CN101973976B - Method for extracting dihydromyricetin, ampelopsis grossedentata amylase and ampelopsis grossedentata polyphenol from ampelopsis grossedentata - Google Patents
Method for extracting dihydromyricetin, ampelopsis grossedentata amylase and ampelopsis grossedentata polyphenol from ampelopsis grossedentata Download PDFInfo
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Abstract
The invention relates to a method for extracting dihydromyricetin, ampelopsis grossedentata amylase and ampelopsis grossedentata polyphenol from ampelopsis grossedentata. The method comprises the following processing steps of: (a) crushing raw materials: drying and crushing fresh ampelopsis grossedentata stem leaves; (b) leaching: extracting with hot water and centrifugally collecting supernatant fluid; (c) concentrating: decompressing and concentrating the supernatant fluid I, cooling, standing still and centrifugally collecting precipitate and supernatant fluid II; (d) extracting dihydromyricetin: recrystallizing and drying the precipitate in vacuum to obtain dihydromyricetin; (e) alcoholizing: decompressing and concentrating the supernatant fluid II, depositing in alcohol and centrifugally collecting a precipitate and supernatant fluid III; (f) extracting crude polysaccharides: purifying the precipitate, drying in vacuum or spraying and drying to obtain ampelopsis grossedentata amylase; and (g) extracting ampelopsis grossedentata polyphenol: decompressing and concentrating the supernatant fluid III, extracting with ethyl acetate, concentrating and drying to obtain ampelopsis grossedentata polyphenol or absorbing with AB-8 resin, eluting with distilled water, eluting with ethanol and collecting eluate, concentrating and drying in vacuum to obtain ampelopsis grossedentata polyphenol. The method has the advantages of low cost, higher product yield and environmental protection, is suitable for industrial continuous production and can comprehensively develop and utilize ampelopsis grossedentata resources and improve the economic benefits.
Description
Technical field
The invention belongs to the natural product extracting technique field, be specifically related to a kind of method of from vine tea, extracting dibydro myricetrin, vine tea polysaccharide and vine tea polyphenol.
Background technology
Vine tea, a kind of with the Vitaceae ampelopsis---the young stem and leaf of Ampelopsis grossedentata [Ampelopsis-grossedentata (Hand-Mazz) W.T.Wang] is the alternative tea that raw material is made.This plant mainly is distributed in provinces such as Hubei, Hunan, Guangdong, Guangxi, Jiangxi, Fujian, aboundresources.Historical and the modern pharmacological research of conventional use to vine tea among the people shows, contains compositions such as abundant flavones (is main with dibydro myricetrin), vine tea polysaccharide, vine tea polyphenol in the vine tea, and has stronger biological activity such as anti-oxidant, hypoglycemic.Extraction and bioactivity research about the vine tea bioactive ingredients mainly concentrates on vine tea flavone and separated component dibydro myricetrin aspect thereof at present; Relevant patent documentation has: a kind of method CN200510032918.5 that from vine tea, extracts dibydro myricetrin; The compsn CN03138054.9 that contains dibydro myricetrin and ampelopsin; The preparation method CN200510114557 of vine tea flavone micro capsule, etc.; Non-patent literature then more (Qin Jieping etc., contemporary Chinese application pharmaceutical journal, 2000, No.3; Li Wei etc., Food science, 2004, No.11, etc.); Next be vine tea polysaccharide aspect pertinent literature (Luo Zuyou etc., Food science, 2007, No.1, etc.); About the research of vine tea polyphenol report few (the white equality of bear, the Chinese food journal, 2004, No.3).
With the vine tea is the independent extraction that raw material carries out any composition in dibydro myricetrin, vine tea polysaccharide, the vine tea polyphenol, and resource utilization is not high, certainly will influence the economic benefit of comprehensive development and utilization vine tea resource.
Summary of the invention
The problem that the present invention will solve provides a kind of method that adopts with batch raw material substep extraction separation dibydro myricetrin, vine tea polysaccharide and vine tea polyphenol.
A kind of method of from vine tea, extracting dibydro myricetrin, vine tea polysaccharide and vine tea polyphenol of the present invention includes in the procedure of processing: a. raw material pulverizing: fresh vine tea cauline leaf is dry, pulverizing; B. lixiviate: use raw material powder: clear water is 90～100 ℃ in temperature and soaks 30～40min with 1: 8～12 solid-liquid ratios, filters and collect supernatant I; C. concentrate: supernatant I concentrating under reduced pressure is left standstill crystallization, respectively collecting precipitation A and supernatant II; D. extract dibydro myricetrin: precipitate A is added the water recrystallization handle, dry to crystallisate again, get the dibydro myricetrin product; E. add alcohol: the supernatant II of enrichment step is added ethanol, and the limit adds the ethanol limit stirs, after leaving standstill, and difference collecting precipitation B and supernatant III; F. extract Crude polysaccharides: precipitate B after washing with alcohol, is got the water-soluble Crude polysaccharides of vine tea; G. extract the vine tea polyphenol: will add the supernatant III concentrating under reduced pressure of pure step, and reclaim ethanol, and collect the spissated aqueous solution, and use ethyl acetate extraction, again that ethyl acetate extraction is concentrated mutually, drying can obtain vine tea polyphenol product; Or,, collect elutriant and concentrating under reduced pressure through AB-8 macroporous resin adsorption → wash-out impurity elimination → wash-out with the spissated aqueous solution, and reclaiming ethanol, the liquid concentrator drying obtains vine tea polyphenol product.
A kind of method of from vine tea, extracting dibydro myricetrin, vine tea polysaccharide and vine tea polyphenol of the present invention is characterized in that including in the described procedure of processing: a. raw material pulverizing: fresh vine tea cauline leaf is dry, pulverizing; B. lixiviate: use raw material powder: clear water is with 1: 8～12 solid-liquid ratios, be 90～100 ℃ in temperature and soak 30～40min, while hot by filter cloth filter the back, it is centrifugal again that (4000rpm 15min), collects supernatant I, and filter residue is repeated to extract 1 time, merges supernatant I; C. concentrate: with supernatant I concentrating under reduced pressure, under 4 ℃, left standstill 10-15 hour, with the centrifugal 15min of 4000rpm, distinguish collecting precipitation A and supernatant II after the crystallization; D. extract dibydro myricetrin: precipitate A is handled through adding the water recrystallization for 2～3 times, again to crystallisate in 35～45 ℃ of vacuum-dryings, the dibydro myricetrin product; E. add alcohol: the supernatant II of enrichment step is evaporated to 1/3～1/5 of original volume under 50～60 ℃; 95% ethanol that adds 3 times of volumes again, limit add the ethanol limit and stir, behind placement 8～24h; The centrifugal 15min of 4000rpm, collecting precipitation B and supernatant III respectively; F. extract Crude polysaccharides: precipitate B after 95% washing with alcohol, is got the water-soluble Crude polysaccharides of vine tea; G. extract the vine tea polyphenol: will add the supernatant III concentrating under reduced pressure of pure step, and reclaim ethanol, and collect the spissated aqueous solution, and use ethyl acetate extraction, again that ethyl acetate extraction is concentrated mutually, drying can obtain vine tea polyphenol product; Or with the spissated aqueous solution; Through AB-8 macroporous resin dynamic adsorption → zero(ppm) water wash-out impurity elimination → 70% ethanol attitude wash-out that surges; Or leave standstill through the static wash-out of AB-8 resin Static Adsorption → zero(ppm) water wash-out impurity elimination → 70% ethanol; Collect elutriant and concentrating under reduced pressure, reclaim ethanol, liquid concentrator obtains vine tea polyphenol product through low-temperature vacuum drying.
A kind of method of from vine tea, extracting dibydro myricetrin, vine tea polysaccharide and vine tea polyphenol of the present invention; Crude polysaccharides can further carry out purification process in procedure of processing f. extraction Crude polysaccharides; Crude polysaccharides adds water redissolution → sevag method deproteinated → with ydrogen peroxide 50 decolouring → ethanol sedimentation → centrifugal; Collecting precipitation → add water redissolution → ethanol sedimentation → centrifugal, collecting precipitation is in 35～45 ℃ of vacuum-dryings or the back spraying drying that is dissolved in water.
The present invention and prior art compare, owing to adopt with criticizing the raw material combined extraction method of the multiple composition of extraction separation step by step, this method cost is low; The extraction yield is higher; Environmental protection is fit to the industriallization continuous production, can realize the comprehensive development and utilization of vine tea resource and the raising of economic benefit.
Embodiment
Embodiment 1
Raw material powder 500g is produced in fresh vine tea cauline leaf is dry, pulverizing, adds 6L zero(ppm) water, is heated to 95 ℃ and extracts 30min down; After filtering with 100 order double-layer filter cloths while hot, centrifugal while hot again (4000rpm, 15min); Collect supernatant I; To filter residue adding distil water 4L, be heated to and repeat to extract under 95 ℃ 1 time, merge supernatant I; Supernatant I is evaporated to 1/3 volume, and fully cooling was left standstill under 4 ℃ 12 hours, after the crystallization, centrifugal (4000rpm, 15min), difference collecting precipitation A and supernatant II; Precipitate A is handled through adding the water recrystallization for 2 times, again to crystallisate in 40 ℃ of vacuum-dryings, dibydro myricetrin, the product yield can reach 11% (dibydro myricetrin is made standard specimen, aluminum nitrate-Sodium Nitrite colorimetric method for determining, product dihydromyricetin cellulose content >=85%); Supernatant II is evaporated to 1/4 of original volume under 60 ℃, adds 95% ethanol of 3 times of volumes again, the limit adds the ethanol limit stirs, behind the placement 24h, centrifugal (4000rpm, 15min), difference collecting precipitation B and supernatant III; Precipitate B after 95% washing with alcohol, add water redissolve polysaccharide liquid, with sevag method deproteinated; Promptly with the liquid glucose of 3: 1 (v/v): mixed solution (4 chloroforms: 1 propyl carbinol, v/v) deproteinated is 1 time, re-adjustment polysaccharide soln pH to pH8; After 50 ℃ dropping 20% ydrogen peroxide 50 decolours down; Add 95% ethanol sedimentation polysaccharide of 3 times of volumes, centrifugal, collecting precipitation; After adding the water redissolution again, add 95% ethanol sedimentation polysaccharide of 3 times of volumes, centrifugal; Collecting precipitation in 40 ℃ of vacuum-dryings, gets the vine tea water-soluble polysaccharide of preliminary purification; About 4% (glucose is made standard specimen, and the sulfuric acid phynol method is measured, product polysaccharide content about 60%) of product yield; Supernatant III through concentrating under reduced pressure, is reclaimed ethanol, collect the spissated aqueous solution; To the aqueous solution with ethyl acetate extraction (1: 1.2, V/V), again with ETHYLE ACETATE phase concentrating under reduced pressure; Reclaim ETHYLE ACETATE, enriched material obtains the vine tea polyphenol through vacuum-drying, and (gallic acid is made standard specimen to the product yield more than 5%; The ferrous tartrate colorimetric method for determining, product polyphenol content 41%).
Embodiment 2
Raw material powder 1000g is produced in fresh vine tea cauline leaf is dry, pulverizing, adds 12L zero(ppm) water, is heated to 95 ℃ and extracts 30min down; After filtering with 100 order double-layer filter cloths while hot, centrifugal while hot again (4000rpm, 15min); Collect supernatant I; To filter residue adding distil water 8L, be heated to and repeat to extract under 95 ℃ 1 time, merge supernatant I; Supernatant I is evaporated to 1/3 volume, and fully cooling was left standstill under 4 ℃ 12 hours, after the crystallization, centrifugal (4000rpm, 15min), difference collecting precipitation A and supernatant II; Precipitate A is handled through adding the water recrystallization for 2 times, again to crystallisate in 40 ℃ of vacuum-dryings, the dibydro myricetrin product, the product yield can reach 11% (dibydro myricetrin is made standard specimen, aluminum nitrate-Sodium Nitrite colorimetric method for determining, product dihydromyricetin cellulose content >=85%); Supernatant II is evaporated to 1/4 of original volume under 60 ℃, adds 95% ethanol of 3 times of volumes again, the limit adds the ethanol limit stirs, behind the placement 24h, centrifugal (4000rpm, 15min), difference collecting precipitation B and supernatant III; Precipitate B after 95% washing with alcohol, add water redissolve polysaccharide liquid, with sevag method deproteinated; Promptly with the liquid glucose of 3: 1 (v/v): mixed solution (4 chloroforms: 1 propyl carbinol, v/v) deproteinated is 1 time, re-adjustment polysaccharide soln pH to pH8; After 50 ℃ dropping 20% ydrogen peroxide 50 decolours down; Add 95% ethanol sedimentation polysaccharide of 3 times of volumes, centrifugal, collecting precipitation; After adding the water redissolution again, add 95% ethanol sedimentation polysaccharide of 3 times of volumes, centrifugal; Collecting precipitation, after redissolving again, spray-dried (the 180 ℃ of inlet temperatures of the aqueous solution; 80 ℃ of temperature outs), get light grey Powdered vine tea water-soluble polysaccharide, (glucose is made standard specimen to product yield about 4%; The sulfuric acid phynol method is measured, product polysaccharide content about 60%); With supernatant III concentrating under reduced pressure, reclaim ethanol, the static wash-out of 70% ethanol that the spissated aqueous solution spends the night through AB-8 resin Static Adsorption---impurity elimination of zero(ppm) water wash-out---; Collect elutriant and concentrating under reduced pressure; Reclaim ethanol, liquid concentrator obtains the vine tea polyphenol through low-temperature vacuum drying, and the product yield can reach 3%, and (gallic acid is made standard specimen; The ferrous tartrate colorimetric method for determining, product polyphenol content 34%).
Embodiment 3
Fresh vine tea cauline leaf is dry, pulverize as the extraction raw material; Raw material powder 1500g adds 18L zero(ppm) water, is heated to 95 ℃ and extracts 30min down, after filtering with 100 order double-layer filter cloths while hot; It is centrifugal while hot again that (4000rpm 15min), collects supernatant I; To filter residue adding distil water 12L, be heated to and repeat to extract under 95 ℃ 1 time, merge supernatant I; Supernatant I is evaporated to 1/3 volume, and fully cooling was left standstill under 4 ℃ 12 hours, after the crystallization, centrifugal (4000rpm, 15min), difference collecting precipitation A and supernatant II; Precipitate A is handled through adding the water recrystallization for 2 times, again to crystallisate in 40 ℃ of vacuum-dryings, dibydro myricetrin, the product yield can reach 11% (dibydro myricetrin is made standard specimen, aluminum nitrate-Sodium Nitrite colorimetric method for determining, product dihydromyricetin cellulose content >=85%); Supernatant II is evaporated to 1/4 of original volume under 60 ℃, adds 95% ethanol of 3 times of volumes again, the limit adds the ethanol limit stirs, behind the placement 24h, centrifugal (4000rpm, 15min), difference collecting precipitation B and supernatant III; Precipitate B after 95% washing with alcohol, add water redissolve polysaccharide liquid, with sevag method deproteinated; Promptly with the liquid glucose of 3: 1 (v/v): mixed solution (4 chloroforms: 1 propyl carbinol, v/v) deproteinated is 1 time, re-adjustment polysaccharide soln pH to pH8; After 50 ℃ dropping 20% ydrogen peroxide 50 decolours down; Add 95% ethanol sedimentation polysaccharide of 3 times of volumes, centrifugal, collecting precipitation; After adding the water redissolution again, add 95% ethanol sedimentation polysaccharide of 3 times of volumes, centrifugal; Collecting precipitation in 40 ℃ of vacuum-dryings, gets the vine tea water-soluble polysaccharide of preliminary purification; About 4% (glucose is made standard specimen, and the sulfuric acid phynol method is measured, product polysaccharide content about 60%) of product yield; Supernatant III through concentrating under reduced pressure, is reclaimed ethanol, collect the spissated aqueous solution; To the aqueous solution with ethyl acetate extraction (1: 1.2, V/V), again with ETHYLE ACETATE phase concentrating under reduced pressure; Reclaim ETHYLE ACETATE, enriched material obtains the vine tea polyphenol through vacuum-drying, and (gallic acid is made standard specimen to the product yield more than 5%; The ferrous tartrate colorimetric method for determining, product polyphenol content 41%).
Claims (3)
1. a method of from vine tea, extracting dibydro myricetrin, vine tea polysaccharide and vine tea polyphenol is characterized in that including in the procedure of processing: a. raw material pulverizing: fresh vine tea cauline leaf is dry, pulverizing;
B. lixiviate: use raw material powder: clear water is 90～100 ℃ in temperature and soaks 30～40min with 1: 8～12 solid-liquid ratios, filters and collect supernatant I;
C. concentrate: supernatant I concentrating under reduced pressure is left standstill crystallization, respectively collecting precipitation A and supernatant II;
D. extract dibydro myricetrin: precipitate A is added the water recrystallization handle, dry to crystallisate again, get the dibydro myricetrin product;
E. add alcohol: the supernatant II of enrichment step is added ethanol, and the limit adds the ethanol limit stirs, after leaving standstill, and difference collecting precipitation B and supernatant III;
F. extract Crude polysaccharides: precipitate B after washing with alcohol, is got the water-soluble Crude polysaccharides of vine tea;
G. extract the vine tea polyphenol: will add the supernatant III concentrating under reduced pressure of pure step, and reclaim ethanol, and collect the spissated aqueous solution, and use ethyl acetate extraction, again that ethyl acetate extraction is concentrated mutually, drying can obtain vine tea polyphenol product; Or,, collect elutriant and concentrating under reduced pressure through AB-8 macroporous resin adsorption → wash-out impurity elimination → wash-out with the spissated aqueous solution, and reclaiming ethanol, the liquid concentrator drying obtains vine tea polyphenol product.
2. according to the described a kind of method of from vine tea, extracting dibydro myricetrin, vine tea polysaccharide and vine tea polyphenol of claim 1, it is characterized in that including in the described procedure of processing: a. raw material pulverizing: fresh vine tea cauline leaf is dry, pulverizing;
B. lixiviate: use raw material powder: clear water is 90～100 ℃ in temperature and soaks 30～40min with 1: 8～12 solid-liquid ratios, after being filtered by filter cloth while hot, again with the centrifugal 15min of 4000rpm, collects supernatant I, and filter residue is repeated to extract 1 time, merges supernatant I;
C. concentrate: with supernatant I concentrating under reduced pressure, under 4 ℃, left standstill 10-15 hour, with the centrifugal 15min of 4000rpm, distinguish collecting precipitation A and supernatant II after the crystallization;
D. extract dibydro myricetrin: precipitate A is handled through adding the water recrystallization for 2～3 times, again to crystallisate in 35～45 ℃ of vacuum-dryings, the dibydro myricetrin product;
E. add alcohol: the supernatant II of enrichment step is evaporated to 1/3～1/5 of original volume under 50～60 ℃; 95% ethanol that adds 3 times of volumes again, limit add the ethanol limit and stir, behind placement 8～24h; The centrifugal 15min of 4000rpm, collecting precipitation B and supernatant III respectively;
F. extract Crude polysaccharides: precipitate B after 95% washing with alcohol, is got the water-soluble Crude polysaccharides of vine tea;
G. extract the vine tea polyphenol: will add the supernatant III concentrating under reduced pressure of pure step, and reclaim ethanol, and collect the spissated aqueous solution, and use ethyl acetate extraction, again that ethyl acetate extraction is concentrated mutually, drying can obtain vine tea polyphenol product; Or with the spissated aqueous solution; Through AB-8 macroporous resin dynamic adsorption → zero(ppm) water wash-out impurity elimination → 70% ethanol attitude wash-out that surges; Or leave standstill through the static wash-out of AB-8 resin Static Adsorption → zero(ppm) water wash-out impurity elimination → 70% ethanol; Collect elutriant and concentrating under reduced pressure, reclaim ethanol, liquid concentrator obtains vine tea polyphenol product through low-temperature vacuum drying.
3. according to the described a kind of method of from vine tea, extracting dibydro myricetrin, vine tea polysaccharide and vine tea polyphenol of claim 2; It is characterized in that Crude polysaccharides can further carry out purification process in the described procedure of processing f. extraction Crude polysaccharides; Crude polysaccharides adds water redissolution → sevag method deproteinated → with ydrogen peroxide 50 decolouring → ethanol sedimentation → centrifugal; Collecting precipitation → add water redissolution → ethanol sedimentation → centrifugal, collecting precipitation is in 35～45 ℃ of vacuum-dryings or the back spraying drying that is dissolved in water.
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