CN102477453A - Method of preparing taxifolin monomer from engelhardtia leaf and application - Google Patents
Method of preparing taxifolin monomer from engelhardtia leaf and application Download PDFInfo
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Abstract
The invention discloses a method of preparing a taxifolin monomer from engelhardtia leaves and an application, and the method comprises the following steps: crushing or cutting dried engelhardtia leaves into segments, adding ethanol with a certain concentration, performing extraction, concentrating the obtained extract liquid to paste; adding a buffer with a pH of 3-6, adding a proper amount of enzymes for enzymatic hydrolysis at a certain temperature, performing separation, enrichment, purification, and recrystallization treatment of the enzymatic hydrolysate to obtain a taxifolin monomer with purity of above 99%. The method of the invention is characterized in that enzymatic hydrolysis of the engelhardtia leaf extract is directly performed to obtain the taxifolin monomer with high purity, and the method of the invention has the advantages of strong reaction specificity, high conversion rate, simple and practical process, less pollution, and suitability for large-scale industrial production, and the like. The disadvantages of great destructive effect on aglycone, poor specificity, low conversion rate, high equipment corrosivity, severe pollution, tedious process and the like in the prior art are overcome. The taxifolin monomer prepared in the invention is applicable to the prevention and treatment of fatty liver.
Description
Technical field:
The invention belongs to medical technical field, relate to and adopt zymolysis technique that Folium Engelhardia roxburghina extrat is carried out enzymolysis, from enzymolysis product, separate preparation Taxifolin monomer methods, and use it for prevention and treatment fatty liver.
Background technology:
Folium Engelhardia roxburghina is the dry leave of the yellow Qi of juglandaceae plant (Engeihardtia roxburghiana Wall), has the effect of clearing heat and detoxicating pain relieving.The normal do health care tea-drinking among the people in Guangdong and Guangxi Provinces areas, have certain promoting blood circulation and removing blood stasis, reducing blood-fat, hypoglycemic and improve nourishing function such as immunizing power and anti-oxidant, suppress multiple physiologically actives such as fat, antianaphylaxis and preventing cancer.
Taxifolin has another name called Taxifoliol, taxifolin, and its chemistry is called-the penta hydroxy group flavanone, English name Taxifolin, and Dihydroquercetin, 3,5,7,3 ', 4 '-Pentahydroxyflavanone, molecular formula is C
15H
12O
7, molecular weight is 304.26, structural formula is following:
Taxifolin is one of natural flavone active component, has many important BAs, can suppress and activate plurality of enzymes, thereby produces different physiological effects.Because it contains more phenolic hydroxyl group, has powerful antioxygenation.It can eliminate excessive radical (Free Radical Biological Medicine, 1999,27 (11-12): 1313-1323), promote the perviousness of capillary vessel effectively from human body; Efficient recovery capillary vessel elasticity (Journalof Biomedical Science, 2006,13 (1): 127-141) are arranged; Improve immunologic function (Biological& Pharmaceutical Bulletin, 2007,30 (6); 1074-1079), stop the formation of inflammation and lump, reduce generation (the Cancer Letter of cancer; 1993,69 (3): 191-196, Anti-cancer Research, 1999; 19 (2A): 1261-1269, Anti-cancer Drugs, 1992,3 (5): 525-530).Bibliographical information, Taxifolin can be through suppressing DGAT enzyme and MTP enzyme the secretion of activity reduction apolipoprotein B reduce triglyceride level and SUV in serum and the liver cell (Atherosclerosis, 2004,176 (2), 247-253).Be used for clinically cerebral thrombosis, cerebral infarction sequela (Clin.Hemorheol.Microcirc., 2004,30:449-452), coronary heart disease (Phytother.Res., 2003, the prevention and the treatment that 17:276-278) wait.In addition, Taxifolin can be used as sanitas as a kind of natural antioxidants of strong effect in foodstuffs industry, prolong the quality guaranteed period of vegetables oil, animal tallow, dried milk, fatty sweet goods etc. and can make food performance be improved significantly.
Through the patent inquiry, the many preparation Taxifolins that from tamarack and Folium Engelhardia roxburghina, extract of prior art." a kind of method of from tamarack, extracting Taxifoliol " disclosed like Chinese patent 200610035280.5; 200610035752.7 disclose " a kind of method that adopts absorption method from tamarack, to extract Taxifoliol "; 200710144650.3 disclose " a kind of method of from tamarack and processing residue material thereof, extracting Taxifoliol and arabogalactan ", all be to extract Taxifolin as raw materials for production with tamarack.But tamarack resource itself is comparatively rare, and the content of Taxifolin is also lower in the tamarack, and general less than 2% is used for producing Taxifolin, and not only the tamarack resource to shortage causes waste and destruction, and yield is lower.Patent 200710105982.0 discloses the method for extraction separation Taxifoliol " a kind of from Folium Engelhardia roxburghina "; Adopted resourceful Folium Engelhardia roxburghina as the raw material of producing Taxifolin; Its technological process is for isolating the precursor astilbin of Taxifolin earlier, and the hydrolysis astilbin prepares Taxifolin again.Chemical ingredients relative complex because of Folium Engelhardia roxburghina; Contain a series of and astilbin structure and similar flavonoid glycoside thereof; Like different astilbin, new astilbin, engelitin, isoengelitin, new engelitin, new isoengelitin etc.; The pure article difficulty of direct separation astilbin is bigger, moreover separates the astilbin obtain and just can obtain Taxifolin through hydrolysis again, and technology is comparatively loaded down with trivial details and yield is lower.Patent 101781277A discloses " a kind of separation purification method of dihydroquercetin monomer "; Its main technique is carried out acid hydrolysis for direct alcohol extract to Folium Engelhardia roxburghina; Obtain the Taxifolin bullion, adopt macroporous resin etc. that bullion is carried out purification enrichment again, get the pure article of method at last with recrystallization.More than two the invention in hydrolysis all adopt acid hydrolysis, reaction conditions is violent, destroys product easily, often can not get complete aglycon.Have four enantiomers owing to fir in addition, the acid hydrolysis poor selectivity, the hydrolysate that very difficult assurance obtains is the simplification compound.In addition, acid hydrolysis is big to equipment corrosion, and a large amount of acid waste liquid environmental pollutions is bigger, is not suitable for suitability for industrialized production.At present, Shang Weijian adopts enzymolysis process to prepare the monomeric report of Taxifolin.
Summary of the invention:
The objective of the invention is to be to provide a kind of and from Folium Engelhardia roxburghina, prepare the Taxifolin monomer methods, and the Taxifolin monomer is used for the prevention and the treatment of fatty liver.That this method has is easy and simple to handle, process stabilizing, mild condition, environmental pollution are little, can realize the monomeric preparation of a large amount of Taxifolins, is easy to realize characteristics such as suitability for industrialized production.
In order to realize the foregoing invention purpose, the present invention adopts following technical scheme:
A kind of Taxifolin monomer methods that from Folium Engelhardia roxburghina, prepares is characterized in that may further comprise the steps:
A, extraction:
(1) the exsiccant Folium Engelhardia roxburghina is pulverized or segment; Add mass percent concentration and be the alcohol reflux 3 times of 30%-90%, add the solvent of 8-20 times of quality of medicinal material the 1st time, add the solvent of 6-10 times of quality of medicinal material the 2nd, 3 time; Extraction time is 2h; United extraction liquid, the concentrating under reduced pressure solvent gets medicinal extract.
(2) the exsiccant Folium Engelhardia roxburghina is pulverized or segment, added 3-20 times of quality of medicinal material, mass percent concentration is the ethanol of 30%-90%, supersound process 2-3 time, and each 1h, united extraction liquid, the concentrating under reduced pressure solvent gets medicinal extract.
(3) the exsiccant Folium Engelhardia roxburghina is pulverized or segment, added 6-20 times of quality of medicinal material, mass percent concentration is that the ethanol of 30%-90% oozes rumble, collects to ooze rumble liquid, and concentrating under reduced pressure gets medicinal extract.
(4) the exsiccant Folium Engelhardia roxburghina is pulverized or segment, is added 6-20 times of quality of medicinal material, mass percent concentration is the alcohol immersion 3 times of 30%-90%, 12h at every turn.Merge soak solution, concentrating under reduced pressure gets medicinal extract.
B, enzymolysis: the paste of steps A gained adds enzyme by enzyme/paste=0.01-5 mass ratio and carries out enzymic hydrolysis with the damping fluid of the ratio adding pH=3-6 of every gram adding 30-100ml, and the temperature of reaction is 15-70 ℃, and the reaction times is 4-72h.Wherein, the pH value is preferably 4.5-5.5, and the mass ratio of enzyme/paste is preferably 0.1-0.5, and temperature of reaction is preferably 37-50 ℃.
Above-mentioned preparation method, the damping fluid described in the step B can be any one in Sodium phosphate, dibasic-citrate buffer solution, phosphate buffered saline buffer, acetic acid-sodium-acetate buffer, citric acid-sodium citrate damping fluid or the Hydrocerol A-sodium hydroxide-hydrochloride buffer.Described enzyme can be one or more mixtures in beta-glycosidase, cellulase, Snailase, naringinase, the polygalacturonase, preferred beta-glycosidase and cellulase.
Add a certain amount of ethanol or methyl alcohol termination reaction in C, the above-mentioned enzymolysis solution, centrifugal or remove by filter deposition, supernatant or filtrating, being evaporated to does not have the alcohol flavor.
(1) supernatant or filtrating extract fully through ETHYLE ACETATE or propyl carbinol at first through petroleum ether degreasing more repeatedly, and concentrated extract is to there being solid to separate out, and placement is spent the night, and leaches solid and promptly gets the Taxifolin bullion.
(2) supernatant or filtrating are passed through macroporous adsorptive resins or polyamide column with the flow velocity of 2～4 times of column volumes, and is saturated up to absorption; With pure water and 10% ethanol elution impurity, closely colourless up to the wash fluid; Use 40% ethanol elution to Taxifolin wash-out complete again, collect the ethanolic soln elutriant, reclaim ethanol, continue to concentrate and reclaim solvent to there being solid to separate out, placement is spent the night, and leaches solid and promptly gets the Taxifolin bullion.
(3) supernatant or filtrating evaporate to dryness, ethanol redissolves, through silica gel column chromatography; The ester of the haloalkane of sherwood oil, low carbon chain, the pure and mild low carbon chain of low carbon chain; The ketone of low carbon chain, or wherein the mixture wash-out or gradient elution to the Taxifolin wash-out of two kind solvents is complete, reclaims solvent to there being solid to separate out; Placement is spent the night, and leaches solid and promptly gets the Taxifolin bullion.
Above-mentioned preparation method, described macroporous resin model can be D101, D201, HPD-100, HPD-200, AB-8, FL-1, NK-9, any one among the NK-II.The preferred resin model is FL-1, D101, HPD-100 and AB-8.The eluting solvent of described silica gel chromatography is that the haloalkane of low carbon chain comprises methyl halide, halo ethane etc., like trichloromethane, methylene dichloride, ethylene dibromide, tribromoethane etc.The alcohol of low carbon chain refers to C
1-3Alcohol, like methyl alcohol, ethanol, propyl alcohol, Virahol etc.The ester of low carbon chain refers to C
1-5Ester, like methyl-formiate or ethyl ester, methyl acetate or ethyl ester etc.The ketone of low carbon chain refers to C
1-5Ketone, like acetone, butanone etc.Wherein preferred: trichlorine methyl alcohol-methyl alcohol, methylene chloride-methanol, trichlorine methyl alcohol-acetone, methylene dichloride-acetone, ethyl acetate-ethanol system, the most preferably minimum ethyl acetate-ethanol system of toxicity.
D, recrystallization: the Taxifolin bullion promptly gets the Taxifolin monomer of purity＞99% through alcohol-water, methanol-water or acetone-water recrystallization.
The present invention compared with prior art; Maximum characteristics are to adopt one or more mixed enzyme in beta-glycosidase, cellulase, Snailase, naringinase, the polygalacturonase directly the ethanol extraction of Folium Engelhardia roxburghina to be carried out enzymolysis to prepare the Taxifolin monomer; Simplified operation steps, and the reaction specificity is strong, transformation efficiency is high, have pollution is easy to realize advantages such as industriallization for a short time.Overcome existing in prior technology to the aglycon destructiveness is big, seriously polluted, purpose is poor, transformation efficiency is low, serious to equipment corrosion, be inappropriate for shortcoming such as suitability for industrialized production.
The monomeric purposes of Taxifolin of the present invention is prevention and the treatment that is used for fatty liver.The effect experiment result is following:
Experiment one: the foundation of hyperlipemia rat model
After rat flexibility raised for 1 week, be divided into high lipoprotein emulsion group (model group), high lipoprotein emulsion+high dosage Taxifolin set of monomers, high lipoprotein emulsion+middle dosage Taxifolin set of monomers, high lipoprotein emulsion+low dosage Taxifolin set of monomers, positive controls (SV) and blank group (every group each 10) at random by quality.All animals are given the standard particle feed every day, and except that blank control group, all the other are respectively organized rat and give the high lipoprotein emulsion of 2mL (containing 20% lard, 10% SUV, 2% Sodium cholic acid, 1% propylthiouracil, with dissolved in distilled water) filling stomach every day, and water is freely drunk.The blank control group rat experiment begins equivalent zero(ppm) water filling every day stomach, and gives tap water and freely drink.After modeling continued for 2 weeks, get blood examination and survey high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), triglyceride level (TG) and total cholesterol (TC).Model group, high, normal, basic dose groups, positive controls are compared with the blank group, and LDL-C, TC all obviously increase (P＜0.01), and HDL-C then significantly reduces (P＜0.01), and TG slightly raises.
Experiment two: the Taxifolin monomer is to the influence of hyperlipidemia animal model
High, medium and low dosage Taxifolin set of monomers is respectively according to 100mg/kg, 50mg/kg and 25mg/kg administration, and positive controls is pressed the 4.0mg/kg administration.Successive administration 28 days.
All animals is last weekly with the blood sampling of truncation method, spinning serum after the natural coagulation after administration.The mensuration of serum lipid adopts the Beckman automatic clinical chemistry analyzer, detects TC, TG, LDL-C and HDL-C.
Detected result is following:
The above results shows that Taxifolin has good effect for reducing fat, and effect is superior to commercially available lipid lowerers SV, therefore the preparation blood lipid-lowering medicine is had a good application prospect.
Experiment three, Taxifolin monomer are to the influence of ethionine induced mice fatty liver model
48 of ICR mouse, male, body weight 21.2 ± 1.2 is divided into 6 groups at random, 8 every group.High, medium and low dose groups is respectively according to 100mg/kg, 50mg/kg and 25mg/kg; Positive control drug (SLB) group is pressed 150mg/kg respectively at 0h, 8h, 16h, 24h, 32h, 48h, 56h and 64h administration, except that blank control group, all irritates the ethionine modeling that stomach gives 300mg/kg behind the fasting 8h.Be administered three times respectively at 1h, 10h and 18h after the modeling.Modeling 24 as a child back (last beginning fasting in evening) is all put to death, and takes out liver, testing index TG.
Detected result is following:
*Expression P＜0.01
*Expression P＜0.05
Above-mentioned drug effect result shows; Taxifolin has good liver-protecting and blood fat-reducing effect; And effect is superior to commercially available hepatoprotective lipid lowerers SLB, and the result is dosage and relies on, so Taxifolin has a good application prospect to preparation prevention and treatment fatty liver medicine.
Description of drawings:
Fig. 1 is the embodiment of the invention 1 a gained Taxifolin
1The H-NMR collection of illustrative plates
Fig. 2 is the embodiment of the invention 1 a gained Taxifolin
13The C-NMR collection of illustrative plates
Embodiment:
Through following examples the present invention is done further detailed description, but should notice that scope of the present invention does not receive any restriction of these embodiment.
Among following each embodiment, the monomeric purity detecting of finished product Taxifolin all adopts anti-phase analysis mode liquid chromatography (RP-HPLC) method, and chromatographic condition is following:
With the octadecylsilane chemically bonded silica is weighting agent; With methyl alcohol: water: Glacial acetic acid min. 99.5 (35: 65: 0.3) is a moving phase; 30 ℃ of column temperatures; Flow velocity 1.0ml/min; The detection wavelength is 290nm.
The exsiccant Folium Engelhardia roxburghina is pulverized, placed multi-function extractor, add 70% alcohol reflux 2h of 10 times of amounts for the 1st time, add 70% alcohol reflux 2h of 8 times of amounts for the 2nd, 3 time, united extraction liquid, concentrating under reduced pressure gets paste.The every gram of gained paste adds Sodium phosphate, dibasic-citrate buffer solution of the pH=5.0 of 100ml, presses enzyme/paste=0.05 and adds beta-glycosidase, and 50 ℃ are reacted 48h down, add 4 times of volume of ethanol termination reactions, centrifugal removal deposition.Supernatant concentration is adsorbed through the FL-1 macroporous resin to there not being the alcohol flavor, and water and 10% ethanol elution to effluent color are extremely shallow respectively, remove impurity, uses 40% ethanol elution to effluent colourless again.Elutriant is merged, and decompression recycling ethanol is to there being deposition to separate out, and placement is spent the night, and leaches deposition.Precipitate the ethyl alcohol recrystallization with 60%, obtain the off-white color needle-like crystal, purity is 99.4%, and total rate of transform is 35.06%.
The exsiccant Folium Engelhardia roxburghina is pulverized, added 90% ethanol ultrasonic extraction 1h of 5 times of amounts, leach soup, the dregs of a decoction are used 60% ethanol ultrasonic extraction 1h again, merge filtrating twice, are evaporated to medicinal extract.The every gram of gained paste adds Sodium phosphate, dibasic-citrate buffer solution of the pH=5.0 of 50ml, presses enzyme/paste=0.05 and adds beta-glycosidase, and 50 ℃ are reacted 48h down, add 4 times of volume of ethanol termination reactions, centrifugal removal deposition.Supernatant concentration is adsorbed through the FL-1 macroporous resin to there not being the alcohol flavor, and water and 10% ethanol elution to effluent color are extremely shallow respectively, remove impurity, uses 40% ethanol elution to effluent colourless again.Elutriant is merged, and decompression recycling ethanol is to there being deposition to separate out, and placement is spent the night, and leaches deposition.Precipitate the ethyl alcohol recrystallization with 60%, obtain the off-white color needle-like crystal, purity is 99.2%, and total rate of transform is 33.26%.
Embodiment 3
The exsiccant Folium Engelhardia roxburghina is pulverized, placed multi-function extractor, add 90% alcohol reflux 2h of 10 times of amounts for the 1st time, add 60% alcohol reflux 2h of 8 times of amounts for the 2nd, 3 time, united extraction liquid, concentrating under reduced pressure gets paste.The every gram of gained paste adds acetic acid-sodium-acetate buffer of the pH=4.8 of 50ml, presses enzyme/paste=1 and adds beta-glycosidase, and 50 ℃ of reaction 4h add 4 times of volume of ethanol termination reactions, centrifugal removal deposition.Supernatant concentration is adsorbed through FL-1 to there not being the alcohol flavor, and water and 10% ethanol elution to effluent color are extremely shallow respectively, remove impurity, uses 40% ethanol elution to effluent colourless again.Elutriant is merged, and decompression recycling ethanol is to there being deposition to separate out, and placement is spent the night, and leaches deposition.Ethyl alcohol recrystallization with 60% obtains the off-white color needle-like crystal, and purity is 99.5%, and total rate of transform is 36.24%.
Embodiment 4
The exsiccant Folium Engelhardia roxburghina is pulverized, and 60% ethanol of 15 times of amounts oozes rumble, collects to ooze rumble liquid, and concentrating under reduced pressure gets paste.The every gram of gained paste adds Hydrocerol A-sodium hydroxide-hydrochloride buffer of the pH=5.8 of 30ml, presses enzyme/paste=0.2 and adds cellulase, and 50 ℃ are reacted 48h down, add 4 times of volume of ethanol termination reactions, centrifugal removal deposition.Supernatant concentration is adsorbed through the D101 macroporous resin to there not being the alcohol flavor, and water and 10% ethanol elution to effluent color are extremely shallow respectively, remove impurity, uses 40% ethanol elution to effluent colourless again.Elutriant is merged, and decompression recycling ethanol is to there being deposition to separate out, and placement is spent the night, and leaches deposition.Ethyl alcohol recrystallization with 40% obtains the off-white color needle-like crystal, and purity is 99.1%, and total rate of transform is 33.12%.
The exsiccant Folium Engelhardia roxburghina is pulverized, placed multi-function extractor, add 90% alcohol reflux 2h of 10 times of amounts for the 1st time, add 60% alcohol reflux 2h of 8 times of amounts for the 2nd, 3 time, united extraction liquid, concentrating under reduced pressure gets paste.The every gram of gained paste adds the phosphate buffered saline buffer of the pH=5.0 of 50ml; Press the mixed enzyme (mass ratio 1: 1) of enzyme/paste=0.2 adding beta-glycosidase and cellulase; Place 37 ℃ of following 100r/min reaction 48h in the constant-temperature shaking water-bath; Add 4 times of volume of ethanol termination reactions, centrifugal removal deposition.Supernatant concentration is adsorbed through the AB-8 macroporous resin to there not being the alcohol flavor, and water and 10% ethanol elution to effluent color are extremely shallow respectively, remove impurity, uses 40% ethanol elution to effluent colourless again.Elutriant is merged, and decompression recycling ethanol is to there being deposition to separate out, and placement is spent the night, and leaches deposition.Recrystallizing methanol with 50% obtains the off-white color needle-like crystal, and purity is 99.4%, and total rate of transform is 33.72%.
The exsiccant Folium Engelhardia roxburghina is pulverized, placed multi-function extractor, add 80% alcohol reflux 2h of 10 times of amounts for the 1st time, add 80% alcohol reflux 2h of 8 times of amounts for the 2nd, 3 time, united extraction liquid, concentrating under reduced pressure gets paste.The every gram of gained paste adds the citric acid-sodium citrate damping fluid of the pH=4.8 of 50ml, presses enzyme/paste=0.5 and adds Snailase, and 55 ℃ are reacted 8h down, add 4 times of volume of ethanol termination reactions, centrifugal removal deposition.Supernatant concentration is adsorbed through the NK-9 macroporous resin column to there not being the alcohol flavor, and water and 10% ethanol elution to effluent color are extremely shallow respectively, remove impurity, uses 40% ethanol elution to effluent colourless again.Elutriant is merged, and decompression recycling ethanol is to there being deposition to separate out, and placement is spent the night, and leaches deposition.Ethyl alcohol recrystallization with 70% obtains the off-white color needle-like crystal, and purity is 99.1%, and total rate of transform is 31.16%.
Embodiment 7
The exsiccant Folium Engelhardia roxburghina is pulverized, and adds 10 times of amount 70% alcohol immersion 3 times, and each 12h crosses and filters soak solution, merges soak solution, and concentrating under reduced pressure gets medicinal extract.The every gram of gained medicinal extract adds acetic acid-sodium-acetate buffer of the pH=5.5 of 50ml; Press enzyme/paste=0.2 and add polygalacturonase, beta-glycosidase and cellulase mixed enzyme (mass ratio 2: 2: 1); 50 ℃ of reaction 24h add 4 times of volume of ethanol termination reactions, centrifugal removal deposition.Supernatant concentration is adsorbed through polyamide column to there not being the alcohol flavor, and water and 10% ethanol elution to effluent color are extremely shallow respectively, remove impurity, uses 40% ethanol elution to effluent colourless again.Elutriant is merged, and decompression recycling ethanol is to there being deposition to separate out, and placement is spent the night, and leaches deposition.Ethyl alcohol recrystallization with 60% obtains the off-white color needle-like crystal, and purity is 99.3%, and total rate of transform is 31.93%.
Embodiment 8
The exsiccant Folium Engelhardia roxburghina is pulverized, placed multi-function extractor, add 80% alcohol reflux 2h of 10 times of amounts for the 1st time, add 80% alcohol reflux 2h of 8 times of amounts for the 2nd, 3 time, united extraction liquid, concentrating under reduced pressure gets paste.The every gram of gained paste adds the citric acid-sodium citrate damping fluid of the pH=5.0 of 50ml, presses enzyme/paste=0.1 and adds naringinase, and 50 ℃ of reaction 48h add 4 times of volume of ethanol termination reactions, centrifugal removal deposition.The supernatant solvent evaporated, ethanol redissolves, and silica gel mixed sample dries naturally; Through silica gel column chromatography, sample/silica gel mass ratio 1: 5, ETHYLE ACETATE: 10: 1 wash-outs of ethanol, TLC follows the tracks of detection; Merge the cut that contains Taxifolin, reclaim solvent, use 45% acetone recrystallization again; Obtain the off-white color needle-like crystal, purity is 99.1%, and total rate of transform is 31.20%.
Embodiment 9
The exsiccant Folium Engelhardia roxburghina is pulverized, placed multi-function extractor, add 50% alcohol reflux 2h of 10 times of amounts for the 1st time, add 50% alcohol reflux 2h of 8 times of amounts for the 2nd, 3 time, united extraction liquid, concentrating under reduced pressure gets paste.The every gram of gained paste adds acetic acid-sodium-acetate buffer of the pH=5.5 of 50ml; Press enzyme/paste=3 and add beta-glycosidase, naringinase, cellulase mixed enzyme (mass ratio 1: 1: 3); 50 ℃ of reaction 4h add 4 times of volume of ethanol termination reactions, centrifugal removal deposition.Supernatant concentration is adsorbed through the HPD-100 macroporous resin to there not being the alcohol flavor, and water and 10% ethanol elution to effluent color are extremely shallow respectively, remove impurity, uses 40% ethanol elution to effluent colourless again.Elutriant is merged, and decompression recycling ethanol is to there being deposition to separate out, and placement is spent the night, and leaches deposition.Recrystallizing methanol with 50% obtains the off-white color needle-like crystal, and purity is 99.2%, and total rate of transform is 32.96%.
The exsiccant Folium Engelhardia roxburghina is pulverized, placed multi-function extractor, add 90% alcohol reflux 2h of 10 times of amounts for the 1st time, add 60% alcohol reflux 2h of 8 times of amounts for the 2nd, 3 time, united extraction liquid, concentrating under reduced pressure gets paste.The every gram of gained paste adds Sodium phosphate, dibasic-citrate buffer solution of the pH=5.0 of 50ml, presses enzyme/paste=0.1 and adds beta-glycosidase, and 50 ℃ are reacted 36h down, add 4 times of volume of ethanol termination reactions, centrifugal removal deposition.Supernatant concentration is to there not being the alcohol flavor, and at first through petroleum ether degreasing, it is extremely shallow to be extracted to the extraction liquid color repeatedly with the ETHYLE ACETATE of 2 times of volumes subsequently, and combining extraction liquid has been evaporated to solid and has separated out, and placement is spent the night, and leaches deposition.Precipitate the ethyl alcohol recrystallization with 70%, obtain the off-white color needle-like crystal, purity is 99.1%, and total rate of transform is 32.26%.
The exsiccant Folium Engelhardia roxburghina is pulverized, 70% ethanol ultrasonic extraction of 3 times of amounts 2 times, each 1h, united extraction liquid, concentrating under reduced pressure gets paste.The every gram of gained paste adds Sodium phosphate, dibasic-citrate buffer solution of the pH=5.0 of 50ml, presses enzyme/paste=0.5 and adds cellulase, and 50 ℃ are reacted 24h down, add 4 times of volume of ethanol termination reactions, centrifugal removal deposition.Supernatant concentration is to there not being the alcohol flavor, and at first through petroleum ether degreasing, it is extremely shallow to be extracted to the extraction liquid color repeatedly with the propyl carbinol of 2 times of volumes subsequently, and combining extraction liquid has been evaporated to solid and has separated out, and placement is spent the night, and leaches deposition.Precipitate the ethyl alcohol recrystallization with 60%, obtain the off-white color needle-like crystal, purity is 99.2%, and total rate of transform is 32.19%.
The exsiccant Folium Engelhardia roxburghina is pulverized, placed multi-function extractor, add 90% alcohol reflux 2h of 10 times of amounts for the 1st time, add 60% alcohol reflux 2h of 8 times of amounts for the 2nd, 3 time, united extraction liquid, concentrating under reduced pressure gets paste.The every gram of gained paste adds Sodium phosphate, dibasic-citrate buffer solution of the pH=6 of 50ml; The mixture (both mass ratioes are 3: 2) that adds naringinase and polygalacturonase by enzyme/paste=0.2; 50 ℃ are reacted 24h down, add 4 times of volume of ethanol termination reactions, centrifugal removal deposition.Supernatant concentration is adsorbed through the D201 macroporous resin column to there not being the alcohol flavor, and water and 10% ethanol elution to effluent color are extremely shallow respectively; Remove impurity, use 40% ethanol elution to effluent colourless again, concentrate eluant is to there being solid to separate out; Placement is spent the night, and leaches deposition, the acetone recrystallization with 40%; Obtain the off-white color needle-like crystal, purity is 99.5%, and total rate of transform is 32.19%.
The exsiccant Folium Engelhardia roxburghina is pulverized, placed multi-function extractor, add 70% alcohol reflux 2h of 10 times of amounts for the 1st time, add 70% alcohol reflux 2h of 8 times of amounts for the 2nd, 3 time, united extraction liquid, concentrating under reduced pressure gets paste.The every gram of gained paste adds Sodium phosphate, dibasic-citrate buffer solution of the pH=5.0 of 50ml, presses enzyme/paste=0.1 and adds beta-glycosidase, and 50 ℃ are reacted 24h down, add 4 times of volume of ethanol termination reactions, centrifugal removal deposition.Supernatant concentration is adsorbed through the NK-II macroporous resin to there not being the alcohol flavor, and water and 10% ethanol elution to effluent color are extremely shallow respectively, remove impurity, uses 40% ethanol elution to effluent colourless again.Elutriant is merged, and decompression recycling ethanol is to there being deposition to separate out, and placement is spent the night, and leaches deposition.Precipitate the recrystallizing methanol with 60%, obtain the off-white color needle-like crystal, purity is 99.4%, and total rate of transform is 33.06%.
Embodiment 14
The exsiccant Folium Engelhardia roxburghina is pulverized, placed multi-function extractor, add 70% alcohol reflux 2h of 10 times of amounts for the 1st time, add 70% alcohol reflux 2h of 8 times of amounts for the 2nd, 3 time, united extraction liquid, concentrating under reduced pressure gets paste.The every gram of gained paste adds Sodium phosphate, dibasic-citrate buffer solution of the pH=5.0 of 50ml, presses enzyme/paste=0.3 and adds polygalacturonase, and 50 ℃ are reacted 24h down, add 4 times of volume of ethanol termination reactions, centrifugal removal deposition.The supernatant solvent evaporated, ethanol redissolves, and silica gel mixed sample dries naturally; Through silica gel column chromatography, sample/silica gel mass ratio 1: 10, chloroform: 9: 1 wash-outs of methyl alcohol, TLC follows the tracks of detection; Merge the cut that contains Taxifolin, reclaim solvent, use 45% acetone recrystallization again; Obtain the off-white color needle-like crystal, purity is 99.3%, and total rate of transform is 28.96%.
Claims (14)
1. one kind prepares the Taxifolin monomer methods from Folium Engelhardia roxburghina, may further comprise the steps:
A, extraction: dry Folium Engelhardia roxburghina is pulverized or segment, is added solvent extraction, extracting solution concentrate medicinal extract.
B, enzymolysis:, add an amount of enzyme at a certain temperature and carry out enzyme digestion reaction with of the damping fluid suspendible or the dissolving of above-mentioned medicinal extract in the ratio adding certain pH value of every gram adding 30-100ml.
C, isolation and purification: the removal of impurities of enzymolysis solution alcohol precipitation, get supernatant or filtrating, supernatant or filtrating are concentrated into does not have the alcohol flavor.Gained supernatant or filtrating adopt certain separation purification method can obtain the Taxifolin bullion.
D, recrystallization: the Taxifolin bullion promptly gets the Taxifolin monomer of purity＞99% through recrystallization.
2. preparation method according to claim 1 is characterized in that: process for extracting can be reflux, ultrasonic, ooze rumble, immersion etc., wherein preferred refluxing extraction; Solvent for use is that mass percent concentration is the ethanol of 30%-90%, the ethanol of preferred 50%-80%.
3. preparation method according to claim 1 is characterized in that: the pH value of the damping fluid of enzyme digestion reaction is 3-6 in the B step, and preferred pH value is 4.5-5.5.
4. preparation method according to claim 1 is characterized in that: selected damping fluid can be any one in Sodium phosphate, dibasic-citrate buffer solution, phosphate buffered saline buffer, acetic acid-sodium-acetate buffer, citric acid-sodium citrate damping fluid and the Hydrocerol A-sodium hydroxide-hydrochloride buffer in the B step.
5. preparation method according to claim 1 is characterized in that: used enzyme can be one or more mixing in beta-glycosidase, cellulase, Snailase, naringinase, the polygalacturonase in the B step.Wherein preferred beta-glycosidase and cellulase.
6. preparation method according to claim 1 is characterized in that: add enzyme by enzyme/paste quality ratio=0.01-5 in the B step and carry out enzymolysis, wherein preferred enzyme/paste quality ratio=0.1-0.5.The temperature of reaction is 15-70 ℃, and preferred 37-50 ℃, the reaction times is 4-72h.
7. preparation method according to claim 1 is characterized in that: the alcohol precipitation removal of impurities in the C step, used alcohol are ethanol or methyl alcohol.
8. preparation method according to claim 1 is characterized in that: the certain separation purification method in the C step can adopt the separation purification method of organic solvent extraction.Enzymolysis supernatant or filtrating are earlier with sherwood oil or the polar phase low polar organic solvent degreasing of working as with it; And then with ETHYLE ACETATE, propyl carbinol or with it the polar phase organic solvent of working as extract repeatedly fully; Concentrated extract is to there being solid to separate out; Placement is spent the night, and leaches solid and is the Taxifolin bullion.
9. preparation method according to claim 1 is characterized in that: the certain separation purification method in the C step can adopt the method for chromatographic separation and purification.Its chromatography separation media comprises macroporous resin, polymeric amide and silica gel.Wherein used macroporous resin model can be D101, D201, HPD-100, HPD-200, AB-8, NK-9, NK-II, any one among the FL-1.Wherein the preferred resin model is FL-1, D101, HPD-100 and AB-8.
10. method according to claim 9; It is characterized in that: the concrete separation purification method of macroporous resin and polyamide column for the supernatant after the hydrolysis or filtrating with the flow velocity of 2～4 times of column volumes through macroporous adsorptive resins or polyamide column, saturated up to absorption; With pure water and 10% ethanol elution impurity, closely colourless up to the wash fluid; Use 40% ethanol elution to Taxifolin wash-out complete again, collect the ethanolic soln elutriant, reclaim ethanol, continue to concentrate and reclaim solvent to there being solid to separate out, this solid is the Taxifolin bullion.
11. method according to claim 9; It is characterized in that: the method for the separation and purification of silica gel chromatography is with supernatant after the hydrolysis or filtrating evaporate to dryness; It is complete to mix appearance, dress post, organic solvent wash-out or gradient elution to Taxifolin wash-out; Reclaim solvent to there being solid to separate out, this solid is the Taxifolin bullion.
12. method according to claim 11 is characterized in that the used organic solvent of wash-out is the ester of pure and mild low carbon chain of haloalkane, the low carbon chain of sherwood oil, low carbon chain, the ketone of low carbon chain, or the mixture of two kind solvents wherein.Wherein, the haloalkane of low carbon chain comprises methyl halide, halo ethane etc., like trichloromethane, methylene dichloride, ethylene dibromide, tribromoethane etc.The alcohol of low carbon chain refers to C
1-3Alcohol, like methyl alcohol, ethanol, propyl alcohol, Virahol etc.The ester of low carbon chain refers to C
1-5Ester, like methyl-formiate, ethyl formate, methyl acetate, ETHYLE ACETATE etc.The ketone of low carbon chain refers to C
1-5Ketone, like acetone, butanone etc.
13. preparation method according to claim 1 is characterized in that: recrystallization solvent is the mixing solutions of ethanol, methyl alcohol, acetone and water.
14. the monomeric purposes of Taxifolin is characterized in that: be used for the prevention and the treatment of fatty liver.
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| WO2020262703A1 (en) * | 2019-06-25 | 2020-12-30 | 独立行政法人国立病院機構 | Hepatic fibrosis-inhibiting agent and brown fat cell-activating agent containing taxifolin |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105175378A (en) * | 2015-07-16 | 2015-12-23 | 苏州禾研生物技术有限公司 | Preparation method of taxifolin |
| CN107198080A (en) * | 2016-03-18 | 2017-09-26 | 陈夫 | A kind of medicinal and edible plant extract effervescent tablet and preparation method thereof |
| CN106244644A (en) * | 2016-08-29 | 2016-12-21 | 珀莱雅化妆品股份有限公司 | A kind of method extracting Taxifolin from Acacia farnesiana Willd. bark |
| CN109771411A (en) * | 2018-11-19 | 2019-05-21 | 延边大学 | Dihydroquercetin is used to prepare the purposes in the drug for the treatment of fatty liver |
| WO2020262703A1 (en) * | 2019-06-25 | 2020-12-30 | 独立行政法人国立病院機構 | Hepatic fibrosis-inhibiting agent and brown fat cell-activating agent containing taxifolin |
| JPWO2020262703A1 (en) * | 2019-06-25 | 2020-12-30 | ||
| JP7058031B2 (en) | 2019-06-25 | 2022-04-21 | 独立行政法人国立病院機構 | Hepatic fibrosis inhibitor and brown adipocyte activator containing taxifolin |
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