CN114315781A - Fermentation pretreatment method for extracting fisetin from cotinus coggygria - Google Patents
Fermentation pretreatment method for extracting fisetin from cotinus coggygria Download PDFInfo
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- CN114315781A CN114315781A CN202210062836.9A CN202210062836A CN114315781A CN 114315781 A CN114315781 A CN 114315781A CN 202210062836 A CN202210062836 A CN 202210062836A CN 114315781 A CN114315781 A CN 114315781A
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- XHEFDIBZLJXQHF-UHFFFAOYSA-N fisetin Chemical compound C=1C(O)=CC=C(C(C=2O)=O)C=1OC=2C1=CC=C(O)C(O)=C1 XHEFDIBZLJXQHF-UHFFFAOYSA-N 0.000 title claims abstract description 134
- 235000011990 fisetin Nutrition 0.000 title claims abstract description 66
- 241000134400 Cotinus coggygria Species 0.000 title claims abstract description 58
- 238000000855 fermentation Methods 0.000 title claims abstract description 49
- 230000004151 fermentation Effects 0.000 title claims abstract description 49
- 238000002203 pretreatment Methods 0.000 title claims abstract description 11
- 239000000463 material Substances 0.000 claims abstract description 45
- 239000007787 solid Substances 0.000 claims abstract description 21
- 239000002994 raw material Substances 0.000 claims abstract description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 37
- 239000000843 powder Substances 0.000 claims description 24
- 238000000605 extraction Methods 0.000 claims description 11
- 238000004519 manufacturing process Methods 0.000 abstract description 4
- 241000894006 Bacteria Species 0.000 abstract description 3
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- 230000009286 beneficial effect Effects 0.000 abstract description 3
- 230000036983 biotransformation Effects 0.000 abstract 1
- 239000000523 sample Substances 0.000 description 19
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 15
- 238000001035 drying Methods 0.000 description 8
- 238000004128 high performance liquid chromatography Methods 0.000 description 8
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- 239000000243 solution Substances 0.000 description 4
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- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
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- 206010023126 Jaundice Diseases 0.000 description 3
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- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 description 2
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- STECJAGHUSJQJN-USLFZFAMSA-N LSM-4015 Chemical compound C1([C@@H](CO)C(=O)OC2C[C@@H]3N([C@H](C2)[C@@H]2[C@H]3O2)C)=CC=CC=C1 STECJAGHUSJQJN-USLFZFAMSA-N 0.000 description 1
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Abstract
The invention provides a fermentation pretreatment method for extracting fisetin from cotinus coggygria, which utilizes self biological enzyme and beneficial bacteria in the cotinus coggygria medicinal materials to carry out solid natural fermentation, generates more fisetin through biotransformation, obviously improves the fisetin content in the extracted raw materials, and reduces the production cost of fisetin.
Description
Technical Field
The invention relates to a pretreatment method for extracting natural products, in particular to a pretreatment method for fermentation for extracting fisetin from cotinus coggygria, and belongs to the field of natural product extraction.
Background
Cotinus coggygria Scop is a Cotinus L-plant of Cotinus genus of Anacardiaceae family, and is mainly distributed in southern Europe, Iran, Pakistan, northern India, southwest, North China, Zhejiang river and the like. Cotinus coggygria is generally used as a water and soil conservation shelter and landscaping plant and has the reputation of "tobacco trees". The Cotinus coggygria contains various chemical components such as flavone and gallic acid, is also a traditional medicinal plant in China, and can be used for treating jaundice hepatitis, scald caused by hot water or fire and dermatitis rhus; the branches are mainly used for removing dysphoria with smothery sensation, dispelling cellulitis, and removing yellow eyes; the leaf contains aromatic oil, has antiinflammatory, antibacterial and astringent effects, and is mainly used for clearing away heat and toxic materials, relieving fatigue and pain, and treating jaundice hepatitis, scald, dermatitis due to fire, and gastroenteritis by oral administration, and external application for treating trauma; cotinus coggygria root bark Cotinus coggygria medicinal part for treating postpartum strain of women is dry leaves and twigs of Cotinus coggygria, is collected in summer, and is used for clearing away damp-heat and treating acute infectious hepatitis.
Fisetin (Fisetin) is a natural flavonoid compound separated from cotinus coggygria and has the chemical name of 3,3',4', 7-tetrahydroxyflavone, which is also called Fisetin, Fisetin and Fisetin. Research shows that fisetin has pharmacological effects of inhibiting atherosclerosis, regulating immunity, improving cognitive impairment, resisting inflammation, resisting oxidation, resisting tumor, etc., has protective effect on myocardial ischemia reperfusion injury, and can be used for treating common cold, hypertension, jaundice, etc. The smoke tree has high economic value, so the smoke tree has high price. The molecular structural formula of fisetin is as follows:
CN104072465A discloses a process for extracting fisetin from cotinus coggygria, which comprises the steps of cotinus coggygria raw material crushing, ethanol pretreatment, ultrasonic extraction, macroporous resin adsorption and elution, high-speed counter-current chromatography separation and purification, crystallization and the like.
CN102924419A discloses a method for extracting fisetin from ramulus Buxi Sinicae (ramulus Cotinus coggygria), which comprises the steps of raw material crushing, extraction, crystallization and the like.
CN102442987A discloses a method for extracting fisetin, which comprises the steps of crushing dry branches and leaves of cotinus coggygria, soaking and extracting by saturated lime water solution, adsorbing by macroporous resin column, eluting by ethanol, crystallizing, dissolving by 5% sodium carbonate solution, extracting by n-butyl alcohol and ethyl acetate in sequence, recrystallizing precipitates and the like.
In order to better develop and utilize the medicinal value of cotinus coggygria, improve the yield of fisetin of cotinus coggygria and reduce the production cost of fisetin, the extraction technology of fisetin needs to be deeply researched. The extraction technology is used for directly extracting the cotinus coggygria medicinal material, and the yield of fisetin are both limited by the content of fisetin in the medicinal material. The inventor researches the extraction and preparation method of fisetin for a long time, and surprisingly discovers that the fisetin content in the medicinal materials can be obviously improved by performing solid natural fermentation pretreatment on the pulverized fresh cotinus coggygria medicinal materials by using own biological enzyme and beneficial bacteria in the cotinus coggygria medicinal materials, so that the fisetin yield of the cotinus coggygria is improved, and the invention is completed.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a fermentation pretreatment method for extracting fisetin from cotinus coggygria.
The technical scheme of the invention is as follows:
the method comprises the steps of crushing cotinus coggygria medicinal materials and naturally fermenting the solid to obtain the fisetin extraction raw material.
In some embodiments, natural solid fermentation is carried out with fresh or dried cotinus coggygria, and the raw materials are pulverized into 10-60 mesh coarse powder, preferably 20-40 mesh coarse powder before fermentation.
In some embodiments, the temperature of the solid state fermentation is from 40 ℃ to 60 ℃, preferably from 45 ℃ to 55 ℃, more preferably from 48 ℃ to 52 ℃.
In some embodiments, the air relative humidity of the solid fermentation is 75% to 98%, preferably 85% to 95%, more preferably 90% to 92%.
In some embodiments, the period of the solid natural fermentation is 3 to 15 days, preferably 8 to 13 days, and more preferably 10 to 11 days.
In some embodiments, water is added to the comminuted cotinus coggygria medicinal material fermentation material in an amount such that the water content of said fermentation material is between 40% and 80%, preferably between 50% and 70%, more preferably 60%; optionally, a proper amount of water is sprayed during the fermentation process to maintain the above water content in the fermented material.
In some embodiments, fresh cotinus coggygria medicinal material is crushed into 30-40 meshes of coarse powder, a proper amount of water is added to enable the water content of the coarse powder to reach 60%, and the coarse powder is subjected to solid natural fermentation for 10-11 days under the conditions of 50 ℃ and 90% of air relative humidity to obtain the fisetin extraction raw material.
In some embodiments, the smoke tree dry medicinal materials are crushed into 30-40 mesh coarse powder, a proper amount of water is added to enable the water content to reach 60%, and the solid is naturally fermented for 10-11 days under the conditions of 50 ℃ and 90% of air relative humidity, so that the fisetin extraction raw material is obtained.
The preparation method of the fisetin crude extract comprises the following steps: weighing appropriate amount of Cotinus coggygria (fermented or unfermented sample), reflux-extracting with 80-100% methanol for 2-3 times, each for 90min, mixing extractive solutions, concentrating under reduced pressure to dry, and vacuum-drying to obtain fisetin crude extract.
HPLC detection method of fisetin content:
firstly, chromatographic conditions:
1. a chromatographic column: ZORBAX extended C-184.6X 250mm 5 μm
2. Mobile phase: acetonitrile: 0.1% phosphoric acid solution: 26: 74
3. Flow rate: 1ml/min
4. Detection wavelength: 368nm
5. Sample introduction amount: 20 μ l
6. Column temperature: 30 deg.C
Secondly, sample preparation:
1. preparing a reference substance: precisely weighing fisetin reference 10 mg in a 100ml volumetric flask, adding 50ml of 50% methanol, ultrasonically dissolving for 10min, adding 50% methanol, ultrasonically dissolving for 20min, cooling, and fixing the volume.
2. Preparing a test article: precisely weighing a proper amount of a sample (weighing the sample amount according to the content) in a 100ml volumetric flask, adding 50ml of 50% methanol, ultrasonically dissolving for 10min, adding 50% methanol, ultrasonically dissolving for 20min, cooling and fixing the volume.
Third, sample determination
The determination method comprises precisely sucking about 10ml of reference solution and sample solution, discarding the initial filtrate, injecting into liquid chromatograph, quantifying with 20 μ l quantitative loop, determining, and calculating fisetin content with external standard method.
Fourthly, calculating the content:
wherein: mStandard sample-is the weight of the fisetin standard,
Cstandard sample-is the content of the fisetin standard,
Asample (I)-is the area of the peak of the sample,
Astandard sample-is the peak area of the fisetin standard,
Msample (I)-is the sample weight.
During the fermentation process, the content of fisetin is sampled and detected, an HPLC (high performance liquid chromatography) spectrum of a sample shows two main peaks, the peak retention time is about 10min, the fisetin peak and the peak retention time is about 15min, the relative change of the areas of the peak I and the peak II can be observed along with the extension of the fermentation time, the fisetin peak is shown, namely the area of one peak is relatively increased, the area of the other peak is correspondingly relatively reduced, namely the component of the peak II is obviously observed to be converted into fisetin of the peak I.
The pretreatment method for extracting fisetin from cotinus coggygria provided by the invention has the following advantages:
(1) the natural fermentation of the self biological enzyme and beneficial bacteria in the cotinus coggygria medicinal material can improve the content of fisetin in the medicinal material for the first time, and the biological conversion of related components to fisetin is generated by performing the pretreatment of solid natural fermentation on the crushed cotinus coggygria medicinal material, so that the content of fisetin in the fermentation material is obviously improved, the fisetin yield is greatly improved, and the production cost of fisetin is reduced;
(2) the treatment process is simple and easy to implement, the cost is low, and the economic value is obvious;
(3) reduces the using amount of the cotinus coggygria medicinal material in the production of fisetin and saves resources.
Drawings
FIG. 1 is a HPLC profile and associated analytical data tabulation of a sample of the feedstock prior to fermentation in example 1.
FIG. 2 is a HPLC profile and associated analytical data tabulation of day 3 samples from the fermentation of example 1.
FIG. 3 is a HPLC profile and associated analytical data tabulation of the day 5 samples of the fermentation of example 1.
FIG. 4 is a HPLC profile and associated analytical data tabulation of the day 8 samples of the fermentation of example 1.
FIG. 5 is a HPLC profile and associated analytical data tabulation of the sample at day 10 of fermentation of example 1.
Detailed Description
The present invention is described in further detail with reference to the following examples, but the present invention is not limited thereto, and any equivalent replacement in the field made in accordance with the present disclosure is included in the scope of the present invention.
Example 1
100kg of fresh cotinus coggygria medicinal material (the water content is 30 percent, the content of fisetin in a dried medicinal material sample is measured to be 0.76 percent) is taken and crushed into coarse powder of 30-40 meshes, proper amount of water is added to ensure that the water content reaches 60 percent, solid natural fermentation is carried out for 10 days at the relative humidity of 90 percent and the temperature of 50 ℃, and the content of fisetin in the cotinus coggygria is detected to be 3.90 percent after drying.
Example 2
100kg of dry cotinus coggygria medicinal material (the content of fisetin in a dry medicinal material sample is measured to be 0.78 percent) is taken and crushed into coarse powder of 40 meshes, a proper amount of water is added for stirring and wetting to ensure that the water content reaches 65 percent, solid natural fermentation is carried out for 11 days at the relative humidity of 85 percent and the temperature of 45 ℃, and the content of fisetin in the cotinus coggygria is detected to be 3.19 percent after drying.
Example 3
Taking 50kg of dry cotinus coggygria medicinal material (the content of fisetin in a dry medicinal material sample is measured to be 0.75%), crushing the dry cotinus coggygria medicinal material into 20-mesh coarse powder, adding a proper amount of water, stirring the coarse powder to wet the coarse powder to ensure that the water content of the coarse powder reaches 40%, carrying out solid natural fermentation for 8 days at the relative humidity of 95% and the temperature of 55 ℃ (spraying a proper amount of water to ensure that the water content of a fermentation material is kept at 40%) in the fermentation process, and detecting that the fisetin content in the cotinus coggygria is 3.02% after drying.
Example 4
Taking 200kg of fresh cotinus coggygria medicinal material (the water content is 30 percent, and the content of fisetin in a dried medicinal material sample is measured to be 0.73 percent after drying), crushing the cotinus coggygria medicinal material into 10-20 meshes of coarse powder, adding a proper amount of water to ensure that the water content reaches 75 percent, performing solid natural fermentation for 4 days at the relative humidity of 80 percent and the temperature of 60 ℃, and detecting the content of fisetin in the cotinus coggygria to be 2.88 percent after drying.
Example 5
Taking 50kg of fresh cotinus coggygria medicinal material (the water content is 30 percent, and the content of fisetin in a dried medicinal material sample is measured to be 0.80 percent after drying), crushing the cotinus coggygria medicinal material into coarse powder of 40-60 meshes, adding a proper amount of water to ensure that the water content reaches 40 percent, performing solid natural fermentation for 13 days at the relative humidity of 98 percent and the temperature of 40 ℃ (spraying a proper amount of water to ensure that the water content of the fermented material is kept at 40 percent during the fermentation process), and detecting the content of fisetin in the cotinus coggygria to be 3.27 percent after drying.
Example 6
Taking 150kg of dry cotinus coggygria medicinal material (the content of fisetin in a dry medicinal material sample is measured to be 0.81 percent), crushing the dry cotinus coggygria medicinal material into coarse powder of 30 meshes, adding a proper amount of water, stirring the coarse powder to wet the coarse powder to ensure that the water content of the coarse powder reaches 45 percent, carrying out solid natural fermentation for 9 days at the relative humidity of 75 percent and the temperature of 48 ℃, and detecting the content of fisetin in the cotinus coggygria to be 3.33 percent after drying.
Claims (9)
1. A fermentation pretreatment method for extracting fisetin from cotinus coggygria is characterized by comprising the steps of crushing cotinus coggygria medicinal materials and naturally fermenting the solid to obtain fisetin extraction raw materials.
2. The pretreatment for fermentation of claim 1, wherein said cotinus coggygria is selected from the group consisting of fresh cotinus coggygria and dry cotinus coggygria.
3. The pretreatment method for fermentation according to claim 2, wherein the medicinal material is pulverized into 10 to 60 mesh coarse powder, preferably 20 to 40 mesh coarse powder.
4. The pretreatment for fermentation according to claims 1 to 3, wherein the temperature of the solid natural fermentation is 40 ℃ to 60 ℃, preferably 45 ℃ to 55 ℃, and more preferably 48 ℃ to 52 ℃.
5. The pretreatment for fermentation according to claims 1 to 3, wherein the air relative humidity for the natural fermentation of the solid is 75 to 98%, preferably 85 to 95%, and more preferably 90 to 92%.
6. The pretreatment for fermentation according to claims 1 to 3, wherein the period of the solid natural fermentation is 3 to 15 days, preferably 8 to 13 days, and more preferably 10 to 11 days.
7. The pretreatment for fermentation as claimed in claims 1-3, wherein a proper amount of water is added to the pulverized cotinus coggygria medicinal material fermented material to make the water content of said fermented material 40-80%, preferably 50-70%, more preferably 60%; optionally, a proper amount of water is sprayed during the fermentation process to maintain the above water content in the fermented material.
8. A fermentation pretreatment method for extracting fisetin from cotinus coggygria is characterized in that fresh cotinus coggygria medicinal materials are crushed into coarse powder of 30-40 meshes, a proper amount of water is added to enable the water content of the coarse powder to reach 60%, and solid natural fermentation is carried out for 10-11 days at the temperature of 50 ℃ and the relative humidity of air of 90% to obtain fisetin extraction raw materials.
9. A fermentation pretreatment method for extracting fisetin from cotinus coggygria is characterized in that dry cotinus coggygria medicinal materials are crushed into coarse powder of 30-40 meshes, a proper amount of water is added to enable the water content of the coarse powder to reach 60%, and solid natural fermentation is carried out for 10-11 days at the temperature of 50 ℃ and the relative humidity of air of 90% to obtain fisetin extraction raw materials.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120283322A1 (en) * | 2010-08-11 | 2012-11-08 | Azi Company Ltd. | Rhus Verniciflua Stokes Extract Having Increased Content Of Active Flavonoid Compound And Method For Preparing Same |
| CN106147288A (en) * | 2015-04-08 | 2016-11-23 | 黄武 | A kind of preparation method of Ramulus et Folium Cotini Coggygiae vegetable colour |
| CN109369589A (en) * | 2018-10-12 | 2019-02-22 | 王刚 | The synchronization extraction process of smoke tree flavonoid glycoside and its application |
| CN113754626A (en) * | 2021-07-12 | 2021-12-07 | 雅安职业技术学院 | Method for preparing fisetin by enzyme method |
-
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- 2022-01-19 CN CN202210062836.9A patent/CN114315781A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20120283322A1 (en) * | 2010-08-11 | 2012-11-08 | Azi Company Ltd. | Rhus Verniciflua Stokes Extract Having Increased Content Of Active Flavonoid Compound And Method For Preparing Same |
| CN106147288A (en) * | 2015-04-08 | 2016-11-23 | 黄武 | A kind of preparation method of Ramulus et Folium Cotini Coggygiae vegetable colour |
| CN109369589A (en) * | 2018-10-12 | 2019-02-22 | 王刚 | The synchronization extraction process of smoke tree flavonoid glycoside and its application |
| CN113754626A (en) * | 2021-07-12 | 2021-12-07 | 雅安职业技术学院 | Method for preparing fisetin by enzyme method |
Non-Patent Citations (1)
| Title |
|---|
| 徐怀德等: "天然产物提取工艺学", vol. 2, 31 July 2021, 中国轻工业出版社, pages: 29 - 30 * |
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