CN104447896A - Extraction-separation method and application of corilagin - Google Patents

Abstract

The invention discloses an extraction-separation method and an application of corilagin. The method comprises the steps of treating longan kernel powder once by using anhydrous acetone and extracting by using acetone aqueous solution again for the second time; uniformly mixing a proper amount of chromatography polyamide with the acetone aqueous solution, recycling acetone and applying to a polyamide chromatography column; eluting by sequentially using pure water, methyl alcohol aqueous solutions with different concentrations and methyl alcohol; gathering 40-60 percent of methyl alcohol eluant in a separate bottle form, condensing and separating out corilagin crystals; mixing mother liquid with the rest of eluant containing corilagin, condensing to be dry, dissolving into methyl alcohol and applying to a polydextran gel LH-20 column; eluting by methyl alcohol; gathering eluant in a separate bottle form and standing; mixing with the eluants in which corilagin crystals are separated out; standing again until the orilagin crystals which are separated out are not increased; mixing the obtained corilagin crystals with corilagin crystals obtained by chromatography polyamide; performing recrystallization by using ethyl alcohol to obtain corilagin with high purity. The corilagin subjected to extraction and separation has a function of inhibiting tyrosinase.

Description

The extraction and separation method of corilagin and application thereof

Technical field:

The invention belongs to biomedicine field, be specifically related to a kind of extraction and separation method and application thereof of corilagin.

Background technology:

Rangkadilok N etc. report that dry longan fruit stone is containing corilagin 3.7-8.6mg/g; Soong and Barlow utilizes HPLC-ESI-MS coupling technique to determine in seed of Arillus Longan containing 13 kinds of aldehydes matters such as corilagin (corilagin), gallic acid (gallic acid), ellagic acids (ellagic acid).

Corilagin (Corilagin) is a kind of Nutgalls acyl hexahydroxy-biphenyl glucopyranoside compound, belongs to hydrolyzable tannin, and energy is water-soluble, the large polar solvent such as methyl alcohol, ethanol, acetone, and toxicity is little.As far back as nineteen fifty-two Schmidt, O.T. and Lademann, R. have just found that it is the powerful scavenging agent of free radical, and comprising hypertension, atherosclerosis, apoplexy, congestive heart failure and ischemic cardiac disease to many cardiovascular disordeies has good effect; Good effect is had equally to antitumor, antiviral; It still treats one of main component of the Yexiazhu tablet for treating hepatism of hepatitis B.

Extraction conditions for longan fruit stone polyphenol has bibliographical information.As: acetone concentration 50% (v/v), solid-liquid ratio l:140 (g/mL), extraction temperature 50 DEG C, extraction time 2h, ultrasonic 20min, lixiviate three times, seed of Arillus Longan polyphenol yield is 62.1mg/g (PB method) (Wang Zhiyuan, the separation and purification Structural Identification of Pericarpium Longan and seed of Arillus Longan many points and anti-oxidant activity [D]. Xiamen: Xiamen University, 2007.).Extraction time 106min, volume fraction of ethanol is 58%, Extracting temperature 64 DEG C, and solid-liquid ratio is 1: 9, and with this understanding, polyphenol yield is 42.690mg/g (Forint phenol method) (Wu Lanlan etc.Response phase method optimizes the research [J] of seed of Arillus Longan polyphenol extraction technology. Collects The American University's journal (natural science edition), 15 (5): 22-26).

Dalian Mei Lun Bioisystech Co., Ltd website marketing information display: 20mg corilagin standard substance (HPLC purity assay >=98%) price 960 yuan.

Longan mainly distribute and cultivate in Fujian, Guangdong and Guangxi, the plump succulence of pulp, nutritious, there are very high nutritive value and food therapy value, just there is the saying of " strong soul is clever, old, being proficient in the law of natural movement of making light of one's life by commiting suicide " from ancient times, modern pharmacology has also proved that longan aril extracting solution has anti-ageing, anticancer and effect such as immunomodulatory and promotion intelligent growth, is praised highly as " fruit Zisco product " by people.China is the country of origin and the producing country of longan, and cultivated area and output account for more than 70% and 50% of the world respectively, is maximum YEAST IN LONGAN PRODUCTION state.Over nearly 10 years, ultimate production increases substantially, and be increased to 110.65 ten thousand tons in 2006 by 36.53 ten thousand tons in 1997, amplification reaches 202.90%, wherein 47.56 ten thousand tons, Guangdong in 2006,38.57 ten thousand tons, Guangxi, 21.28 ten thousand tons, Fujian.But because fruit listing is more concentrated, storing condition is limited and storage and freshness-retaining technology is not enough, and fresh fruit consumption is restricted, and a large amount of fresh fruits is used for processing as longan pulp, wine of longan, longan sweet can etc., and the fruit stone produced is not used, and causes huge waste.

Summary of the invention:

First object of the present invention is to provide a kind of extraction and separation method of corilagin.

The extraction and separation method of corilagin of the present invention, is characterized in that, comprises the following steps:

(1), the extraction of corilagin: first longan fruit stone powder is added anhydrous propanone according to certain mass volume ratio, stirring at room temperature carries out pre-treatment, leaves standstill and separates out upper liquid, abandon this upper liquid; Again longan fruit stone powder is added aqueous acetone solution according to certain mass volume ratio second time, control temperature stirs, and leaves standstill and separates out upper liquid and filter, and obtains second time filtrate; Finally longan fruit stone powder is added aqueous acetone solution according to certain mass volume ratio third time, operation, with second time, obtains third time filtrate; Merge second and third filtrate, obtain extracting solution, this extracting solution is used for next step and is separated;

(2), extracting solution is separated prepares corilagin: chromatography column on pretreated chromatography polymeric amide of learning from else's experience, pretreated chromatography polymeric amide of learning from else's experience again mixes in the extracting solution of step (1) gained, obtain mixture, reclaim acetone transpiring moisture to mixture becomes thick, afterwards mixture is added to polyamide chromatography post, use pure water successively, 5% ~ 15% methyl alcohol, 25% ~ 35% methyl alcohol, 40% ~ 60% methyl alcohol, anhydrous methanol carries out wash-out, 40% ~ 60% meoh eluate sub-bottle is collected and concentrates and check corilagin elution profile (because in this gradient elution system, corilagin can not be washed out by the methanol aqueous solution that volume fraction is less than 35%, and can wash out completely with volume fraction 40% ~ 60% methyl alcohol, therefore only subsequent disposal need be carried out to this part), this sub-bottle is collected eluate that corilagin solids content in concentrated eluate is more than or equal to 60% merge concentrated after have corilagin crystal to separate out, after leaching crystal, mother liquor and other are concentrated into dry containing the eluate merging of corilagin after testing, upper sephadex column after being dissolved in anhydrous methanol again, wash-out is carried out with anhydrous methanol, eluate sub-bottle is collected, leave standstill, be associated with the eluate that corilagin crystal is separated out, leave standstill again to precipitation corilagin crystal without increase, separate corilagin crystal, the corilagin crystal that itself and chromatography polyamide column 40% ~ 60% methanol-eluted fractions are got is merged, again with dehydrated alcohol recrystallization, finally obtain highly purified corilagin.

Described longan fruit stone powder, its preparation method is: collect longan in longan fruit field and process the fruit stone abandoned, naturally dry, pulverized 20 mesh sieves and obtain longan fruit stone powder.

Preferably, be first that 1:15 adds anhydrous propanone by longan fruit stone powder according to mass volume ratio in step (1).

Preferably, be that 1:15 second time adds 80% aqueous acetone solution by longan fruit stone powder according to mass volume ratio again in step (1), control temperature is 55 DEG C and carries out stirrings 2h, leaves standstill and separates out upper liquid also 0.04 ~ 0.05MPa vacuum filtration.

Preferably, be finally 1:15 third time add 60% aqueous acetone solution by longan fruit stone powder according to mass volume ratio in step (1).

Chromatography column on pretreated chromatography polymeric amide of learning from else's experience described in step (2), the mass ratio (w/w) of the longan fruit stone powder be extracted in the pretreated chromatography polymeric amide got and step (1) is preferably 0.8:1; Described pretreated chromatography polymeric amide of learning from else's experience again mixes in the extracting solution of step (1) gained, then the mass ratio (w/w) of the longan fruit stone powder be extracted in learn from else's experience pretreated chromatography polymeric amide and the step (1) of getting is preferably 0.2:1; Described pre-treatment, concrete grammar is: get polymeric amide with 90 ~ 95% (V) alcohol immersion, continuous stirring, load in post after removing bubble, with 90 ~ 95% (V) ethanol elution of 3 ~ 4BV, be washed till elutriant transparent and without residue (or few residue) after evaporate to dryness, use 10% (W) aqueous acetic acid wash-out of the distilled water of 2 ~ 2.5BV 5% (W) the NaOH aqueous solution, 1BV, 2 ~ 2.5BV more successively, be finally eluted to neutrality with distilled water.

Recovery acetone described in step (2), is preferably normal pressure rotary evaporation and reclaims acetone, described transpiring moisture, is preferably decompression rotary evaporation moisture.

Preferably, carry out wash-out with pure water 3BV, 5% ~ 15% methyl alcohol 4BV, 25% ~ 35% methyl alcohol 4BV, 40% ~ 60% methyl alcohol 4BV, anhydrous methanol 8BV successively in step (2), elution flow rate is 0.5BV/h.

40% ~ 60% meoh eluate sub-bottle being collected described in step (2) is concentrated, preferably 40% ~ 60% meoh eluate is pressed 1/9BV sub-bottle and collects concentrated.

Preferably, after leaching crystal in step (2), mother liquor and other are concentrated into dry containing the eluate merging of corilagin after testing, again with mass volume ratio (g/ml) for 1:5 is dissolved in upper sephadex column after anhydrous methanol, described sephadex column is sephadex lh-20 post, the mass volume ratio (g/ml) of applied sample amount and sephadex lh-20 is 1:100, wash-out is carried out with 4BV anhydrous methanol, elution flow rate is 1ml/min, elutriant sub-bottle is collected, preferably presses 1/5BV sub-bottle and collect.

The extraction and separation method of corilagin of the present invention, the yield adopting the method in step (1) to extract corilagin in longan fruit stone is 7.1mg/g dry fruit core.The present invention adopts RP-HPLC method to measure the yield extracting corilagin from longan fruit stone and is separated with track and extract thing the situation preparing corilagin, and evaluates the effect of corilagin restraint of tyrosinase.

With the L-DOPA of 1.0mmo1/L for substrate, first 1.0mL 3mol/L L-DOPA solution (solution is the phosphate buffer soln of pH=6.8) is placed in cuvette, add the phosphate buffer soln of 0.85mL pH=6.8, constant temperature l0min in 30 DEG C of waters bath with thermostatic control, add 1.0mL corilagin solution (solution is in the phosphate buffer soln of pH=6.8) and the 0.15mL 0.15g/L tyrosine oxidase aqueous solution, measure OD ₄₇₅.This surveys in live body system, and the whole mass concentration of enzyme is 7.5mg/L.Can formula be defined as to the relative inhibition (I) of enzyme:

$I=(1-\frac{{\mathrm{OD}}_1-{\mathrm{OD}}_3}{{\mathrm{OD}}_2-{\mathrm{OD}}_4})\×100\%$

In formula: OD _lrefer to the absorbance of the survey live body system containing substrate, tyrosine oxidase, corilagin; OD ₂refer to containing substrate, tyrosine oxidase, but not containing the absorbance of the survey live body system of corilagin; OD ₃refer to containing substrate, corilagin, but not containing the absorbance of the survey live body system of tyrosine oxidase; OD ₄refer to containing substrate, but not containing the absorbance of the survey live body system of tyrosine oxidase, corilagin.

Result is the inhibiting rate of 0.5mg/mL (0.79mmol/L) corilagin to tyrosine oxidase is 51.38%.And the IC of the tyrosine oxidase of makeup whitening additive arbutin ₅₀value is 5.3mmol/L.

Therefore, second object of the present invention is to provide the application of corilagin in restraint of tyrosinase, and this application can be used for preparing skin-lightening cosmetic.

Technique effect of the present invention: adopt aqueous acetone solution to extract, extraction effect is good, the yield of corilagin is more than 6mg/g dry fruit core, solvent is easy to reclaim, adopting anhydrous propanone pre-treatment longan fruit stone powder not only can obtain essential oil (separately dealing with) but also can also reduce oil-soluble ingredient in extracting solution therefore can simplify processes step, extract does not need namely to extract respectively with opposed polarity solvent with ordinary method process and direct upper polyamide chromatography post, primary column chromatography just can obtain high-purity corilagin, the purity of corilagin crystal reaches more than 98%, simple to operate, simultaneously for corilagin provides a new application, also for the Application and Development of longan fruit stone provides an effective way.The corilagin obtained by method extraction and isolation of the present invention has obvious restraining effect to tyrosine oxidase, can be used for preparing skin-lightening cosmetic.

Embodiment:

Following examples further illustrate of the present invention, instead of limitation of the present invention.Other technological steps do not described in detail are the method known by those skilled in the art.

Embodiment 1:

(1), the extraction of corilagin: first collect longan in longan fruit field and process the fruit stone abandoned, naturally dry, pulverized 20 mesh sieves and obtained longan fruit stone powder, and got longan fruit stone powder 100g in 2000mL there-necked flask, then add 1500mL anhydrous propanone, the whipping appts of mounting strap sealing, all the other two mouthfuls of jam-packs, stirring at room temperature carries out pre-treatment 1h, leaves standstill 1h, separate out upper liquid (substantially clarifying), abandon this upper liquid; Second time adds 1500mL 80% aqueous acetone solution, and installs thermometer and reflux, and being placed in that water-bath heat and control solution temperature is 55 DEG C, stirs 2h, leaves standstill 1h afterwards, separate out upper liquid and 0.04 ~ 0.05MPa vacuum filtration, obtain filtrate for the second time; Third time adds 1500mL 60% aqueous acetone solution, and operation, with second time, obtains third time filtrate; Merge second and third filtrate, obtain extracting solution, this extracting solution is used for next step and is separated;

(2), extracting solution is separated and prepares corilagin: by the method pre-treatment chromatography polymeric amide (80-100 order) of regulation, concrete grammar is: get polymeric amide with 90 ~ 95% (V) alcohol immersion, continuous stirring, load in post after removing bubble, with 90 ~ 95% (V) ethanol elution of 3 ~ 4BV, be washed till elutriant transparent and without residue (or few residue) after evaporate to dryness, use 10% (W) aqueous acetic acid wash-out of the distilled water of 2 ~ 2.5BV5% (W) the NaOH aqueous solution, 1BV, 2 ~ 2.5BV more successively, be finally eluted to neutrality with distilled water.Pretreated chromatography polymeric amide 80g wet method of learning from else's experience dress post, chromatography column specification is Φ 30 × 300mm, the pretreated chromatography polymeric amide 20g that learns from else's experience again mixes in the extracting solution of step (1) gained, obtain mixture, normal pressure rotary evaporation reclaims acetone rotary evaporation moisture to the mixture that then reduces pressure and becomes thick again, afterwards mixture is added to polyamide chromatography post, secondary pure water 3BV, 10% methyl alcohol 4BV, 30% methyl alcohol 4BV, 50% methyl alcohol 4BV, anhydrous methanol 8BV carries out wash-out, elution flow rate is 0.5BV/h, by 50% meoh eluate by 1/9BV sub-bottle collect concentrated and respectively with thin layer polyamide board (with chloroform: methyl alcohol: water=15:3:1 is for developping agent, the Rf value of corilagin is 0.23) be aided with RP-HPLC method (chromatographic condition: Inertsil ODS-3C ₁₈reverse-phase chromatographic column (4.6mm × 250mm, 5 μm, Japan's Shimadzu), moving phase: acetonitrile-volume fraction 0.1% phosphate aqueous solution, gradient elution program is 0 ~ 10min 5% ~ 11%, 10 ~ 35min 11% ~ 13%, 35 ~ 45min 13% ~ 16%, flow velocity: 1.0mL/min, determined wavelength 280nm, column temperature 30 DEG C, flow velocity 1.0mL/min, corilagin retention time is 32.17min) check corilagin content situation in eluate, being merged by the eluate of corilagin solids content>=60% in eluate concentrated for the collection of this sub-bottle after concentrating has corilagin crystal to separate out, the eluate merging after leaching crystal, mother liquor and other being contained after testing corilagin is concentrated into dry, obtain 118.4mg material, upper sephadex lh-20 post after being dissolved in 1mL anhydrous methanol again, sephadex lh-20 post specification is Φ 10 × 200mm, wash-out is carried out with anhydrous methanol, elution flow rate is 1mL/min, every for eluate 2mL sub-bottle is collected a stream part, leave standstill 1 day, 44th ~ 49 stream parts have corilagin crystal to separate out, be associated with the eluate that corilagin crystal is separated out, leave standstill again to precipitation corilagin crystal without increase, separate corilagin crystal, the corilagin crystal that itself and chromatography polyamide column 50% methanol-eluted fractions are got is merged, 83.6mg corilagin crystal is obtained again with dehydrated alcohol recrystallization.

The yield of the corilagin that method in the present embodiment step (1) is extracted is 7.1mg/g dry fruit core, then by after the separation and purification of step (2), and the purity of the corilagin crystal obtained through the analysis of RP-HPLC method is 98.32%.

With the L-DOPA of 1.0mmo1/L for substrate, first 1.0mL 3mol/L L-DOPA solution (solution is the phosphate buffer soln of pH=6.8) is placed in cuvette, add the phosphate buffer soln of 0.85mL pH=6.8, constant temperature l0min in 30 DEG C of waters bath with thermostatic control, add 1.0mL corilagin solution (solution is in the phosphate buffer soln of pH=6.8) and the 0.15mL 0.15g/L tyrosine oxidase aqueous solution, measure OD ₄₇₅.This surveys in live body system, and the whole mass concentration of enzyme is 7.5mg/L.Can formula be defined as to the relative inhibition (I) of enzyme:

$I=(1-\frac{{\mathrm{OD}}_1-{\mathrm{OD}}_3}{{\mathrm{OD}}_2-{\mathrm{OD}}_4})\×100\%$

In formula: OD _lrefer to the absorbance of the survey live body system containing substrate, tyrosine oxidase, corilagin; OD ₂refer to containing substrate, tyrosine oxidase, but not containing the absorbance of the survey live body system of corilagin; OD ₃refer to containing substrate, corilagin, but not containing the absorbance of the survey live body system of tyrosine oxidase; OD ₄refer to containing substrate, but not containing the absorbance of the survey live body system of tyrosine oxidase, corilagin.

Result is the inhibiting rate of 0.5mg/mL (0.79mmol/L) corilagin to tyrosine oxidase is 51.38%.

Embodiment 2:

(1), the extraction of corilagin: first collect longan in longan fruit field and process the fruit stone abandoned, naturally dry, pulverized 20 mesh sieves and obtained longan fruit stone powder, and got longan fruit stone powder 100g in 2000mL there-necked flask, then add 1500mL anhydrous propanone, the whipping appts of mounting strap sealing, all the other two mouthfuls of jam-packs, stirring at room temperature carries out pre-treatment 1h, leaves standstill 1h, separate out upper liquid (substantially clarifying), abandon this upper liquid; Second time adds 1500mL 80% aqueous acetone solution, and installs thermometer and reflux, and being placed in that water-bath heat and control solution temperature is 55 DEG C, stirs 2h, leaves standstill 1h afterwards, separate out upper liquid and 0.04 ~ 0.05MPa vacuum filtration, obtain filtrate for the second time; Third time adds 1500mL 60% aqueous acetone solution, and operation, with second time, obtains third time filtrate; Merge second and third filtrate, obtain extracting solution, this extracting solution is used for next step and is separated;

(2), extracting solution is separated and prepares corilagin: by the method pre-treatment chromatography polymeric amide (80-100 order) of regulation, concrete grammar is: get polymeric amide with 90 ~ 95% (V) alcohol immersion, continuous stirring, load in post after removing bubble, with 90 ~ 95% (V) ethanol elution of 3 ~ 4BV, be washed till elutriant transparent and without residue (or few residue) after evaporate to dryness, use 10% (W) aqueous acetic acid wash-out of the distilled water of 2 ~ 2.5BV5% (W) the NaOH aqueous solution, 1BV, 2 ~ 2.5BV more successively, be finally eluted to neutrality with distilled water.Pretreated chromatography polymeric amide 80g wet method of learning from else's experience dress post, chromatography column specification is Φ 30 × 300mm, the pretreated chromatography polymeric amide 20g that learns from else's experience again mixes in the extracting solution of step (1) gained, obtain mixture, normal pressure rotary evaporation reclaims acetone rotary evaporation moisture to the mixture that then reduces pressure and becomes thick again, afterwards mixture is added to polyamide chromatography post, secondary pure water 3BV, 5% methyl alcohol 4BV, 25% methyl alcohol 4BV, 40% methyl alcohol 4BV, anhydrous methanol 8BV carries out wash-out, elution flow rate is 0.5BV/h, by 40% meoh eluate by 1/9BV sub-bottle collect concentrated and respectively with thin layer polyamide board (with chloroform: methyl alcohol: water=15:3:1 is for developping agent, the Rf value of corilagin is 0.23) be aided with RP-HPLC method (chromatographic condition: Inertsil ODS-3C ₁₈reverse-phase chromatographic column (4.6mm × 250mm, 5 μm, Japan's Shimadzu), moving phase: acetonitrile-volume fraction 0.1% phosphate aqueous solution, gradient elution program is 0 ~ 10min 5% ~ 11%, 10 ~ 35min 11% ~ 13%, 35 ~ 45min 13% ~ 16%, flow velocity: 1.0mL/min, determined wavelength 280nm, column temperature 30 DEG C, flow velocity 1.0mL/min, corilagin retention time is 32.17min) check corilagin content situation in eluate, being merged by the eluate of corilagin solids content>=60% in eluate concentrated for the collection of this sub-bottle after concentrating has corilagin crystal to separate out, the eluate merging after leaching crystal, mother liquor and other being contained after testing corilagin is concentrated into dry, obtain 113.8mg material, upper sephadex lh-20 post after being dissolved in 1mL anhydrous methanol again, sephadex lh-20 post specification is Φ 10 × 200mm, wash-out is carried out with anhydrous methanol, elution flow rate is 1mL/min, every for eluate 2mL sub-bottle is collected a stream part, leave standstill 1 day, 44th ~ 49 stream parts have corilagin crystal to separate out, be associated with the eluate that corilagin crystal is separated out, leave standstill again to precipitation corilagin crystal without increase, separate corilagin crystal, the corilagin crystal that itself and chromatography polyamide column 50% methanol-eluted fractions are got is merged, 80.3mg corilagin crystal is obtained again with dehydrated alcohol recrystallization.

The yield of the corilagin that method in the present embodiment step (1) is extracted is 6.8mg/g dry fruit core, then by after the separation and purification of step (2), and the purity of the corilagin crystal obtained through the analysis of RP-HPLC method is 98.20%.

With the L-DOPA of 1.0mmo1/L for substrate, first 1.0mL 3mol/L L-DOPA solution (solution is the phosphate buffer soln of pH=6.8) is placed in cuvette, add the phosphate buffer soln of 0.85mL pH=6.8, constant temperature l0min in 30 DEG C of waters bath with thermostatic control, add 1.0mL corilagin solution (solution is in the phosphate buffer soln of pH=6.8) and the 0.15mL 0.15g/L tyrosine oxidase aqueous solution, measure OD ₄₇₅.This surveys in live body system, and the whole mass concentration of enzyme is 7.5mg/L.Can formula be defined as to the relative inhibition (I) of enzyme:

$I=(1-\frac{{\mathrm{OD}}_1-{\mathrm{OD}}_3}{{\mathrm{OD}}_2-{\mathrm{OD}}_4})\×100\%$

In formula: OD _lrefer to the absorbance of the survey live body system containing substrate, tyrosine oxidase, corilagin; OD ₂refer to containing substrate, tyrosine oxidase, but not containing the absorbance of the survey live body system of corilagin; OD ₃refer to containing substrate, corilagin, but not containing the absorbance of the survey live body system of tyrosine oxidase; OD ₄refer to containing substrate, but not containing the absorbance of the survey live body system of tyrosine oxidase, corilagin.

Result is the inhibiting rate of 0.5mg/mL (0.79mmol/L) corilagin to tyrosine oxidase is 51.21%.

Embodiment 3:

(1), the extraction of corilagin: first collect longan in longan fruit field and process the fruit stone abandoned, naturally dry, pulverized 20 mesh sieves and obtained longan fruit stone powder, and got longan fruit stone powder 100g in 2000mL there-necked flask, then add 1500mL anhydrous propanone, the whipping appts of mounting strap sealing, all the other two mouthfuls of jam-packs, stirring at room temperature carries out pre-treatment 1h, leaves standstill 1h, separate out upper liquid (substantially clarifying), abandon this upper liquid; Second time adds 1500mL 80% aqueous acetone solution, and installs thermometer and reflux, and being placed in that water-bath heat and control solution temperature is 55 DEG C, stirs 2h, leaves standstill 1h afterwards, separate out upper liquid and 0.04 ~ 0.05MPa vacuum filtration, obtain filtrate for the second time; Third time adds 1500mL 60% aqueous acetone solution, and operation, with second time, obtains third time filtrate; Merge second and third filtrate, obtain extracting solution, this extracting solution is used for next step and is separated;

(2), extracting solution is separated and prepares corilagin: by the method pre-treatment chromatography polymeric amide (80-100 order) of regulation, concrete grammar is: get polymeric amide with 90 ~ 95% (V) alcohol immersion, continuous stirring, load in post after removing bubble, with 90 ~ 95% (V) ethanol elution of 3 ~ 4BV, be washed till elutriant transparent and without residue (or few residue) after evaporate to dryness, use 10% (W) aqueous acetic acid wash-out of the distilled water of 2 ~ 2.5BV5% (W) the NaOH aqueous solution, 1BV, 2 ~ 2.5BV more successively, be finally eluted to neutrality with distilled water.Pretreated chromatography polymeric amide 80g wet method of learning from else's experience dress post, chromatography column specification is Φ 30 × 300mm, the pretreated chromatography polymeric amide 20g that learns from else's experience again mixes in the extracting solution of step (1) gained, obtain mixture, normal pressure rotary evaporation reclaims acetone rotary evaporation moisture to the mixture that then reduces pressure and becomes thick again, afterwards mixture is added to polyamide chromatography post, secondary pure water 3BV, 15% methyl alcohol 4BV, 35% methyl alcohol 4BV, 60% methyl alcohol 4BV, anhydrous methanol 8BV carries out wash-out, elution flow rate is 0.5BV/h, by 60% meoh eluate by 1/9BV sub-bottle collect concentrated and respectively with thin layer polyamide board (with chloroform: methyl alcohol: water=15:3:1 is for developping agent, the Rf value of corilagin is 0.23) be aided with RP-HPLC method (chromatographic condition: Inertsil ODS-3C ₁₈reverse-phase chromatographic column (4.6mm × 250mm, 5 μm, Japan's Shimadzu), moving phase: acetonitrile-volume fraction 0.1% phosphate aqueous solution, gradient elution program is 0 ~ 10min 5% ~ 11%, 10 ~ 35min 11% ~ 13%, 35 ~ 45min 13% ~ 16%, flow velocity: 1.0mL/min, determined wavelength 280nm, column temperature 30 DEG C, flow velocity 1.0mL/min, corilagin retention time is 32.17min) check corilagin content situation in eluate, being merged by the eluate of corilagin solids content>=60% in eluate concentrated for the collection of this sub-bottle after concentrating has corilagin crystal to separate out, the eluate merging after leaching crystal, mother liquor and other being contained after testing corilagin is concentrated into dry, obtain 115.2mg material, upper sephadex lh-20 post after being dissolved in 1mL anhydrous methanol again, sephadex lh-20 post specification is Φ 10 × 200mm, wash-out is carried out with anhydrous methanol, elution flow rate is 1mL/min, every for eluate 2mL sub-bottle is collected a stream part, leave standstill 1 day, 44th ~ 49 stream parts have corilagin crystal to separate out, be associated with the eluate that corilagin crystal is separated out, leave standstill again to precipitation corilagin crystal without increase, separate corilagin crystal, the corilagin crystal that itself and chromatography polyamide column 50% methanol-eluted fractions are got is merged, 81.4mg corilagin crystal is obtained again with dehydrated alcohol recrystallization.

The yield of the corilagin that method in the present embodiment step (1) is extracted is 6.9mg/g dry fruit core, then by after the separation and purification of step (2), and the purity of the corilagin crystal obtained through the analysis of RP-HPLC method is 98.26%.

With the L-DOPA of 1.0mmo1/L for substrate, first 1.0mL 3mol/L L-DOPA solution (solution is the phosphate buffer soln of pH=6.8) is placed in cuvette, add the phosphate buffer soln of 0.85mL pH=6.8, constant temperature l0min in 30 DEG C of waters bath with thermostatic control, add 1.0mL corilagin solution (solution is in the phosphate buffer soln of pH=6.8) and the 0.15mL 0.15g/L tyrosine oxidase aqueous solution, measure OD ₄₇₅.This surveys in live body system, and the whole mass concentration of enzyme is 7.5mg/L.Can formula be defined as to the relative inhibition (I) of enzyme:

$I=(1-\frac{{\mathrm{OD}}_1-{\mathrm{OD}}_3}{{\mathrm{OD}}_2-{\mathrm{OD}}_4})\×100\%$

In formula: OD _lrefer to the absorbance of the survey live body system containing substrate, tyrosine oxidase, corilagin; OD ₂refer to containing substrate, tyrosine oxidase, but not containing the absorbance of the survey live body system of corilagin; OD ₃refer to containing substrate, corilagin, but not containing the absorbance of the survey live body system of tyrosine oxidase; OD ₄refer to containing substrate, but not containing the absorbance of the survey live body system of tyrosine oxidase, corilagin.

Result is the inhibiting rate of 0.5mg/mL (0.79mmol/L) corilagin to tyrosine oxidase is 51.29%.

Claims (10)

1. an extraction and separation method for corilagin, is characterized in that, comprises the following steps:

(1), the extraction of corilagin: first longan fruit stone powder is added anhydrous propanone according to certain mass volume ratio, stirring at room temperature carries out pre-treatment, leaves standstill and separates out upper liquid, abandon this upper liquid; Again longan fruit stone powder is added aqueous acetone solution according to certain mass volume ratio second time, control temperature stirs, and leaves standstill and separates out upper liquid and filter, and obtains second time filtrate; Finally longan fruit stone powder is added aqueous acetone solution according to certain mass volume ratio third time, operation, with second time, obtains third time filtrate; Merge second and third filtrate, obtain extracting solution, this extracting solution is used for next step and is separated;

(2), extracting solution is separated prepares corilagin: chromatography column on pretreated chromatography polymeric amide of learning from else's experience, pretreated chromatography polymeric amide of learning from else's experience again mixes in the extracting solution of step (1) gained, obtain mixture, reclaim acetone transpiring moisture to mixture becomes thick, afterwards mixture is added to polyamide chromatography post, use pure water successively, 5% ~ 15% methyl alcohol, 25% ~ 35% methyl alcohol, 40% ~ 60% methyl alcohol, anhydrous methanol carries out wash-out, 40% ~ 60% meoh eluate sub-bottle is collected and concentrates and check corilagin elution profile, being merged by the eluate of corilagin solids content >=60% in eluate concentrated for the collection of this sub-bottle after concentrating has corilagin crystal to separate out, the eluate merging after leaching crystal, mother liquor and other being contained after testing corilagin is concentrated into dry, upper sephadex column after being dissolved in anhydrous methanol again, wash-out is carried out with anhydrous methanol, eluate sub-bottle is collected, leave standstill, be associated with the eluate that corilagin crystal is separated out, leave standstill again to precipitation corilagin crystal without increase, separate corilagin crystal, the corilagin crystal that itself and chromatography polyamide column 40% ~ 60% methanol-eluted fractions are got is merged, again with dehydrated alcohol recrystallization, finally obtain highly purified corilagin.

2. an extraction and separation method for corilagin according to claim 1, is characterized in that, is first that 1:15 adds anhydrous propanone by longan fruit stone powder according to mass volume ratio in step (1).

3. the extraction and separation method of a corilagin according to claim 1, it is characterized in that, in step (1) again by longan fruit stone powder according to mass volume ratio be 1:15 second time add 80% aqueous acetone solution, control temperature is 55 DEG C and carries out stirring 2h, leaves standstill and separates out upper liquid and 0.04 ~ 0.05MPa vacuum filtration.

4. an extraction and separation method for corilagin according to claim 1, is characterized in that, is finally 1:15 third time add 60% aqueous acetone solution by longan fruit stone powder according to mass volume ratio in step (1).

5. the extraction and separation method of a corilagin according to claim 1, it is characterized in that, chromatography column on pretreated chromatography polymeric amide of learning from else's experience described in step (2), the mass ratio of the longan fruit stone powder be extracted in the pretreated chromatography polymeric amide got and step (1) is 0.8:1; Described pretreated chromatography polymeric amide of learning from else's experience again mixes in the extracting solution of step (1) gained, then the mass ratio of the longan fruit stone powder be extracted in learn from else's experience pretreated chromatography polymeric amide and the step (1) of getting is 0.2:1; Described pre-treatment, concrete grammar is: getting polymeric amide with volume fraction is the alcohol immersion of 90 ~ 95%, continuous stirring, load in post after removing bubble, be the ethanol elution of 90 ~ 95% by the volume fraction of 3 ~ 4BV, be washed till elutriant transparent and without residue or few residue after evaporate to dryness, then be the aqueous acetic acid wash-out of 10% with the NaOH aqueous solution of 2 ~ 2.5BV massfraction 5%, the distilled water of 1BV, the massfraction of 2 ~ 2.5BV successively, be finally eluted to neutrality with distilled water.

6. an extraction and separation method for corilagin according to claim 1, is characterized in that, the recovery acetone described in step (2) is that normal pressure rotary evaporation reclaims acetone, and described transpiring moisture is decompression rotary evaporation moisture.

7. the extraction and separation method of a corilagin according to claim 1, it is characterized in that, carry out wash-out with pure water 3BV, 5% ~ 15% methyl alcohol 4BV, 25% ~ 35% methyl alcohol 4BV, 40% ~ 60% methyl alcohol 4BV, anhydrous methanol 8BV successively in step (2), elution flow rate is 0.5BV/h.

8. an extraction and separation method for corilagin according to claim 1, is characterized in that, 40% ~ 60% meoh eluate sub-bottle being collected described in step (2) is concentrated, collects concentrated for 40% ~ 60% meoh eluate being pressed 1/9BV sub-bottle.

9. the extraction and separation method of a corilagin according to claim 1, it is characterized in that, after leaching crystal in step (2), mother liquor and other sub-bottles being collected concentrated eluate merging is concentrated into dry, be upper sephadex column after 1:5 is dissolved in anhydrous methanol again with mass volume ratio, described sephadex column is sephadex lh-20 post, the mass volume ratio of applied sample amount and sephadex lh-20 is 1:100, wash-out is carried out with 4BV anhydrous methanol, elution flow rate is 1ml/min, elutriant sub-bottle being collected, collecting for elutriant being pressed 1/5BV sub-bottle.

10. by the application of corilagin in restraint of tyrosinase of the method extraction and isolation of claim 1.