CN109867651B - Method for extracting sciadopitysin from ginkgo leaves - Google Patents
Method for extracting sciadopitysin from ginkgo leaves Download PDFInfo
- Publication number
- CN109867651B CN109867651B CN201910278270.1A CN201910278270A CN109867651B CN 109867651 B CN109867651 B CN 109867651B CN 201910278270 A CN201910278270 A CN 201910278270A CN 109867651 B CN109867651 B CN 109867651B
- Authority
- CN
- China
- Prior art keywords
- enzymolysis
- sciadopitysin
- eluent
- concentrated solution
- extracting
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Abstract
The invention discloses a method for extracting sciadopitysin from ginkgo leaves. The method uses enzymolysis to carry out early-stage treatment of ginkgo leaves, and carries out multiple extraction and separation on the crude extract, and obtains the sciadopitysin extract with higher yield by simple process conditions. The method uses enzymolysis for pretreatment, improves the extraction rate of the product, the extraction rate is higher than 96%, the enzymolysis can be carried out in a large area, the operation is convenient, the use of organic solvents can be reduced by the enzymolysis reaction of the enzyme preparation, the discharge of organic wastewater is reduced, and the production concept of environmental protection is facilitated. The product has the advantages of full extraction, high purity of the product, and high content of over 99.5%, improves the action effect of the sciadopitysin, and is beneficial to the wide application of the sciadopitysin.
Description
Technical Field
The invention relates to a method for extracting sciadopitysin from ginkgo leaves, and relates to the field of traditional Chinese medicine extraction processes.
Background
The diabetic heart disease refers to heart disease which is complicated or accompanied by diabetic patients, 70 to 80 percent of the diabetic patients die from cardiovascular complications, the pathogenesis of the diabetic heart disease is not completely clarified, but macroangiopathy and microangiopathy are very critical factors found from diabetic metabolic disorder, pathophysiology, noninvasive cardiac function examination, pathological anatomy data and the like. Diabetic macroangiopathy, which is the accelerated development of atherosclerosis and is responsible for the increased incidence of myocardial infarction in diabetic patients, has been shown by the well-known Heart Preservation Study (HPS) to be a group of patients who have been given statins with tight control of blood lipids, especially LDL, with a significantly lower incidence of cardiovascular events than in the poorly lipid-controlled group, from which diabetic patients benefit more. The microangiopathy refers to the diabetic cardiomyopathy which is caused by extensive myocardial ischemia and focal necrosis fibrosis caused by capillary vessels, small artery lesion before the capillary vessels, myocardial microangiopathy and myocardial metabolic disorder due to thickening of a basement membrane of the capillary vessels. Clinical observations show that some diabetic patients can develop severe heart failure and congestive cardiomyopathy but no coronary artery lesions are seen in arteriography, no coronary artery obstruction and myocardial infarction are seen even after autopsy, and extensive myocardial lesions (focal necrosis) are seen in some cases, suggesting possible association with intramyocardial microangiopathy. Hyperglycemia is responsible for the biochemical abnormalities that lead to diabetic complications, and the pathways such as glycolysis are interrelated with the PKC pathway, which are thought to cause dysfunction and damage to retinal vascular endothelial cells, leading to abnormal cell growth and survival, increased vascular permeability, altered blood flow, ischemia, abnormal angiogenesis.
The sciadopitysin has a molecular formula of C33H24O10The molecular weight is 580.53766, it is yellow crystal at normal temperature and pressure, it is dissolved in acetone, ethyl acetate, slightly soluble in petroleum ether and water, and it has better stability at room temperature, and it needs to be stored in shade. Patent 201310032630.2 discloses the application of sciadopitysin in preparing medicine for preventing and treating diabetes, and experimental research shows that sciadopitysin has good effect of treating diabetes and has good therapeutic effect on diabetes, diabetes complicated with heart disease, diabetes complicated with hyperlipidemia, and other related diseases. At present, when the sciadopitysin is extracted from the ginkgo leaves, the extraction is not thorough, the cost is high, and the market demand cannot be met.
Disclosure of Invention
The invention overcomes the defects of the prior art and provides a method for extracting sciadopitysin from ginkgo leaves. The method uses enzymolysis to carry out early-stage treatment of ginkgo leaves, and carries out multiple extraction and separation on the crude extract, and obtains the sciadopitysin extract with higher yield by simple process conditions.
A method for extracting sciadopitysin from folium Ginkgo comprises the following steps:
1) cleaning and drying folium Ginkgo, pulverizing, sieving with 40 mesh sieve, adding water of equal mass, and making into leaf pulp;
2) adding cellulase into the leaf pulp obtained in the step 1), wherein the mass ratio of the cellulase to the leaf pulp is 1: 400-;
3) adjusting the pH value of the enzymolysis liquid A obtained in the step 2) to 4.5-5.0, adding a complex enzyme preparation with the mass ratio of hemicellulase to ligninase of 1.5-2:1, performing enzymolysis for 1-2h at 48-50 ℃ and inactivating enzyme at 90 ℃ to obtain an enzymolysis crude extract, wherein the mass ratio of the complex enzyme preparation to the enzymolysis liquid A is 1: 380-400;
4) fully mixing the enzymolysis crude extract obtained in the step 3) with methanol with the same volume, extracting for 2-3 times, combining the extract, and concentrating under reduced pressure to obtain a concentrated solution;
5) adding the concentrated solution obtained in the step 4) into a chromatographic column with silica gel as an adsorbent for adsorption, eluting with a mixed solution of dichloromethane and methanol in a volume ratio of 1:3, collecting eluent until a yellow color band completely disappears to obtain a first eluent, concentrating the first eluent under reduced pressure to obtain a first concentrated solution, carrying out chromatography on the first concentrated solution with 100-mesh polyamide, eluting with 40% ethanol solution, collecting eluent until the yellow color band completely disappears to obtain a second eluent, and recrystallizing the second eluent with acetone to obtain yellow needle crystals, wherein the yellow needle crystals are sciadopitysin.
Furthermore, the activity of the cellulase is more than or equal to 10000 IU/g.
Furthermore, the activity of the hemicellulase is more than or equal to 10000IU/g, and the activity of the ligninase is more than or equal to 10000 IU/g.
Further, the extracted sciadopitysin is combined with a pharmaceutically acceptable carrier or excipient to prepare various clinically acceptable preparations.
Further, the preparation is tablets, capsules, granules, injections or dropping pills.
Has the advantages that:
(1) the enzymolysis is used for carrying out pretreatment, the extraction rate of products is improved, the extraction rate is higher than 96%, the enzymolysis can be carried out in a large area, the use of organic solvents can be reduced by the enzymolysis reaction of an enzyme preparation, the discharge of organic wastewater is reduced, and the production concept of environmental protection is facilitated.
(2) The method has the advantages of simple process operation, good repeatability, high yield, and improved production efficiency, and is beneficial to large-scale popularization.
(3) The product has the advantages of full extraction, high purity of the product, and high content of over 99.5%, improves the action effect of the sciadopitysin, and is beneficial to the wide application of the sciadopitysin.
Detailed Description
EXAMPLE 1 cellulase enzyme hydrolysis timing
Crushing ginkgo leaves, sieving the crushed ginkgo leaves with a 40-mesh sieve, taking 100g of samples, adding water with equal mass to prepare leaf pulp, adding cellulase, wherein the mass ratio of the cellulase to the leaf pulp is 1:400, carrying out enzymolysis for 2 hours, 2.5 hours, 3 hours, 3.5 hours and 4 hours at 37-40 ℃, inactivating enzyme for 5min at 90 ℃ to obtain an enzymolysis liquid A, adjusting the pH of the enzymolysis liquid A to be 4.7, adding a complex enzyme preparation with the mass ratio of hemicellulase to ligninase of 1.5-2:1, wherein the mass ratio of the complex enzyme preparation to the enzymolysis liquid A is 1:400, carrying out enzymolysis for 1.0 hour at 48-50 ℃, and inactivating enzyme for 5min at 90 ℃ to obtain an enzymolysis crude extract; mixing the enzymolysis crude extract with methanol of the same volume, extracting for 2-3 times, mixing the extractive solutions, and concentrating under reduced pressure to obtain concentrated solution; adding the obtained concentrated solution into a chromatographic column using silica gel as an adsorbent for adsorption, eluting by using a mixed solution of dichloromethane and methanol in a volume ratio of 1:3, collecting eluent until a yellow color band completely disappears to obtain a first eluent, concentrating the first eluent under reduced pressure at 500Pa for 62-70min to obtain a first concentrated solution, carrying out chromatography on the first concentrated solution by using 100-mesh polyamide, eluting and eluting by using a 40% ethanol solution, collecting eluent until the yellow color band completely disappears to obtain a second eluent, concentrating the second eluent and recrystallizing by using acetone to obtain yellow needle crystals, wherein the yellow needle crystals are sciadopitysin.
TABLE 1 Effect of different cellulase enzymolysis times on extraction yield
| Enzymolysis time of cellulase | 2h | 2.5h | 3h | 3.5h | 4h |
| Extraction rate of sciadopitysin | 76.6％ | 85.4％ | 95.6％ | 95.6％ | 95.5％ |
As can be seen from Table 1, the extraction rate of the product extracted with 3h enzymolysis time can reach 95%, and the effect is better.
Example 2 hemicellulase and ligninase enzymatic hydrolysis time selection
Crushing ginkgo leaves, sieving the crushed ginkgo leaves with a 40-mesh sieve, taking 100g of a sample, adding water with equal mass to prepare leaf pulp, adding cellulase, wherein the mass ratio of the cellulase to the leaf pulp is 1:400, carrying out enzymolysis for 3h at 37-40 ℃, inactivating enzyme for 5min at 90 ℃ to obtain an enzymolysis liquid A, adjusting the pH of the enzymolysis liquid A to be 4.7, adding a complex enzyme preparation with the mass ratio of hemicellulase to ligninase of 1.5-2:1, carrying out enzymolysis for 0.5h, 1.0h, 1.5h, 2h and inactivating enzyme for 5min at 48-50 ℃ to obtain an enzymolysis crude extract; mixing the enzymolysis crude extract with methanol of the same volume, extracting for 2-3 times, mixing the extractive solutions, and concentrating under reduced pressure; adding the obtained concentrated solution into a chromatographic column using silica gel as an adsorbent for adsorption, eluting by using a mixed solution of dichloromethane and methanol in a volume ratio of 1:3, collecting eluent until a yellow color band completely disappears to obtain a first eluent, concentrating the first eluent under reduced pressure at 500Pa for 62-70min to obtain a first concentrated solution, carrying out chromatography on the first concentrated solution by using 100-mesh polyamide, eluting by using a 40% ethanol solution, collecting eluent until the yellow color band completely disappears to obtain a second eluent, and concentrating the second eluent by using acetone for recrystallization to obtain yellow needle crystals, wherein the yellow needle crystals are sciadopitysin.
TABLE 2 influence of different enzymatic hydrolysis times of hemicellulase and ligninase on the extraction yield of sciadopitysin
| Time of enzymolysis | 0.5h | 1h | 1.5h | 2h |
| Extraction rate of sciadopitysin | 90.9％ | 95.6％ | 96.2％ | 96.2％ |
As can be seen from Table 2, the extraction rate of sciadopitysin is better when the enzymolysis time of hemicellulase and ligninase is 1.5 h.
EXAMPLE 3 selection of eluent ratios
Crushing ginkgo leaves, sieving the crushed ginkgo leaves with a 40-mesh sieve, taking 100g of a sample, adding water with equal mass to prepare leaf pulp, adding cellulase, wherein the mass ratio of the cellulase to the leaf pulp is 1:400, performing enzymolysis for 3h at 37-40 ℃, inactivating enzyme for 5min at 90 ℃ to obtain an enzymolysis solution A, adjusting the pH of the enzymolysis solution A to 4.7, adding a complex enzyme preparation with the mass ratio of hemicellulase to ligninase of 1.5-2:1, performing enzymolysis for 1.5h at 48-50 ℃ and inactivating enzyme for 5min at 90 ℃ to obtain enzymolysis crude extract; mixing the enzymolysis crude extract with methanol of the same volume, extracting for 2-3 times, mixing the extractive solutions, and concentrating under reduced pressure; adsorbing the obtained concentrated solution on a chromatographic column with silica gel as an adsorbent, performing gradient elution with a mixed solution of dichloromethane and methanol in a volume ratio of 1:1-5 (the gradient is selected from 1:1, 1:2, 1:3, 1:4 and 1:5 respectively), collecting eluent by volume of 1/40 total eluent, detecting by HPLC method, and collecting sciadopitysin when the mixed solution of 1:3 dichloromethane and methanol is used for elution.
Example 4 repeatability experiments
Crushing ginkgo leaves, sieving the crushed ginkgo leaves with a 40-mesh sieve, taking 100g of a sample, adding water with equal mass to prepare leaf pulp, adding cellulase, wherein the mass ratio of the cellulase to the leaf pulp is 1:400, carrying out enzymolysis for 3h at 37-40 ℃, inactivating enzyme for 5min at 90 ℃ to obtain an enzymolysis liquid A, adjusting the pH of the enzymolysis liquid A to be 4.7, adding a complex enzyme preparation with the mass ratio of hemicellulase to ligninase of 1.5-2:1, carrying out enzymolysis for 1.5h at 48-50 ℃, and inactivating enzyme for 5min at 90 ℃ to obtain an enzymolysis crude extract; mixing the enzymolysis crude extract with methanol of the same volume, extracting for 2-3 times, mixing the extractive solutions, and concentrating under reduced pressure; adding the obtained concentrated solution into a chromatographic column using silica gel as an adsorbent for adsorption, eluting by using a mixed solution of dichloromethane and methanol in a volume ratio of 1:3, collecting eluent until a yellow color band completely disappears to obtain a first eluent, concentrating the first eluent under reduced pressure at 500Pa for 62-70min to obtain a first concentrated solution, carrying out chromatography on the first concentrated solution by using 100-mesh polyamide, eluting by using a 40% ethanol solution, collecting eluent until the yellow color band completely disappears to obtain a second eluent, and concentrating the second eluent by using acetone for recrystallization to obtain yellow needle crystals, wherein the yellow needle crystals are sciadopitysin. The parallel detection is repeated for 3 times, the extraction rates of the sciadopitysin are respectively 96.3%, 96.2% and 96.3%, and the contents are respectively 99.7%, 99.7% and 99.6%, which shows that the experiment repeatability is good.
Example 5 structural characterization
EI-MS m/z(％)580[M]+，579[M-H]+，565[M-CH3]+，549[M-OCH3]+，534[M-OCH3-CH3]+；H-NMR(500Hz,DMSO-d6)：6.80、6.35、7.70、7.61、6.88、6.79、6.96、6.32、6.80、8.09、7.34、3.31、3.78、3.82、10.77；13C-NMR(125MHz,DMSO-d6) 16.08, 103.3, 181.8, 103.5, 160.6, 98.5, 161.8, 103.7, 154.3, 122.7, 127.7, 114.5, 162.3, 114.7, 128.7, 163.4, 103.9, 181.9, 104.6, 161.1, 98.3, 165.2, 92.6, 157.4, 122.7, 130.8, 121.7, 160.4, 111.8, 128.3, 55.4, 55.8, 55.9. The spectrum data is basically consistent with that of the sciadopitysin reported in the literature, and the structure of the sciadopitysin is determined to be the sciadopitysin.
Claims (6)
1. A method for extracting sciadopitysin from ginkgo leaves is characterized by comprising the following steps:
1) cleaning folium Ginkgo, sun drying, pulverizing, and adding 0.8-1.2 times of water to obtain leaf pulp;
2) adding cellulase into the leaf pulp obtained in the step 1), wherein the mass ratio of the cellulase to the leaf pulp is 1: 400-;
3) adjusting the pH value of the enzymolysis liquid A obtained in the step 2) to 4.5-5.0, adding a complex enzyme preparation containing hemicellulase and ligninase, wherein the mass ratio of the complex enzyme preparation to the enzymolysis liquid A is 1:380-400, and carrying out enzymolysis under proper conditions to obtain an enzymolysis crude extract;
4) extracting the enzymolysis crude extract obtained in the step 3) by using methanol, and concentrating the extract under reduced pressure to obtain a concentrated solution;
5) adding the concentrated solution obtained in the step 4) into a chromatographic column with silica gel as an adsorbent for adsorption, eluting with a mixed solution of dichloromethane and methanol in a volume ratio of 1:3, collecting eluent until a yellow color band completely disappears to obtain a first eluent, concentrating the first eluent under reduced pressure to obtain a first concentrated solution, carrying out chromatography on the first concentrated solution with 100-mesh polyamide, eluting with 40% ethanol solution, collecting eluent until the yellow color band completely disappears to obtain a second eluent, recrystallizing the second eluent with acetone to obtain yellow needle crystals, wherein the yellow needle crystals are sciadopitysin;
the mass ratio of the hemicellulase to the ligninase in the compound enzyme preparation is 1.5-2: 1;
the proper conditions in the step 2) refer to enzymolysis at 37-40 ℃ for 3-3.5h, and enzyme deactivation at 90-95 ℃ after enzymolysis;
the proper conditions in the step 3) refer to enzymolysis for 1-2h at 48-50 ℃, and enzyme deactivation at 90-95 ℃ after enzymolysis.
2. The method as claimed in claim 1, wherein the cellulase activity in step 2) is not less than 10000 IU/g.
3. The method of claim 1, wherein the hemicellulase activity in step 3) is greater than or equal to 10000IU/g, and the ligninase activity is greater than or equal to 10000 IU/g.
4. The method as claimed in claim 1, wherein the volume ratio of the methanol to the enzymolysis crude extract in the step 4) is 1:0.8-1, the extraction is carried out for 2-3 times, and the extracts are combined.
5. The method of any one of claims 1-4, wherein the extracted sciadopitysin is combined with a pharmaceutically acceptable carrier or excipient to prepare a clinically acceptable formulation.
6. The method of claim 5, wherein the formulation is a tablet, capsule, granule, injection or drop pill.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910278270.1A CN109867651B (en) | 2019-04-09 | 2019-04-09 | Method for extracting sciadopitysin from ginkgo leaves |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910278270.1A CN109867651B (en) | 2019-04-09 | 2019-04-09 | Method for extracting sciadopitysin from ginkgo leaves |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109867651A CN109867651A (en) | 2019-06-11 |
| CN109867651B true CN109867651B (en) | 2021-01-05 |
Family
ID=66922138
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910278270.1A Active CN109867651B (en) | 2019-04-09 | 2019-04-09 | Method for extracting sciadopitysin from ginkgo leaves |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109867651B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113651791B (en) * | 2021-09-01 | 2023-03-24 | 湖南华诚生物资源股份有限公司 | Method for separating hesperetin from immature bitter orange |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2007008900A (en) * | 2005-07-04 | 2007-01-18 | Bio Yakuhin Kk | Hair growth promoter containing biflavones as active component |
-
2019
- 2019-04-09 CN CN201910278270.1A patent/CN109867651B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2007008900A (en) * | 2005-07-04 | 2007-01-18 | Bio Yakuhin Kk | Hair growth promoter containing biflavones as active component |
Non-Patent Citations (3)
| Title |
|---|
| 从银杏叶中制备金松双黄酮对照品的研究;孔繁晟 等;《中成药》;20100531;第32卷(第5期);第878页 左栏 * |
| 酶解-溶剂提取银杏叶活性成分工艺条件;张小清 等;《应用化工》;20051031;第34卷(第10期);摘要，第635页左栏第1段，第635页右栏，第637页左栏 3.结论 * |
| 银杏叶，银杏叶提取物及其注射液中化学成分及酚酸含量的研究;陈晶;《中国优秀硕士学位论文全文数据库 医药卫生科技辑》;20130815;正文第17页 4.2部分至第18页，第21-23页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109867651A (en) | 2019-06-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20220194915A1 (en) | Method for extracting and separating dihydromyricetin from rattan tea | |
| CN102000138B (en) | Method for purifying effective ingredients of cicer ariethnum L. bean sprouts | |
| US20170360802A1 (en) | Enrichment Method of Ergosterol Peroxide from Sporoderm-Broken Ganoderma Lucidum Spore Powder | |
| CN108659068A (en) | A kind of isolation and purification method of Cyanidin -3-O- rutinosides and its application | |
| CN102925497A (en) | Method for preparing high-purity resveratrol from polygonum cuspidatum | |
| CN109867651B (en) | Method for extracting sciadopitysin from ginkgo leaves | |
| CN101244190A (en) | Method of preparing allium macrostemon total sterides saponin extract and uses thereof | |
| CN109694366B (en) | Method for separating and purifying active ingredients of clematis filamentosa dunn | |
| CN108409805B (en) | Separation and purification method of delphinidin-3-O-galactoside and application thereof | |
| CN109369733B (en) | Method for simultaneously extracting multiple flavonoid compounds from tartary buckwheat leaves | |
| CN108864224B (en) | Separation and purification method of malvidin-3-O-arabinoside and application thereof | |
| CN112028959A (en) | Preparation method and application of triterpenoid with anti-diabetic activity in sessile ganoderma lucidum | |
| CN105601693B (en) | Ginseng saponin F1Preparation and its antitumor action | |
| CN112125944A (en) | Preparation method and application of triterpene compound with alpha glucosidase inhibitory activity in ganoderma sessiliflorum | |
| CN108822168B (en) | Separation and purification method of malvidin-3-O-galactoside and application thereof | |
| CN109400665B (en) | Method for preparing four triterpenoid compound reference substances from pubescent holly root | |
| CN108771690B (en) | A Balanophora japonica L extract with blood sugar or blood lipid reducing effect, and its preparation method and application | |
| CN107375356B (en) | Method for simultaneously preparing high-purity total flavonol glycosides and ginkgolides | |
| KR20110093477A (en) | Antitumor composition comprising fermented red ginseng | |
| CN111297847B (en) | Application of amomum kravanh extract in preparation of alpha-glucosidase inhibitor medicine | |
| CN114470101A (en) | Traditional Chinese medicine composition, preparation method thereof and oral agent containing traditional Chinese medicine composition | |
| KR101802979B1 (en) | Mehod for producing ginsenoside F1 using bioconversion | |
| CN108558645B (en) | Method for extracting crocin from gardenia | |
| CN106822071B (en) | Chinese medicinal effective component for treating coronary heart disease and hyperlipidemia, its preparation method and method for separating effective component from the same | |
| KR102056163B1 (en) | Method for producing high amount of ginsenoside Rg1 and compound K |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20201208 Address after: 250355, 4655, University Road, University Science Park, Changqing District, Shandong, Ji'nan Applicant after: SHANDONG University OF TRADITIONAL CHINESE MEDICINE Address before: 250011 Department of Endocrinology, Affiliated Hospital of Shandong University of Traditional Chinese Medicine, 42 Cultural West Road, Lixia District, Jinan City, Shandong Province Applicant before: AFFILIATED HOSPITAL OF SHANDONG University OF TRADITIONAL CHINESE MEDICINE |
|
| TA01 | Transfer of patent application right | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |