CN101974059A - Preparation of oleanolic acid structure analogues and semisynthesis method of oleanolic acid - Google Patents
Preparation of oleanolic acid structure analogues and semisynthesis method of oleanolic acid Download PDFInfo
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Abstract
The invention relates to preparation of oleanolic acid structure analogues and a semisynthesis method of oleanolic acid, which belongs to a manufacturing method of a chemical substance or medicament. The invention solves the problem that the traditional extraction and preparation of oleanolic acid has low efficiency due to the limit of the oleanolic acid content in plants, prepares oleanolic acid structure analogues contained in the plants by separation, and further realizes the semisynthesis of the oleanolic acid by using the oleanolic acid structure analogues as raw materials so as to solve the problem of low extraction yield in the prior art. In the method, leaves of olea europaea l. (an oleaceae plant) or fruits of ligustrum lucidum ait. are extracted and separated into two oleanolic acid structure analogues, and the two oleanolic acid structure analogues are used to carry out the semisynthesis of the oleanolic acid. The method comprises the steps of pretreatment, extraction, crude separation, purification and semisynthesis. Thus, the invention ensures the quality of the finished product, saves the production cost and improves the production efficiency.
Description
Technical field
The invention belongs to the manufacture method of chemical substance or medicine, the leaf or glossy privet (the ligustrum lucidum ait.) fruit that are specifically related to Oleaceae plant olea (olea europaea l.) are raw material, behind extraction separation, obtain two kinds of Oleanolic Acid analogs, and be the method for the semi-synthetic Oleanolic Acid of raw material with this analog.
Background technology
Oleanolic Acid is a pentacyclic triterpenoid, is present in the various plants with the form of episome and glycoside.Oleanolic Acid is white needle; Odorless, tasteless, dissolve in methyl alcohol, ethanol, benzene, ether, acetone and chloroform, water-soluble hardly, all unstable to soda acid.Oleanolic Acid has mainly that the liver of protecting falls enzyme, promotes liver cell regeneration, anti-inflammatory, cardiac stimulant, diuresis, effect such as antitumor, also has hypoglycemic, reducing blood-fat, calm effect, is exploitation treatment hepatopathy and effective ingredient such as hypoglycemic.
Oleanolic Acid is the hepatopathy adjuvant, and the clinical infectivity acute icterohepatitis that is used for the treatment of has tangible reduction gpt and removing jaundice effect, improves viral and the symptom chronic persistent hepatitis patient, sign and liver function; Also can be used for psoriatic, rheumatic arthritis, nephritic edema, hepatic ascites, acute, chronic hepatitis, the stomachache stranguria with turbid discharge, metrorrhagia, wound, carbuncle is swollen, soreness of the waist and knees, diseases such as threatened abortion.
At present, Oleanolic Acid is mainly derived from the leaf or glossy privet (the ligustrum lucidum ait.) fruit of Oleaceae plant olea (olea europaea l.), obtains behind the extraction separation purifying.But, because the content of Oleanolic Acid in plant is usually less than 1%, thereby cause its separation and purification difficulty, and the preparation yield is low.
At present, the preparation method for Oleanolic Acid has, and: Wang Zhongzhuan equals to be called " a kind of novel method of producing Oleanolic Acid " in publication number CN1303850, name in 1999 and adopts: raw material through 70% ethanol heat extract, Acid precipitation separates total saponin, the total saponin of separate acid hydrolysis, make with extra care steps such as purifying and obtain Oleanolic Acid;
Zou Yuansheng equals to be called " a kind of novel method of extraction separation Oleanolic Acid " in employing in publication number CN1552722, name in 2004: with Fructus Hippophae, seabuckthorn fruit peel slag, sea-buckthorn seed or Folium Hippophae is raw material, through supercritical extraction, membrane filtration obtains Oleanolic Acid after drying under reduced pressure or the spraying drying; The supercritical extraction step that this method adopts is difficult for amplifying, and the product purity that obtains is lower;
Wang Youcai was called employing in " method of extraction Oleanolic Acid from cockle pawpaw " in application number 200610116160, name in 2006: the dried powder after cockle pawpaw or its pulverizing be impregnated in the aqueous ethanolic solution, diacolation then, collect percolate, filter, the filtrate decompression distillation gets medicinal extract, medicinal extract adds anhydrous alcohol solution, add gac, decolouring, the silica gel separation obtains Oleanolic Acid; This method leaching process efficient is lower and consuming time, and this technology utilization activated carbon decolorizing causes the partial loss of main component.
Hu Changqiang equals to be called " a kind of method of extracting high purity oleanolic acid " in application number 200910273265, name in 2009 and adopts: with the wooden skin of Aralia is raw material, acidolysis, and alkaline hydrolysis, alcohol immersion obtains finished product behind the Acid precipitation; This technology is used soda acid in a large number, and environmental pollution is more serious, and efficient is lower.
Liu Dongfeng equals to be called " a kind of preparation method of Oleanolic Acid " in employing in application number 201010111779, name in 2010: with the Glossy Privet Fruit medicinal material is raw material, the supersaturation limewater extraction, be acidified with acid, suction filtration gets crude product, crude product extracts with methanol eddy, filtered while hot, nanofiltration concentrates, and drying obtains product; The same a large amount of use soda acids of this process, and nanofiltration process cost height, it is difficult to amplify.
At present, also have many about extraction, purifying, prepare the method for Oleanolic Acid, but these methods all are subjected to the lower effects limit of Oleanolic Acid content in plant material basically, thereby efficient is not high, raw material availability is lower.
Summary of the invention
The invention provides a kind of preparation of Oleanolic Acid analog and the method for semi-synthetic Oleanolic Acid thereof, solve existing extraction and preparation Oleanolic Acid owing to be subjected to its content restriction in plant, and cause the problem of inefficiency, by separating the Oleanolic Acid analog that contains in the preparation plant, and, solve the low problem of prior art extract yield as the semi-synthetic Oleanolic Acid of raw material.
The preparation of a kind of Oleanolic Acid analog of the present invention and the method for semi-synthetic Oleanolic Acid thereof, leaf or glossy privet (ligustrum lucidum ait.) fruit with Oleaceae plant olea (olea europaea l.) are raw material, behind extraction separation, obtain two kinds of Oleanolic Acid analogs, and be the semi-synthetic Oleanolic Acid of raw material with this analog, comprising:
(1) raw material pre-treatment step; After the raw material pulverizing, add the aqueous solution that contains cellulase and polygalacturonase and carry out the broken wall reaction, obtain extracting raw material after the oven dry;
(2) extraction step; The extraction raw material that step (1) is obtained, 50～80% extraction using alcohols, 60～90 ℃ are extracted down, solid-liquid ratio 1: 5～1: 10 extracts three times, behind the united extraction liquid, add the chitosan precipitation, the quality that chitosan adds and the volume ratio of extracting solution are 1～3: 100 (mass volume ratios), filter, and the filtrate evaporate to dryness gets extract;
(3) roughing out step; With the extract that step (2) obtains, after separating, the process silicagel column obtains the roughing out product;
(4) purification step; To the roughing out product that the roughing out step obtains, adopt Sephadex LH-20 post refining, obtain Oleanolic Acid two kinds of analog 12-OH-Oleanolic Acid and Oleanolic Acid methyl esters simultaneously;
(5) semi-synthetic step; In nonionic weakly alkaline solvent, is dewatering agent with the strong lewis acid with the 12-OH-Oleanolic Acid that obtains in the step (4), carries out obtaining Oleanolic Acid after the dehydration reaction; With the Oleanolic Acid methyl esters that obtains in the step (4), join in a certain amount of water and dissolve, add acid hydrolysis solution, carry out at a certain temperature obtaining Oleanolic Acid behind the acid hydrolytic reaction.
In the described raw material pre-treatment step, the aqueous solution of described cellulase and polygalacturonase is: the volume of the aqueous solution and the mass ratio of raw material are 0.5～2: 1 (volume mass ratio), the amount of cellulase and the mass ratio of raw material are 0.5～3: 1000 (mass ratioes), and the quality of polygalacturonase and the mass ratio of raw material are 0.5～2: 1000 (mass ratioes); Described broken wall reaction is: 30～60 ℃ of temperature of reaction, 1～3 hour reaction times.
In the described roughing out step, described silicagel column is: be filled with silica gel based filler, 5: 1～20: 1 device of aspect ratio, the silica gel based filler is made up of silica gel and diatomite, and the mass percent of diatomite and silica gel is 10%～20%; The silica gel particle diameter is 30 μ m～80 μ m, and the silica gel activity is II～III level; Described through the silicagel column separation, its process is: sample is to silicagel column, with methyl alcohol on the extract that step (2) is obtained: normal hexane=90: 10～98: 2 (V/V) is an elutriant, and flow velocity 0.5～2 column volume per hour, collect positive component, evaporate to dryness obtains the roughing out product.
Described purification step is: with the roughing out product that obtains, utilize Sephadex LH-20 post refining, behind the last sample, methanol aqueous solution with 40～80% (V/V) wash-out, flow velocity be 0.5～2 column volume per hour, Fractional Collections obtains 12-OH-Oleanolic Acid and Oleanolic Acid methyl esters respectively behind the concentrated evaporate to dryness.
In the described semi-synthetic step, described strong lewis acid is BF
3, SOCl
2, POCl
3, C
2O
2Cl
2Wherein a kind of; Described nonionic weakly alkaline solvent is wherein a kind of of pyridine, dimethyl formamide, trolamine, N-xylidene(s), hexamethylenetetramine; Its process of described dehydration reaction is: the 12-OH-Oleanolic Acid that obtains in the step (4) is dissolved in the nonionic weakly alkaline solvent, under nitrogen protection, slowly be added drop-wise in the strong lewis acid, 4～30 ℃, reacted 6～12 hours, reaction finishes the back and adds frozen water, and ammoniacal liquor is regulated pH 9.0～12.0, ether or n-hexane extraction three times obtain Oleanolic Acid behind the combining extraction liquid evaporate to dryness; The volume ratio of the quality of 12-OH-Oleanolic Acid and nonionic weakly alkaline solvent is 1: 5～1: 20 (mass volume ratio), and the volume ratio of strong lewis acid and nonionic weakly alkaline solvent is 1: 5～1: 10; The volume ratio of nonionic weakly alkaline solvent and frozen water is 1: 3～1: 5.
In the described semi-synthetic step, described joining in a certain amount of water is dissolved as: the volume of institute's water and the mass ratio of Oleanolic Acid methyl esters are 5～20: 1 (volume mass ratio); Described acid hydrolysis solution is: 3～6mol/LHCl, SOCl
2, C
2Cl
2O
2In a kind of, its volume is 1: 2～1: 8 (volume ratio) with the ratio of the volume of institute water; Carry out acid hydrolytic reaction under the described certain temperature, its process is: under 60～95 ℃, and stirring reaction 2～8 hours, reaction finishes back ammoniacal liquor and regulates pH9.0～12.0, and ether or n-hexane extraction three times obtain Oleanolic Acid behind the combining extraction liquid evaporate to dryness.
The invention has the advantages that, conception rationally, method is easy, promote easily, can solve the existing practical problems of extracting and preparing the Oleanolic Acid inefficiency, realized utilizing the Oleanolic Acid analog in the leaching process,, thereby solved the existing low problem of Oleanolic Acid bulk drug extract yield by semi-synthetic Oleanolic Acid.The present invention includes raw material pre-treatment step, extraction step, roughing out step, purification step, semi-synthetic step, guaranteed the quality of the final product of utilization the present invention preparation, saved production cost, improved production efficiency.The present invention is a raw material with the leaf or glossy privet (the ligustrum lucidum ait.) fruit of Oleaceae plant olea (olea europaea l.), behind extraction separation, obtain two kinds of Oleanolic Acid analogs, and be the semi-synthetic Oleanolic Acid of raw material with this analog.Technology provided by the invention is easy and be easy to be amplified to suitability for industrialized production, can improve raw material availability greatly simultaneously.
Description of drawings
Fig. 1 is the semi-synthetic step synoptic diagram of Oleanolic Acid analog.
Embodiment
Fig. 1 is the semi-synthetic step synoptic diagram of Oleanolic Acid analog; In nonionic weakly alkaline solvent, is dewatering agent with the strong lewis acid with the 12-OH-Oleanolic Acid that obtains in the step (4), carries out obtaining Oleanolic Acid after the dehydration reaction; With the Oleanolic Acid methyl esters that obtains in the step (4), join in a certain amount of water and dissolve, add acid hydrolysis solution, carry out at a certain temperature obtaining Oleanolic Acid behind the acid hydrolytic reaction.
Embodiment 1
(1) raw material pre-treatment step:
Getting Oleaceae plant olea (olea europaea l.) leaf is raw material, behind the oven dry crushing screening, accurately take by weighing 1000g, add the aqueous solution 500ml that contains cellulase 0.50g, polygalacturonase 0.50g, wherein the mass ratio of the volume of the aqueous solution and raw material is 0.5: 1 (a volume mass ratio), the amount of cellulase and the mass ratio of raw material are 0.5: 1000 (mass ratio), the quality of polygalacturonase and the mass ratio of raw material are 0.5: 1000 (mass ratio), be heated to 60 ℃, react after 1 hour, oven dry obtains extracting raw material.
(2) extraction step
Extraction raw material with gained in the step (1), 80% extraction using alcohol, 60 ℃ are extracted down, solid-liquid ratio 1: 10 extracts three times, and united extraction liquid amounts to 30L, add 300g chitosan precipitation, the quality that chitosan adds and the volume ratio of extracting solution are 1: 100 (mass volume ratio), filter, and the filtrate evaporate to dryness gets extract 56.72g.
(3) roughing out step
Be filled with silica gel based filler, 5: 1 silicagel column of aspect ratio, the silica gel based filler is made up of silica gel and diatomite, and the mass percent of diatomite and silica gel is 10%; The silica gel particle diameter is 30 μ m, and the silica gel activity is the II level.The extract 56.72g that step (2) is obtained goes up sample to silicagel column, and with methyl alcohol: normal hexane=90: 10 (V/V) is an elutriant, and flow velocity 2 column volumes are per hour collected positive component, and evaporate to dryness obtains roughing out product 28.90g.
(4) purification step
Roughing out product 28.90g with obtaining is dissolved in the 20.0ml methyl alcohol, and last sample is to Sephadex LH-20, methanol aqueous solution with 40% (V/V) wash-out, flow velocity be 1 column volume per hour, Fractional Collections, concentrate behind the evaporate to dryness respectively 12-OH-Oleanolic Acid 7.79g and Oleanolic Acid methyl esters 6.35g.
(5) semi-synthetic step
The 12-OH-Oleanolic Acid 7.79g that obtains in the step (4) is dissolved in the 155.80ml dimethyl formamide, under nitrogen protection, slowly is added drop-wise to 15.58ml SOCl
2In, 4 ℃ were reacted 12 hours down, and reaction finishes the back and adds frozen water 779ml, ammoniacal liquor adjusting pH 12.0, extracted with diethyl ether three times obtains Oleanolic Acid 7.08g behind the combining extraction liquid evaporate to dryness.Wherein, the volume ratio of the quality of 12-OH-Oleanolic Acid and nonionic weakly alkaline solvent is 1: 20 (mass volume ratio), and the volume ratio of strong lewis acid and nonionic weakly alkaline solvent is 1: 10, and the volume ratio of nonionic weakly alkaline solvent and frozen water is 1: 5.
The Oleanolic Acid methyl esters 6.35g that obtains in the step (4) is dissolved in the 127ml water, the volume of institute's water and the mass ratio of Oleanolic Acid methyl esters are 20: 1 (volume mass ratio), add 6mol/LHCl 63.50ml, its volume is 1: 2 (volume ratio) with the ratio of the volume of institute water, under 95 ℃, stirring reaction 2 hours, reaction finishes back ammoniacal liquor and regulates pH 9.0, n-hexane extraction three times obtains Oleanolic Acid 5.88g behind the combining extraction liquid evaporate to dryness.
Embodiment 2
(1) raw material pre-treatment step:
Get glossy privet (ligustrum lucidum ait.) fruit raw material, behind the oven dry crushing screening, accurately take by weighing 2000g, add the aqueous solution 4000ml that contains cellulase 6.0g, polygalacturonase 4.0g, wherein the mass ratio of the volume of the aqueous solution and raw material is 2: 1 (a volume mass ratio), the amount of cellulase and the mass ratio of raw material are 3: 1000 (mass ratio), the quality of polygalacturonase and the mass ratio of raw material are 2: 1000 (mass ratio), are heated to 30 ℃, react after 3 hours, oven dry obtains extracting raw material.
(2) extraction step
Extraction raw material with gained in the step (1), 50% extraction using alcohol, 90 ℃ are extracted down, solid-liquid ratio 1: 5 extracts three times, and united extraction liquid amounts to 30L, add 900g chitosan precipitation, the quality that chitosan adds and the volume ratio of extracting solution are 3: 100 (mass volume ratio), filter, and the filtrate evaporate to dryness gets extract 112.50g.
(3) roughing out step
Be filled with silica gel based filler, 20: 1 silicagel column of aspect ratio, the silica gel based filler is made up of silica gel and diatomite, and the mass percent of diatomite and silica gel is 20%; The silica gel particle diameter is 80 μ m, and the silica gel activity is the III level.The extract 112.50g that step (2) is obtained goes up sample to silicagel column, and with methyl alcohol: normal hexane=98: 2 (V/V) is an elutriant, and flow velocity 0.5 column volume is per hour collected positive component, and evaporate to dryness obtains roughing out product 56.60g.
(4) purification step
With the roughing out product 56.60g that obtains, be dissolved in the 30.0ml methyl alcohol, last sample is to Sephadex LH-20, methanol aqueous solution with 80% (V/V) wash-out, flow velocity be 0.5 column volume per hour, Fractional Collections gets 12-OH-Oleanolic Acid 15.15g and Oleanolic Acid methyl esters 12.10g respectively behind the concentrated evaporate to dryness.
(5) semi-synthetic step
The 12-OH-Oleanolic Acid 15.15g that obtains in the step (4) is dissolved in the 75.75ml pyridine, under nitrogen protection, slowly is added drop-wise to 15.15ml POCl
3In, 30 ℃ were reacted 6 hours down, and reaction finishes the back and adds frozen water 227.25ml, ammoniacal liquor adjusting pH 9.0, n-hexane extraction three times obtains Oleanolic Acid 13.85g behind the combining extraction liquid evaporate to dryness.Wherein, the volume ratio of the quality of 12-OH-Oleanolic Acid and nonionic weakly alkaline solvent is 1: 5 (mass volume ratio), and the volume ratio of strong lewis acid and nonionic weakly alkaline solvent is 1: 5, and the volume ratio of nonionic weakly alkaline solvent and frozen water is 1: 3.
The Oleanolic Acid methyl esters 12.10g that obtains in the step (4) is dissolved in the 60.50ml water, the volume of institute's water and the mass ratio of Oleanolic Acid methyl esters are 5: 1 (volume mass ratio), add 3mol/L HCl 15.13ml, its volume is 1: 4 (volume ratio) with the ratio of the volume of institute water, under 60 ℃, stirring reaction 8 hours, reaction finishes back ammoniacal liquor and regulates pH 12.0, extracted with diethyl ether three times obtains Oleanolic Acid 11.17g behind the combining extraction liquid evaporate to dryness.
(1) raw material pre-treatment step:
Get the short-tube lycoris raw material, behind the oven dry crushing screening, accurately take by weighing 3000g, add the aqueous solution 3000ml that contains cellulase 3.0g, polygalacturonase 3.0g, wherein the mass ratio of the volume of the aqueous solution and short-tube lycoris raw material is 1: 1 (a volume mass ratio), the mass ratio of the amount of cellulase and short-tube lycoris raw material is 1: 1000 (mass ratio), the quality of polygalacturonase and the mass ratio of short-tube lycoris are 1: 1000 (mass ratio), are heated to 50 ℃, react after 2 hours, oven dry obtains the supercritical extraction raw material.
(2) extraction step
Extraction raw material with gained in the step (1), 60% extraction using alcohol, 80 ℃ are extracted down, solid-liquid ratio 1: 8 extracts three times, and united extraction liquid amounts to 72L, add 1440g chitosan precipitation, the quality that chitosan adds and the volume ratio of extracting solution are 2: 100 (mass volume ratio), filter, and the filtrate evaporate to dryness gets extract 171.45g.
(3) roughing out step
Be filled with silica gel based filler, 10: 1 silicagel column of aspect ratio, the silica gel based filler is made up of silica gel and diatomite, and the mass percent of diatomite and silica gel is 15%; The silica gel particle diameter is 50 μ m, and the silica gel activity is the II level.The extract 171.45g that step (2) is obtained goes up sample to silicagel column, and with methyl alcohol: normal hexane=95: 5 (V/V) is an elutriant, and flow velocity 1 column volume is per hour collected positive component, and evaporate to dryness obtains roughing out product 87.62g.
(4) purification step
With the roughing out product 87.62g that obtains, be dissolved in the 40.0ml methyl alcohol, last sample is to Sephadex LH-20, methanol aqueous solution with 60% (V/V) wash-out, flow velocity be 2 column volumes per hour, Fractional Collections gets 12-OH-Oleanolic Acid 24.35g and Oleanolic Acid methyl esters 19.42g respectively behind the concentrated evaporate to dryness.
(5) semi-synthetic step
The 12-OH-Oleanolic Acid 24.35g that obtains in the step (4) is dissolved in the 243.5ml trolamine, under nitrogen protection, slowly is added drop-wise to 30.44ml BF
3In, 15 ℃ were reacted 8 hours down, and reaction finishes the back and adds frozen water 974ml, ammoniacal liquor adjusting pH 10.0, extracted with diethyl ether three times obtains Oleanolic Acid 20.07g behind the combining extraction liquid evaporate to dryness.Wherein, the volume ratio of the quality of 12-OH-Oleanolic Acid and nonionic weakly alkaline solvent is 1: 10 (mass volume ratio), and the volume ratio of strong lewis acid and nonionic weakly alkaline solvent is 1: 8, and the volume ratio of nonionic weakly alkaline solvent and frozen water is 1: 4.
The Oleanolic Acid methyl esters 19.42g that obtains in the step (4) is dissolved in the 194.20ml water, the volume of institute's water and the mass ratio of Oleanolic Acid methyl esters are 10: 1 (volume mass ratio), add 4mol/L HCl 97.10ml, its volume is 1: 2 (volume ratio) with the ratio of the volume of institute water, under 80 ℃, stirring reaction 6 hours, reaction finishes back ammoniacal liquor and regulates pH 11.0, n-hexane extraction three times obtains Oleanolic Acid 17.82g behind the combining extraction liquid evaporate to dryness.
Embodiment 4
(1) raw material pre-treatment step:
Get the short-tube lycoris raw material, behind the oven dry crushing screening, accurately take by weighing 1500g, add the aqueous solution 750ml that contains cellulase 3.0g, polygalacturonase 3.0g, wherein the mass ratio of the volume of the aqueous solution and short-tube lycoris raw material is 0.5: 1 (a volume mass ratio), the mass ratio of the amount of cellulase and short-tube lycoris raw material is 2: 1000 (mass ratio), the quality of polygalacturonase and the mass ratio of short-tube lycoris are 2: 1000 (mass ratio), are heated to 60 ℃, react after 2 hours, oven dry obtains the supercritical extraction raw material.
(2) extraction step
Extraction raw material with gained in the step (1), 70% extraction using alcohol, 70 ℃ are extracted down, solid-liquid ratio 1: 6 extracts three times, and united extraction liquid amounts to 27L, add 270g chitosan precipitation, the quality that chitosan adds and the volume ratio of extracting solution are 1: 100 (mass volume ratio), filter, and the filtrate evaporate to dryness gets extract 85.57g.
(3) roughing out step
Be filled with silica gel based filler, 15: 1 silicagel column of aspect ratio, the silica gel based filler is made up of silica gel and diatomite, and the mass percent of diatomite and silica gel is 20%; The silica gel particle diameter is 80 μ m, and the silica gel activity is the III level.The extract 85.57g that step (2) is obtained goes up sample to silicagel column, and with methyl alcohol: normal hexane=94: 6 (V/V) is an elutriant, and flow velocity 2 column volumes are per hour collected positive component, and evaporate to dryness obtains roughing out product 43.75g.
(4) purification step
With the roughing out product 43.75g that obtains, be dissolved in the 30.0ml methyl alcohol, last sample is to Sephadex LH-20, methanol aqueous solution with 70% (V/V) wash-out, flow velocity be 1 column volume per hour, Fractional Collections gets 12-OH-Oleanolic Acid 11.83g and Oleanolic Acid methyl esters 9.78g respectively behind the concentrated evaporate to dryness.
(5) semi-synthetic step
The 12-OH-Oleanolic Acid 11.83g that obtains in the step (4) is dissolved in the 59.15ml N-xylidene(s), under nitrogen protection, slowly is added drop-wise to 5.92ml C
2O
2Cl
2In, 20 ℃ were reacted 12 hours down, and reaction finishes the back and adds frozen water 295.75ml, ammoniacal liquor adjusting pH 11.0, n-hexane extraction three times obtains Oleanolic Acid 11.13g behind the combining extraction liquid evaporate to dryness.Wherein, the volume ratio of the quality of 12-OH-Oleanolic Acid and nonionic weakly alkaline solvent is 1: 5 (mass volume ratio), and the volume ratio of strong lewis acid and nonionic weakly alkaline solvent is 1: 10, and the volume ratio of nonionic weakly alkaline solvent and frozen water is 1: 5.
The Oleanolic Acid methyl esters 9.78g that obtains in the step (4) is dissolved in the 97.80ml water, and the volume of institute's water and the mass ratio of Oleanolic Acid methyl esters are 10: 1 (volume mass ratio), add SOCl
212.23ml its volume is 1: 8 (volume ratio) with the ratio of the volume of institute water, under 95 ℃, and stirring reaction 4 hours, reaction finishes back ammoniacal liquor and regulates pH 9.0, and extracted with diethyl ether three times obtains Oleanolic Acid 8.91g behind the combining extraction liquid evaporate to dryness.
Embodiment 5
(1) raw material pre-treatment step:
Get the short-tube lycoris raw material, behind the oven dry crushing screening, accurately take by weighing 2500g, add the aqueous solution 2500ml that contains cellulase 2.5g, polygalacturonase 5.0g, wherein the mass ratio of the volume of the aqueous solution and short-tube lycoris raw material is 1: 1 (a volume mass ratio), the mass ratio of the amount of cellulase and short-tube lycoris raw material is 1: 1000 (mass ratio), the quality of polygalacturonase and the mass ratio of short-tube lycoris are 2: 1000 (mass ratio), are heated to 50 ℃, react after 2 hours, oven dry obtains the supercritical extraction raw material.
(2) extraction step
Extraction raw material with gained in the step (1), 50% extraction using alcohol, 80 ℃ are extracted down, solid-liquid ratio 1: 10 extracts three times, and united extraction liquid amounts to 75L, add 1500g chitosan precipitation, the quality that chitosan adds and the volume ratio of extracting solution are 2: 100 (mass volume ratio), filter, and the filtrate evaporate to dryness gets extract 140.56g.
(3) roughing out step
Be filled with silica gel based filler, 5: 1 silicagel column of aspect ratio, the silica gel based filler is made up of silica gel and diatomite, and the mass percent of diatomite and silica gel is 20%; The silica gel particle diameter is 30 μ m, and the silica gel activity is the II level.The extract 140.56g that step (2) is obtained goes up sample to silicagel column, and with methyl alcohol: normal hexane=92: 8 (V/V) is an elutriant, and flow velocity 1 column volume is per hour collected positive component, and evaporate to dryness obtains roughing out product 73.48g.
(4) purification step
With the roughing out product 73.48g that obtains, be dissolved in the 40.0ml methyl alcohol, last sample is to Sephadex LH-20, methanol aqueous solution with 80% (V/V) wash-out, flow velocity be 0.5 column volume per hour, Fractional Collections gets 12-OH-Oleanolic Acid 19.95g and Oleanolic Acid methyl esters 16.15g respectively behind the concentrated evaporate to dryness.
(5) semi-synthetic step
The 12-OH-Oleanolic Acid 19.95g that obtains in the step (4) is dissolved in the 399.00ml hexamethylenetetramine, under nitrogen protection, slowly is added drop-wise to 39.90ml SOCl
2In, 4 ℃ were reacted 6 hours down, and reaction finishes the back and adds frozen water 1197.00ml, ammoniacal liquor adjusting pH 12.0, extracted with diethyl ether three times obtains Oleanolic Acid 17.15g behind the combining extraction liquid evaporate to dryness.Wherein, the volume ratio of the quality of 12-OH-Oleanolic Acid and nonionic weakly alkaline solvent is 1: 20 (mass volume ratio), and the volume ratio of strong lewis acid and nonionic weakly alkaline solvent is 1: 10, and the volume ratio of nonionic weakly alkaline solvent and frozen water is 1: 3.
The Oleanolic Acid methyl esters 16.15g that obtains in the step (4) is dissolved in the 161.50ml water, and the volume of institute's water and the mass ratio of Oleanolic Acid methyl esters are 10: 1 (volume mass ratio), add C
2Cl
2O
226.92ml its volume is 1: 6 (volume ratio) with the ratio of the volume of institute water, under 75 ℃, and stirring reaction 6 hours, reaction finishes back ammoniacal liquor and regulates pH 10.0, and n-hexane extraction three times obtains Oleanolic Acid 14.80g behind the combining extraction liquid evaporate to dryness.
Claims (6)
1. the method for the preparation of an Oleanolic Acid analog and semi-synthetic Oleanolic Acid thereof, leaf or glossy privet (ligustrum lucidum ait.) fruit with Oleaceae plant olea (olea europaea l.) are raw material, behind extraction separation, obtain two kinds of Oleanolic Acid analogs, and be the semi-synthetic Oleanolic Acid of raw material with this analog, comprising:
(1) raw material pre-treatment step; After the raw material pulverizing, add the aqueous solution that contains cellulase and polygalacturonase and carry out the broken wall reaction, obtain extracting raw material after the oven dry;
(2) extraction step; The extraction raw material that step (1) is obtained, 50～80% extraction using alcohols, 60～90 ℃ are extracted down, solid-liquid ratio 1: 5～1: 10 extracts three times, behind the united extraction liquid, add the chitosan precipitation, the quality that chitosan adds and the volume ratio of extracting solution are 1～3: 100 (mass volume ratios), filter, and the filtrate evaporate to dryness gets extract;
(3) roughing out step; With the extract that step (2) obtains, after separating, the process silicagel column obtains the roughing out product;
(4) purification step; To the roughing out product that the roughing out step obtains, adopt Sephadex LH-20 post refining, obtain Oleanolic Acid two kinds of analog 12-OH-Oleanolic Acid and Oleanolic Acid methyl esters simultaneously;
(5) semi-synthetic step; In nonionic weakly alkaline solvent, is dewatering agent with the strong lewis acid with the 12-OH-Oleanolic Acid that obtains in the step (4), carries out obtaining Oleanolic Acid after the dehydration reaction; With the Oleanolic Acid methyl esters that obtains in the step (4), join in a certain amount of water and dissolve, add acid hydrolysis solution, carry out at a certain temperature obtaining Oleanolic Acid behind the acid hydrolytic reaction.
2. the preparation of a kind of Oleanolic Acid analog as claimed in claim 1 and the method for semi-synthetic Oleanolic Acid thereof, it is characterized in that: in the described raw material pre-treatment step, the aqueous solution of described cellulase and polygalacturonase is: the volume of the aqueous solution and the mass ratio of raw material are 0.5～2: 1 (volume mass ratio), the amount of cellulase and the mass ratio of raw material are 0.5～3: 1000 (mass ratioes), and the quality of polygalacturonase and the mass ratio of raw material are 0.5～2: 1000 (mass ratioes); Described broken wall reaction is: 30～60 ℃ of temperature of reaction, 1～3 hour reaction times.
3. the preparation of a kind of Oleanolic Acid analog as claimed in claim 1 and the method for semi-synthetic Oleanolic Acid thereof, it is characterized in that: in the described roughing out step, described silicagel column is: be filled with silica gel based filler, 5: 1～20: 1 device of aspect ratio, the silica gel based filler is made up of silica gel and diatomite, and the mass percent of diatomite and silica gel is 10%～20%; The silica gel particle diameter is 30 μ m～80 μ m, and the silica gel activity is II～III level; Described through the silicagel column separation, its process is: sample is to silicagel column, with methyl alcohol on the extract that step (2) is obtained: normal hexane=90: 10～98: 2 (V/V) is an elutriant, and flow velocity 0.5～2 column volume per hour, collect positive component, evaporate to dryness obtains the roughing out product.
4. the preparation of a kind of Oleanolic Acid analog as claimed in claim 1 and the method for semi-synthetic Oleanolic Acid thereof, it is characterized in that: described purification step is: with the roughing out product that obtains, utilize Sephadex LH-20 post refining, behind the last sample, methanol aqueous solution with 40～80% (V/V) wash-out, flow velocity be 0.5～2 column volume per hour, Fractional Collections obtains 12-OH-Oleanolic Acid and Oleanolic Acid methyl esters respectively after concentrating evaporate to dryness.
5. the preparation of a kind of Oleanolic Acid analog as claimed in claim 1 and the method for semi-synthetic Oleanolic Acid thereof is characterized in that: in the described semi-synthetic step, described strong lewis acid is BF
3, SOCl
2, POCl
3, C
2O
2Cl
2Wherein a kind of; Described nonionic weakly alkaline solvent is wherein a kind of of pyridine, dimethyl formamide, trolamine, N-xylidene(s), hexamethylenetetramine; Its process of described dehydration reaction is: the 12-OH-Oleanolic Acid that obtains in the step (4) is dissolved in the nonionic weakly alkaline solvent, under nitrogen protection, slowly be added drop-wise in the strong lewis acid, 4～30 ℃, reacted 6～12 hours, reaction finishes the back and adds frozen water, and ammoniacal liquor is regulated pH 9.0～12.0, ether or n-hexane extraction three times obtain Oleanolic Acid behind the combining extraction liquid evaporate to dryness; The volume ratio of the quality of 12-OH-Oleanolic Acid and nonionic weakly alkaline solvent is 1: 5～1: 20 (mass volume ratio), and the volume ratio of strong lewis acid and nonionic weakly alkaline solvent is 1: 5～1: 10; The volume ratio of nonionic weakly alkaline solvent and frozen water is 1: 3～1: 5.
6. the preparation of a kind of Oleanolic Acid analog as claimed in claim 1 and the method for semi-synthetic Oleanolic Acid thereof, it is characterized in that: in the described semi-synthetic step, described joining in a certain amount of water is dissolved as: the volume of institute's water and the mass ratio of Oleanolic Acid methyl esters are 5～20: 1 (volume mass ratio); Described acid hydrolysis solution is: 3～6mol/L HCl, SOCl
2, C
2Cl
2O
2In a kind of, its volume is 1: 2～1: 8 (volume ratio) with the ratio of the volume of institute water; Carry out acid hydrolytic reaction under the described certain temperature, its process is: under 60～95 ℃, and stirring reaction 2～8 hours, reaction finishes back ammoniacal liquor and regulates pH 9.0～12.0, and ether or n-hexane extraction three times obtain Oleanolic Acid behind the combining extraction liquid evaporate to dryness.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013079018A1 (en) * | 2011-12-01 | 2013-06-06 | 杭州本生药业有限公司 | 2-substituted oleanolic acid derivative, method preparing for same, and application thereof |
| CN103945852A (en) * | 2011-12-01 | 2014-07-23 | 杭州本生药业有限公司 | 2-substituted oleanolic acid derivative, method preparing for same, and application thereof |
| CN111789873A (en) * | 2020-08-04 | 2020-10-20 | 中国科学院西北高原生物研究所 | Method for extracting high-content seabuckthorn triterpenic acid extract |
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|---|---|---|---|---|
| CN1303850A (en) * | 1999-10-26 | 2001-07-18 | 上海长海医院 | Method for producing oleanolic acid |
| CN1449372A (en) * | 2000-08-08 | 2003-10-15 | 日清奥利友株式会社 | Process for producing oleanolic acid and/or maslinic acid |
| CN1552722A (en) * | 2003-05-26 | 2004-12-08 | 河北神兴沙棘研究院 | Method for extracting and separating oleanolic acid |
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2010
- 2010-09-10 CN CN 201010278123 patent/CN101974059A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1303850A (en) * | 1999-10-26 | 2001-07-18 | 上海长海医院 | Method for producing oleanolic acid |
| CN1449372A (en) * | 2000-08-08 | 2003-10-15 | 日清奥利友株式会社 | Process for producing oleanolic acid and/or maslinic acid |
| CN1552722A (en) * | 2003-05-26 | 2004-12-08 | 河北神兴沙棘研究院 | Method for extracting and separating oleanolic acid |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013079018A1 (en) * | 2011-12-01 | 2013-06-06 | 杭州本生药业有限公司 | 2-substituted oleanolic acid derivative, method preparing for same, and application thereof |
| CN103945852A (en) * | 2011-12-01 | 2014-07-23 | 杭州本生药业有限公司 | 2-substituted oleanolic acid derivative, method preparing for same, and application thereof |
| US9156801B2 (en) | 2011-12-01 | 2015-10-13 | Hangzhou Bensheng Pharmaceutical Co., Ltd. | 2-substituted oleanolic acid derivative, method preparing for same, and application thereof |
| CN103945852B (en) * | 2011-12-01 | 2016-10-19 | 杭州本生药业有限公司 | The substituted oleanolic acid derivate in 2-position, and its preparation method and application |
| CN111789873A (en) * | 2020-08-04 | 2020-10-20 | 中国科学院西北高原生物研究所 | Method for extracting high-content seabuckthorn triterpenic acid extract |
| CN111789873B (en) * | 2020-08-04 | 2022-05-27 | 中国科学院西北高原生物研究所 | Method for extracting high-content seabuckthorn triterpenic acid extract |
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