CN103044417A - Novel technology method for purifying berberine in wild berberis sibirica - Google Patents
Novel technology method for purifying berberine in wild berberis sibirica Download PDFInfo
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- CN103044417A CN103044417A CN2012105956385A CN201210595638A CN103044417A CN 103044417 A CN103044417 A CN 103044417A CN 2012105956385 A CN2012105956385 A CN 2012105956385A CN 201210595638 A CN201210595638 A CN 201210595638A CN 103044417 A CN103044417 A CN 103044417A
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Abstract
The invention belongs to the field of natural organic chemistry, and relates to a method for extracting and purifying berberine from wild berberis roots in great khingan, in particular to a novel method for purifying berberis by a biological enzymolysis, ultrasonic extraction and column chromatography purification combined technique. The method has the advantages that 1, a biological enzymolysis technique is applied to allow that berberine in cells is dissolved out fully; 2, an ultrasonic extraction technique is adopted; the heating time for an effective component is shortened; the activity loss is less; the bioactivity of a product is high; and 3, a silica gel column chromatography and recrystallization purification technique is adopted, so that the purity of the effective component, namely berberine is further increased; the operation is simple and easy; the production cost is lowered greatly; and the method can be applicable to industrial use. According to the method, the biological enzymolysis technique is adopted, the effective component is dissolved out as much as possible, then ultrasonic extraction is conducted, alcohol serves as a solvent, then column chromatography purification is conducted, and finally recrystallization is conducted for secondary purification, so that the purity of berberine is increased to 98.4%.
Description
Technical field:
The invention belongs to the natural organic chemistry field, relate to a kind of method of extracting the purifying Berberine in the wild Root of Chinese Barberry in Daxing'an Mountainrange, particularly relate to a kind of biological enzymolysis, ultrasonic extraction column chromatography purification technology, novel method of purifying Berberine utilized.
Background technology:
Thorn leaf barberry, another name northeast Root of Chinese Barberry, machaka is born in high height above sea level chad hillside.Originate in Daxing'an Mountainrange and get the ground such as the large bald mountain of boolean forestry bureau and Tahe Forestry Bureau.Up to more than 100 centimetres.Bark dull gray black has thin longitudinal furrow on the branch; The branch taupe has the thorns of 5～8 forks in the base portion of brachyplast; The thorn bifurcated of branch bottom is more, and is thin and sharp, is lead.Leafage is born on the brachyplast, and obovate is fallen lanceolar or the shape of falling ovum Long Circle, and long 1～1.8 centimetre of leaf is wide 5～9 millimeters, the base portion wedge shape, and tip is justified or is blunt nosed, and the edge has fine acicular to dredge teeth, and the leaf two sides is yellow-green colour, and the food value of leaf is keratin more a little; Sepal 6 has 2～3 little buds under it.Hua Dan is born in the brachyplast end leafage, and 2 flowers are seldom arranged, and bennet is about 1 centimetre, and flower is pistac; Petal 6 is little than sepal, base portion tool 2 sweet arteries and veins; Stamen 6 touches its thing and then plays vibration, and medicine is 2 to split.Berry, obovate is about 8 millimeters, ripe rear garnet, June at florescence; The fruit September phase.
Composition: rhizome contains Berbamine, berbamunine, jateorhizine, magnoflorine, Berberine, palmatine, the different sinomenine of controlling, palmatine, oxyberberine, vinetine etc.In addition, complete stool all contains alkaloid, tonka bean camphor, flavones, vanillic acid, forulic acid etc.Berberine has the multiple efficacies such as calmness, anti-inflammatory, antibiotic, anti-diabetic, antiulcer agent, lowering blood glucose.
Domestic extraction Berberine technique mainly is that acid extraction method and direct purification of ethanol decoction form by the salt Acid precipitation, and its purification effect is nothing like the utilization biological enzymolysis, and intensified by ultrasonic wave extracts the effect of carrying out again silica gel column chromatography.
Summary of the invention:
The object of the invention is to overcome the shortcomings such as the routine techniques extraction yield is lower, product purity is low, a kind of biological enzymolysis that utilizes is provided, intensified by ultrasonic wave extracts the method for carrying out again purification by silica gel column chromatography Root of Chinese Barberry Berberine, for achieving the above object, the technical solution used in the present invention is: a kind of biological enzymolysis lotus root connection intensified by ultrasonic wave that utilizes extracts, the novel method of column chromatography purification Berberine again, its step is as follows:
(1) enzymolysis:
After Root of Chinese Barberry is pulverized, adopt complex cellulase, at 40-50 ℃ of lower enzymolysis, insulation precipitation 1-3h;
(2) supersound extraction:
Add ethanol, ultrasonic extraction, ultrasonic frequency 50-80KHZ, solid-liquid ratio 1: 8-10, ultrasonic time 50-80min, thin-layer chromatography detect and analyze, and Recycled ethanol gets crude extract;
(3) get meal:
With obtaining primary extract ethanol heating for dissolving, transferring its pH value with acid is 2.0～2.5, leaves standstill, filters and be washed with water to neutrality, and concentrate drying gets Berberine and slightly carries powder;
(4) silica gel column chromatography:
The gained meal is dissolved in hot ethanol adds the proper silica gel stirring, dried the 60-80 mesh sieve, dry method loading, upper 200-300 order silica gel column chromatography;
(5) wash-out:
Carry out wash-out with ethyl acetate-ethanol solution with certain proportion, tlc is followed the tracks of and is detected, and collects each stepwise elution liquid of Berberine;
(6) concentrated:
Recycled ethanol, each stepwise elution liquid of Berberine is concentrated, get content and be the berberine hydrochloride powder more than 80%;
(7) recrystallization
Get the barberry ground caustic and add ethanol by certain amount, heating for dissolving is concentrated to 1/10th of original volume, and placement is spent the night, and crystallization filters isolation of crystalline; With residuum with as above method heating for dissolving, recrystallization, so repeatedly twice, the purity that obtains making with extra care is at the Berberine finished product more than 98%.
Embodiment:
Case study on implementation 1: a kind of biological enzymolysis lotus root connection intensified by ultrasonic wave that utilizes extracts, the novel method of column chromatography purification Berberine again, and its step is as follows:
(1) enzymolysis:
100g is crushed to 20 purpose Root of Chinese Barberry, adopts complex cellulase, and enzyme dosage is 0.2% of substrate, 40 ℃ of hydrolysis temperatures, and insulation 1h:
(2) supersound extraction:
Add 50% ethanol, solid-liquid ratio 1: 8, ultrasonic extraction, ultrasonic frequency 50KHZ, ultrasonic time 50min, thin-layer chromatography detect and analyze, and Recycled ethanol gets crude extract;
(3) get meal:
With obtaining primary extract ethanol heating for dissolving, transferring its pH value with acid is 2.0, leaves standstill, filters and be washed with water to neutrality, and concentrate drying gets Berberine and slightly carries powder;
(4) silica gel column chromatography:
The gained meal is dissolved in hot ethanol adds the proper silica gel stirring, dried 60 mesh sieves, dry method loading, upper 200 order silica gel column chromatographies;
(5) wash-out:
Carry out wash-out with ethyl acetate-ethanol solution with 5: 1 ratios, tlc is followed the tracks of and is detected, and collects each stepwise elution liquid of Berberine;
(6) concentrated:
Recycled ethanol, each stepwise elution liquid of berberine hydrochloride is concentrated, get content and be 87.8% berberine hydrochloride powder;
(7) recrystallization:
Get the barberry ground caustic and add ethanol by certain amount, heating for dissolving is concentrated to 1/10th of original volume, and placement is spent the night, and crystallization filters isolation of crystalline; With residuum with as above method heating for dissolving, recrystallization, so repeatedly twice, the purity that obtains making with extra care is at 984% Berberine finished product.
Case study on implementation 2: a kind of biological enzymolysis lotus root connection intensified by ultrasonic wave that utilizes extracts, the novel method of column chromatography purification Berberine again, and its step is as follows:
(1) enzymolysis:
100g is crushed to 30 purpose Root of Chinese Barberry, adopts complex cellulase, and enzyme dosage is 0.4% of substrate, 45 ℃ of hydrolysis temperatures, and insulation 2h:
(2) supersound extraction:
The ethanol that adds 60% concentration, ultrasonic extraction, ultrasonic frequency 70KHZ, solid-liquid ratio 1: 9, ultrasonic time 60min, thin-layer chromatography detect and analyze, and Recycled ethanol gets crude extract;
(3) get meal:
With obtaining primary extract ethanol heating for dissolving, transferring its pH value with hydrochloric acid is 2.5, leaves standstill, filters and be washed with water to neutrality, and concentrate drying gets Berberine and slightly carries powder;
(4) silica gel column chromatography:
The gained meal is dissolved in hot ethanol adds the proper silica gel stirring, dried 80 mesh sieves, dry method loading, upper 300 order silica gel column chromatographies;
(5) wash-out:
Carry out wash-out with ethyl acetate-ethanol solution with 8: 1 ratios, tlc is followed the tracks of and is detected, and collects each stepwise elution liquid of Berberine;
(6) concentrated:
Recycled ethanol, each stepwise elution liquid of Berberine is concentrated, get content and be 87% Berberine powder;
(7) recrystallization
Get the barberry ground caustic and add ethanol by certain amount, heating for dissolving is concentrated to 1/10th of original volume, and placement is spent the night, crystallization filters, and isolation of crystalline is with as above method heating for dissolving of residuum, recrystallization, so repeatedly twice, the purity that obtains making with extra care is at 982% Berberine finished product.
Case study on implementation 3: a kind of biological enzymolysis lotus root connection intensified by ultrasonic wave that utilizes extracts, the novel method of column chromatography purification Berberine again, and its step is as follows:
(1) enzymolysis:
100g is crushed to 40 purpose Root of Chinese Barberry, adopts complex cellulase, and enzyme dosage is 0.6% of substrate, 50 ℃ of hydrolysis temperatures, insulation 3h;
(2) supersound extraction:
The ethanol that adds 70% concentration, ultrasonic extraction, ultrasonic frequency 80KHZ, solid-liquid ratio 1: 10, ultrasonic time 80min, thin-layer chromatography detect and analyze, and Recycled ethanol gets crude extract;
(3) get meal:
With obtaining primary extract ethanol heating for dissolving, transferring its pH value with hydrochloric acid is 2.0, leaves standstill, filters and be washed with water to neutrality, and concentrate drying gets Berberine and slightly carries powder;
(4) silica gel column chromatography:
The gained meal is dissolved in hot ethanol adds the proper silica gel stirring, dried 60 mesh sieves, dry method loading, upper 200 order silica gel column chromatographies;
(5) wash-out:
Carry out wash-out with ethyl acetate-ethanol solution with 10: 1 ratios, tlc is followed the tracks of and is detected, and collects each stepwise elution liquid of Berberine;
(6) concentrated:
Recycled ethanol, each stepwise elution liquid of Berberine is concentrated, get content and be 85.4% Berberine powder;
(7) recrystallization:
Get the barberry ground caustic and add ethanol by certain amount, heating for dissolving is concentrated to 1/10th of original volume, and placement is spent the night, and crystallization filters isolation of crystalline; With residuum with as above method heating for dissolving, recrystallization, so repeatedly twice, the purity that obtains making with extra care is at 98.32% Berberine finished product.
Claims (4)
1. one kind is utilized biological enzymolysis lotus root connection intensified by ultrasonic wave to extract, the novel method of column chromatography purification Berberine again, and its step is as follows:
(1) enzymolysis:
After Root of Chinese Barberry is pulverized, adopt complex cellulase, at 40-50 ℃ of lower enzymolysis, insulation precipitation 1-3h;
(2) supersound extraction:
Add the ethanol ultrasonic extraction, ultrasonic frequency 50-80KHZ, solid-liquid ratio 1: 8-10, ultrasonic time 50-80min, thin-layer chromatography detect and analyze, and Recycled ethanol gets crude extract;
(3) get meal:
With obtaining primary extract ethanol heating for dissolving, transferring its pH value with acid is 2.0～2.5, leaves standstill, filters and be washed with water to neutrality, and concentrate drying gets berberine hydrochloride and slightly carries powder;
(4) silica gel column chromatography:
The gained meal is dissolved in hot ethanol adds the proper silica gel stirring, dried the 60-80 mesh sieve, dry method loading, upper 200-300 order silica gel column chromatography;
(5) wash-out:
Carry out wash-out with ethyl acetate-ethanol solution with certain proportion, tlc is followed the tracks of and is detected, and collects each stepwise elution liquid of Berberine;
(6) concentrated:
Recycled ethanol, each stepwise elution liquid of berberine hydrochloride is concentrated, get content and be the berberine hydrochloride powder more than 80%;
(7) recrystallization:
Get the barberry ground caustic and add ethanol by certain amount, heating for dissolving is concentrated to 1/10th of original volume, and placement is spent the night, and crystallization filters isolation of crystalline; With residuum with as above method heating for dissolving, recrystallization, so repeatedly twice, the purity that obtains making with extra care is at the Berberine finished product more than 98%.
2. the biological enzymolysis lotus root connection intensified by ultrasonic wave that utilizes according to claim 1 extracts, the novel method of column chromatography purification Berberine again, it is characterized in that: in the described step (1), cross 20～40 mesh sieves after Root of Chinese Barberry is pulverized, enzyme dosage is the 0.2-06% of substrate.
3. the biological enzymolysis lotus root connection intensified by ultrasonic wave that utilizes according to claim 2 extracts, the novel method of column chromatography purification bark of a cork tree alkali again, and it is characterized in that: in the described step (2), the volumetric concentration of organic solvent ethanol is 50-70%.
4. the method for utilizing berberine hydrochloride in the enriching and purifying macroporous resin Root of Chinese Barberry according to claim 3 is characterized in that; In the described step (5), ethyl acetate-ethanol solution is with 5～10: 1 ratio is carried out wash-out.
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| CN201210595638.5A CN103044417B (en) | 2012-12-13 | 2012-12-13 | The processing method of a kind of purifying wild thorn leaf barberry Berberine |
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| CN201210595638.5A CN103044417B (en) | 2012-12-13 | 2012-12-13 | The processing method of a kind of purifying wild thorn leaf barberry Berberine |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113372340A (en) * | 2021-04-30 | 2021-09-10 | 西安绿泉科技有限公司 | Method for refining berberine hydrochloride crude product |
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Non-Patent Citations (2)
| Title |
|---|
| 徐艳等: "超声-酶法提取黄柏中小檗碱的工艺研究", 《时珍国医医药》 * |
| 梁柏林等: "酶法提取小檗碱工艺研究", 《应用化工》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113372340A (en) * | 2021-04-30 | 2021-09-10 | 西安绿泉科技有限公司 | Method for refining berberine hydrochloride crude product |
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Address after: 165000 Heilongjiang Province, Jiagedaqi Xinyuan District Changhong community road Beilin Gebe AGA group Applicant after: DAXINGANLING LINGOBERRY BOREAL BIOTECH Co.,Ltd. Address before: 165000 Heilongjiang Province, Jiagedaqi Xinyuan District Changhong community road Beilin Gebe AGA group Applicant before: Daxinganling Lingonberry Organic Foodstuffs Co.,Ltd. |
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Free format text: CORRECT: APPLICANT; FROM: GREAT XINGAN MOUNTAINS LINGEBEI ORGANIC FOOD CO., LTD. TO: GREATER KHINGAN MOUNTAIN LINGONBERRY BIOTECHNOLOGY CO., LTD. |
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Granted publication date: 20151223 Termination date: 20211213 |