
The bitter glycosides rate of recovery.
Rate of recovery %=(oleuropein quality in oleuropein quality/upper prop liquid in ethanol eluate) × 100
Table 4：Factor level table
Note：A quality of liquid medicine concentration (mgmL-1)；B column blade diameter length ratios；C adsorption flow rates (BVh-1)；
D blank
Table 5：Orthogonal experiments
Table 6：Variance analysis
Note:F0.05(2,2)=19.0；F0.1(2,2)=9.0
From the orthogonal experiments of table 4~6, influence size of each factor to the oleuropein rate of recovery is B ＞ A ＞
C, wherein B factors, the i.e. influence of column blade diameter length ratio are significantly (p ＜ 0.01).The optimal combination of three factors is A2B1C2, i.e. liquid
Mass concentration is 3.50mgmL-1, column blade diameter length ratio is 1:3, adsorption flow rate 2BVh-1。
IV, the investigation for washing volume
2g pretreatment polyamide is weighed, is packed into chromatographic column (bed volume 10mL, column blade diameter length ratio 1:3), by pH be 6.0 it is upper
Sample liquid (10mL3.50mgmL-1M2Solution) plus on a column, adsorption flow rate 2BVh-1, water elution is distilled, collects elution
Liquid is collected once per 1/3BV (10mL 1BV), measures Solid content (solute) quality in every part of eluent.Using eluent as horizontal seat
Mark, Solid content accumulated quality draw graph 6 for ordinate.The result shows that during water consumption≤2BV, in the 1st~6 part of eluent
Solid content accumulated quality gradually increases, but does not detect oleuropein, and a small amount of olive is detected since the 7th part of eluent
Bitter glycosides, and Solid content accumulated quality tends to be steady.Consider, it is 2BV to determine water consumption.
V, the influence of eluant, eluent
7 parts of every part of 2g processing polyamide are weighed, are respectively charged into chromatographic column (bed volume 10mL, column blade diameter length ratio 1:3), by pH
6.0 sample solutions (10mL 3.50mgmL-1M2Solution) it is added in chromatographic column, adsorption flow rate 2BVh-1, first distilled and washed with 2BV
It is de-, then with 5BV, 30%, 40%, 50%, 60%, 70%, 80%, 95% ethanol water elutes by volume respectively, collects second
Alcohol eluen, oleuropein mass concentration is measured after being settled to same volume, and eluting rate is calculated according to previously described formula,
The result is shown in Fig. 7.It is believed that 80% ethanol water elution effect is best by volume by Fig. 7 results.
The influence of VI, eluting agent
It weighs 2g processing polyamide and is packed into chromatographic column (bed volume 10mL, column blade diameter length ratio 1:3), by pH6.0 sample solutions
(10mL3.50mg·mL-1M2Solution) it is added in chromatographic column, adsorption flow rate 2BVh-1, first water elution is distilled with 2BV.Again to
80% ethanol water of stereometer elutes, and collects ethanol eluate, is collected once per 1BV, is then settled to root after same volume
Oleuropein mass concentration is measured according to previously described method, eluting rate is calculated according to previously described formula, the result is shown in figures
8.When eluting agent is after 4BV, eluting rate has tended towards stability, it is 4BV to determine eluting agent.
The influence of VII, eluant, eluent flow velocity
6 parts of every part of 2g processing polyamide are weighed, are respectively charged into chromatographic column (bed volume 10mL, column blade diameter length ratio 1:3), will
PH6.0 sample solutions (10mL3.50mgmL-1M2Solution) it is added in chromatographic column, adsorption flow rate 2BVh-1, first with 2BV distilled water
Elution, then 80% ethanol water elutes by volume with 4BV, elution flow rate is respectively 1BVh-1、1.5BV·h-1、2BV·
h-1、2.5BV·h-1、3BV·h-1With 4BVh-1, ethanol eluate is collected, after being settled to same volume, according to previously described
Method measures the mass concentration of oleuropein, calculates the oleuropein rate of recovery according to previously described formula, the result is shown in Fig. 9.
When elution flow rate increases, the oleuropein rate of recovery is integrally on a declining curve, but from 1BVh-1Increase to 2BVh-1When, olive
The bitter glycosides rate of recovery only reduces by 0.79%, and from 2BVh-1Increase to 4BVh-1When, the oleuropein rate of recovery reduces
11.11%.Consider, elution flow rate 2BVh-1To be best.
VIII, checking test
3 parts of every part of 2g processing polyamide are weighed, are respectively charged into chromatographic column, according to above-mentioned optimum process condition (30-90 mesh
Polyamide, applied sample amount 16.25mgg-1(M2/ polyamide), sample solution mass concentration 3.50mgmL-1, sample solution pH is 6.0,
Column blade diameter length ratio 1:3, adsorption flow rate 2BVh-1, first washed with 2BV, then 80% ethanol water elutes by weight with 4BV,
Elution flow rate 2BVh-1) 3 parallel tests are carried out, 80% ethanol water elution liquid by volume is collected respectively, according to front
The method of description measures oleuropein mass concentration, and the oleuropein rate of recovery is calculated according to previously described formula.Ethyl alcohol
Eluent concentrates, dry, obtains off-white powder M3, measure powder in oleuropein percentage composition.Oleuropein is calculated
Average recovery rate is 88.07%, RSD 1.73%, and the average mass fraction of oleuropein is 65.34%, RSD in dried powder
For 1.41%, the average recoveries that oleuropein is calculated by medicinal material are 5.40%.
Table 7：Checking test result
Consider, polycaprolactam column adsorption treatment pale yellow powder M2Solution condition is as follows：
The column blade diameter length ratio 1 of polycaprolactam column:2~4.
Adsorption conditions：The weight ratio of applied sample amount is M2/ polyamide=16.0~16.5mgg-1, sample solution mass concentration is
3.40~3.60mgmL-1, sample solution pH is 5.8~6.2, and adsorption flow rate is 1.8~2.2BVh-1；
Distilled water flushing condition：1.8~2.2BV of washing distillation water consumption；
Elution requirement：Eluting solvent is 75~85% ethanol water by weight, and dosage is 3.5~4.5BV, is eluted
1.8~2.2BVh of flow velocity-1。
D, oleuropein is prepared
The off-white powder M that step C is obtained3It is separated by silica gel column chromatography, is eluted, obtained with chloroform methanol mixed solvent
Eluent solvent is removed under reduced pressure, obtain oleuropein crude product and crystallized in methyl alcohol under conditions of 0~4 DEG C of temperature, filtered, done
It is dry, obtain the oleuropein.
The silica gel column chromatography purification step is as follows：Allow the off-white powder M that step C is obtained3The loading in a manner of dry method,
Applied sample amount M3:Silica gel=1:8~12, it is then 6~8 with weight ratio:1 chloroform methanol mixed solvent elution, Fractional Collections
Eluent detects oleuropein content using previously described HPLC methods, and the content is washed for by weight more than 90%
De- liquid merges, and solvent is recovered under reduced pressure, obtains oleuropein crude product, it is tied under conditions of 0~4 DEG C of temperature in methyl alcohol again
Crystalline substance filters, dry, obtains the oleuropein.
The oleuropein crude product is crystallized under conditions of 0~4 DEG C of temperature in methyl alcohol, is filtered, with anhydrous sulphur
Sour copper is dried at ambient temperature, gas chromatography is used to measure its water content as by weight 0.1~0.3%, before
It is more than 98.2% that the HPLC methods of face description, which measure its purity, according to previously described calculation formula, is calculated according to medicinal material weight
Oleuropein average recoveries are 2.2%.
In this step, the powder M that can also be allowed3Pass through sephadex column chromatographic purifying.
The sephadex column chromatography purification step is as follows：The off-white powder M that step C is obtained3Use ethanol water
Dissolving, is then filtered, and filtrate elutes by sephadex column, then with 30% ethanol water by weight, Fractional Collections
Eluent detects oleuropein content using previously described HPLC, by the eluent that the content is by weight more than 90%
Merging, solvent is recovered under reduced pressure, obtains oleuropein crude product, it is crystallized under conditions of 0~4 DEG C of temperature in methyl alcohol again,
Filtering, it is dry, obtain the oleuropein.The sephadex that the present invention uses is because of biological chromatography technology by Beijing Puri
The product that Co., Ltd is sold with trade name Sephadex LH-20.The actual conditions of sephadex column chromatography：Applied sample amount M3/
Sephadex weight ratio 10mgg-1, sample solution mass concentration 2.0mgmL-1, column blade diameter length ratio 1:80, adsorption flow rate is
2.0BV·h-1。
The invention further relates to the oleuropeins being prepared using high-purity oleuropein method is extracted from jasmine.It adopts
It is determined with previously described analysis method, its purity is 98.0-99.5% by weight.
Below with regard to the steps such as alcohol flooding, macroporous resin adsorption, polycaprolactam, silica gel column chromatography purifying in the method for the present invention
Oleuropein content and the influence of oleuropein recovery rate are tested in rapid combination, and result of the test is listed in the table below in 8.
Table 8：Influence of the different step combination to oleuropein content and oleuropein recovery rate
By the result of table 8 it is clear that the macroporous resin adsorption series connection polycaprolactam purifying that the present invention uses can
Effectively improve oleuropein content and oleuropein recovery rate.
In addition, CN101003557A discloses a kind of preparation side of the extractive of olive growing leaves rich in high-purity oleuropein
Method, this method is that olive growing leaves are first extracted with polar solvent, then centrifugal sedimentation in alkaline conditions, obtain centrifugal clear liquid and
Sediment then carries out film lotus root and closes separation, centrifugal clear liquid is allowed to pass sequentially through ceramic membrane, ultrafiltration membrane and nanofiltration membrane treatment, is obtained
Concentrate is inhaled again with making choice property of blending agent more than one or both of aluminium oxide, macroporous absorbent resin, polyamide
Attached, eluent drying obtains the extractive of olive growing leaves that oleuropein content is more than 50%.CN102228514A discloses one kind
The method that oleuropein is extracted from olive growing leaves, this method are extracted with water or alcohol water mixed solvent, extracting solution concentration, then
Extracting solution is isolated and purified by macroporous resin adsorption, decolorizing resin absorption and organic solvent extraction successively, is finally obtained
Oleuropein content is more than 40% oleuropein extract；CN102659867A discloses a kind of high-content oleuropein extraction
The preparation method of object, this method are that crude extract is allowed to obtain crude extract II through macroporous absorbent resin adsorbing separation, then through solvent extraction
Crude extract III is obtained, is then separated through silica gel or alumina column chromatography, the olive for being finally dried to obtain content up to 80-90% is bitter
Glucoside extract.Although these existing methods can be extracted to obtain the oleuropein extract of certain content, effect is still paid no attention to very much
Think.
The present invention overcomes oleuropein content in oleuropein extract existing for existing preparation method it is not high the problem of,
Pass through the steps such as alcohol flooding, macroporous resin adsorption series connection polycaprolactam, silica gel column chromatography or sephadex column chromatographic purifying
Suddenly, keep oleuropein average recoveries (in terms of medicinal material) on the premise of 2~4%, obtain oleuropein content reach with
The extract of weight meter 98.0-99.5%.
[advantageous effect]
Compared with prior art, the present invention has following advantageous effect：
1st, the present invention provides a kind of methods for being capable of prepare with scale high-purity oleuropein.The method of the present invention passes through second
The extraction of alcohol cold soaking, macroporous resin adsorption, polycaprolactam, silica gel column chromatography, Sephadex LH-20 column chromatographies and recrystallization etc.
Step prepares high-purity oleuropein, and purity reaches 98.0-99.5% by weight, and being averaged for oleuropein is calculated according to medicinal material
Recovery rate is 2~4%.
2nd, the present invention is suitable for the enrichment of oleuropein, is improved preparation effect using macroreticular resin combination polycaprolactam
Rate.
3rd, extraction process of the invention carries out at room temperature, reduces the impurity such as protein, polysaccharide, pigment and dissolves in, and protects
In extraction process active material do not destroyed by pyrocondensation decompose, oxidation or polymerization, ensure that oleuropein stay in grade and content
It is high.
4. method provided by the invention is stablized, favorable reproducibility is simple and efficient, and obtained product purity is high, high income, is suitble to
In industrialized production.
【Description of the drawings】
Fig. 1 is oleuropein standard items HPLC collection of illustrative plates；
Fig. 2 is oleuropein FAB-MS collection of illustrative plates prepared by the present invention；
Fig. 3 is oleuropein prepared by the present invention1HNMR collection of illustrative plates；
Fig. 4 is oleuropein prepared by the present invention13CNMR collection of illustrative plates；
Fig. 5 is the elution curve of polycaprolactam oleuropein；
Fig. 6 is influence figure of the water consumption to polycaprolactam purifying process；
Fig. 7 is influence figure of the eluant strength to polycaprolactam purifying process；
Fig. 8 is influence figure of the eluant, eluent volume to polycaprolactam purifying process；
Fig. 9 is influence figure of the elution flow rate to polycaprolactam purifying process.
【Specific embodiment】
The present invention is will be better understood that by following embodiments.
Embodiment 1：High-purity oleuropein is extracted from jasmine
The implementation steps of the embodiment are as follows：
A, ethanol water extracts
According to the weight ratio 1 of jasmine bud and 60% ethanol water by weight:12, allow water content by weight
2.0% drying jasmine bud raw material impregnates 3 times in ethanol water under conditions of room temperature, and each 50h is obtained every time
Extracting solution be filtered, filtrate merges, and is then concentrated and dried under conditions of 46 DEG C of temperature, obtains jasmine extraction leaching
Cream M1；
B, macroporous resin column is adsorbed
Macroreticular resin pre-processes：The XDA-16 type macroreticular resins sold by Xi'an Sunresin New Materials Co., Ltd.
22h is impregnated using 95% ethanol water by weight, then wet method dress post, then with 95% ethanol aqueous wash by weight
It is de-, until efflux mixes not white muddiness with water, then it is eluted with water to not having ethyl alcohol smell.
Absorption and elution：The jasmine extraction medicinal extract M that step A is obtained1It is dissolved with methanol aqueous solution, the solution allowed exists
Sample solution mass concentration is crude drug 0.06gmL-1, column blade diameter length ratio 1:3rd, applied sample amount M1/ macroreticular resin weight ratio=3:10, absorption
Flow velocity 2.5BVh-1Under conditions of adsorbed by pre-processing macroporous resin column, then with 8BV distilled water flushings, then use
4.2BV by weight 65% ethanol water with elution flow rate 2.5BVh-1Elution, obtained ethanol eluate E1In temperature 46
Dry concentration, obtains pale yellow powder M under conditions of DEG C2；
C, polyamide column adsorbs
Polyamide prepolymer processing：60-90 mesh is by the polyamide that Taizhou City of Zhejiang Province road and bridge tetramethyl biochemistry plastic molding and processing plant produces with weight
22h is impregnated in 95% ethanol water of gauge, is then charged into chromatographic column, 1.8mol/L sodium hydrate aqueous solutions is added in and impregnates
4.0h is then washed with distilled water to neutrality；Then 3.5mol/L aqueous hydrochloric acid solutions are added in and impregnate 7.0h, then with distillation washing
It washs to neutrality；It is eventually adding 95% ethanol water by weight and impregnates 9h, then be washed with distilled water to no alcohol taste.
Polyamide dress column by pretreatment；The pale yellow powder M that step B is obtained2It is dissolved with methanol aqueous solution, allows obtain
Solution in applied sample amount M2/ polyamide weight ratio 16.3mgg-1, sample solution mass concentration 3.40mgmL-1, sample solution pH
5.8th, column blade diameter length ratio 1:2 with adsorption flow rate be 1.8BVh-1Under conditions of adsorbed by pre-processing polyamide column, then use
1.8BV distilled water flushings, then with 4.0BV by weight 80% ethanol water with elution flow rate 1.8BVh-1Elution, obtains
Ethanol eluate E2Dry concentration, obtains off-white powder M under the conditions of 46 DEG C of temperature3；
D, oleuropein is prepared
The off-white powder M that step C is obtained3It is separated with dry method mode by silica gel column chromatography, applied sample amount M3:Silica gel=1:
10, it is then 6 with weight ratio:1 chloroform methanol mixed solvent elution, the eluent of Fractional Collections are described using this specification
HPLC methods detection oleuropein content, by the content for by weight more than 90% eluent merge, obtained elution
Solvent is removed under reduced pressure in liquid, obtains oleuropein crude product and is crystallized in methyl alcohol under conditions of 2 DEG C of temperature, filters, use anhydrous slufuric acid
Copper is dried at ambient temperature, and gas chromatography is used to measure its water content as by weight 0.2%, is retouched using this specification
It is 98.6% that the HPLC methods stated, which measure its purity, and according to the calculation formula that this specification describes, olive is calculated according to medicinal material weight
Bitter glycosides average recoveries are 3.0%.
Embodiment 2：High-purity oleuropein is extracted from jasmine
The implementation steps of the embodiment are as follows：
A, ethanol water extracts
According to the weight ratio 1 of jasmine bud and 55% ethanol water by weight:11, allow water content by weight
1.9% drying jasmine bud raw material impregnates 3 times in ethanol water under conditions of room temperature, and each 47h is obtained every time
Extracting solution be filtered, filtrate merges, and is then concentrated and dried under conditions of 49 DEG C of temperature, obtains jasmine extraction leaching
Cream M1；
B, macroporous resin column is adsorbed
Macroreticular resin pre-processes：The HPD-100 type macroreticular resins sold by Cangzhou Bon Adsorption Material Science and Technology Co., Ltd
25h is impregnated using 90% ethanol water by weight, then wet method dress post, then with 90% ethanol aqueous wash by weight
It is de-, until efflux mixes not white muddiness with water, then it is eluted with water to not having ethyl alcohol smell.
Absorption and elution：The jasmine extraction medicinal extract M that step A is obtained1It is dissolved with methanol aqueous solution, the solution allowed exists
Sample solution mass concentration is crude drug 0.10gmL-1, column blade diameter length ratio 1:2nd, applied sample amount M1/ macroreticular resin weight ratio=3:11, absorption
Flow velocity 1.8BVh-1Under conditions of adsorbed by pre-processing macroporous resin column, then with 6BV distilled water flushings, then use
3.5BV by weight 75% ethanol water with elution flow rate 1.8BVh-1Elution, obtained ethanol eluate E1In temperature 42
Dry concentration, obtains pale yellow powder M under conditions of DEG C2；
C, polyamide column adsorbs
Polyamide prepolymer processing：30-60 mesh by by the polyamide that Taizhou City of Zhejiang Province road and bridge tetramethyl biochemistry plastic molding and processing plant produces with
26h is impregnated in 95% ethanol water of weight meter, is then charged into chromatographic column, 1.8mol/L sodium hydrate aqueous solutions is added in and impregnates
4.2h is then washed with distilled water to neutrality；Then 4.5mol/L aqueous hydrochloric acid solutions are added in and impregnate 8.0h, then with distillation washing
It washs to neutrality；It is eventually adding 95% ethanol water by weight and impregnates 11h, then be washed with distilled water to no alcohol taste.
Polyamide dress column by pretreatment；The pale yellow powder M that step B is obtained2It is dissolved with methanol aqueous solution, allows obtain
Solution in applied sample amount M2/ polyamide weight ratio 16.0mgg-1, sample solution mass concentration 3.50mgmL-1, sample solution pH
6.2nd, column blade diameter length ratio 1:4 with adsorption flow rate be 2.0BVh-1Under conditions of adsorbed by pre-processing polyamide column, then use
2.0BV distilled water flushings, then with 4.2BV by weight 75% ethanol water with elution flow rate 2.0BVh-1Elution, obtains
Ethanol eluate E2Dry concentration, obtains off-white powder M under the conditions of 44 DEG C of temperature3；
D, oleuropein is prepared
The off-white powder M that step C is obtained3With dry method mode loading, separated by silica gel column chromatography.Applied sample amount M3:Silica gel
=1:8, it is then 7 with weight ratio:1 chloroform methanol mixed solvent elution, the eluent of Fractional Collections are retouched using this specification
The HPLC methods detection oleuropein content stated merges the content for by weight more than 90% eluent, and what is obtained washes
Solvent is removed under reduced pressure in de- liquid, obtains oleuropein crude product and is crystallized in methyl alcohol under conditions of 4 DEG C of temperature, is filtered, with anhydrous sulphur
Sour copper is dried at ambient temperature, gas chromatography is used to measure its water content as by weight 0.3%, using this specification
It is 98.0% that the HPLC methods of description, which measure its purity, and according to the calculation formula that this specification describes, olive is calculated according to medicinal material weight
Olive hardship glycosides average recoveries are 2.6%.
Embodiment 3：High-purity oleuropein is extracted from jasmine
The implementation steps of the embodiment are as follows：
A, ethanol water extracts
According to the weight ratio 10 of jasmine bud and 65% ethanol water by weight:1, allow water content by weight
1.6% drying jasmine bud raw material impregnates 4 times in ethanol water under conditions of room temperature, and each 46h is obtained every time
Extracting solution be filtered, filtrate merge, be then concentrated and dried under conditions of temperature 50 C, obtain jasmine extraction leaching
Cream M1；
B, macroporous resin column is adsorbed
Macroreticular resin pre-processes：The XDA-1 type macroreticular resins sold by Xi'an Sunresin New Materials Co., Ltd.
26h is impregnated using 92% ethanol water by weight, then wet method dress post, then with 92% ethanol aqueous wash by weight
It is de-, until efflux mixes not white muddiness with water, then it is eluted with water to not having ethyl alcohol smell.
Absorption and elution：The jasmine extraction medicinal extract M that step A is obtained1It is dissolved with methanol aqueous solution, the solution allowed exists
Sample solution mass concentration is crude drug 0.08gmL-1, column blade diameter length ratio 1:4th, applied sample amount M1/ macroreticular resin weight ratio=3:8, absorption
Flow velocity 2.2BVh-1Under conditions of adsorbed by pre-processing macroporous resin column, then with 10BV distilled water flushings, then use
4.0BV by weight 70% ethanol water with elution flow rate 2.2BVh-1Elution, obtained ethanol eluate E1In temperature 44
Dry concentration, obtains pale yellow powder M under conditions of DEG C2；
C, polyamide column adsorbs
Polyamide prepolymer processing：The polyamide that 60-90 mesh is produced by Shanghai Yuan Ye bio tech ltd is by weight
It impregnates for 24 hours, is then charged into chromatographic column in 90% ethanol water, add in 2.2mol/L sodium hydrate aqueous solutions and impregnate 3.5h,
Then it is washed with distilled water to neutrality；Then 4.0mol/L aqueous hydrochloric acid solutions are added in and impregnate 9.0h, are then washed with distilled water to
It is neutral；It is eventually adding 90% ethanol water by weight and impregnates 10h, then be washed with distilled water to no alcohol taste.
Polyamide dress column by pretreatment；The pale yellow powder M that step B is obtained2It is dissolved with methanol aqueous solution, allows obtain
Solution in applied sample amount M2/ polyamide weight ratio 16.2mgg-1, sample solution mass concentration 3.60mgmL-1, sample solution pH
6.0th, column blade diameter length ratio 1:3 with adsorption flow rate be 2.2BVh-1Under conditions of adsorbed by pre-processing polyamide column, then use
2.2BV distilled water flushings, then with 3.5BV by weight 78% ethanol water with elution flow rate 2.2BVh-1Elution, obtains
Ethanol eluate E2Dry concentration, obtains off-white powder M under the conditions of 42 DEG C of temperature3；
D, oleuropein is prepared
The off-white powder M that step C is obtained3It with dry method mode loading, is separated by silica gel column chromatography, applied sample amount M3:Silica gel
=1:12, it is then 8 with weight ratio:1 chloroform methanol mixed solvent elution, the eluent of Fractional Collections use this specification
The content for by weight more than 90% eluent is merged, obtained by the HPLC methods detection oleuropein content of description
Solvent is removed under reduced pressure in eluent, obtains oleuropein crude product and is crystallized in methyl alcohol under conditions of 4 DEG C of temperature, filtered, use is anhydrous
Copper sulphate is dry under conditions of room temperature, gas chromatography is used to measure its water content as by weight 0.1%, using this theory
It is more than 99.4% that the HPLC methods of bright book description, which measure its purity, according to the calculation formula that this specification describes, according to medicinal material weight
It is 2.0% that amount, which calculates oleuropein average recoveries,.
Embodiment 4：High-purity oleuropein is extracted from jasmine
The implementation steps of the embodiment are as follows：
A, ethanol water extracts
According to the weight ratio 1 of jasmine bud and 55% ethanol water by weight:14, allow water content by weight
1.8% drying jasmine bud raw material impregnates 3 times in ethanol water under conditions of room temperature, and each 48h is obtained every time
Extracting solution be filtered, filtrate merges, and is then concentrated and dried under conditions of 47 DEG C of temperature, obtains jasmine extraction leaching
Cream M1；
B, macroporous resin column is adsorbed
Macroreticular resin pre-processes：The HPD-400 type macroreticular resins sold by Cangzhou Bon Adsorption Material Science and Technology Co., Ltd
It is impregnated for 24 hours using 95% ethanol water by weight, then wet method dress post, then with 95% ethanol aqueous wash by weight
It is de-, until efflux mixes not white muddiness with water, then it is eluted with water to not having ethyl alcohol smell.
Absorption and elution：The jasmine extraction medicinal extract M that step A is obtained1It is dissolved with methanol aqueous solution, the solution allowed exists
Sample solution mass concentration is crude drug 0.09gmL-1, column blade diameter length ratio 1:3rd, applied sample amount M1/ macroreticular resin weight ratio=3:12, absorption
Flow velocity 1.5BVh-1Under conditions of adsorbed by pre-processing macroporous resin column, then with 9BV distilled water flushings, then use
4.5BV by weight 75% ethanol water with elution flow rate 1.5BVh-1Elution, obtained ethanol eluate E1In temperature 42
Dry concentration, obtains pale yellow powder M under conditions of DEG C2；
C, polyamide column adsorbs
Polyamide prepolymer processing：30~60 mesh are by the polyamide that Taizhou City of Zhejiang Province road and bridge tetramethyl biochemistry plastic molding and processing plant produces with weight
25h is impregnated in 92% ethanol water of gauge, is then charged into chromatographic column, 2.0mol/L sodium hydrate aqueous solutions is added in and impregnates
3.8h is then washed with distilled water to neutrality；Then 3.5mol/L aqueous hydrochloric acid solutions are added in and impregnate 7.5h, then with distillation washing
It washs to neutrality；It is eventually adding 92% ethanol water by weight and impregnates 9h, then be washed with distilled water to no alcohol taste.
Polyamide dress column by pretreatment；The pale yellow powder M that step B is obtained2It is dissolved with methanol aqueous solution, allows obtain
Solution in applied sample amount M2/ polyamide weight ratio 16.5mgg-1, sample solution mass concentration 3.40mgmL-1, sample solution pH
5.8th, column blade diameter length ratio 1:2 with adsorption flow rate be 1.8BVh-1Under conditions of adsorbed by pre-processing polyamide column, then use
1.8BV distilled water flushings, then with 3.8BV by weight 85% ethanol water with elution flow rate 1.8BVh-1Elution, obtains
Ethanol eluate E2Dry concentration, obtains off-white powder M under the conditions of 48 DEG C of temperature3；
D, oleuropein is prepared
The off-white powder M that step C is obtained3With dry method mode loading, separated by silica gel column chromatography.Applied sample amount M3:Silica gel
=1:11, it is then 6 with weight ratio:1 chloroform methanol mixed solvent elution, the eluent of Fractional Collections are retouched using this specification
The HPLC methods detection oleuropein content stated merges the content for by weight more than 90% eluent, and what is obtained washes
Solvent is removed under reduced pressure in de- liquid, obtains oleuropein crude product and is crystallized in methyl alcohol under conditions of 0 DEG C of temperature, is filtered, with anhydrous sulphur
Sour copper is dried at ambient temperature, gas chromatography is used to measure its water content as by weight 0.1%, using this specification
It is 99.0% that the HPLC methods of description, which measure its purity, and according to the calculation formula that this specification describes, olive is calculated according to medicinal material weight
Olive hardship glycosides average recoveries are 3.4%.
Embodiment 5：High-purity oleuropein is extracted from jasmine
The implementation steps of the embodiment are as follows：
A, ethanol water extracts
According to the weight ratio 1 of jasmine bud and 60% ethanol water by weight:13, allow water content by weight
1.9% drying jasmine bud raw material impregnates 2 times in ethanol water under conditions of room temperature, and each 49h is obtained every time
Extracting solution be filtered, filtrate merges, and is then concentrated and dried under conditions of 48 DEG C of temperature, obtains jasmine extraction leaching
Cream M1；
B, macroporous resin column is adsorbed
Macroreticular resin pre-processes：By the D-101 type macroreticular resins of Tianjin Xing Nanyun energy Polymer Technologies Co., Ltd sale
23h is impregnated using 92% ethanol water by weight, then wet method dress post, then with 92% ethanol aqueous wash by weight
It is de-, until efflux mixes not white muddiness with water, then it is eluted with water to not having ethyl alcohol smell.
Absorption and elution：The jasmine extraction medicinal extract M that step A is obtained1It is dissolved with methanol aqueous solution, the solution allowed exists
Sample solution mass concentration is crude drug 0.07gmL-1, column blade diameter length ratio 1:2nd, applied sample amount M1/ macroreticular resin weight ratio=3:9, absorption
Flow velocity 2.0BVh-1Under conditions of adsorbed by pre-processing macroporous resin column, then with 7BV distilled water flushings, then use
3.8BV by weight 70% ethanol water with elution flow rate 2.0BVh-1Elution, obtained ethanol eluate E1In temperature 44
Dry concentration, obtains pale yellow powder M under conditions of DEG C2；
C, polyamide column adsorbs
Polyamide prepolymer processing：The polyamide that 30~60 mesh are produced by Shanghai Yuan Ye bio tech ltd is by weight
23h is impregnated in 94% ethanol water, is then charged into chromatographic column, 2.2mol/L sodium hydrate aqueous solutions is added in and impregnates 4.0h,
Then it is washed with distilled water to neutrality；Then 4.5mol/L aqueous hydrochloric acid solutions are added in and impregnate 8.5h, are then washed with distilled water to
It is neutral；It is eventually adding 95% ethanol water by weight and impregnates 11h, then be washed with distilled water to no alcohol taste.
Polyamide dress column by pretreatment；The pale yellow powder M that step B is obtained2It is dissolved with methanol aqueous solution, allows obtain
Solution in applied sample amount M2/ polyamide weight ratio 16.4mgg-1, sample solution mass concentration 3.50mgmL-1, sample solution pH
6.2nd, column blade diameter length ratio 1:4 with adsorption flow rate be 2.0BVh-1Under conditions of adsorbed by pre-processing polyamide column, then use
2.0BV distilled water flushings, then with 4.5BV by weight 85% ethanol water with elution flow rate 2.0BVh-1Elution, obtains
Ethanol eluate E2Dry concentration, obtains off-white powder M under the conditions of 46 DEG C of temperature3；
D, oleuropein is prepared
The off-white powder M that step C is obtained3It is dissolved, then filtered, filtrate is upper with 60% ethanol water by weight
Sample amount M3/ sephadex weight ratio 10mgg-1, sample solution mass concentration 2.0mgmL-1, column blade diameter length ratio 1:80 flow with absorption
Speed is 2.0BVh-1Under conditions of by sephadex column, then with 30% ethanol water by weight elute, Fractional Collections
Eluent using the HPLC detection oleuropein contents of this specification description, be by weight more than 90% by the content
Eluent merges, and solvent is recovered under reduced pressure, obtains oleuropein crude product, it is crystallized under conditions of 2 DEG C of temperature in methyl alcohol, mistake
Filter, with anhydrous cupric sulfate is dried, gas chromatography is used to measure its water content as by weight 0.1% at ambient temperature,
The HPLC methods that this specification describes is used to measure its purity as 99.5%, according to the calculation formula that this specification describes, according to medicine
It is 3.8% that material weight, which calculates oleuropein average recoveries,.
Embodiment 6：High-purity oleuropein is extracted from jasmine
The implementation steps of the embodiment are as follows：
A, ethanol water extracts
According to the weight ratio 1 of jasmine bud and 60% ethanol water by weight:12, allow water content by weight
1.7% drying jasmine bud raw material impregnates 4 times in ethanol water under conditions of room temperature, and each 48h is obtained every time
Extracting solution be filtered, filtrate merges, and is then concentrated and dried under conditions of 48 DEG C of temperature, obtains jasmine extraction leaching
Cream M1；
B, macroporous resin column is adsorbed
Macroreticular resin pre-processes：The XDA-16 type macroreticular resins sold by Xi'an Sunresin New Materials Co., Ltd.
It is impregnated for 24 hours using 95% ethanol water by weight, then wet method dress post, then with 95% ethanol aqueous wash by weight
It is de-, until efflux mixes not white muddiness with water, then it is eluted with water to not having ethyl alcohol smell.
Absorption and elution：The jasmine extraction medicinal extract M that step A is obtained1It is dissolved with methanol aqueous solution, the solution allowed exists
Sample solution mass concentration is crude drug 0.08gmL-1, column blade diameter length ratio 1:4th, applied sample amount M1/ macroreticular resin weight ratio=3:10, absorption
Flow velocity 1.6BVh-1Under conditions of adsorbed by pre-processing macroporous resin column, then with 8BV distilled water flushings, then use
4.0BV by weight 70% ethanol water with elution flow rate 2.1BVh-1Elution, obtained ethanol eluate E1In temperature 48
Dry concentration, obtains pale yellow powder M under conditions of DEG C2；
C, polyamide column adsorbs
Polyamide prepolymer processing：The polyamide that 30~60 mesh are produced by Sinopharm Chemical Reagent Co., Ltd. is by weight
25h is impregnated in 95% ethanol water, is then charged into chromatographic column, 2.0mol/L sodium hydrate aqueous solutions is added in and impregnates 4.5h,
Then it is washed with distilled water to neutrality；Then 4.0mol/L aqueous hydrochloric acid solutions are added in and impregnate 8.0h, are then washed with distilled water to
It is neutral；It is eventually adding 95% ethanol water by weight and impregnates 10h, then be washed with distilled water to no alcohol taste.
Polyamide dress column by pretreatment；The pale yellow powder M that step B is obtained2It is dissolved with methanol aqueous solution, allows obtain
Solution in applied sample amount M2/ polyamide weight ratio 16.2mgg-1, sample solution mass concentration 3.60mgmL-1, sample solution pH
6.0th, column blade diameter length ratio 1:3 with adsorption flow rate be 2.2BVh-1Under conditions of adsorbed by pre-processing polyamide column, then use
2.2BV distilled water flushings, then with 4.0BV by weight 80% ethanol water with elution flow rate 2.2BVh-1Elution, obtains
Ethanol eluate E2Dry concentration, obtains off-white powder M under the conditions of 44 DEG C of temperature3；
D, oleuropein is prepared
The off-white powder M that step C is obtained3It is dissolved, then filtered, filtrate is upper with 60% ethanol water by weight
Sample amount M3/ sephadex weight ratio 10mgg-1, sample solution mass concentration 2.0mgmL-1, column blade diameter length ratio 1:80 flow with absorption
Speed is 2.0BVh-1Under conditions of by sephadex column, then with 30% ethanol water by weight elute, Fractional Collections
Eluent using the HPLC detection oleuropein contents of this specification description, be by weight more than 90% by the content
Eluent merges, and solvent is recovered under reduced pressure, obtains oleuropein crude product, it is crystallized under conditions of 4 DEG C of temperature in methyl alcohol, mistake
Filter, with anhydrous cupric sulfate is dried, gas chromatography is used to measure its water content as by weight 0.1% at ambient temperature,
The HPLC methods that this specification describes is used to measure its purity as 99.0%, according to the calculation formula that this specification describes, according to medicine
It is 4.0% that material weight, which calculates oleuropein average recoveries,.
Claims (6)
- A kind of 1. method that high-purity oleuropein is extracted from jasmine, it is characterised in that the step of this method is as follows：A, ethanol water extractsAccording to the weight ratio 1 of jasmine bud and 55~65% ethanol waters by weight:10~14, take dry jasmine Bud raw material impregnates 2~4 times in ethanol water under conditions of room temperature, every time 46~50h, the extracting solution obtained every time into Row filtering, filtrate merge, and are then concentrated under conditions of 46~50 DEG C of temperature, are dried to obtain jasmine extraction medicinal extract M1；B, macroporous resin column is adsorbedThe jasmine extraction medicinal extract M that step A is obtained1It is dissolved with methanol aqueous solution, the solution allowed is by pre-processing macroreticular resin Column is adsorbed, and then with distilled water flushing, then is eluted with 65~75% ethanol waters by weight, obtained ethanol elution Liquid E1Dry concentration, obtains pale yellow powder M under conditions of 42~48 DEG C of temperature2；Macroporous resin column is adsorbed with elution requirement such as Under：Sample solution mass concentration is 0.06~0.10gmL of crude drug-1, column blade diameter length ratio 1:2~4, the weight ratio of applied sample amount is big for M1/ Hole resin=3:8~12,1.5~2.5BVh of adsorption flow rate-1；6~10BV of washing distillation water consumption；Eluting solvent is with weight 65~75% ethanol waters are counted, dosage is 3.5~4.5BV, and elution flow rate is 1.5~2.5BVh-1；The macroreticular resin is selected from XDA-16, HPD-100, XDA-1 or HPD-400, the macroreticular resin of D-101 types；Described Macroreticular resin pre-treatment step is as follows：Macroreticular resin use 90~95% ethanol waters immersion by weight 22~ 26h, wet method dress post, then with 90~95% ethanol waters by weight elute, until efflux mixed with water it is not white muddy It is turbid, it is then eluted with water to there is no ethyl alcohol smell；C, polyamide column adsorbsPolyamide dress column by pretreatment, the pale yellow powder M that step B is obtained2It is dissolved with methanol aqueous solution, what is allowed is molten Liquid is adsorbed by pre-processing polyamide column, then with distilled water flushing, then with 75~85% ethanol water by weight Elution, obtained ethanol eluate E2Dry concentration, obtains off-white powder M under the conditions of 42~48 DEG C of temperature3；D, oleuropein is preparedThe off-white powder M that step C is obtained3It is separated by silica gel column chromatography, with dry method mode loading, is then mixed with chloroform methanol Bonding solvent elutes, and solvent is removed under reduced pressure in obtained eluent, obtains oleuropein crude product in methyl alcohol in the item of 0~4 DEG C of temperature It crystallizes, filters under part, it is dry, obtain the oleuropein.
- 2. according to the method described in claim 1, it is characterized in that in step C, the granularity of the polyamide is 30~90 Mesh；The polyamide prepolymer processing step is as follows：30~90 mesh polyamide impregnate in 90~95% ethanol waters by weight 22~26h is then charged into chromatographic column, is added in 1.8~2.2mol/L sodium hydrate aqueous solutions and is impregnated 3.5~4.5h, then uses Water washing is distilled to neutrality；Then 3.5~4.5mol/L aqueous hydrochloric acid solutions are added in and impregnate 7~9h, are then washed with distilled water to It is neutral；It is eventually adding 90~95% ethanol waters by weight and impregnates 9~11h, then be washed with distilled water to no alcohol taste.
- 3. according to the method described in claim 1, it is characterized in that in step C, the adsorption conditions of polyamide column are as follows：Loading The weight ratio of amount is M2/ polyamide=16.0-16.5mgg-1, sample solution mass concentration is 3.40~3.60mgmL-1, on Sample liquid pH is 5.8~6.2, column blade diameter length ratio 1:2~4, adsorption flow rate is 1.8~2.2BVh-1；Washing distillation water consumption 1.8~ 2.2BV, eluting solvent are 75~85% ethanol water by weight, and dosage is 3.5~4.5BV, elution flow rate 1.8~ 2.2BV·h-1。
- 4. according to the method described in claim 1, it is characterized in that in step C, the silica gel column chromatography purification step is as follows： Allow the off-white powder M that step C is obtained3The loading in a manner of dry method, applied sample amount M3:Silica gel=1:8~12, it is then 6 with weight ratio ~8:1 chloroform methanol mixed solvent elution, the eluent of Fractional Collections detect oleuropein content using HPLC, contain by described in The eluent merging for by weight more than 90% is measured, solvent is recovered under reduced pressure, obtains oleuropein crude product, it exists in methyl alcohol again It is crystallized, is filtered under conditions of 0~4 DEG C of temperature, it is dry, obtain the oleuropein.
- 5. according to the method described in claim 1, it is characterized in that in step C, off-white powder M that step C is allowed to obtain3It is logical Sephadex column chromatography is crossed to be purified.
- 6. according to the method described in claim 5, it is characterized in that the sephadex column chromatography purification step is as follows：Step The off-white powder M that C is obtained3It is dissolved with ethanol water, then filtered, filtrate is by sephadex column, then to weight Count the elution of 30% ethanol water, the eluent of Fractional Collections detects oleuropein content using HPLC, by the content be with The eluent of weight meter more than 90% merges, and solvent is recovered under reduced pressure, obtains oleuropein crude product, it is again in methyl alcohol in temperature 0 It is crystallized, is filtered under conditions of~4 DEG C, it is dry, obtain the oleuropein.
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| CN109021041A (en) * | 2018-09-13 | 2018-12-18 | 四川奇格曼药业有限公司 | A kind of purification process of oleuropein crude product |
| CN109021042B (en) * | 2018-09-13 | 2022-04-12 | 四川奇格曼药业有限公司 | Method for extracting high-purity oleuropein from olive leaves |
| CN109608510B (en) * | 2018-12-18 | 2021-09-03 | 五邑大学 | Preparation method of oleic acid glycoside-7-ethyl-11-methyl ester |
| CN111840154A (en) * | 2020-07-07 | 2020-10-30 | 五邑大学 | Application of oleuropein or jasminum grandiflorum extract in preparing anti-glycosylation preparation |
| CN115925759A (en) * | 2021-08-18 | 2023-04-07 | 天津理工大学 | Oleuropein purification method and application thereof |
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