CN105130946A - Method for extracting genipin from eucommia folium - Google Patents
Method for extracting genipin from eucommia folium Download PDFInfo
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- CN105130946A CN105130946A CN201510542692.7A CN201510542692A CN105130946A CN 105130946 A CN105130946 A CN 105130946A CN 201510542692 A CN201510542692 A CN 201510542692A CN 105130946 A CN105130946 A CN 105130946A
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- C07D311/00—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings
- C07D311/02—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems
- C07D311/94—Heterocyclic compounds containing six-membered rings having one oxygen atom as the only hetero atom, condensed with other rings ortho- or peri-condensed with carbocyclic rings or ring systems condensed with rings other than six-membered or with ring systems containing such rings
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Abstract
The invention discloses a method for extracting genipin from eucommia folium. The method includes the steps of pre-wetting stacking, raw material treatment, first-time enzymolysis, oil removal, second-time enzymolysis, column chromatography, third-time enzymolysis, column chromatography and drying. According to the method, first, enzymes are adopted for extracting plant grease, the difference of affinity between a non-oil component and oil and affinity between the non-oil component and water and the different proportions of the oil and water are utilized for separating the oil and the non-oil component, in this way, the yield of the product is increased, and the conversion rate is increased.
Description
Technical field
The present invention relates to the extracting method of biological technical field, is the method extracting genipin from Folium Eucommiae specifically.
Background technology
The bark of eucommia (formal name used at school EucommiaulmoidesOliver), has another name called bakelite, is Eucommiaceae plant.Bark of eucommia total free aminoacids is few, a small amount of protein contained, be with most foodstuff like adequate proteins, namely can be hydrolyzed 8 seed amino acids detected needed by human.Determine 15 kinds of mineral elements contained by the bark of eucommia, wherein have the trace elements such as zinc, copper, iron, and the macroelement such as calcium, phosphorus, potassium, magnesium.The medicinal bark of eucommia, is the dry bark of the Eucommiaceae plant bark of eucommia, is Chinese famous and precious tonic herb.Its taste is sweet, warm in nature.Have tonify the liver and kidney, strengthen muscles and bones, recuperating CHONG and REN meridians, solid through antiabortive effect.Can treat the pain in the waist and lower extremities or aching and limp unable that insufficiency of kidney-YANG causes, born of the same parents' tire that deficiency of liver-QI causes is solid, and scrotum is wet the disease such as to itch.Be listed in top grade in Shennong's Herbal.The modern study bark of eucommia has rubbish in purged body, strengthens human body cell substance metabolism, prevents muscle skeleton aging, balanced human's blood pressure, decomposer inner cholesterol, reduces body fat, recovers blood vessel elasticity, diuresis heat-clearing, broad-spectrum antimicrobial, stimulating central nervous system, improves white cell pharmacological action.
Genipin (Genipin) decomposes rear separating-purifying by Geniposide and obtains, its main application has: genipin can with amino acid or proteins react, generate a kind of mast cyanine, this natural pigment is used to the making of food dye because of its nontoxicity; Excellent natural biological linking agent: can with the crosslinked making biomaterial such as protein, collagen, gelatin and chitosan, as artificial skelecton, wound dressing materials etc., its toxicity is far below glutaraldehyde and other conventional chemical cross-linking agents; Can be used for treatment hepatic diseases, step-down, defaecation etc.The result of study of Israel's medical centre shows, genipin can alleviate the symptom of type ii diabetes; Prepare the linking agent of immobilized enzyme; In the iridoid glycoside found so far, suppress mutagen the strongest active material; Also bibliographical information genipin is had to have the effect of antithrombotic inhibited apoptosis; Generally speaking, genipin is widely used, and has potentiality to be exploited, wide market outlook and huge utility value.
Geniposide is mainly present in the natural phant such as mast, the bark of eucommia, be extract the desirable feedstock of Geniposide, in domestic existing extraction process scheme, also exist that extraction yield is not high, product purity is low, energy consumption is large, in a large number with an organic solvent, the problem such as wastewater flow rate is large.
Summary of the invention
The object of this invention is to provide the method extracting genipin from Folium Eucommiae, there is technique simple, be applicable to producing, the advantages such as production cost is low.
The present invention is achieved through the following technical solutions, and specifically comprises the steps:
1) to prewet stacking: Folium Eucommiae is built heap in the cool, water spray, after water flows out from heap bottom, stacking naturally;
2) Feedstock treating: by step 1) Folium Eucommiae that obtains to pulverize or broken, adds the pure water being equivalent to raw material 2-3 times weight, be heated to 50-60 DEG C and maintain 5-10 minute, obtain paste serous material;
3) first time enzymolysis: by step 2) paste serous material that obtains, tunes pH is 4-5, and the N.F,USP MANNITOL of the pectin-cellulose prozyme and 0.001 weight part that add paste serous material 0.05-0.1% weight part carries out enzymolysis 40-60 minute, obtains enzymolysis solution for the first time;
4) deoil: by step 3) the first time enzymolysis solution that obtains, add the pure water of 2-3 times of weight, be heated to 60-70 DEG C of lixiviate 60-120 minute, obtain upper strata oil slick liquid and subnatant, slag mixture, be separated;
5) second time enzymolysis: by step 4) subnatant, the slag mixture that obtain, adjust pH to be 4.5, add the cellulase being equivalent to mixture 0.1-0.2% weight part and carry out enzymolysis 40-60 minute, obtain second time enzymolysis solution, repeat twice;
6) column chromatography: merge second time enzymolysis solution, filter, concentrated, by column chromatography, adsorbing with macroporous adsorbent resin, is colourless with washed with de-ionized water resin to flowing liquid, then is the ethanolic soln wash-out of 15-20% by concentration, collects elutriant;
7) third time enzymolysis: by step 6) elutriant that obtains, adjust pH to be 4.5, add beta-glucosidase and carry out enzymolysis, the consumption of enzyme is the 5-10% of elutriant weight, and the concentration of enzyme is 1mg/ml, and hydrolysis temperature is 50-60 DEG C;
8) column chromatography: by step 7) obtain enzymolysis solution, by column chromatography, adsorbing with macroporous resin, is colourless with washed with de-ionized water resin to flowing liquid, then is the ethanolic soln wash-out of 10-20% by concentration, collects elutriant;
9) dry: by step 8) elutriant that obtains concentrates, then obtains Powdered genipin by drying.
Step 1 of the present invention) described in the stacking of prewetting, preferred 6-12 hour.
Step 3) described in pectin-cellulose prozyme, preferred activity is the solid-state of 1-3 ten thousand activity unit/gram (milliliter) or liquid pectin-fiber composite enzyme.
Step 6) and step 8) described in the preferred D101 type of macroreticular resin, DA201 type.
Compared with prior art, advantage of the present invention:
1, the vegetable cell of Folium Eucommiae is interior containing a large amount of greases, be the mixture of the compound composition of triglyceride level primarily of composition, these greases with elaioleucite and oil body protoplastis form, the existence form such as to be irregularly dispersed in cell in discontinuous particulate state, together with granule protein body and to exist.Existing extracting method, all by mode extracting directly such as water extraction/alcohol extracting, enzymolysis or microwave extraction after directly the raw materials such as Folium Eucommiae being smashed or pulverized, when extraction, grease in cell together with time isolate, the compositions such as the group containing grease and Geniposide, Geniposidic acid, pigment mix, thus affect the separation of follow-up effective constituent, add the intractability of operation.The present invention first adopts enzyme extraction Vegetable oil lipoprotein, utilizes non-oil component different with the avidity difference of water and profit proportion and by oil and non-oil component separating, add product yield and improve extraction yield to oil.
2, the present invention adopts Folium Eucommiae to prewet stacking, adds suitable quantity of water, and build heap fermentation, along with the rising of fermenting process temperature, Folium Eucommiae softens gradually, and moisture enters into inside naturally, is beneficial to the separation of Vegetable oil lipoprotein, is beneficial to follow-up enzymolysis, deoils.
3, the present invention first adopts pectin-cellulose prozyme to remove cell walls, and the grease in release vegetable cell, adds N.F,USP MANNITOL, can keep osmotic pressure, be beneficial to the release of grease.
4, adopt method of the present invention, Geniposide transformation efficiency is high, more than 75%.
Embodiment
With embodiment, the invention will be further described below, but the present invention is not limited to these embodiments.
Embodiment 1:
From Folium Eucommiae, extract the method for genipin, specifically comprise the steps:
1) to prewet stacking: Folium Eucommiae is built heap in the cool, water spray, after water flows out from heap bottom, stacking 12 hours naturally;
2) Feedstock treating: by step 1) Folium Eucommiae that obtains pulverizes, adds the pure water being equivalent to raw material 2-3 times weight, be heated to 50 DEG C and maintain 5 minutes, obtain paste serous material;
3) first time enzymolysis: by step 2) paste serous material that obtains, pH is adjusted to be 4, add paste serous material 0.05% parts by weight of activated be 10,000 activity units/gram pectin-cellulose prozyme and the N.F,USP MANNITOL of 0.001 weight part carry out enzymolysis 40 minutes, obtain first time enzymolysis solution;
4) deoil: by step 3) the first time enzymolysis solution that obtains, add the pure water of 2 times of weight, be heated to 60 DEG C of lixiviates 120 minutes, obtain upper strata oil slick liquid and subnatant, slag mixture, be separated;
5) second time enzymolysis: by step 4) subnatant, the slag mixture that obtain, adjust pH to be 4.5, add the cellulase being equivalent to mixture 0.1% weight part and carry out enzymolysis 60 minutes, obtain second time enzymolysis solution, repeat twice;
6) column chromatography: merge second time enzymolysis solution, filter, concentrated, by column chromatography, adsorbing with D101 type macroporous adsorbent resin, is colourless with washed with de-ionized water resin to flowing liquid, then is the ethanolic soln wash-out of 20% by concentration, collects elutriant;
7) third time enzymolysis: by step 6) elutriant that obtains, adjust pH to be 4.5, add beta-glucosidase and carry out enzymolysis, the consumption of enzyme is 5% of elutriant weight, and the concentration of enzyme is 1mg/ml, and hydrolysis temperature is 60 DEG C;
8) column chromatography: by step 7) obtain enzymolysis solution, by column chromatography, adsorbing with D101 macroporous resin, is colourless with washed with de-ionized water resin to flowing liquid, then is the ethanolic soln wash-out of 20% by concentration, collects elutriant;
9) dry: by step 8) elutriant that obtains concentrates, then obtains Powdered genipin by drying, and Geniposide transformation efficiency is 76.5%.
Embodiment 2:
From Folium Eucommiae, extract the method for genipin, specifically comprise the steps:
1) to prewet stacking: Folium Eucommiae is built heap in the cool, water spray, after water flows out from heap bottom, stacking 10 hours naturally;
2) Feedstock treating: by step 1) Folium Eucommiae that obtains is broken, and add the pure water being equivalent to raw material 2-3 times weight, be heated to 60 DEG C and maintain 8 minutes, obtain paste serous material;
3) first time enzymolysis: by step 2) paste serous material that obtains, pH is adjusted to be 4.5, adding paste serous material 0.1% parts by weight of activated is that the pectin-cellulose prozyme of 20,000 activity units/milliliter and the N.F,USP MANNITOL of 0.001 weight part carry out enzymolysis 50 minutes, obtains first time enzymolysis solution;
4) deoil: by step 3) the first time enzymolysis solution that obtains, add the pure water of 3 times of weight, be heated to 70 DEG C of lixiviates 60 minutes, obtain upper strata oil slick liquid and subnatant, slag mixture, be separated;
5) second time enzymolysis: by step 4) subnatant, the slag mixture that obtain, adjust pH to be 4.5, add the cellulase being equivalent to mixture 0.15% weight part and carry out enzymolysis 50 minutes, obtain second time enzymolysis solution, repeat twice;
6) column chromatography: merge second time enzymolysis solution, filter, concentrated, by column chromatography, adsorbing with DA201 type macroporous adsorbent resin, is colourless with washed with de-ionized water resin to flowing liquid, then is the ethanolic soln wash-out of 15% by concentration, collects elutriant;
7) third time enzymolysis: by step 6) elutriant that obtains, adjust pH to be 4.5, add beta-glucosidase and carry out enzymolysis, the consumption of enzyme is 10% of elutriant weight, and the concentration of enzyme is 1mg/ml, and hydrolysis temperature is 50 DEG C;
8) column chromatography: by step 7) obtain enzymolysis solution, by column chromatography, adsorbing with DA201 type macroporous resin, is colourless with washed with de-ionized water resin to flowing liquid, then is the ethanolic soln wash-out of 15% by concentration, collects elutriant;
9) dry: by step 8) elutriant that obtains concentrates, then obtains Powdered genipin by drying, and Geniposide transformation efficiency is 75.8%.
Embodiment 3:
1) to prewet stacking: Folium Eucommiae is built heap in the cool, water spray, after water flows out from heap bottom, stacking 6 hours naturally;
2) Feedstock treating: by step 1) Folium Eucommiae that obtains pulverizes, adds the pure water being equivalent to raw material 2-3 times weight, be heated to 55 DEG C and maintain 10 minutes, obtain paste serous material;
3) first time enzymolysis: by step 2) paste serous material that obtains, pH is adjusted to be 5, add paste serous material 0.08% parts by weight of activated be 30,000 activity units/gram pectin-cellulose prozyme and the N.F,USP MANNITOL of 0.001 weight part carry out enzymolysis 60 minutes, obtain first time enzymolysis solution;
4) deoil: by step 3) the first time enzymolysis solution that obtains, add the pure water of 2 times of weight, be heated to 65 DEG C of lixiviates 100 minutes, obtain upper strata oil slick liquid and subnatant, slag mixture, be separated;
5) second time enzymolysis: by step 4) subnatant, the slag mixture that obtain, adjust pH to be 4.5, add the cellulase being equivalent to mixture 0.2% weight part and carry out enzymolysis 40 minutes, obtain second time enzymolysis solution, repeat twice;
6) column chromatography: merge second time enzymolysis solution, filter, concentrated, by column chromatography, adsorbing with D101 type macroporous adsorbent resin, is colourless with washed with de-ionized water resin to flowing liquid, then is the ethanolic soln wash-out of 20% by concentration, collects elutriant;
7) third time enzymolysis: by step 6) elutriant that obtains, adjust pH to be 4.5, add beta-glucosidase and carry out enzymolysis, the consumption of enzyme is 8% of elutriant weight, and the concentration of enzyme is 1mg/ml, and hydrolysis temperature is 55 DEG C;
8) column chromatography: by step 7) obtain enzymolysis solution, by column chromatography, adsorbing with D101 type macroporous resin, is colourless with washed with de-ionized water resin to flowing liquid, then is the ethanolic soln wash-out of 10% by concentration, collects elutriant;
9) dry: by step 8) elutriant that obtains concentrates, then obtains Powdered genipin by drying, and Geniposide transformation efficiency is 78.2%.
Claims (4)
1. from Folium Eucommiae, extract the method for genipin, it is characterized in that, comprise the steps:
1) to prewet stacking: Folium Eucommiae is built heap in the cool, water spray, after water flows out from heap bottom, stacking naturally;
2) Feedstock treating: by step 1) Folium Eucommiae that obtains to pulverize or broken, adds the pure water being equivalent to raw material 2-3 times weight, be heated to 50-60 DEG C and maintain 5-10 minute, obtain paste serous material;
3) first time enzymolysis: by step 2) paste serous material that obtains, tunes pH is 4-5, and the N.F,USP MANNITOL of the pectin-cellulose prozyme and 0.001 weight part that add paste serous material 0.05-0.1% weight part carries out enzymolysis 40-60 minute, obtains enzymolysis solution for the first time;
4) deoil: by step 3) the first time enzymolysis solution that obtains, add the pure water of 2-3 times of weight, be heated to 60-70 DEG C of lixiviate 60-120 minute, obtain upper strata oil slick liquid and subnatant, slag mixture, be separated;
5) second time enzymolysis: by step 4) subnatant, the slag mixture that obtain, adjust pH to be 4.5, add the cellulase being equivalent to mixture 0.1-0.2% weight part and carry out enzymolysis 40-60 minute, obtain second time enzymolysis solution, repeat twice;
6) column chromatography: merge second time enzymolysis solution, filter, concentrated, by column chromatography, adsorbing with macroporous adsorbent resin, is colourless with washed with de-ionized water resin to flowing liquid, then is the ethanolic soln wash-out of 15-20% by concentration, collects elutriant;
7) third time enzymolysis: by step 6) elutriant that obtains, adjust pH to be 4.5, add beta-glucosidase and carry out enzymolysis, the consumption of enzyme is the 5-10% of elutriant weight, and the concentration of enzyme is 1mg/ml, and hydrolysis temperature is 50-60 DEG C;
8) column chromatography: by step 7) obtain enzymolysis solution, by column chromatography, adsorbing with macroporous resin, is colourless with washed with de-ionized water resin to flowing liquid, then is the ethanolic soln wash-out of 10-20% by concentration, collects elutriant;
9) dry: by step 8) elutriant that obtains concentrates, then obtains Powdered genipin by drying.
2. the method extracting genipin from Folium Eucommiae according to claim 1, is characterized in that: step 1) described in the stacking of prewetting, be 6-12 hour.
3. the method extracting genipin from Folium Eucommiae according to claim 1, it is characterized in that: step 3) described in pectin-cellulose prozyme, for activity be 1-3 ten thousand activity unit/gram the liquid pectin-fiber composite enzyme of solid-state or 1-3 ten thousand activity units/milliliter.
4. the method extracting genipin from Folium Eucommiae according to claim 1, is characterized in that: step 6) described in macroreticular resin be D101 type, DA201 type.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113133523A (en) * | 2021-05-28 | 2021-07-20 | 湖北工业大学 | Preparation method of kudzu rice rich in protein |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101029066A (en) * | 2007-04-13 | 2007-09-05 | 桂林莱茵生物科技股份有限公司 | Method for extracting genipin and geniposide from gardenia jasminoides |
| CN103788152A (en) * | 2012-10-26 | 2014-05-14 | 湖北老龙洞杜仲开发有限公司 | Method for preparing geniposide in eucommia leaf |
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- 2015-08-31 CN CN201510542692.7A patent/CN105130946A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101029066A (en) * | 2007-04-13 | 2007-09-05 | 桂林莱茵生物科技股份有限公司 | Method for extracting genipin and geniposide from gardenia jasminoides |
| CN103788152A (en) * | 2012-10-26 | 2014-05-14 | 湖北老龙洞杜仲开发有限公司 | Method for preparing geniposide in eucommia leaf |
Non-Patent Citations (1)
| Title |
|---|
| 曹慧等: "酶法提取杜仲中降压活性成分", 《中国生化药物杂志》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113133523A (en) * | 2021-05-28 | 2021-07-20 | 湖北工业大学 | Preparation method of kudzu rice rich in protein |
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