CN104725450A - Method for extracting high-purity oleuropein from jasminum grandiflorum - Google Patents
Method for extracting high-purity oleuropein from jasminum grandiflorum Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
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Abstract
The invention relates to a method for extracting high-purity oleuropein from jasminum grandiflorum. The method includes digestion of ethanol water, adsorption of a large-hole resin column and a polyamide column and preparation of oleuropein. Purity of the prepared oleuropein is up to 98.0-99.5% by weight, extraction rate of oleuropein is up to 2-4%, and the method is stable, good in reproducibility, convenient and very applicable to industrial production.
Description
[technical field]
The invention belongs to extract Technique of Chinese Medicine Efficacious Ingredient field.More specifically, the present invention relates to a kind of method extracting high purity Oleuropein from Largeflower Jasmine Flower.
[background technology]
Largeflower Jasmine Flower is the dry flower of Oleaceae Jasminum plant jasmine (Jasminum officinale L.var.grandiflorum), has another name called jasmine pin, knows tender tea leaves flower, have effect of dispersing the stagnated live-QI to relieve the stagnation of QI, promoting the circulation of QI to relieve pain.China is among the people treats the illnesss such as maldigestion, duodenal bulbar ulcer, chronic hepatitis, liver cirrhosis with it.Jasmine is widely cultivated all over the world, and resource is very abundant.Result of study shows, Largeflower Jasmine Flower is mainly containing iridoid glycosides, triterpene saponin and flavonoid compound, and wherein the content of iridoid glycosides compound Oleuropein is relatively high, has larger research and development and is worth.
Oleuropein molecular formula is C
25h
32o
13, there is multiple biological activity, as antiviral, antibacterial, anti-inflammatory, hypoglycemic etc., have a extensive future in sector applications such as medicine, protective foods, makeup.
At present, Oleuropein mainly extracts and obtains from the plants such as Fructus oleae europaeae (Olea europaea L.), Syringa oblata Lindl. (Syringaoblate Lindl.), daphne lilac (Syringa microphylla Diels).Extracting method mainly contains heat extraction, ultrasonic-assisted extraction, microblogging assisted extraction etc.All there is certain technological deficiency in these methods, such as power consumption is large, consumptive material is many, the high Oleuropein that causes of Extracting temperature easily decomposes.CN101955503A discloses a kind of from spending more the method extracting Oleuropein jasmine (Jasminum polyanthum Franch.): Largeflower Jasmine Flower is spent more in drying and is added to CO
2in supercritical extraction device, with ethanol as entrainment agent, be extracted thing, be dissolved in water, with equivalent extraction into ethyl acetate 1 ~ 3 time, pipette aqueous phase, adsorbed by AB-8 macroporous adsorptive resins, with 35% aqueous ethanolic solution wash-out, collect 3 ~ 8 times amount column volume elutriants, decompression recycling ethanol is also concentrated, refrigerates 12 ~ 36 hours, filters, filter residue adds dehydrated alcohol recrystallization, fractional crystallization, washing, drying, obtain the Oleuropein that purity is greater than 97%.The method does not provide the final extraction yield of Oleuropein, and the supercritical CO used
2extraction instrument investment cost is high, and operational condition is harsh.The present inventor reports the processing condition of Oleuropein in application XDA-16 type macroporous resin enrichment purifying Largeflower Jasmine Flower in " Chinese experimental pharmacology of traditional Chinese medical formulae magazine " the 6th phase (2013), applies Oleuropein average mass fraction in the pressed powder that this technique obtains and is only 32.82%.Have not yet to see the research report of high purity Oleuropein scale manufacturing technique; research and develop new Oleuropein preparation technology, from Largeflower Jasmine Flower, high purity Oleuropein is prepared in mass-producing, for the application and development of Largeflower Jasmine Flower; and for the application and development of Oleuropein, all have great importance.
The present invention is directed to the problem that Oleuropein content that in prior art, enrichment obtains from Largeflower Jasmine Flower is not high, a kind of method extracting high purity Oleuropein from Largeflower Jasmine Flower is provided.
[summary of the invention]
[technical problem that will solve]
The object of this invention is to provide a kind of method extracting high purity Oleuropein from Largeflower Jasmine Flower.
[technical scheme]
The present invention is achieved through the following technical solutions.
The present invention relates to a kind of method extracting high purity Oleuropein from Largeflower Jasmine Flower.
The step of the method is as follows:
A, aqueous ethanolic solution lixiviate
Get dry Largeflower Jasmine Flower bud raw material to soak 2 ~ 4 times under the condition of room temperature in aqueous ethanolic solution, each 46 ~ 50h, the extracting solution at every turn obtained filters, and filtrate merges, then concentrate under the condition of temperature 46 ~ 50 DEG C, drying obtains Largeflower Jasmine Flower and extracts medicinal extract M
1;
B, macroporous resin column are adsorbed
The Largeflower Jasmine Flower that steps A obtains extracts medicinal extract M
1dissolve with methanol aqueous solution, allow the solution obtained be adsorbed by pre-treatment macroporous resin column, then use distilled water flushing, then use 65 ~ 75% aqueous ethanolic solution wash-outs by weight, the ethanol eluate E obtained
1dry concentrated under the condition of temperature 42 ~ 48 DEG C, obtain pale yellow powder M
2;
C, polyamide column adsorb
Through pretreated polymeric amide dress post, the pale yellow powder M that step B obtains
2dissolve with methanol aqueous solution, allow the solution obtained be adsorbed by pre-treatment polyamide column, then use distilled water flushing, then use 75 ~ 85% aqueous ethanolic solution wash-outs by weight, the ethanol eluate E obtained
2dry concentrated under temperature 42 ~ 48 DEG C of conditions, obtain off-white powder M
3;
D, prepare Oleuropein
The off-white powder M that step C obtains
3be separated by silica gel column chromatography, by dry method mode loading, then use chloroform methanol mixed solvent wash-out, the elutriant removal of solvent under reduced pressure obtained, obtains the crystallization under the condition of temperature 0 ~ 4 DEG C in methyl alcohol of Oleuropein crude product, filters, drying, obtains described Oleuropein.
A preferred embodiment of the invention, in step, the weight ratio of Largeflower Jasmine Flower bud and by weight 55 ~ 65% aqueous ethanolic solutions is 1:10 ~ 14.
According to another kind of preferred implementation of the present invention, in stepb, described macroporous resin is selected from the macroporous resin of XDA-16, HPD-100, XDA-1 or HPD-400, D-101 type; Described macroporous resin pre-treatment step is as follows: described macroporous resin uses 90 ~ 95% aqueous ethanolic solutions by weight to soak 22 ~ 26h, wet method dress post, use 90 ~ 95% aqueous ethanolic solution wash-outs by weight again, until effluent liquid mixes with water not in white opacity, then wash with water to there is no ethanol smell.
According to another kind of preferred implementation of the present invention, in stepb, macroporous resin column absorption is as follows with elution requirement: sample solution mass concentration is crude drug 0.06 ~ 0.10gmL
-1, post blade diameter length ratio 1:2 ~ 4, the weight ratio of applied sample amount is M
1/ macroporous resin=3:8 ~ 12, adsorption flow rate 1.5 ~ 2.5BVh
-1, washing distilled water consumption 6 ~ 10BV; Eluting solvent is 65 ~ 75% aqueous ethanolic solutions by weight, and its consumption is 3.5 ~ 4.5BV, and elution flow rate is 1.5 ~ 2.5BVh
-1.
According to another kind of preferred implementation of the present invention, in step C, the granularity of described polymeric amide is 30 ~ 90 orders;
Described polyamide prepolymer treatment step is as follows: 30 ~ 90 order polymeric amide soak 22 ~ 26h in 90 ~ 95% aqueous ethanolic solutions by weight, then load in chromatography column, add 1.8 ~ 2.2mol/L aqueous sodium hydroxide solution and soak 3.5 ~ 4.5h, be then washed with distilled water to neutrality; Then add 3.5 ~ 4.5mol/L aqueous hydrochloric acid and soak 7 ~ 9h, be then washed with distilled water to neutrality; Finally add 90 ~ 95% aqueous ethanolic solutions by weight and soak 9 ~ 11h, then be washed with distilled water to without alcohol taste.
According to another kind of preferred implementation of the present invention, in step C, the adsorption conditions of polyamide column is as follows: the weight ratio of applied sample amount is M
2/ polymeric amide=16.0-16.5mgg
-1, sample solution mass concentration is 3.40 ~ 3.60mgmL
-1, sample solution pH is 5.8 ~ 6.2, post blade diameter length ratio 1:2 ~ 4, and adsorption flow rate is 1.8 ~ 2.2BVh
-1; Washing distilled water consumption 1.8 ~ 2.2BV, eluting solvent is 75 ~ 85% aqueous ethanolic solutions by weight, and its consumption is 3.5 ~ 4.5BV, elution flow rate 1.8 ~ 2.2BVh
-1.
According to another kind of preferred implementation of the present invention, in step D, described purification by silica gel column chromatography step is as follows: allow the off-white powder M that step C obtains
3with dry method mode loading, applied sample amount M
3: silica gel=1:8 ~ 12, then be the chloroform methanol mixed solvent wash-out of 6 ~ 8:1 by weight ratio, the elutriant of Fractional Collections adopts HPLC to detect Oleuropein content, be the elutriant merging of by weight more than 90% by described content, decompression and solvent recovery, obtains Oleuropein crude product, it carries out crystallization again in methyl alcohol under the condition of temperature 0 ~ 4 DEG C, filter, dry, obtain described Oleuropein.
According to another kind of preferred implementation of the present invention, in step D, the off-white powder M that step C obtains is allowed
3purifying is carried out by dextrane gel column chromatography.
According to another kind of preferred implementation of the present invention, described sephadex column chromatography purification step is as follows: the off-white powder M that step C obtains
3dissolve with aqueous ethanolic solution, then filter, filtrate by sephadex column, then uses 30% aqueous ethanolic solution wash-out by weight, the elutriant of Fractional Collections adopts HPLC to detect Oleuropein content, be the elutriant merging of by weight more than 90% by described content, decompression and solvent recovery, obtains Oleuropein crude product, it carries out crystallization again in methyl alcohol under the condition of temperature 0 ~ 4 DEG C, filter, dry, obtain described Oleuropein.
According to the Oleuropein product that the method for the invention prepares, its purity is by weight 98.0 ~ 99.5%.
In more detail the present invention will be described below.
The present invention relates to a kind of method extracting high purity Oleuropein from Largeflower Jasmine Flower.
The step of the method is as follows:
A, aqueous ethanolic solution lixiviate
Get dry Largeflower Jasmine Flower bud raw material to soak 2 ~ 4 times under the condition of room temperature in aqueous ethanolic solution, each 46 ~ 50h, the extracting solution at every turn obtained filters, and filtrate merges, then concentrate under the condition of temperature 46 ~ 50 DEG C, drying obtains Largeflower Jasmine Flower and extracts medicinal extract M
1.
In this step, Largeflower Jasmine Flower bud is 1:10 ~ 14 with the weight ratio of 55 ~ 65% aqueous ethanolic solutions by weight.
The Largeflower Jasmine Flower bud raw material that the present invention uses is product sold in the market, such as purchased from the product of Hui nationality's medicinal material trade center, this product is accredited as the dry flower of Oleaceae (Oleaceous) plant jasmine (Jasminum officinale L.var.grandiflorum) through Institute of Radiation Medicine of Military Medical Science Institute.Employing gas chromatography measures, and the water content of described dry flower is with Largeflower Jasmine Flower bud raw material total weight less than 2%.
In the present invention, with Oleuropein product in contrast, by the content of determined by ultraviolet spectrophotometry total iridoid glycoside, calculating total iridoid glycoside yield (calculating by medicinal material) is evaluation index, adopts L
9(3
4) orthogonal experiment method each factor to following water cold-maceration, decocting cooking method, ethanol cold-maceration and ethanol refluxing process investigates, and determines that 4 kinds of extracting method extraction conditions are as follows:
Often kind of method takes 3 parts of Largeflower Jasmine Flower medicinal materials respectively, and every part of 20g carries out parallel test.
Water cold-maceration: get dry Largeflower Jasmine Flower medicinal material, the 12 times amount water yields, at room temperature soak 3 times, each 72h, filter, and three times filtrate merges, and Conventional concentration, drying, obtain dry extract, weighs.
Ethanol refluxing process: get dry Largeflower Jasmine Flower medicinal material, 12 times amount 60% aqueous ethanolic solution amounts, reflux 3 times, each 2h, filters, and three times filtrate merges, decompression recycling ethanol, and Conventional concentration is dry, obtains dry extract, weighs.
Decocting cooking method: get dry Largeflower Jasmine Flower medicinal material, the 16 times amount water yields, decoct 3 times, each 1h, filter, and three times filtrate merges, and Conventional concentration, drying, obtain dry extract, weighs.
Ethanol cold-maceration: get dry Largeflower Jasmine Flower medicinal material, 12 times amount 60% aqueous ethanolic solution amounts, soaking at room temperature 3 times, each 48h, filters, and three times filtrate merges, and Conventional concentration, drying, obtain dry extract, weighs.
In each extract, total iridoid glycoside content all adopts Conventional UV spectrophotometry.Take 4.0mg dry extract at every turn, be placed in 100ml volumetric flask, add methanol dilution to scale, shake up, make the solution of every 1ml containing 0.040mg dry extract, using this solution as test liquid.Use ultraviolet spectrophotometer to measure optical density under normal conditions at wavelength 232nm place, calculated the total iridoid glycoside concentration C of test liquid by typical curve
1, according to medicinal material by following formulae discovery total iridoid glycoside yield, and variance analysis is carried out to result:
Total iridoid glycoside yield %=(C
1/ 0.0400 × M
dry extract)/20.00 × 100
Test-results shows, the total iridoid glycoside average recovery rate of 4 kinds of extracting method is respectively: water cold-maceration 20.67%, ethanol refluxing process 21.72%, decocting cooking method 17.33%, ethanol cold-maceration 23.26%, wherein the average recovery rate of ethanol cold-maceration is the highest, obviously be better than other method, and simple to equipment requirements, stablize feasible, therefore selection ethanol cold-maceration is the preparation method of Largeflower Jasmine Flower extract.
B, macroporous resin column are adsorbed
The Largeflower Jasmine Flower that steps A obtains extracts medicinal extract M
1dissolve with methanol aqueous solution, allow the solution obtained be adsorbed by pre-treatment macroporous resin column, then use distilled water flushing, then use 65 ~ 75% aqueous ethanolic solution wash-outs by weight, the ethanol eluate E obtained
1dry concentrated under the condition of temperature 42 ~ 48 DEG C, obtain pale yellow powder M
2.
The macroporous resin that the present invention uses is selected from the macroporous resin of XDA-16, HPD-100, XDA-1 or HPD-400, D-101 type.These resins are all product solds in the market, XDA-16, XDA-1 type such as sold by Xi'an Sunresin New Materials Co., Ltd.; HPD-100, HPD-400 type sold by Cangzhou Bon Adsorption Material Science and Technology Co., Ltd; The D-101 type etc. sold by Tianjin Xing Nanyun energy Polymer Technology company limited.
Described macroporous resin all needs to carry out pre-treatment before use, its object is to remove usually contain in resin synthesis process a small amount of and does not complete the oligopolymer of polymerization, do not participate in the material of polyreaction and some soluble impurities.Pre-treatment not only can improve resin stability, can also play activated resin, improves the effect of its operating capacity.
The pre-treatment step of the macroporous resin that the present invention uses is as follows: described macroporous resin uses 90 ~ 95% aqueous ethanolic solutions by weight to soak 22 ~ 26h, wet method dress post, use 90 ~ 95% aqueous ethanolic solution wash-outs by weight again, until effluent liquid mixes with water not in white opacity, then wash with water to there is no ethanol smell.
The present invention uses macroporous resin column process Largeflower Jasmine Flower to extract medicinal extract M
1solution condition is as follows:
Macroporous resin column adsorption conditions is as follows:
In the present invention, the Largeflower Jasmine Flower that steps A obtains extracts medicinal extract M
1dissolve with methanol aqueous solution, obtain solution concentration be generally every milliliter be 0.05 ~ 0.15g crude drug, be preferably 0.06 ~ 0.10g.The excessive concentration of described solution or too low be all disadvantageous, its reason is that excessive concentration easily causes revealing, and concentration is too low can extend the production time, improves production cost.The concentration of described methanol aqueous solution normally by volume 40 ~ 80%, preferably by volume 50 ~ 70%.
Post blade diameter length ratio 1:2 ~ 4 of macroporous resin column.
Applied sample amount for extract in gram Largeflower Jasmine Flower medicinal extract with in the ratio of gram macroporous resin for 3:8 ~ 12.
Adsorption flow rate 1.5 ~ 2.5BVh
-1, BV is column volume, lower same.
Distilled water flushing condition: washing distilled water consumption 6 ~ 10BV.
Elution requirement: eluting solvent is 65 ~ 75% aqueous ethanolic solutions by volume, eluting agent 3.5 ~ 4.5BV, elution flow rate 1.5 ~ 2.5BVh
-1.
The HPLC method measuring method of Oleuropein content is described below:
The preparation of I, reference substance solution
Precision takes 20.2mg Oleuropein reference substance, puts in 50mL volumetric flask, and add methanol dilution to scale, shake up, obtaining concentration is 404 μ gmL
-1oleuropein reference substance solution.
II, chromatographic condition
Use 1200 type high performance liquid chromatographs of U.S. Agilent Company, the ZORBAX SB-C18 chromatographic column (4.6mm × 250mm, 5 μm) of U.S. Agilent Company, flow velocity 1.0mLmin
-1, column temperature 25 DEG C, determined wavelength 232nm, moving phase is acetonitrile-water (by volume, 22: 78).Oleuropein reference substance solution color atlas is shown in accompanying drawing 1.
III, linear relationship are investigated
Accurate draw 0.5,1,2,4,6mL Oleuropein reference substance solution, be diluted to 10mL respectively, sample detection under above-mentioned chromatographic condition.Take concentration as X-coordinate, peak area is that ordinate zou draws out regression curve, tries to achieve following regression equation by its regression curve:
Y＝486803X-3577560，r＝0.9999，
Oleuropein is at 20.2 ~ 242.4 μ gmL
-1in good linear relation.
Previously described HPLC method is adopted to determine M
2in Oleuropein content, and calculate the Oleuropein rate of recovery by following calculation formula:
Oleuropein rate of recovery %=(M
2middle Oleuropein quality/M
1middle Oleuropein quality) × 100
Oleuropein massfraction is calculated by following calculation formula:
Oleuropein massfraction %=(M
2middle Oleuropein quality/M
2quality) × 100
Oleuropein extraction yield is calculated by following calculation formula:
Oleuropein extraction yield %=(M
2middle Oleuropein quality/quality of medicinal material) × 100
Test-results shows, the Oleuropein rate of recovery is more than 90.0%, and Oleuropein massfraction is more than 32.0%, and the extraction yield calculating Oleuropein by medicinal material is more than 6.0%.
C, polyamide column adsorb
Through pretreated polymeric amide dress post, the pale yellow powder M that step B obtains
2dissolve with methanol aqueous solution, allow the solution obtained be adsorbed by pre-treatment polyamide column, then use distilled water flushing, then use 75 ~ 85% aqueous ethanolic solution wash-outs by weight, the ethanol eluate E obtained
2dry concentrated under temperature 42 ~ 48 DEG C of conditions, obtain off-white powder M
3.
In the present invention, described polymeric amide should be the polymeric amide with adsorption and de-adsorption performance.The polymeric amide that the present invention uses is product sold in the market, the polymeric amide such as produced by the biochemical plastic molding and processing plant of Taizhou City of Zhejiang Province road and bridge tetramethyl, Yuan Ye bio tech ltd, Shanghai or Chemical Reagent Co., Ltd., Sinopharm Group, its granularity is 30 ~ 90 orders.
Described polyamide prepolymer treatment step is as follows: 30 ~ 90 order polymeric amide soak 22 ~ 26h in 90 ~ 95% aqueous ethanolic solutions by weight, and the object of immersion is unpolymerized starting monomer and low-molecular(weight)polymer in removing polymeric amide production process; Then polymeric amide is loaded in chromatography column, add 1.8 ~ 2.2mol/L aqueous sodium hydroxide solution and soak 3.5 ~ 4.5h, then be washed with distilled water to neutrality, the effect that aqueous sodium hydroxide solution soaks is to remove the acidic impurities stronger with its effect that polymeric amide adsorbs; Then add 3.5 ~ 4.5mol/L aqueous hydrochloric acid and soak 7 ~ 9h, be then washed with distilled water to neutrality, the effect that aqueous hydrochloric acid soaks is to remove the alkaline impurities stronger with its effect that resin adsorbs; Finally add 95% aqueous ethanolic solution by weight and soak 9 ~ 11h, then be washed with distilled water to without alcohol taste.
Inquire into affecting polycaprolactam factor below.
One, polymeric amide particle size influences research
I, Static Adsorption ability are investigated
Take 9 parts of pre-treatment, 3 kinds of each 0.5g of order number polymeric amide (3 often kind repetitions), be placed in tool plug triangular flask respectively, respectively add 15mL mass concentration 750.4 μ g/mL M
2solution (containing Oleuropein 246.2 μ g/mL), at room temperature carry out Static Adsorption 18h, every 2h jolting once.Pipette supernatant liquor, measure wherein Oleuropein mass concentration, calculate polycaprolactam amount and adsorption rate.The polymeric amide of different adsorption saturation is placed in tool plug triangular flask, respectively adds 15mL 95% ethanol and carry out static wash-out 18h in room temperature, every 2h jolting once, is separated and obtains elutriant.Then measure the Oleuropein mass concentration of elutriant, calculate eluting rate, and average to group process.The results are shown in following table 1.
Q＝(C
0－C
r)V
1/W
D＝(C
0－C
r)/C
0×100％
E＝2C
eV
2/Q×100％
In formula:
Q adsorptive capacity (mg/g), C
0initial mass concentration (mg/mL), C
rabsorption supernatant liquor mass concentration (mg/mL), V
1sample solution volume (mL), W resin quality (g), D adsorption rate (%), E eluting rate and V
2elute soln volume (mL)
Table 1: granularity is on the impact (n=3) of polymeric amide Static Adsorptive capacity
The investigation of II, dynamic adsorption capacity
Take 9 parts of pre-treatment, 3 kinds of each 0.5g of order number polymeric amide (3 often kind repetitions), precision weighing, loading internal diameter in a wet process is respectively in 1.2cm chromatographic column.Respectively get 15mL 750.4 μ g/mL M
2(containing Oleuropein 246.2 μ g/mL) solution is with flow velocity 2BVh
-1upper prop adsorbs, and collects effluent liquid, measures the mass concentration of Oleuropein, calculates polycaprolactam amount and adsorption rate.Each post 30mL by volume 95% ethanol with flow velocity 2BVh
-1wash-out, measures Oleuropein mass concentration in elutriant, and calculate eluting rate, test-results is listed in table 2.
Table 2: granularity is on the impact (n=3) of polymeric amide dynamic adsorption
Table 1 and table 2 result show, polymeric amide granularity has certain influence to polymeric amide Static Adsorption and desorption performance, but difference is little.Consider subsequent operations feasibility, determine that the granularity selected is 30-90 order polymeric amide.
Two, polymeric amide enrichment and purification method research
The impact of I, applied sample amount
Take 2g process polymeric amide and load chromatography column, a certain amount of sample solution (concentration 750.4 μ g/mL) is passed through polyamide column, collect effluent liquid, every 1/3BV (10mL is 1BV) collects once, measure the Oleuropein mass concentration of every part of effluent liquid, its mass concentration curve is shown in shown in Fig. 5.This result shows, occur a small amount of Oleuropein in the 14th part of effluent liquid, best applied sample amount is 13/3BV, 43.3mL, and namely best applied sample amount is 16.25mgg
-1(M
2/ polymeric amide)
The impact of II, sample solution pH
Take 8 parts of processed polymeric amide of every part of 2g, load chromatography column, by 8 of different pH parts of sample solutions, (concentration is 3.50mgmL
-1, volume is 10mL) be added to respectively in polyamide resin column, other step is identical with A's.Measure Oleuropein mass concentration, according to adsorptive capacity and the adsorption rate of previously described formulae discovery Oleuropein, the results are shown in Table 3 for this.This result shows, sample solution pH Oleuropein adsorption rate 6.0 time is the highest, therefore the Optimal pH of sample solution is 6.0.
Table 3: sample solution pH is on the impact of Oleuropein adsorption rate
The orthogonal design of III, sample solution concentration, post blade diameter length ratio and adsorption flow rate
Adopt L
9(3
4) orthogonal, with the Oleuropein rate of recovery for evaluation index, screening is optimized to purifying process herb liquid mass concentration, post blade diameter length ratio and adsorption flow rate 3 conditions.
Take 2g process polymeric amide (column volume 10mL), 10mL mass concentration 3.50mgmL
-1, pH 6.0 M
2sample liquid is added in polyamide resin column, first uses 3BV distilled water flushing, and with 5BV 95% aqueous ethanolic solution wash-out, other step is identical with A's.The mass content of Oleuropein in ethanol eluate is measured, by the following formulae discovery Oleuropein rate of recovery according to previously described method.
Rate of recovery %=(in ethanol eluate in Oleuropein quality/upper prop liquid Oleuropein quality) × 100
Table 4: level of factor table
Note: A quality of liquid medicine concentration (mgmL
-1); B post blade diameter length ratio; C adsorption flow rate (BVh
-1);
D is blank
Table 5: orthogonal experiments
Table 6: variance analysis
Note: F
0.05(2,2)=19.0; F
0.1(2,2)=9.0
From the orthogonal experiments of table 4 ~ 6, the affect size of each factor on the Oleuropein rate of recovery is B > A > C, wherein B factor, namely the impact of post blade diameter length ratio significantly (p < 0.01).The best of breed of Three factors is A
2b
1c
2, namely quality of liquid medicine concentration is 3.50mgmL
-1, post blade diameter length ratio is 1:3, and adsorption flow rate is 2BVh
-1.
The investigation of IV, washing volume
Take 2g pre-treatment polymeric amide, loading chromatographic column (column volume 10mL, post blade diameter length ratio 1:3), is the sample solution (10mL3.50mgmL of 6.0 by pH
-1m
2solution) add on a column, adsorption flow rate 2BVh
-1, distilled water wash-out, collects elutriant, and every 1/3BV (10mL is 1BV) collects once, measures in every part of elutriant admittedly containing thing (solute) quality.Take elutriant as X-coordinate, admittedly be ordinate zou curve plotting Fig. 6 containing thing accumulated quality.Result shows, during water consumption≤2BV, admittedly increases gradually containing thing accumulated quality, but Oleuropein do not detected, from the 7th part of elutriant, a small amount of Oleuropein detected in 1st ~ 6 parts of elutriants, and admittedly tend to be steady containing thing accumulated quality.Consider, determine that water consumption is 2BV.
The impact of V, eluent
Take 7 parts every part 2g process polymeric amide, be respectively charged into chromatographic column (column volume 10mL, post blade diameter length ratio 1:3), by pH 6.0 sample solution (10mL 3.50mgmL
-1m
2solution) be added in chromatographic column, adsorption flow rate 2BVh
-1first use 2BV distilled water wash-out, again respectively with 5BV 30%, 40%, 50%, 60%, 70%, 80%, 95% aqueous ethanolic solution wash-out by volume, collect ethanol eluate, Oleuropein mass concentration is measured after being settled to same volume, according to previously described formulae discovery eluting rate, it the results are shown in Figure 7.Can be thought by Fig. 7 result, 80% aqueous ethanolic solution wash-out effect is best by volume.
The impact of VI, eluting agent
Take 2g process polymeric amide and load chromatographic column (column volume 10mL, post blade diameter length ratio 1:3), by pH6.0 sample solution (10mL3.50mgmL
-1m
2solution) be added in chromatographic column, adsorption flow rate 2BVh
-1, first use 2BV distilled water wash-out.Use 80% aqueous ethanolic solution wash-out by volume again, collect ethanol eluate, every 1BV collects once, measures Oleuropein mass concentration after being then settled to same volume according to previously described method, according to previously described formulae discovery eluting rate, it the results are shown in Figure 8.After eluting agent is 4BV, eluting rate tends towards stability, determines that eluting agent is 4BV.
The impact of VII, eluent flow velocity
Take 6 parts every part 2g process polymeric amide, be respectively charged into chromatographic column (column volume 10mL, post blade diameter length ratio 1:3), by pH6.0 sample solution (10mL3.50mgmL
-1m
2solution) be added in chromatographic column, adsorption flow rate 2BVh
-1, first use 2BV distilled water wash-out, then with 4BV 80% aqueous ethanolic solution wash-out by volume, elution flow rate is respectively 1BVh
-1, 1.5BVh
-1, 2BVh
-1, 2.5BVh
-1, 3BVh
-1with 4BVh
-1, collect ethanol eluate, after being settled to same volume, measure the mass concentration of Oleuropein according to previously described method, according to the previously described formulae discovery Oleuropein rate of recovery, it the results are shown in Figure 9.When elution flow rate increases, Oleuropein rate of recovery entirety is on a declining curve, but from 1BVh
-1increase to 2BVh
-1time, the Oleuropein rate of recovery only reduces by 0.79%, and from 2BVh
-1increase to 4BVh
-1time, the Oleuropein rate of recovery reduces 11.11%.Consider, elution flow rate 2BVh
-1for best.
VIII, proof test
Take 3 parts every part 2g process polymeric amide, be respectively charged in chromatographic column, according to above-mentioned optimum process condition (30-90 order polymeric amide, applied sample amount 16.25mgg
-1(M
2/ polymeric amide), sample solution mass concentration 3.50mgmL
-1, sample solution pH is 6.0, post blade diameter length ratio 1:3, and adsorption flow rate is 2BVh
-1, first with 2BV washing, then with 4BV 80% aqueous ethanolic solution wash-out by weight, elution flow rate 2BVh
-1) carry out 3 parallel tests, collect 80% aqueous ethanolic solution wash-out liquid by volume respectively, measure Oleuropein mass concentration according to previously described method, calculate the Oleuropein rate of recovery according to previously described formulae discovery.Ethanol eluate concentrates, dry, obtains off-white powder M
3, measure the percentage composition of Oleuropein in powder.To calculate Oleuropein average recovery rate be 88.07%, RSD is 1.73%, and in dried powder, the average mass fraction of Oleuropein is 65.34%, RSD is 1.41%, and the average recoveries calculating Oleuropein by medicinal material is 5.40%.
Table 7: proof test result
Consider, polycaprolactam post adsorption treatment pale yellow powder M
2solution condition is as follows:
Post blade diameter length ratio 1:2 ~ 4 of polycaprolactam post.
Adsorption conditions: the weight ratio of applied sample amount is M
2/ polymeric amide=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: washing distilled water consumption 1.8 ~ 2.2BV;
Elution requirement: eluting solvent is 75 ~ 85% aqueous ethanolic solutions by weight, and its consumption is 3.5 ~ 4.5BV, elution flow rate 1.8 ~ 2.2BVh
-1.
D, prepare Oleuropein
The off-white powder M that step C obtains
3be separated by silica gel column chromatography, by chloroform methanol mixed solvent wash-out, the elutriant removal of solvent under reduced pressure obtained, obtain the crystallization under the condition of temperature 0 ~ 4 DEG C in methyl alcohol of Oleuropein crude product, filter, dry, obtain described Oleuropein.
Described purification by silica gel column chromatography step is as follows: allow the off-white powder M that step C obtains
3with dry method mode loading, its applied sample amount M
3: silica gel=1:8 ~ 12, then be the chloroform methanol mixed solvent wash-out of 6 ~ 8:1 by weight ratio, the elutriant of Fractional Collections adopts previously described HPLC method to detect Oleuropein content, be the elutriant merging of by weight more than 90% by described content, decompression and solvent recovery, obtains Oleuropein crude product, it carries out crystallization again in methyl alcohol under the condition of temperature 0 ~ 4 DEG C, filter, dry, obtain described Oleuropein.
Described Oleuropein crude product carries out crystallization in methyl alcohol under the condition of temperature 0 ~ 4 DEG C, filter, dry at ambient temperature with anhydrous cupric sulfate, gas chromatography is adopted to measure its water-content for by weight 0.1 ~ 0.3%, adopting previously described HPLC method to measure its purity is more than 98.2%, according to previously described calculation formula, be 2.2% according to medicinal material Weight computation Oleuropein average recoveries.
In this step, the powder M obtained can also be allowed
3by dextrane gel column chromatography purification.
Described sephadex column chromatography purification step is as follows: the off-white powder M that step C obtains
3dissolve with aqueous ethanolic solution, then filter, filtrate by sephadex column, then uses 30% aqueous ethanolic solution wash-out by weight, the elutriant of Fractional Collections adopts previously described HPLC to detect Oleuropein content, be the elutriant merging of by weight more than 90% by described content, decompression and solvent recovery, obtains Oleuropein crude product, it carries out crystallization again in methyl alcohol under the condition of temperature 0 ~ 4 DEG C, filter, dry, obtain described Oleuropein.The dextrane gel that the present invention uses is with trade(brand)name Sephadex LH-20 product sold by Beijing Pu Ruiyin biological chromatography Technology Co., Ltd..The actual conditions of dextrane gel column chromatography: applied sample amount M
3/ dextrane gel weight ratio 10mgg
-1, sample solution mass concentration 2.0mgmL
-1, post blade diameter length ratio 1:80, adsorption flow rate is 2.0BVh
-1.
The invention still further relates to the Oleuropein adopting and extract high purity Oleuropein method and prepare from Largeflower Jasmine Flower.Adopt previously described analytical procedure to determine, its purity is 98.0-99.5% by weight.
Test the impact of Oleuropein content and Oleuropein extraction yield with regard to the combination of the step such as alcohol flooding, macroporous resin adsorption, polycaprolactam, purification by silica gel column chromatography in the inventive method below, its test-results is listed in the table below in 8.
Table 8: different step combines the impact on Oleuropein content and Oleuropein extraction yield
Can be known by the result of table 8 and find out, the macroporous resin adsorption series connection polycaprolactam purifying that the present invention adopts effectively can improve Oleuropein content and Oleuropein extraction yield.
In addition, CN101003557A discloses a kind of preparation method being rich in the olive leaf extract of high purity Oleuropein, the method is that leaf of Fructus oleae europaeae first extracts with polar solvent, centrifugal settling in the basic conditions again, obtain centrifugal clear liquid and throw out, then carry out film lotus root and close separation, centrifugal clear liquid is allowed to pass through ceramic membrane successively, ultra-filtration membrane and nanofiltration membrane treatment, the concentrated solution obtained uses aluminum oxide again, macroporous adsorbent resin, one or more blending agent in polymeric amide carries out selective adsorption, elutriant is dry, obtain the olive leaf extract that Oleuropein content is greater than 50%.CN102228514A discloses a kind of method extracting Oleuropein from leaf of Fructus oleae europaeae, the method is extracting with water or alcohol water mixed solvent, extracting solution concentrates, through macroporous resin adsorption, decolorizing resin absorption and organic solvent extraction, separation and purification is carried out to extracting solution successively again, finally obtain the oleuropein extract that Oleuropein content is greater than 40%; CN102659867A discloses a kind of preparation method of high-content oleuropein extract, the method allows crude extract be separated through absorption with macroporous adsorbent resin obtain crude extract II, crude extract III is obtained again through solvent extraction, then be separated through silica gel or alumina column chromatography, final drying obtains the oleuropein extract that content reaches 80-90%.Although these existing methods can extract the oleuropein extract obtaining certain content, effect is still very undesirable.
Instant invention overcomes the problem that in the oleuropein extract of existing preparation method existence, Oleuropein content is not high, by steps such as alcohol flooding, macroporous resin adsorption series connection polycaprolactam, silica gel column chromatography or dextrane gel column chromatography purifications, under maintenance Oleuropein average recoveries (in medicinal material) is the prerequisite of 2 ~ 4%, obtain the extract that Oleuropein content reaches 98.0-99.5% by weight.
[beneficial effect]
Compared with prior art, the present invention has following beneficial effect:
1, the invention provides and a kind ofly mass-producing can prepare the method for high purity Oleuropein.The inventive method prepares high purity Oleuropein by steps such as the extraction of ethanol cold soaking, macroporous resin adsorption, polycaprolactam, silica gel column chromatography, SephadexLH-20 column chromatography and recrystallizations, purity reaches 98.0-99.5% by weight, and the average recoveries calculating Oleuropein according to medicinal material is 2 ~ 4%.
2, the present invention adopts macroporous resin in conjunction with polycaprolactam, is suitable for the enrichment of Oleuropein, improves preparation efficiency.
3, leaching process of the present invention at room temperature carries out, and reduces the impurity such as protein, polysaccharide, pigment and dissolves in, to protect in leaching process active substance by pyrocondensation destroy decompose, oxidation or polymerization, ensure that Oleuropein stay in grade and content is high.
4. method provided by the invention is stablized, and favorable reproducibility, simple and efficient, the product purity obtained is high, and yield is high, is suitable for suitability for industrialized production.
[accompanying drawing explanation]
Fig. 1 is Oleuropein standard substance 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 invention
1hNMR collection of illustrative plates;
Fig. 4 is Oleuropein prepared by the present invention
13cNMR collection of illustrative plates;
Fig. 5 is the elution curve of polycaprolactam Oleuropein;
Fig. 6 is the effect diagram of water consumption to polycaprolactam purifying process;
Fig. 7 is the effect diagram of eluant strength to polycaprolactam purifying process;
Fig. 8 is the effect diagram of eluent volume to polycaprolactam purifying process;
Fig. 9 is the effect diagram of elution flow rate to polycaprolactam purifying process.
[embodiment]
The present invention can be understood better by following embodiment.
Embodiment 1: extract high purity Oleuropein from Largeflower Jasmine Flower
The implementation step of this embodiment is as follows:
A, aqueous ethanolic solution lixiviate
According to the weight ratio 1:12 of Largeflower Jasmine Flower bud with 60% aqueous ethanolic solution by weight, allow water-content by weight 2.0% dry Largeflower Jasmine Flower bud raw material in aqueous ethanolic solution under the condition of room temperature soak 3 times, each 50h, the extracting solution at every turn obtained filters, filtrate merges, then under the condition of temperature 46 DEG C, carry out concentrate drying, obtain Largeflower Jasmine Flower and extract medicinal extract M
1;
B, macroporous resin column are adsorbed
Macroporous resin pre-treatment: the XDA-16 type macroporous resin sold by Xi'an Sunresin New Materials Co., Ltd. uses 95% aqueous ethanolic solution by weight to soak 22h, then wet method dress post, use 95% aqueous ethanolic solution wash-out by weight again, until effluent liquid mixes with water not in white opacity, then wash with water to there is no ethanol smell.
Absorption and wash-out: the Largeflower Jasmine Flower that steps A obtains extracts medicinal extract M
1dissolve with methanol aqueous solution, allow the solution obtained be crude drug 0.06gmL in sample solution mass concentration
-1, post blade diameter length ratio 1:3, applied sample amount M
1/ macroporous resin weight ratio=3:10, adsorption flow rate 2.5BVh
-1condition under adsorbed by pre-treatment macroporous resin column, then use 8BV distilled water flushing, then with 4.2BV by weight 65% aqueous ethanolic solution with elution flow rate 2.5BVh
-1wash-out, the ethanol eluate E obtained
1dry concentrated under the condition of temperature 46 DEG C, obtain pale yellow powder M
2;
C, polyamide column adsorb
Polyamide prepolymer process: the polymeric amide produced by the biochemical plastic molding and processing plant of Taizhou City of Zhejiang Province road and bridge tetramethyl of 60-90 order soaks 22h in 95% aqueous ethanolic solution by weight, then load in chromatography column, add 1.8mol/L aqueous sodium hydroxide solution and soak 4.0h, be then washed with distilled water to neutrality; Then add 3.5mol/L aqueous hydrochloric acid and soak 7.0h, be then washed with distilled water to neutrality; Finally add 95% aqueous ethanolic solution by weight and soak 9h, then be washed with distilled water to without alcohol taste.
Through pretreated polymeric amide dress post; The pale yellow powder M that step B obtains
2dissolve with methanol aqueous solution, allow the solution obtained at applied sample amount M
2/ polymeric amide weight ratio 16.3mgg
-1, sample solution mass concentration 3.40mgmL
-1, sample solution pH 5.8, post blade diameter length ratio 1:2 and adsorption flow rate be 1.8BVh
-1condition under adsorbed by pre-treatment polyamide column, then use 1.8BV distilled water flushing, then with 4.0BV by weight 80% aqueous ethanolic solution with elution flow rate 1.8BVh
-1wash-out, the ethanol eluate E obtained
2dry concentrated under temperature 46 DEG C of conditions, obtain off-white powder M
3;
D, prepare Oleuropein
The off-white powder M that step C obtains
3be separated by silica gel column chromatography by dry method mode, applied sample amount M
3: silica gel=1:10, then be the chloroform methanol mixed solvent wash-out of 6:1 by weight ratio, the elutriant of Fractional Collections, the HPLC method adopting this specification sheets to describe detects Oleuropein content, it is the elutriant merging of by weight more than 90% by described content, the elutriant removal of solvent under reduced pressure obtained, obtain the crystallization under the condition of temperature 2 DEG C in methyl alcohol of Oleuropein crude product, filter, dry at ambient temperature with anhydrous cupric sulfate, gas chromatography is adopted to measure its water-content for by weight 0.2%, it is 98.6% that the HPLC method adopting this specification sheets to describe measures its purity, according to the calculation formula that this specification sheets describes, be 3.0% according to medicinal material Weight computation Oleuropein average recoveries.
Embodiment 2: extract high purity Oleuropein from Largeflower Jasmine Flower
The implementation step of this embodiment is as follows:
A, aqueous ethanolic solution lixiviate
According to the weight ratio 1:11 of Largeflower Jasmine Flower bud with 55% aqueous ethanolic solution by weight, allow water-content by weight 1.9% dry Largeflower Jasmine Flower bud raw material in aqueous ethanolic solution under the condition of room temperature soak 3 times, each 47h, the extracting solution at every turn obtained filters, filtrate merges, then under the condition of temperature 49 DEG C, carry out concentrate drying, obtain Largeflower Jasmine Flower and extract medicinal extract M
1;
B, macroporous resin column are adsorbed
Macroporous resin pre-treatment: the HPD-100 type macroporous resin sold by Cangzhou Bon Adsorption Material Science and Technology Co., Ltd uses 90% aqueous ethanolic solution by weight to soak 25h, then wet method dress post, use 90% aqueous ethanolic solution wash-out by weight again, until effluent liquid mixes with water not in white opacity, then wash with water to there is no ethanol smell.
Absorption and wash-out: the Largeflower Jasmine Flower that steps A obtains extracts medicinal extract M
1dissolve with methanol aqueous solution, allow the solution obtained be crude drug 0.10gmL in sample solution mass concentration
-1, post blade diameter length ratio 1:2, applied sample amount M
1/ macroporous resin weight ratio=3:11, adsorption flow rate 1.8BVh
-1condition under adsorbed by pre-treatment macroporous resin column, then use 6BV distilled water flushing, then with 3.5BV by weight 75% aqueous ethanolic solution with elution flow rate 1.8BVh
-1wash-out, the ethanol eluate E obtained
1dry concentrated under the condition of temperature 42 DEG C, obtain pale yellow powder M
2;
C, polyamide column adsorb
Polyamide prepolymer process: 30-60 order soaks 26h by the polymeric amide produced by the biochemical plastic molding and processing plant of Taizhou City of Zhejiang Province road and bridge tetramethyl in 95% aqueous ethanolic solution by weight, then load in chromatography column, add 1.8mol/L aqueous sodium hydroxide solution and soak 4.2h, be then washed with distilled water to neutrality; Then add 4.5mol/L aqueous hydrochloric acid and soak 8.0h, be then washed with distilled water to neutrality; Finally add 95% aqueous ethanolic solution by weight and soak 11h, then be washed with distilled water to without alcohol taste.
Through pretreated polymeric amide dress post; The pale yellow powder M that step B obtains
2dissolve with methanol aqueous solution, allow the solution obtained at applied sample amount M
2/ polymeric amide weight ratio 16.0mgg
-1, sample solution mass concentration 3.50mgmL
-1, sample solution pH 6.2, post blade diameter length ratio 1:4 and adsorption flow rate be 2.0BVh
-1condition under adsorbed by pre-treatment polyamide column, then use 2.0BV distilled water flushing, then with 4.2BV by weight 75% aqueous ethanolic solution with elution flow rate 2.0BVh
-1wash-out, the ethanol eluate E obtained
2dry concentrated under temperature 44 DEG C of conditions, obtain off-white powder M
3;
D, prepare Oleuropein
The off-white powder M that step C obtains
3by dry method mode loading, be separated by silica gel column chromatography.Applied sample amount M
3: silica gel=1:8, then be the chloroform methanol mixed solvent wash-out of 7:1 by weight ratio, the HPLC method that the elutriant of Fractional Collections adopts this specification sheets to describe detects Oleuropein content, it is the elutriant merging of by weight more than 90% by described content, the elutriant removal of solvent under reduced pressure obtained, obtain the crystallization under the condition of temperature 4 DEG C in methyl alcohol of Oleuropein crude product, filter, dry at ambient temperature with anhydrous cupric sulfate, gas chromatography is adopted to measure its water-content for by weight 0.3%, it is 98.0% that the HPLC method adopting this specification sheets to describe measures its purity, according to the calculation formula that this specification sheets describes, be 2.6% according to medicinal material Weight computation Oleuropein average recoveries.
Embodiment 3: extract high purity Oleuropein from Largeflower Jasmine Flower
The implementation step of this embodiment is as follows:
A, aqueous ethanolic solution lixiviate
According to the weight ratio 10:1 of Largeflower Jasmine Flower bud with 65% aqueous ethanolic solution by weight, allow water-content by weight 1.6% dry Largeflower Jasmine Flower bud raw material in aqueous ethanolic solution under the condition of room temperature soak 4 times, each 46h, the extracting solution at every turn obtained filters, filtrate merges, then under the condition of temperature 50 C, carry out concentrate drying, obtain Largeflower Jasmine Flower and extract medicinal extract M
1;
B, macroporous resin column are adsorbed
Macroporous resin pre-treatment: the XDA-1 type macroporous resin sold by Xi'an Sunresin New Materials Co., Ltd. uses 92% aqueous ethanolic solution by weight to soak 26h, then wet method dress post, use 92% aqueous ethanolic solution wash-out by weight again, until effluent liquid mixes with water not in white opacity, then wash with water to there is no ethanol smell.
Absorption and wash-out: the Largeflower Jasmine Flower that steps A obtains extracts medicinal extract M
1dissolve with methanol aqueous solution, allow the solution obtained be crude drug 0.08gmL in sample solution mass concentration
-1, post blade diameter length ratio 1:4, applied sample amount M
1/ macroporous resin weight ratio=3:8, adsorption flow rate 2.2BVh
-1condition under adsorbed by pre-treatment macroporous resin column, then use 10BV distilled water flushing, then with 4.0BV by weight 70% aqueous ethanolic solution with elution flow rate 2.2BVh
-1wash-out, the ethanol eluate E obtained
1dry concentrated under the condition of temperature 44 DEG C, obtain pale yellow powder M
2;
C, polyamide column adsorb
Polyamide prepolymer process: the polymeric amide produced by Yuan Ye bio tech ltd, Shanghai of 60-90 order soaks 24h in 90% aqueous ethanolic solution by weight, then load in chromatography column, add 2.2mol/L aqueous sodium hydroxide solution and soak 3.5h, be then washed with distilled water to neutrality; Then add 4.0mol/L aqueous hydrochloric acid and soak 9.0h, be then washed with distilled water to neutrality; Finally add 90% aqueous ethanolic solution by weight and soak 10h, then be washed with distilled water to without alcohol taste.
Through pretreated polymeric amide dress post; The pale yellow powder M that step B obtains
2dissolve with methanol aqueous solution, allow the solution obtained at applied sample amount M
2/ polymeric amide weight ratio 16.2mgg
-1, sample solution mass concentration 3.60mgmL
-1, sample solution pH 6.0, post blade diameter length ratio 1:3 and adsorption flow rate be 2.2BVh
-1condition under adsorbed by pre-treatment polyamide column, then use 2.2BV distilled water flushing, then with 3.5BV by weight 78% aqueous ethanolic solution with elution flow rate 2.2BVh
-1wash-out, the ethanol eluate E obtained
2dry concentrated under temperature 42 DEG C of conditions, obtain off-white powder M
3;
D, prepare Oleuropein
The off-white powder M that step C obtains
3by dry method mode loading, be separated by silica gel column chromatography, applied sample amount M
3: silica gel=1:12, , then be the chloroform methanol mixed solvent wash-out of 8:1 by weight ratio, the HPLC method that the elutriant of Fractional Collections adopts this specification sheets to describe detects Oleuropein content, it is the elutriant merging of by weight more than 90% by described content, the elutriant removal of solvent under reduced pressure obtained, obtain the crystallization under the condition of temperature 4 DEG C in methyl alcohol of Oleuropein crude product, filter, dry under the condition of room temperature with anhydrous cupric sulfate, gas chromatography is adopted to measure its water-content for by weight 0.1%, it is more than 99.4% that the HPLC method adopting this specification sheets to describe measures its purity, according to the calculation formula that this specification sheets describes, be 2.0% according to medicinal material Weight computation Oleuropein average recoveries.
Embodiment 4: extract high purity Oleuropein from Largeflower Jasmine Flower
The implementation step of this embodiment is as follows:
A, aqueous ethanolic solution lixiviate
According to the weight ratio 1:14 of Largeflower Jasmine Flower bud with 55% aqueous ethanolic solution by weight, allow water-content by weight 1.8% dry Largeflower Jasmine Flower bud raw material in aqueous ethanolic solution under the condition of room temperature soak 3 times, each 48h, the extracting solution at every turn obtained filters, filtrate merges, then under the condition of temperature 47 DEG C, carry out concentrate drying, obtain Largeflower Jasmine Flower and extract medicinal extract M
1;
B, macroporous resin column are adsorbed
Macroporous resin pre-treatment: the HPD-400 type macroporous resin sold by Cangzhou Bon Adsorption Material Science and Technology Co., Ltd uses 95% aqueous ethanolic solution by weight to soak 24h, then wet method dress post, use 95% aqueous ethanolic solution wash-out by weight again, until effluent liquid mixes with water not in white opacity, then wash with water to there is no ethanol smell.
Absorption and wash-out: the Largeflower Jasmine Flower that steps A obtains extracts medicinal extract M
1dissolve with methanol aqueous solution, allow the solution obtained be crude drug 0.09gmL in sample solution mass concentration
-1, post blade diameter length ratio 1:3, applied sample amount M
1/ macroporous resin weight ratio=3:12, adsorption flow rate 1.5BVh
-1condition under adsorbed by pre-treatment macroporous resin column, then use 9BV distilled water flushing, then with 4.5BV by weight 75% aqueous ethanolic solution with elution flow rate 1.5BVh
-1wash-out, the ethanol eluate E obtained
1dry concentrated under the condition of temperature 42 DEG C, obtain pale yellow powder M
2;
C, polyamide column adsorb
Polyamide prepolymer process: the polymeric amide produced by the biochemical plastic molding and processing plant of Taizhou City of Zhejiang Province road and bridge tetramethyl of 30 ~ 60 orders soaks 25h in 92% aqueous ethanolic solution by weight, then load in chromatography column, add 2.0mol/L aqueous sodium hydroxide solution and soak 3.8h, be then washed with distilled water to neutrality; Then add 3.5mol/L aqueous hydrochloric acid and soak 7.5h, be then washed with distilled water to neutrality; Finally add 92% aqueous ethanolic solution by weight and soak 9h, then be washed with distilled water to without alcohol taste.
Through pretreated polymeric amide dress post; The pale yellow powder M that step B obtains
2dissolve with methanol aqueous solution, allow the solution obtained at applied sample amount M
2/ polymeric amide weight ratio 16.5mgg
-1, sample solution mass concentration 3.40mgmL
-1, sample solution pH 5.8, post blade diameter length ratio 1:2 and adsorption flow rate be 1.8BVh
-1condition under adsorbed by pre-treatment polyamide column, then use 1.8BV distilled water flushing, then with 3.8BV by weight 85% aqueous ethanolic solution with elution flow rate 1.8BVh
-1wash-out, the ethanol eluate E obtained
2dry concentrated under temperature 48 DEG C of conditions, obtain off-white powder M
3;
D, prepare Oleuropein
The off-white powder M that step C obtains
3by dry method mode loading, be separated by silica gel column chromatography.Applied sample amount M
3: silica gel=1:11, then be the chloroform methanol mixed solvent wash-out of 6:1 by weight ratio, the HPLC method that the elutriant of Fractional Collections adopts this specification sheets to describe detects Oleuropein content, it is the elutriant merging of by weight more than 90% by described content, the elutriant removal of solvent under reduced pressure obtained, obtain the crystallization under the condition of temperature 0 DEG C in methyl alcohol of Oleuropein crude product, filter, dry at ambient temperature with anhydrous cupric sulfate, gas chromatography is adopted to measure its water-content for by weight 0.1%, it is 99.0% that the HPLC method adopting this specification sheets to describe measures its purity, according to the calculation formula that this specification sheets describes, be 3.4% according to medicinal material Weight computation Oleuropein average recoveries.
Embodiment 5: extract high purity Oleuropein from Largeflower Jasmine Flower
The implementation step of this embodiment is as follows:
A, aqueous ethanolic solution lixiviate
According to the weight ratio 1:13 of Largeflower Jasmine Flower bud with 60% aqueous ethanolic solution by weight, allow water-content by weight 1.9% dry Largeflower Jasmine Flower bud raw material in aqueous ethanolic solution under the condition of room temperature soak 2 times, each 49h, the extracting solution at every turn obtained filters, filtrate merges, then under the condition of temperature 48 DEG C, carry out concentrate drying, obtain Largeflower Jasmine Flower and extract medicinal extract M
1;
B, macroporous resin column are adsorbed
Macroporous resin pre-treatment: the D-101 type macroporous resin sold by Tianjin Xing Nanyun energy Polymer Technology company limited uses 92% aqueous ethanolic solution by weight to soak 23h, then wet method dress post, use 92% aqueous ethanolic solution wash-out by weight again, until effluent liquid mixes with water not in white opacity, then wash with water to there is no ethanol smell.
Absorption and wash-out: the Largeflower Jasmine Flower that steps A obtains extracts medicinal extract M
1dissolve with methanol aqueous solution, allow the solution obtained be crude drug 0.07gmL in sample solution mass concentration
-1, post blade diameter length ratio 1:2, applied sample amount M
1/ macroporous resin weight ratio=3:9, adsorption flow rate 2.0BVh
-1condition under adsorbed by pre-treatment macroporous resin column, then use 7BV distilled water flushing, then with 3.8BV by weight 70% aqueous ethanolic solution with elution flow rate 2.0BVh
-1wash-out, the ethanol eluate E obtained
1dry concentrated under the condition of temperature 44 DEG C, obtain pale yellow powder M
2;
C, polyamide column adsorb
Polyamide prepolymer process: the polymeric amide produced by Yuan Ye bio tech ltd, Shanghai of 30 ~ 60 orders soaks 23h in 94% aqueous ethanolic solution by weight, then load in chromatography column, add 2.2mol/L aqueous sodium hydroxide solution and soak 4.0h, be then washed with distilled water to neutrality; Then add 4.5mol/L aqueous hydrochloric acid and soak 8.5h, be then washed with distilled water to neutrality; Finally add 95% aqueous ethanolic solution by weight and soak 11h, then be washed with distilled water to without alcohol taste.
Through pretreated polymeric amide dress post; The pale yellow powder M that step B obtains
2dissolve with methanol aqueous solution, allow the solution obtained at applied sample amount M
2/ polymeric amide weight ratio 16.4mgg
-1, sample solution mass concentration 3.50mgmL
-1, sample solution pH 6.2, post blade diameter length ratio 1:4 and adsorption flow rate be 2.0BVh
-1condition under adsorbed by pre-treatment polyamide column, then use 2.0BV distilled water flushing, then with 4.5BV by weight 85% aqueous ethanolic solution with elution flow rate 2.0BVh
-1wash-out, the ethanol eluate E obtained
2dry concentrated under temperature 46 DEG C of conditions, obtain off-white powder M
3;
D, prepare Oleuropein
The off-white powder M that step C obtains
3with 60% aqueous ethanolic solution dissolving by weight, then filter, filtrate is at applied sample amount M
3/ dextrane gel weight ratio 10mgg
-1, sample solution mass concentration 2.0mgmL
-1, post blade diameter length ratio 1:80 and adsorption flow rate be 2.0BVh
-1condition under pass through sephadex column, use 30% aqueous ethanolic solution wash-out by weight again, the HPLC that the elutriant of Fractional Collections adopts this specification sheets to describe detects Oleuropein content, it is the elutriant merging of by weight more than 90% by described content, decompression and solvent recovery, obtain Oleuropein crude product, it is crystallization under the condition of temperature 2 DEG C in methyl alcohol, filter, dry at ambient temperature with anhydrous cupric sulfate, gas chromatography is adopted to measure its water-content for by weight 0.1%, it is 99.5% that the HPLC method adopting this specification sheets to describe measures its purity, according to the calculation formula that this specification sheets describes, be 3.8% according to medicinal material Weight computation Oleuropein average recoveries.
Embodiment 6: extract high purity Oleuropein from Largeflower Jasmine Flower
The implementation step of this embodiment is as follows:
A, aqueous ethanolic solution lixiviate
According to the weight ratio 1:12 of Largeflower Jasmine Flower bud with 60% aqueous ethanolic solution by weight, allow water-content by weight 1.7% dry Largeflower Jasmine Flower bud raw material in aqueous ethanolic solution under the condition of room temperature soak 4 times, each 48h, the extracting solution at every turn obtained filters, filtrate merges, then under the condition of temperature 48 DEG C, carry out concentrate drying, obtain Largeflower Jasmine Flower and extract medicinal extract M
1;
B, macroporous resin column are adsorbed
Macroporous resin pre-treatment: the XDA-16 type macroporous resin sold by Xi'an Sunresin New Materials Co., Ltd. uses 95% aqueous ethanolic solution by weight to soak 24h, then wet method dress post, use 95% aqueous ethanolic solution wash-out by weight again, until effluent liquid mixes with water not in white opacity, then wash with water to there is no ethanol smell.
Absorption and wash-out: the Largeflower Jasmine Flower that steps A obtains extracts medicinal extract M
1dissolve with methanol aqueous solution, allow the solution obtained be crude drug 0.08gmL in sample solution mass concentration
-1, post blade diameter length ratio 1:4, applied sample amount M
1/ macroporous resin weight ratio=3:10, adsorption flow rate 1.6BVh
-1condition under adsorbed by pre-treatment macroporous resin column, then use 8BV distilled water flushing, then with 4.0BV by weight 70% aqueous ethanolic solution with elution flow rate 2.1BVh
-1wash-out, the ethanol eluate E obtained
1dry concentrated under the condition of temperature 48 DEG C, obtain pale yellow powder M
2;
C, polyamide column adsorb
Polyamide prepolymer process: the polymeric amide produced by Chemical Reagent Co., Ltd., Sinopharm Group of 30 ~ 60 orders soaks 25h in 95% aqueous ethanolic solution by weight, then load in chromatography column, add 2.0mol/L aqueous sodium hydroxide solution and soak 4.5h, be then washed with distilled water to neutrality; Then add 4.0mol/L aqueous hydrochloric acid and soak 8.0h, be then washed with distilled water to neutrality; Finally add 95% aqueous ethanolic solution by weight and soak 10h, then be washed with distilled water to without alcohol taste.
Through pretreated polymeric amide dress post; The pale yellow powder M that step B obtains
2dissolve with methanol aqueous solution, allow the solution obtained at applied sample amount M
2/ polymeric amide weight ratio 16.2mgg
-1, sample solution mass concentration 3.60mgmL
-1, sample solution pH 6.0, post blade diameter length ratio 1:3 and adsorption flow rate be 2.2BVh
-1condition under adsorbed by pre-treatment polyamide column, then use 2.2BV distilled water flushing, then with 4.0BV by weight 80% aqueous ethanolic solution with elution flow rate 2.2BVh
-1wash-out, the ethanol eluate E obtained
2dry concentrated under temperature 44 DEG C of conditions, obtain off-white powder M
3;
D, prepare Oleuropein
The off-white powder M that step C obtains
3with 60% aqueous ethanolic solution dissolving by weight, then filter, filtrate is at applied sample amount M
3/ dextrane gel weight ratio 10mgg
-1, sample solution mass concentration 2.0mgmL
-1, post blade diameter length ratio 1:80 and adsorption flow rate be 2.0BVh
-1condition under pass through sephadex column, use 30% aqueous ethanolic solution wash-out by weight again, the HPLC that the elutriant of Fractional Collections adopts this specification sheets to describe detects Oleuropein content, it is the elutriant merging of by weight more than 90% by described content, decompression and solvent recovery, obtain Oleuropein crude product, it is crystallization under the condition of temperature 4 DEG C in methyl alcohol, filter, dry at ambient temperature with anhydrous cupric sulfate, gas chromatography is adopted to measure its water-content for by weight 0.1%, it is 99.0% that the HPLC method adopting this specification sheets to describe measures its purity, according to the calculation formula that this specification sheets describes, be 4.0% according to medicinal material Weight computation Oleuropein average recoveries.
Claims (10)
1. from Largeflower Jasmine Flower, extract a method for high purity Oleuropein, it is characterized in that the step of the method is as follows:
A, aqueous ethanolic solution lixiviate
Get dry Largeflower Jasmine Flower bud raw material to soak 2 ~ 4 times under the condition of room temperature in aqueous ethanolic solution, each 46 ~ 50h, the extracting solution at every turn obtained filters, and filtrate merges, then concentrate under the condition of temperature 46 ~ 50 DEG C, drying obtains Largeflower Jasmine Flower and extracts medicinal extract M
1;
B, macroporous resin column are adsorbed
The Largeflower Jasmine Flower that steps A obtains extracts medicinal extract M
1dissolve with methanol aqueous solution, allow the solution obtained be adsorbed by pre-treatment macroporous resin column, then use distilled water flushing, then use 65 ~ 75% aqueous ethanolic solution wash-outs by weight, the ethanol eluate E obtained
1dry concentrated under the condition of temperature 42 ~ 48 DEG C, obtain pale yellow powder M
2;
C, polyamide column adsorb
Through pretreated polymeric amide dress post, the pale yellow powder M that step B obtains
2dissolve with methanol aqueous solution, allow the solution obtained be adsorbed by pre-treatment polyamide column, then use distilled water flushing, then use 75 ~ 85% aqueous ethanolic solution wash-outs by weight, the ethanol eluate E obtained
2dry concentrated under temperature 42 ~ 48 DEG C of conditions, obtain off-white powder M
3;
D, prepare Oleuropein
The off-white powder M that step C obtains
3be separated by silica gel column chromatography, by dry method mode loading, then use chloroform methanol mixed solvent wash-out, the elutriant removal of solvent under reduced pressure obtained, obtains the crystallization under the condition of temperature 0 ~ 4 DEG C in methyl alcohol of Oleuropein crude product, filters, drying, obtains described Oleuropein.
2. method according to claim 1, is characterized in that in step, and the weight ratio of Largeflower Jasmine Flower bud and by weight 55 ~ 65% aqueous ethanolic solutions is 1:10 ~ 14.
3. method according to claim 1, is characterized in that in stepb, and described macroporous resin is selected from the macroporous resin of XDA-16, HPD-100, XDA-1 or HPD-400, D-101 type; Described macroporous resin pre-treatment step is as follows: described macroporous resin uses 90 ~ 95% aqueous ethanolic solutions by weight to soak 22 ~ 26h, wet method dress post, use 90 ~ 95% aqueous ethanolic solution wash-outs by weight again, until effluent liquid mixes with water not in white opacity, then wash with water to there is no ethanol smell.
4. method according to claim 1, is characterized in that in stepb, and macroporous resin column absorption is as follows with elution requirement: sample solution mass concentration is crude drug 0.06 ~ 0.10gmL
-1, post blade diameter length ratio 1:2 ~ 4, the weight ratio of applied sample amount is M
1/ macroporous resin=3:8 ~ 12, adsorption flow rate 1.5 ~ 2.5BVh
-1; Washing distilled water consumption 6 ~ 10BV; Eluting solvent is 65 ~ 75% aqueous ethanolic solutions by weight, and its consumption is 3.5 ~ 4.5BV, and elution flow rate is 1.5 ~ 2.5BVh
-1.
5. method according to claim 1, is characterized in that in step C, and the granularity of described polymeric amide is 30 ~ 90 orders;
Described polyamide prepolymer treatment step is as follows: 30 ~ 90 order polymeric amide soak 22 ~ 26h in 90 ~ 95% aqueous ethanolic solutions by weight, then load in chromatography column, add 1.8 ~ 2.2mol/L aqueous sodium hydroxide solution and soak 3.5 ~ 4.5h, be then washed with distilled water to neutrality; Then add 3.5 ~ 4.5mol/L aqueous hydrochloric acid and soak 7 ~ 9h, be then washed with distilled water to neutrality; Finally add 90 ~ 95% aqueous ethanolic solutions by weight and soak 9 ~ 11h, then be washed with distilled water to without alcohol taste.
6. method according to claim 1, is characterized in that in step C, and the adsorption conditions of polyamide column is as follows: the weight ratio of applied sample amount is M
2/ polymeric amide=16.0-16.5mgg
-1, sample solution mass concentration is 3.40 ~ 3.60mgmL
-1, sample solution pH is 5.8 ~ 6.2, post blade diameter length ratio 1:2 ~ 4, and adsorption flow rate is 1.8 ~ 2.2BVh
-1; Washing distilled water consumption 1.8 ~ 2.2BV, eluting solvent is 75 ~ 85% aqueous ethanolic solutions by weight, and its consumption is 3.5 ~ 4.5BV, elution flow rate 1.8 ~ 2.2BVh
-1.
7. method according to claim 1, is characterized in that in step C, and described purification by silica gel column chromatography step is as follows: allow the off-white powder M that step C obtains
3with dry method mode loading, applied sample amount M
3: silica gel=1:8 ~ 12, then be the chloroform methanol mixed solvent wash-out of 6 ~ 8:1 by weight ratio, the elutriant of Fractional Collections adopts HPLC to detect Oleuropein content, be the elutriant merging of by weight more than 90% by described content, decompression and solvent recovery, obtains Oleuropein crude product, it carries out crystallization again in methyl alcohol under the condition of temperature 0 ~ 4 DEG C, filter, dry, obtain described Oleuropein.
8. method according to claim 1, is characterized in that in step C, allows the off-white powder M that step C obtains
3purifying is carried out by dextrane gel column chromatography.
9. method according to claim 8, is characterized in that described sephadex column chromatography purification step is as follows: the off-white powder M that step C obtains
3dissolve with aqueous ethanolic solution, then filter, filtrate by sephadex column, then uses 30% aqueous ethanolic solution wash-out by weight, the elutriant of Fractional Collections adopts HPLC to detect Oleuropein content, be the elutriant merging of by weight more than 90% by described content, decompression and solvent recovery, obtains Oleuropein crude product, it carries out crystallization again in methyl alcohol under the condition of temperature 0 ~ 4 DEG C, filter, dry, obtain described Oleuropein.
10. the Oleuropein product that method prepares according to claim arbitrary in claim 1-9, the purity that it is characterized in that it is by weight 98.0 ~ 99.5%.
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