CN102451235A - Preparation method of olive leaf extract - Google Patents
Preparation method of olive leaf extract Download PDFInfo
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- CN102451235A CN102451235A CN2010105230567A CN201010523056A CN102451235A CN 102451235 A CN102451235 A CN 102451235A CN 2010105230567 A CN2010105230567 A CN 2010105230567A CN 201010523056 A CN201010523056 A CN 201010523056A CN 102451235 A CN102451235 A CN 102451235A
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- oleae europaeae
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- 229940114496 olive leaf extract Drugs 0.000 title claims abstract description 73
- 238000002360 preparation method Methods 0.000 title abstract description 6
- RFWGABANNQMHMZ-HYYSZPHDSA-N Oleuropein Chemical compound O([C@@H]1OC=C([C@H](C1=CC)CC(=O)OCCC=1C=C(O)C(O)=CC=1)C(=O)OC)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O RFWGABANNQMHMZ-HYYSZPHDSA-N 0.000 claims abstract description 73
- RFWGABANNQMHMZ-UHFFFAOYSA-N 8-acetoxy-7-acetyl-6,7,7a,8-tetrahydro-5H-benzo[g][1,3]dioxolo[4',5':4,5]benzo[1,2,3-de]quinoline Natural products CC=C1C(CC(=O)OCCC=2C=C(O)C(O)=CC=2)C(C(=O)OC)=COC1OC1OC(CO)C(O)C(O)C1O RFWGABANNQMHMZ-UHFFFAOYSA-N 0.000 claims abstract description 72
- HKVGJQVJNQRJPO-UHFFFAOYSA-N Demethyloleuropein Natural products O1C=C(C(O)=O)C(CC(=O)OCCC=2C=C(O)C(O)=CC=2)C(=CC)C1OC1OC(CO)C(O)C(O)C1O HKVGJQVJNQRJPO-UHFFFAOYSA-N 0.000 claims abstract description 72
- RFWGABANNQMHMZ-CARRXEGNSA-N oleuropein Natural products COC(=O)C1=CO[C@@H](O[C@H]2O[C@@H](CO)[C@H](O)[C@@H](O)[C@@H]2O)C(=CC)[C@H]1CC(=O)OCCc3ccc(O)c(O)c3 RFWGABANNQMHMZ-CARRXEGNSA-N 0.000 claims abstract description 72
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Abstract
The invention discloses a preparation method of an olive leaf extract. The preparation method comprises the following steps of: putting dried olive leaves into a polar solvent for extracting; recovering the solvent from an extracting solution, adding water and auxiliary materials, mixing uniformly, standing, filtering and combining filtrates; adsorbing a filtrate with macroporous resin, desorbing with an ethanol-water mixed solution, and collecting a desorption solution; and recovering the solvent from the desorption solution, and drying to obtain the olive leaf extract. The method for preparing the olive leaf extract by adding auxiliary materials and purifying with the macroporous resin has the advantages of simple process, low cost and high content of oleuropein in the obtained olive leaf extract, and is suitable for large-scale production.
Description
Technical field
The present invention relates to the extractive technique field of natural materials, more specifically, relate to a kind of method for preparing of olive leaf extract, relate in particular to a kind of method for preparing that is rich in the olive leaf extract of oleuropein.
Background technology
Fructus oleae europaeae (Olea europaea L.) belongs to Oleaceae (Oleaceae) Olea (Olea) aiphyllium, is world-renowned woody oil tree species, and the cultivation history in more than 4000 year is arranged.Fructus oleae europaeae mainly is distributed in 45 ° of north latitude between 37 ° in the south latitude, and the central distribution district is a mediterranean country, and Greece, Italy, Tunisia, Spain are for concentrating the place of production, and all there is introducing and planting countries in the world now.The used Fructus Canarii albi of China's Chinese medicine is the fruit of olive subject plant Fructus Canarii albi (Canarium album Raeusch), also claims wild olive, with Fructus oleae europaeae (Oleaceae) be not same platymiscium, the purposes of the two is obviously different.
Since China introduced a fine variety Fructus oleae europaeae in 1964; Through the researcher years of researches; Confirm that China is suitable for the Fructus oleae europaeae growth and blocky best normal region is: low mountain, white Dragon River, the upper reaches, Jia Lingjiang River valley area is the center with Gansu Province's Wudu County, about 40,000 hectares of cultivated area; Jinsha jiang River dry-hot valley is the center with Xichang, Sichuan Province, about 100,000 hectares of cultivated area; Low mountain, Yangtze River Gorges valley area comprises Wan County, Sichuan Province, Wushan, Fengjie; Hubei Province's the east of Sichuan, Zigui, Yichang etc., about 150,000 hectares of Fructus oleae europaeae area; Qinling Mountains band is the center with Hanzhong and Guangyuan, Sichuan, has 150,000 hectares approximately.Over past ten years; Under the development of the West Regions policy support, the plantation of Fructus oleae europaeae development is very fast, and the construction of base of Gansu, Sichuan Er Sheng begins to take shape; And set up olive oil processing factory; The Fructus oleae europaeae fruit is fully used, but annual because of the depleted leaf of Fructus oleae europaeae of pruning more than thousands of tons of, and do not obtain fine utilization.
Oleuropein (oleuropein) is the bitter principle in the Fructus Canarii albi; It is a kind of phenol secoiridoid glycoside; Extensively be present in Oleaceae Olea vegetable oil Fructus Canarii albi (olive) (Olea europaea L.) and the card Cen platymiscium Japan Chinese ash leaves of plants such as (Fraxinus Japanlca Blune), and in Ligustrum plant Fructus Ligustri Lucidi (Ligustrum lucidum Ait) and the Ligustrum japonicum Thunb. fruits such as (Ligustnun japanica Thumb).Because its importance in multiple natural product biological approach, secoiridoid is considered to the labelled compound on the Olea europaea section plant chemotaxonomy, is one of main component of polyphenol.
Oleuropein molecular formula C
25H
32O
13, molecular weight 540.52,87～89 ℃ of fusing points are prone to moisture absorption, are soluble in ethanol, acetone, pyridine etc., and water soluble, butanols, ethyl acetate etc. are dissolved in ether, petroleum ether, chloroform etc. hardly, and its structural formula is as follows:
Oleuropein is a kind of bioactive substance; Have extremely strong oxidation resistance, be used for skin care item and can promote the former protein renaturation of skin, correct aging vestige; Naturally resisting the skin that is caused by oxidation destroys; Avoid uv damage, effectively keep the tender and elasticity of skin, interpolation is all arranged in the cosmetics of super quality of external many famous brand names.Its antioxidant activity is confirmed by numerous scholar that oleuropein has powerful antibacterium and ntiviral characteristic simultaneously, can be used for making the new drug of caused diseases such as treating virus, antibacterial, protozoa, parasite and blood sucker and treatment flu.Olive leaf extract is used as dietary supplement with raise immunity in America and Europe.
Chinese patent CN101003557 discloses a kind of method for preparing that is rich in the olive leaf extract of high-purity oleuropein; Obtain concentrated solution through steps such as the extraction of Folium olive polar solvent, alkaline centrifugal sedimentation, centrifugal clear liquid, film coupling separation through NF membrane; Medium absorption then, desorption obtain eluent; The vacuum concentration after drying obtains oleuropein content greater than 50% the olive leaf extract that is rich in the high-purity oleuropein.The method that this method adopts alkaline sedimentation, membrance separation and medium absorption to combine has obtained purity and has been the olive leaf extract greater than 50% oleuropein; But the practical operation step is too much; Be unfavorable for industrialized great production, and the membrane separation plant first-phase investment is big, has improved product cost.One Chinese patent application CN101084945 adopts film device microfiltration, ultrafiltration, nanofiltration to carry out purification equally, and its content that obtains the effective ingredient oleuropein reaches more than 40%, but its yield is lower, and the unit cost of production is high.
Chinese patent CN1583052 discloses a kind of Olive leaf P.E and extracts method for preparing; With the rinsing of Folium olive disinfection by chlorine dioxide, dry back alcohol extraction, vacuum concentration; In concentrated solution, add the tea polyphenols antioxidation; Through the dry Olive leaf P.E that obtains, the Olive leaf P.E active constituent content of its preparation is low, and yield is low.Adopt alcohol-water solution heat to carry more than the secondary in the U.S. Pat 5714150, spray drying or vacuum drying behind the extracting solution vacuum concentration, it prepares the Olive leaf P.E that contains oleuropein 30-40%.TOHKEMY 2002128678 usefulness aqueous solvents extract, and after synthetic resin was handled, drying obtained containing the extract of oleuropein more than 25%.The content of oleuropein is too low in these extracts, all can not satisfy market demands.
In addition, also have one Chinese patent application CN101781344 to disclose a kind of method for preparing of Olive leaf P.E, the Folium olive polar solvent extracts; Water precipitating, centrifugal; Centrifugal liquid is used petroleum ether, ethyl acetate extraction respectively; After ethyl acetate reclaims solvent mutually, add the dilution of water or pure water, 0.45 μ m NF membrane filters, and the preparative high performance liquid chromatography post separates, and obtains containing the Olive leaf P.E of oleuropein＞90% after the drying.In the European patent EP 1795201 Folium olive worn into the fine powder of 0.05 to 1 millimeter of diameter, the pH8.0 potass extraction; The water extract is through organic solvent extraction, and the extract vacuum dehydrating at lower temperature obtains containing the crude extract of oleuropein 40-45%; This crude extract is collected the stripping liquid that contains the oleuropein part again through the fast silica gel chromatogram column purification, obtains containing the amorphous solid of 95-98% oleuropein behind the recovery solvent.Though oleuropein content is higher in these two kinds of methods, they all are more suitable in scientific research, and cost is higher, is difficult to accomplish scale production.
Summary of the invention
The technical problem that the present invention will solve is; To the defective that extraction step in the prior art is complicated, cost is high, oleuropein content is low; The method for preparing of the olive leaf extract that a kind of technology is simple, cost is low is provided, utilizes the oleuropein content in the olive leaf extract that this method obtains higher.
The technical solution adopted for the present invention to solve the technical problems is: a kind of method for preparing of olive leaf extract may further comprise the steps:
Step 1: the Fructus oleae europaeae cured leaf is dosed in the polar solvent extracts;
Step 2: extracting solution adds water and adjuvant water precipitating after reclaiming solvent, mixing, leave standstill, filter pressing, merging filtrate;
Step 3: adopt macroporous resin column chromatography method purification, the filtrating of step 2 is collected stripping liquid after macroporous resin adsorption, ethanol water mixed liquid desorption;
Step 4: stripping liquid reclaims the solvent final vacuum and concentrates, and drying obtains olive leaf extract.
The method for preparing of olive leaf extract of the present invention, wherein, said Fructus oleae europaeae cured leaf is that fresh leaf of Fructus oleae europaeae obtains through drying in the shade, perhaps dry 1-3h obtains under 70-110 ℃.
The method for preparing of olive leaf extract of the present invention, wherein, the water content in the said Fructus oleae europaeae cured leaf is lower than 10%, and oleuropein content is not less than 4%.
The method for preparing of olive leaf extract of the present invention, wherein, said Fructus oleae europaeae cured leaf also will pass through remove impurity, broken step.
The method for preparing of olive leaf extract of the present invention, wherein, said polar solvent is the ethanol water of alcoholic degree 50-85%, the w/v of Fructus oleae europaeae cured leaf and polar solvent is 1: 5-15.
The method for preparing of olive leaf extract of the present invention, wherein, the method for distilling of said step 1 is reflux, dipping or percolation, the number of times of said reflux is a secondary or more than the secondary.
The method for preparing of olive leaf extract of the present invention, wherein, in the said step 2, the w/v that makes water and Fructus oleae europaeae cured leaf during water precipitating is 5-12: 1.
The method for preparing of olive leaf extract of the present invention, wherein, the adjuvant of said step 2 is a kieselguhr.
The method for preparing of olive leaf extract of the present invention, wherein, the ethanol water mixed liquid in the said step 3 is the ethanol water of alcoholic degree 20-70%.
The method for preparing of olive leaf extract of the present invention, wherein, the drying means in the said step 4 is at least a in vacuum drying, spray drying, the forced air drying.
The method for preparing of olive leaf extract of the present invention; Adopt the oleuropein in the polar solvent Extraction oil Folium olive, and carry out pretreatment, adopt the macroporous resin adsorption oleuropein then through ethanol extract from water precipitation; Concentrated, dry at last, obtain olive leaf extract.
The method for preparing of olive leaf extract of the present invention; When the water precipitating of ethanol extract from water precipitation; Add adjuvant kieselguhr (diatomite); Utilize physical method to replace the chemical method absorb oil, the solvent contamination of having avoided chemical method to bring has prevented grease contamination macroporous resin and the purification and the regeneration that influence macroporous resin effectively.
In the method for preparing of olive leaf extract of the present invention; Through utilizing diatomite adsorption oils and fats and the combination of adopting macroporous resin enrichment target product oleuropein; Improved the content of oleuropein in the olive leaf extract that finally obtains effectively, the mass percent of oleuropein in extract can reach more than 55%.
The method for preparing of olive leaf extract of the present invention, the olive leaf extract that not only obtains can be met the need of market, and processing step is few, and technology is simple, and whole flow process is easy to operate, has reduced cost.In addition, the method for preparing of olive leaf extract of the present invention consumes energy less, basic non-environmental-pollution, and product yield that obtains and purity are high, are suitable for large-scale production.
Description of drawings
To combine accompanying drawing and embodiment that the present invention is described further below, in the accompanying drawing:
Fig. 1 is the flow chart of the method for preparing of olive leaf extract of the present invention;
Fig. 2 is the HPLC collection of illustrative plates of oleuropein reference substance;
Fig. 3 is the HPLC collection of illustrative plates of flow rate 2BV/h effluent;
Fig. 4 is a HPLC collection of illustrative plates of going up appearance concentration 6mg/mL effluent;
Fig. 5 is the HPLC collection of illustrative plates of 70% ethanolysis imbibition;
Fig. 6 is the HPLC collection of illustrative plates of the olive leaf extract of the embodiment of the invention 1.
Fig. 7 is the HPLC collection of illustrative plates of the olive leaf extract of the embodiment of the invention 2.
Fig. 8 is the HPLC collection of illustrative plates of the olive leaf extract of the embodiment of the invention 3.
Fig. 9 is the HPLC collection of illustrative plates of the olive leaf extract of the embodiment of the invention 4.
The specific embodiment
At this, for fear of obscuring, the applicant thinks and it is important to point out that leaf of Fructus oleae europaeae described in this description or Fructus oleae europaeae cured leaf are the leaf of Oleaceae Olea Fructus oleae europaeae, but not the leaf of the used olive subject plant Fructus Canarii albi of China's Chinese medicine.
In addition, it is important to point out that the percentage of using in this description if no special instructions, generally is meant mass fraction.At this, phrase " w/v " is meant with kg to be the solid matter of measurement unit and to be the ratio between the liquid substance of measurement unit with L, perhaps is meant with L to be the liquid substance of measurement unit and to be the ratio between the solid matter of measurement unit with kg.Should be understood that no matter solid matter pro-or liquid substance pro-, in " w/v ", " weight " is meant that solid matter is the weight of measurement unit with kg, " volume " is meant that liquid substance is the volume of measurement unit with L.And if the solid matter pro-, " w/v " is the ratio between the volume of weight and liquid substance of solid matter; Otherwise if the liquid substance pro-, " w/v " is the ratio between the weight of volume and solid matter of liquid substance.Should look like in order more clearly to express; For example the w/v of Fructus oleae europaeae cured leaf and polar solvent is that the w/v of 1: 5 or polar solvent and Fructus oleae europaeae cured leaf is 5: 1; All be meant 1kg Fructus oleae europaeae cured leaf and 5L polar solvent, 2kg Fructus oleae europaeae cured leaf and 10L polar solvent, 5kg Fructus oleae europaeae cured leaf and 25L polar solvent or 100kg Fructus oleae europaeae cured leaf and 500L polar solvent or 200kg Fructus oleae europaeae cured leaf and 1000L polar solvent, and other exists the Fructus oleae europaeae cured leaf and the polar solvent of similarity relation.
Before the olive leaf extract of oleuropein is rich in the method for preparing production that utilizes olive leaf extract, to select and certain pretreatment leaf of Fructus oleae europaeae usually.
Usually, receive the influence of growth cycle, the content of oleuropein is minimum in the leaf of Fructus oleae europaeae of plucking in April and November; Therefore; According to the present invention, oleuropein content receives excessive influence in the final extract in order not make, and employed leaf of Fructus oleae europaeae is not gathered in this two month.In addition, 1 year At All Other Times in, though because of growth cycle influence, oleuropein content has fluctuation in the leaf of Fructus oleae europaeae, and the minimum content of the oleuropein in the olive leaf extract that obtains is not had too big influence.For in olive leaf extract, obtain content higher oleuropein, also can be according to the suggestion among the Chinese patent CN101003557, gather leaf of Fructus oleae europaeae in February or May.
From the fresh leaf of Fructus oleae europaeae that olive tree is gathered, need be through super-dry, the side is used for the preparation of olive leaf extract then.Be to be understood that; " fresh leaf of Fructus oleae europaeae " described herein is meant that leaf of Fructus oleae europaeae is no more than the leaf of Fructus oleae europaeae of 48h after tree is adopted down; Because the leaf of Fructus oleae europaeae under adopting is in case unprocessed through the long period; The oleuropein that wherein contains will decompose in a large number, causes that content significantly reduces in the extract, does not reach the contents level of the market demand.For this reason, the fresh leaf of Fructus oleae europaeae under suggestion is adopted from olive tree is used for the preparation of olive leaf extract at once, to reduce the degraded of oleuropein as much as possible.
In addition, to the dried that fresh leaf of Fructus oleae europaeae carries out, be in order to remove the moisture in the leaf of Fructus oleae europaeae, oleuropein proportion in leaf of Fructus oleae europaeae to be promoted.Usually, through according to method of the present invention to the leaf of Fructus oleae europaeae drying after, in the Fructus oleae europaeae cured leaf that obtains, moisture is lower than 10%, oleuropein content is not less than 4%.Preferably, moisture is lower than 6% in the Fructus oleae europaeae cured leaf.Though the drying means of existing many leaf of Fructus oleae europaeae in the prior art,, at this preferably, the method that this place adopts is: fresh leaf of Fructus oleae europaeae is dried in the shade, and perhaps dry 1-3h obtains the Fructus oleae europaeae cured leaf under 70-110 ℃.The time of drying in the shade does not have special qualification, measures the moisture in the leaf of Fructus oleae europaeae through oven dry algoscopy described below, and the processing of when it is lower than 10%, representing to dry in the shade finishes.In high temperature and rapid drying is handled, be heat drying under the high temperature of 70 ℃, 75 ℃, 80 ℃, 85 ℃, 90 ℃, 95 ℃, 100 ℃, 105 ℃ or 110 ℃ for example in temperature, for example be drying time such as 1h, 1.5h, 2h, 2.5h, 3h etc.Through behind the drying steps, the moisture in the Fructus oleae europaeae cured leaf and the content of oleuropein all can reach above-mentioned standard.
In addition, for the further solvent extraction of removing impurity and being convenient to oleuropein, also the Fructus oleae europaeae cured leaf is carried out remove impurity and broken step.On the one hand, removal step has reduced impurity content, helps to promote the quality of extract finished product; On the other hand; Broken step is broken into coarse powder or smaller particles with the Fructus oleae europaeae cured leaf, is convenient to it and amasss with the contact surface that extracts solvent, oleuropein is extracted in the solvent more easily go.Because in follow-up filtration step; Coarse powder or granule may stop up the hole on the filter screen, thus can be or not whole leaf of Fructus oleae europaeae are not broken yet, reserve a few part and place on filter screen or the filter membrane; To prevent the obstruction of filter screen or filter membrane eyelet, influence tapping, backflow, percolation efficient.
Fig. 1 is the flow chart of the method for preparing of olive leaf extract of the present invention.In this description, the method for preparing of olive leaf extract may further comprise the steps:
A) the Fructus oleae europaeae cured leaf is dosed in the polar solvent extracts.
In this step; Polar solvent is meant one or more in methanol, ethanol, water, acetone, propanol, the butanols isopolarity solvent; Here preferably, polar solvent is that alcoholic degree is the ethanol water of 50-85%, and the w/v of Fructus oleae europaeae cured leaf and polar solvent is 1: 5-15.For example; Polar solvent is the ethanol water of alcoholic degree 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, and the w/v of Fructus oleae europaeae cured leaf and polar solvent is 1: 5,1: 6,1: 7,1: 8,1: 9,1: 10,1: 11,1: 12,1: 13,1: 14,1: 15.In addition; The inventor finds that polar solvent is if adopt methanol, and extraction effect can be got well than other solvent; But consider that methanol has stronger toxicity to human body; And the removal fully of methanol has certain degree of difficulty in the olive leaf extract in operation, is the health risk that further reduces cost and possibly exist, so the present inventor preferably uses the mixed liquor of second alcohol and water to use as polar solvent.
In addition, method for distilling is preferred, a kind of in reflux, dipping, the percolation.In the reflux method, to extract temperature and be preferably 70-90 ℃, extraction time is 4-8h, and extracts at least twice.In dipping or percolation method, the time is 24-48h.The reflux that this place mentions, dipping or percolation are known method, and those skilled in the art can easily obtain concrete operations step and other details of these methods after consulting related tool book or scientific and technical literature.Therefore, this description no longer is elaborated to it, but " pharmacy of Chinese materia medica " front page that equal reference example of last art method such as China Traditional Chinese Medicine Publishing House published in 2003.The example that here provides provides in order more easily to understand the present invention, if no special instructions, the present invention is not constituted any restriction.
B) will go up the extracting solution that obtains of step and reclaim solvent after, add water and adjuvant water precipitating, mixing, leave standstill, filter pressing, merging filtrate.
At first the polar solvent in the extracting solution is reclaimed in this step; Carry out water precipitating then; The purpose that adds water is 5-12 in order to make the w/v of water and Fructus oleae europaeae cured leaf when the water precipitating promptly: 1, and for example, w/v is 5: 1,6: 1,7: 1,8: 1,9: 1,10: 1,11: 1,12: 1.In addition, also will add adjuvant, the water precipitating time is preferably 4-20h, and water precipitating liquid all filters.The mixing that this place refers to is the known meaning of those skilled in the art, even the distribution homogeneous of each constituent in the mixed liquor forms solution or the mixed liquor of sparing matter.Here, adjuvant is meant kieselguhr, and it is used for through the physical method absorb oil, and the solvent contamination problem of avoiding chemical method to cause prevents the grease contamination macroporous resin in the extracting solution effectively.
C) adopt macroporous resin column chromatography method purification, promptly go up filtrating in the step after macroporous resin adsorption, ethanol water mixed liquid desorption, the collection stripping liquid.
In the macroporous resin adsorption process, preferably, the weight ratio of macroporous resin and Fructus oleae europaeae cured leaf is 1-3: 1, absorption flow velocity be 0.5-3 times of column volume per hour.In addition, preferably, the desorption solvent is the ethanol water of 20-70%, and the volume ratio of itself and column volume is 3-5: 1.
D) stripping liquid recovery solvent final vacuum concentrates, and drying obtains olive leaf extract.
In this step, it is 10-15 ° of B é that vacuum concentration generally is concentrated into Bo Meidu (Baume degrees), dry then, dryly adopts at least a in vacuum drying, spray drying, the forced air drying.
The screening of macroporous resin
Macroporous resin is also referred to as macroporous adsorbent resin, and the cardinal principle of its isolation technics is that macroporous resin has optionally adsorption to the different organic substance compositions in the solution.Through selecting suitable desorption solvent and condition, can reach the purpose of separation and purification.The polarity of macroporous resin and space structure are the principal elements of influence absorption.Because characteristics such as physicochemical property are stablized, can be used repeatedly, unit cost is low, macroporous resin is suitable for commercial production very much.But a subject matter of macroporous resin isolation technics is that the specificity of macroporous resin absorption, centrifugation is higher.Usually, the adsorbent resin of a model only has good separation refining effect to the organic substance of a certain (class), even has changed model, separates refining effect and just has a greatly reduced quality.For this reason, the present inventor screens the macroporous resin that is suitable for the oleuropein extraction on the basis of macroporous resins such as existing Semen Vitis viniferae, Carlina acaulis technology.
1. chromatographic condition is confirmed
Chromatographic condition is as follows.
Chromatographic column: YMC ODS C
18Post, specification 150*4.6mm, 5 μ m; Temperature: room temperature; Detect wavelength: 230nm; Mobile phase: acetonitrile: 0.2% acetic acid water (v: v=24: 76); Flow velocity: 1mL/min.
The HPLC collection of illustrative plates of the oleuropein reference substance that uses in the chromatograph in addition, is as shown in Figure 2.
2. resin screening
The present inventor adopts dynamic adsorption method, has compared the adsorption effect of several kinds of different resins such as HPD-100, AB-8, D101, LSA-8, LSA-21, LS-300B.Than upper column quantity (S) with than adsorbance (A), the adsorption effect of D101, LSA-21, three kinds of resins of LS-300B is better according to the adsorpting characteristic parameter of each resin.The result is as shown in table 1.
Table 1 resin screening S, A value
The adsorpting characteristic parameter have than upper column quantity (S, saturationratio) and than adsorbance (A, absorptionratio).When adsorbing terminal point for reaching than upper column quantity, the summation that composition is carried in the absorption of unit mass dried resin secretly, the overall capacity of expression resin absorption, carrying, S is big more, and bearing capacity is strong more, is the key parameter of confirming resin demand.Than adsorbance is the summation of unit mass dried resin adsorption component, the true absorbability of expression resin, and A is big more, and absorbability is strong more, is the important parameter of selecting resin kind and evaluating resin regeneration effect.Computing formula is following:
S=(M
OnOne M
Residual)/M
A=(M
OnOne M
ResidualOne M
Washing)/M
Wherein, M: the dried resin quality is commercial resins and is dried to the quality that constant weight records;
M
On: the quality of oleuropein in the upper prop liquid is the product of last column volume and oleuropein concentration;
M
Residual: cross the quality of oleuropein in the post effluent, be the product of effluent volume and oleuropein concentration;
M
Washing: upper prop is finished, and initial water elutes the quality of oleuropein, is the product of water lotion volume and oleuropein concentration.
3. go up confirming of appearance concentration and flow rate
3.1 flow rate
Get pretreated resin, wet method dress post (column volume 100mL).Add leaf of Fructus oleae europaeae water precipitating filtered solution, control flow rate 0.5BV/h (column volume/hour), 1BV/h, 2BV/h, 3BV/h, every 1BV collects 1 bottle, detects the amount of oleuropein in the calculated flow fluid through HPLC.The result shows does not have outflow below the 2BV/h basically, and 3BV/h has a small amount of outflow, flow rate with 2BV/h for well.Fig. 3 is the HPLC collection of illustrative plates of flow rate 2BV/h effluent.
3.2 last appearance concentration
The pretreatment resin; High concentration water precipitating filtered solution is diluted to 10mg/mL, 8mg/mL, 6mg/mL, 4mg/mL, 2mg/mL respectively; With the flow rate upper prop of 2BV/h, HPLC detects oleuropein content in the effluent, does not have outflow below the 6mg/mL basically; 10mg/mL has part to flow out, so confirm to go up appearance concentration 6mg/mL or following.As shown in Figure 4, be the HPLC collection of illustrative plates of last appearance concentration 6mg/mL effluent.
4. eluant strength and consumption
Use each 500mL of ethanol (being equivalent to 5 column volumes) eluting of alcoholic degree 30%, 40%, 50%, 60%, 70% after the last appearance washing respectively, every 50mL collects 1 bottle, detects through HPLC, calculates the amount of oleuropein in each flow point.50% ethanol gets final product fine desorbing, but consumption is big; 60% ethanol and 70% ethanol 3BV can complete desorbings.Increase the concentration of alcohol again, 80% ethanol does not increase the response rate of product and improves content.As shown in Figure 5, be the HPLC collection of illustrative plates of 70% ethanolysis imbibition.
Table 2 different concentration ethanol elute effect
5. conclusion
Oleuropein has certain polarity, and according to " similar mixing " principle, several resin columns of low pole all have stronger absorbability, and wherein the adsorption effect of LS-300B resin is best, and eluting is more complete, is suitable in suitability for industrialized production, using.Semi-polarity LSA-21 resin also has absorption and elute effect preferably.In addition, nonpolar D101 resin has also shown the good adsorption effect, maybe be relevant with its bigger specific surface area.
Through the repetition test research to above a plurality of naval stores, we have obtained olive leaf extract higher yields, that content is high, and wherein the oleuropein proportion reaches more than 55%.Producing the result who obtains unanimity in the post equipment (high 3.5 meters, 0.5 meter of internal diameter) that amplifies, therefore, this has verified method for preparing Application feasibility in large-scale production of olive leaf extract of the present invention.
Measuring method
1. the measurement of moisture
Fresh leaf of Fructus oleae europaeae needs to measure the content of moisture in the Fructus oleae europaeae cured leaf that obtains after drying, make it be lower than 10%.Moisture is generally through " Chinese pharmacopoeia version in 2005 appendix IX H aquametry first method (oven drying method) measures.
2. the measurement of alcoholic degree
Alcoholic degree is measured through alcohol meter in the ethanol water.
3. the measurement of oleuropein content
The content measurement of oleuropein in leaf of Fructus oleae europaeae or olive leaf extract adopts HPLC (HPLC) to accomplish.Chromatographic condition is:
Chromatographic column: YMC ODS C
18Post, specification 150*4.6mm, 5 μ m;
Detected temperatures: room temperature;
Detect wavelength: 230nm;
Mobile phase: acetonitrile: 0.2% acetic acid water (v: v=24: 76);
Flow velocity: 1mL/min.
4. the calculating of yield and oleuropein content
The yield of olive leaf extract and the wherein calculating of oleuropein content are calculated according to following formula.
Yield=the M of olive leaf extract
Extract/ M
Cured leaf* 100%;
Wherein, M
Extract: the dried weight of olive leaf extract (kg), M
Cured leaf: the Fructus oleae europaeae cured leaf weight (kg) that feeds intake.
Oleuropein content={ (A
Sample/ A
Reference substance) * (V
Reference substance advances/ V
Sample advances) * C
Reference substance* V
Sample}/M
Get The appearance amount* 100%
Wherein, A
Sample: sample peak area, A
Reference substance: reference substance peak area, V
Reference substance advances: reference substance sample size (μ l), V
Sample advances: sample feeding amount (μ l), C
Reference substance: reference substance concentration (mg/ml), V
Sample: sample volume (ml), M
Sampling amount: the sampling weight (mg) of sample.
In addition, the new resin of buying need be used 95% soak with ethanol usually, and washes, and just normally uses then.
Embodiment 1
Through exsiccant leaf of Fructus oleae europaeae 200kg, through detecting, moisture is less than 6%, and oleuropein content is greater than 5%.Folium olive is broken into coarse powder, and with the alcoholic degree 75% ethanol water heating and refluxing extraction twice of 1400L, each 4 hours, extracting solution concentrated after reclaiming ethanol; Concentrated solution adds water 2000L water precipitating, adds adjuvant kieselguhr simultaneously, and mixing left standstill 12 hours; Filter pressing, 400kg D101 macroporous resin column on the filtrating, flow velocity 0.5BV/h, alcoholic degree 70% ethanol water desorbing; Stripping liquid reclaims the ethanol final vacuum and concentrates, and is concentrated into 10 ° of B é, spray drying; Obtain olive leaf extract 10.3kg, yield 5.15%, oleuropein content 61%.Among this embodiment, the HPLC collection of illustrative plates of the oleuropein content of detection olive leaf extract is as shown in Figure 6.
Embodiment 2
Through exsiccant leaf of Fructus oleae europaeae 200kg, through detecting, moisture is less than 6%, and oleuropein content is greater than 5%.The part leaf of Fructus oleae europaeae is broken into coarse powder, does not pulverize on a small quantity, earlier with an amount of alcoholic degree 70% ethanol water moistening; Make it abundant expansion; Fitted tube evenly flattens, and opens the piston of percolate outlet; Add alcoholic degree 70% ethanol water to percolate from top and flow out, solvent immersed surface number centimetre from outlet.Flood and begin percolation with 2000L70% ethanol after 24 hours, flow velocity 100L/h merges percolate, and percolate reclaims ethanol, and concentrated solution adds water 1600L water precipitating; Add adjuvant kieselguhr simultaneously, mixing left standstill 8 hours, filter pressing, 400kg D101 macroporous resin column on the filtrating; Flow velocity 0.5BV/h, alcoholic degree 70% ethanol-water solution desorbing, stripping liquid reclaims the ethanol final vacuum and concentrates, and is concentrated into 15 ° of B é; Vacuum drying obtains olive leaf extract 9.4kg, yield 4.7%, oleuropein content 64%.Among this embodiment, the HPLC collection of illustrative plates of the oleuropein content of detection olive leaf extract is as shown in Figure 7.
Embodiment 3
Through exsiccant leaf of Fructus oleae europaeae 200kg, through detecting, moisture is less than 10%, and oleuropein content is greater than 5%.The part Folium olive is broken into coarse powder, stays on a small quantity and do not pulverize, with alcoholic degree 70% ethanol water of 2000L dipping 48 hours, every at a distance from stirring in two hours once; Reclaim ethanol, concentrated solution adds water 2000L water precipitating, adds adjuvant kieselguhr simultaneously, mixing; Left standstill filter pressing, 400kg D101 macroporous resin column on the filtrating, flow velocity 2BV/h 12 hours; Alcoholic degree 70% ethanol water desorbing, stripping liquid reclaims the ethanol final vacuum and concentrates, and is concentrated into 15 ° of B é, vacuum drying; Obtain olive leaf extract 6.6kg, yield 3.3%, oleuropein content 59%.Among this embodiment, the HPLC collection of illustrative plates of the oleuropein content of detection olive leaf extract is as shown in Figure 8.
Embodiment 4
Through exsiccant leaf of Fructus oleae europaeae 200kg, through detecting, moisture is less than 4%, and oleuropein content is greater than 4%.Folium olive is broken into coarse powder, extracts twice with 1400L alcoholic degree 75% alcohol heating reflux, each 4 hours, extracting solution concentrated after reclaiming ethanol; Concentrated solution adds water 2000L water precipitating, adds adjuvant kieselguhr simultaneously, and mixing left standstill 12 hours; Filter pressing, 400kg LSA-21 macroporous resin column on the filtrating, flow velocity 1BV/h, alcoholic degree 70% ethanol water desorbing; Stripping liquid reclaims the ethanol final vacuum and concentrates, and is concentrated into 10 ° of B é, spray drying; Obtain olive leaf extract 10.7kg, yield 5.35%, oleuropein content 60%.Among this embodiment, the HPLC collection of illustrative plates of the oleuropein content of detection olive leaf extract is as shown in Figure 9.
The above is merely representative embodiment of the present invention, does not limit the present invention in any way, and all any modifications of within spirit of the present invention and principle, being done, is equal to replacement or improvement etc., all should be included within protection scope of the present invention.
Claims (10)
1. the method for preparing of an olive leaf extract is characterized in that, may further comprise the steps:
Step 1: the Fructus oleae europaeae cured leaf is dosed in the polar solvent extracts;
Step 2: extracting solution adds water and adjuvant water precipitating after reclaiming solvent, mixing, leave standstill, filter pressing, merging filtrate;
Step 3: adopt macroporous resin column chromatography method purification, the filtrating of step 2 is collected stripping liquid after macroporous resin adsorption, ethanol water mixed liquid desorption;
Step 4: stripping liquid reclaims the solvent final vacuum and concentrates, and drying obtains olive leaf extract.
2. the method for preparing of olive leaf extract according to claim 1 is characterized in that, said Fructus oleae europaeae cured leaf is that fresh leaf of Fructus oleae europaeae obtains through drying in the shade, and perhaps dry 1-3h obtains under 70-110 ℃.
3. the method for preparing of olive leaf extract according to claim 2 is characterized in that, the water content in the said Fructus oleae europaeae cured leaf is lower than 10%, and oleuropein content is not less than 4%.
4. according to the method for preparing of each described olive leaf extract of claim 1-3, it is characterized in that said Fructus oleae europaeae cured leaf also will pass through remove impurity, broken step.
5. the method for preparing of olive leaf extract according to claim 1 is characterized in that, said polar solvent is the ethanol water of alcoholic degree 50-85%, and the w/v of Fructus oleae europaeae cured leaf and polar solvent is 1: 5-15.
6. the method for preparing of olive leaf extract according to claim 1 is characterized in that, the method for distilling of said step 1 is reflux, dipping or percolation, and the number of times of said reflux is a secondary or more than the secondary.
7. the method for preparing of olive leaf extract according to claim 1 is characterized in that, in the said step 2, the w/v that makes water and Fructus oleae europaeae cured leaf during water precipitating is 5-12: 1.
8. according to the method for preparing of claim 1 or 7 described olive leaf extract, it is characterized in that the adjuvant of said step 2 is a kieselguhr.
9. the method for preparing of olive leaf extract according to claim 1 is characterized in that, the ethanol water mixed liquid in the said step 3 is the ethanol water of alcoholic degree 20-70%.
10. the method for preparing of olive leaf extract according to claim 1 is characterized in that, the drying means in the said step 4 is at least a in vacuum drying, spray drying, the forced air drying.
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