CN111991440A - Method for extracting flavonoid compounds in cinnamomum camphora by polymer aqueous two-phase extraction - Google Patents
Method for extracting flavonoid compounds in cinnamomum camphora by polymer aqueous two-phase extraction Download PDFInfo
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- CN111991440A CN111991440A CN202010858188.9A CN202010858188A CN111991440A CN 111991440 A CN111991440 A CN 111991440A CN 202010858188 A CN202010858188 A CN 202010858188A CN 111991440 A CN111991440 A CN 111991440A
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/54—Lauraceae (Laurel family), e.g. cinnamon or sassafras
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D11/00—Solvent extraction
- B01D11/04—Solvent extraction of solutions which are liquid
- B01D11/0492—Applications, solvents used
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/333—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using mixed solvents, e.g. 70% EtOH
Abstract
The invention relates to a method for extracting flavonoid compounds in cinnamomum camphora by using a polymer aqueous two-phase method, which comprises the following steps: (1) dissolving a water-soluble polymer in water, adding inorganic salt for dissolving, and then carrying out phase separation to form a polymer double-water-phase extractant; (2) adjusting the temperature of the polymer double-aqueous-phase extractant to 20-70 ℃ and the pH value to 3-9, adding crushed camphor roots to enable the material-liquid ratio of the crushed camphor roots to the polymer double-aqueous-phase extractant to be 1g (5-40) mL, stirring and extracting for 30-150 min, and standing, wherein the upper phase is a polymer phase containing flavonoid compounds, and the lower phase is an inorganic salt aqueous phase containing impurities; (3) collecting the extract in a volumetric flask, and measuring the total content of the flavonoid compounds extracted from the cinnamomum camphora by adopting a spectrophotometry. The method has the advantages of high extraction rate and high quality of the flavonoid compounds in the cinnamomum camphora.
Description
Technical Field
The invention relates to the technical field of plant extraction, and particularly relates to a method for extracting flavonoid compounds in cinnamomum camphora by using a polymer aqueous two-phase method.
Background
The flavonoid compounds are important natural organic compounds generated in the plant metabolic process, have the effects of resisting aging, enhancing the immunity of the organism, resisting and preventing cancers, regulating the endocrine system, protecting the liver, resisting inflammation, resisting viruses and the like, and are the hot spots of research and development at home and abroad for nearly 30 years. The camphor tree is evergreen arbor plant of camphor tree, is one of main tree species of subtropical green broad-leaved forest in China, and has rich resources.
The prior method for extracting flavonoids from cinnamomum camphora generally adopts a solvent with large polarity such as ethanol water solution and the like for extraction. The methods have the disadvantages of complicated operation, time consumption, low extraction rate, low quality of extracted flavone in the cinnamomum camphora, and the like. Therefore, an efficient, nontoxic and pollution-free green extraction and purification method needs to be established.
The aqueous two-phase extraction is a mixed solution of two polymers with different water-solubility or a polymer and inorganic salt, and under a certain concentration, the system can be naturally divided into two mutually incompatible phases. After the separated substance enters the aqueous two-phase system, due to the influence of factors such as surface properties, charge interaction, various acting forces (hydrophobic bond, hydrogen bond and ionic bond) and the like, the distribution coefficients between the two phases are different, so that the concentration of the separated substance in the upper phase and the concentration of the separated substance in the lower phase are different, and the separation purpose is achieved.
Disclosure of Invention
In order to solve the technical problems of low extraction rate and low quality of the prior flavonoid compounds in the cinnamomum camphora, a method for extracting the flavonoid compounds in the cinnamomum camphora by using a polymer aqueous two-phase is provided. The method has the advantages of high extraction rate and high quality of the flavonoid compounds in the cinnamomum camphora.
In order to achieve the purpose, the invention is realized by the following technical scheme:
a method for extracting flavonoid compounds in cinnamomum camphora by using polymer aqueous two-phase comprises the following steps:
(1) dissolving a water-soluble polymer in water, adding inorganic salt for dissolving, and then carrying out phase separation to form a polymer double-water-phase extractant;
(2) adjusting the temperature of the polymer double-aqueous-phase extractant to 20-70 ℃ and the pH value to 3-9, adding crushed camphor roots to enable the material-liquid ratio of the crushed camphor roots to the polymer double-aqueous-phase extractant to be 1g (5-40) mL, stirring and extracting for 30-150 min, and standing, wherein the upper phase is a polymer phase containing flavonoid compounds, and the lower phase is an inorganic salt aqueous phase containing impurities;
(3) collecting the extract in a volumetric flask, and measuring the total content of the flavonoid compounds extracted from the cinnamomum camphora by adopting a spectrophotometry.
Further, the inorganic salt in the step (1) is ammonium sulfate; in the step (1), the water-soluble polymer is polyethylene glycol, and the relative molecular mass of the polyethylene glycol is less than or equal to 2000 g/mol.
Preferably, the polyethylene glycol in step (1) has a relative molecular mass of 600 g/mol.
Further, the step (2) also comprises a process of adding sodium chloride into the polymer aqueous two-phase extractant before adding the crushed camphor roots. The inorganic ammonium sulfate in the polymer aqueous two-phase extractant has the function of forming a phase, and the function of sodium chloride can improve the extraction rate of the flavonoid compounds.
Still further, the dosage of the sodium chloride is 1-5% of the total weight of the polymer aqueous two-phase extractant.
Further, in the step (1), the mass fraction of the ammonium sulfate in the polymer aqueous two-phase extractant is 5-30%, and the mass fraction of the water-soluble polymer in the polymer aqueous two-phase extractant is 10-30%.
Further, the temperature of the polymer aqueous two-phase extractant in the step (2) is adjusted to be 25 ℃, the pH value is 9, the material-liquid ratio is 1g:20mL, stirring extraction is carried out for 120min, and the standing time is more than or equal to 2 min.
Further, the spectrophotometry in the step (3) is NaNO2-Al(NO3)3-NaOH complex spectrophotometry.
The beneficial technical effects are as follows:
according to the method, ammonium sulfate is used as inorganic salt in a polymer aqueous two-phase system, so that a phase forming effect is achieved, polyethylene glycol is used as a polymer phase, the condition of phase formation is changed by adjusting the extraction condition by utilizing the solubility difference of the flavonoid compounds and impurities in the camphor root in the aqueous phase and the polymer phase, so that the flavonoid compounds in the camphor root are distributed in the upper-phase polymer phase, the impurities such as saccharides are distributed in the lower-phase inorganic salt aqueous phase, and before the extraction process occurs, sodium chloride can be added into the polymer aqueous two-phase extractant, so that the extraction rate of the flavonoid compounds can be improved, and the high-efficiency selective separation of the flavonoid compounds is realized; the invention adopts the polymer aqueous two-phase to extract the flavonoid compounds in the cinnamomum camphora, and has the advantages of small loss, high extraction rate, few separation steps and mild operation conditions; in addition, the crude extract containing the flavonoid compound is obtained without pre-treating the camphor root, and the camphor root and the polymer aqueous two-phase extractant are directly mixed for extraction, so that the extraction steps are reduced, and the extraction rate of the flavonoid compound is improved.
Drawings
FIG. 1 shows the results of example 1 using Al (NO)3)3A standard curve chart established by measuring flavonoid compounds extracted from cinnamomum camphora by a complex spectrophotometry.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention and the accompanying drawings, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. The following description of at least one exemplary embodiment is merely illustrative in nature and is in no way intended to limit the invention, its application, or uses. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Unless specifically stated otherwise, the numerical values set forth in these examples do not limit the scope of the invention. Techniques, methods known to those of ordinary skill in the relevant art may not be discussed in detail, but are intended to be part of the specification where appropriate. In all examples shown and discussed herein, any particular value should be construed as merely illustrative, and not limiting. Thus, other examples of the exemplary embodiments may have different values.
Example 1
A method for extracting flavonoid compounds in cinnamomum camphora by using polymer aqueous two-phase comprises the following steps:
(1) dissolving 5g of PEG600 in 10g of water, adding 5g of ammonium sulfate, and after dissolution, carrying out phase separation to form a polymer double-water-phase extractant;
(2) adding 0.6g of sodium chloride into a polymer double-aqueous-phase extractant, dissolving and phase-splitting, adjusting the temperature of the polymer double-aqueous-phase extractant to 20 ℃ and the pH value to 7, adding 0.5g of crushed camphor root to ensure that the material-liquid ratio of the crushed camphor root to the polymer double-aqueous-phase extractant is 1g to 20mL (based on the volume of water), stirring and extracting for 60min, standing, wherein the upper phase is a polymer phase containing flavonoid compounds, and the lower phase is an inorganic salt aqueous phase containing impurities;
(3) collecting the upper phase, diluting to a constant volume of 25mL to obtain a to-be-detected solution of flavonoid compounds extracted from Cinnamomum camphora, and extracting with Al (NO)3)3And (3) measuring the content of the total flavone in the upper phase solution to be measured by a complex spectrophotometry.
Al(NO3)3The principle of the complex spectrophotometry is as follows: in neutral or weakly alkaline and NaNO2Under the existing condition, the flavonoid compound and the aluminum salt generate a chelate, the chelate is orange red after being added with a sodium hydroxide solution, and the chelate has an absorption peak at the wavelength of 510nm and accords with the Lambert-beer law;
the specific method comprises the following steps: taking 8 25mL volumetric flasks, sucking rutin standard solution 0.0mL, 1.0mL, 2.0mL, 3.0mL, 4.0mL, 5.0mL, 6.0mL, 7.0mL with a pipette, and adding 5% NaNO respectively2Shaking the solution 1mL, standing for 6min, adding 10% Al (NO) respectively3)31mL of the solution is shaken up and then placed for 6min, 4% NaOH solution l 0mL is added respectively, 50% v/v ethanol is added respectively to fix the volume to the scale, shaken up and placed for 15 min; obtaining a series of rutin standard solutions with different concentrations;
measuring rutin standard solutions with different concentrations at 510nm, taking the concentration of the rutin standard solution as an abscissa and the absorbance as an ordinate, wherein a standard curve is shown in figure 1, and a linear regression equation can be seen from figure 1: a is 12.95238C-0.01033, the correlation coefficient r is 0.99947, and the concentration of the rutin standard solution has a good linear relation with the absorbance in the range of 0.00-0.06 mg/mL.
Taking the flavonoid compound with constant volume to replace rutin standard solution, measuring the absorbance value at 510nm according to the method, and substituting the absorbance value into the standard curve to calculate the total flavone content in the cinnamomum camphora.
In this example, the total extraction rate of the flavonoid compounds in the camphor root is 8.74 wt%.
Example 2
A method for extracting flavonoid compounds in cinnamomum camphora by using polymer aqueous two-phase comprises the following steps:
(1) dissolving 5g of PEG600 in 10g of water, adding 5g of ammonium sulfate, and after dissolution, carrying out phase separation to form a polymer double-water-phase extractant;
(2) adding 0.6g of sodium chloride into a polymer double-aqueous-phase extractant, dissolving and phase-splitting, adjusting the temperature of the polymer double-aqueous-phase extractant to 40 ℃ and the pH value to 7, adding 0.5g of crushed camphor root to ensure that the material-liquid ratio of the crushed camphor root to the polymer double-aqueous-phase extractant is 1g:20mL, stirring and extracting for 60min, standing, wherein the upper phase is a polymer phase containing flavonoid compounds, and the lower phase is an inorganic salt aqueous phase containing impurities;
(3) collecting the upper phase, diluting to a constant volume of 25mL to obtain a to-be-detected solution of flavonoid compounds extracted from Cinnamomum camphora, and extracting with Al (NO)3)3And (3) measuring the content of the total flavone in the upper phase solution to be measured by a complex spectrophotometry.
Al(NO3)3The complexation spectrophotometry method was the same as example 1.
In this example, the total extraction rate of the flavonoid compounds in the camphor root is 8.76 wt%.
Example 3
A method for extracting flavonoid compounds in cinnamomum camphora by using polymer aqueous two-phase comprises the following steps:
(1) dissolving 5g of PEG600 in 10g of water, adding 5g of ammonium sulfate, and after dissolution, carrying out phase separation to form a polymer double-water-phase extractant;
(2) adding 0.6g of sodium chloride into a polymer double-aqueous-phase extractant, dissolving and phase-splitting, adjusting the temperature of the polymer double-aqueous-phase extractant to 25 ℃ and the pH value to 9, adding 0.5g of crushed camphor roots to ensure that the material-liquid ratio of the crushed camphor roots to the polymer double-aqueous-phase extractant is 1g:20mL, stirring and extracting for 100min, standing, wherein the upper phase is a polymer phase containing flavonoid compounds, and the lower phase is an inorganic salt aqueous phase containing impurities;
(3) collecting the upper phase, diluting to a constant volume of 25mL to obtain a to-be-detected solution of flavonoid compounds extracted from Cinnamomum camphora, and extracting with Al (NO)3)3And (3) measuring the content of the total flavone in the upper phase solution to be measured by a complex spectrophotometry method.
Al(NO3)3The complexation spectrophotometry method was the same as example 1.
In this example, the total extraction rate of the flavonoid compounds in the camphor root is 8.92 wt%.
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be considered to be within the technical scope of the present invention, and the technical solutions and the inventive concepts thereof according to the present invention should be equivalent or changed within the scope of the present invention.
Claims (8)
1. A method for extracting flavonoid compounds in cinnamomum camphora by using polymer aqueous two-phase is characterized by comprising the following steps:
(1) dissolving a water-soluble polymer in water, adding inorganic salt for dissolving, and then carrying out phase separation to form a polymer double-water-phase extractant;
(2) adjusting the temperature of the polymer double-aqueous-phase extractant to 20-70 ℃ and the pH value to 3-9, adding crushed camphor roots to enable the material-liquid ratio of the crushed camphor roots to the polymer double-aqueous-phase extractant to be 1g (5-40) mL, stirring and extracting for 30-150 min, and standing, wherein the upper phase is a polymer phase containing flavonoid compounds, and the lower phase is an inorganic salt aqueous phase containing impurities;
(3) collecting the extract in a volumetric flask, and measuring the total content of the flavonoid compounds extracted from the cinnamomum camphora by adopting a spectrophotometry.
2. The method for extracting the flavonoid compounds in the cinnamomum camphora by using the polymer aqueous two-phase method according to claim 1, wherein the inorganic salt in the step (1) is ammonium sulfate; in the step (1), the water-soluble polymer is polyethylene glycol, and the relative molecular mass of the polyethylene glycol is less than or equal to 2000 g/mol.
3. The method for extracting the flavonoid compounds in the cinnamomum camphora by using the polymer aqueous two-phase method according to claim 2, wherein the relative molecular mass of the polyethylene glycol in the step (1) is 600 g/mol.
4. The method for extracting the flavonoid compounds in the cinnamomum camphora by using the polymer aqueous two-phase extraction agent according to any one of claims 1 to 3, wherein the step (2) further comprises a process of adding sodium chloride into the polymer aqueous two-phase extraction agent before adding the crushed cinnamomum camphora roots.
5. The method for extracting the flavonoid compounds in the cinnamomum camphora by using the polymer aqueous two-phase extraction agent as claimed in claim 4, wherein the amount of the sodium chloride is 1-5% of the total weight of the polymer aqueous two-phase extraction agent.
6. The method for extracting the flavonoid compound in the cinnamomum camphora by using the polymer aqueous two-phase extraction agent according to any one of claims 1 to 3, wherein the mass fraction of the ammonium sulfate in the polymer aqueous two-phase extraction agent in the step (1) is 5-30%, and the mass fraction of the water-soluble polymer in the polymer aqueous two-phase extraction agent is 10-30%.
7. The method for extracting the flavonoid compounds in the cinnamomum camphora by using the polymer aqueous two-phase extraction agent according to any one of claims 1 to 3, wherein the temperature of the polymer aqueous two-phase extraction agent in the step (2) is adjusted to be 25 ℃, the pH value is 9, the material-liquid ratio is 1g:20mL, the mixture is stirred and extracted for 120min, and the standing time is not less than 2 min.
8. The method for extracting the flavonoid compound in the cinnamomum camphora by using the polymer aqueous two-phase method according to any one of claims 1 to 3, wherein the spectrophotometric method in the step (3) is NaNO2-Al(NO3)3-NaOH complex spectrophotometry.
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| CN1709542A (en) * | 2004-06-17 | 2005-12-21 | 西北工业大学 | Method for extracting flavone substance from leaves of Diospyms kaki by double water-phase extraction |
| US20090304824A1 (en) * | 2000-07-05 | 2009-12-10 | Capricorn Pharma, Inc. | Rapid-Melt Compositions, Methods of Making Same and Methods of Using Same |
| CN109169726A (en) * | 2018-09-03 | 2019-01-11 | 兰溪市奥而特农业科技有限公司 | A kind of compounding agent containing bactericidal effect |
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090304824A1 (en) * | 2000-07-05 | 2009-12-10 | Capricorn Pharma, Inc. | Rapid-Melt Compositions, Methods of Making Same and Methods of Using Same |
| CN1709542A (en) * | 2004-06-17 | 2005-12-21 | 西北工业大学 | Method for extracting flavone substance from leaves of Diospyms kaki by double water-phase extraction |
| CN109169726A (en) * | 2018-09-03 | 2019-01-11 | 兰溪市奥而特农业科技有限公司 | A kind of compounding agent containing bactericidal effect |
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| 储炬: "《现代生物工艺学》", 30 September 2007, 华东理工大学出版社 * |
| 刘宝亮: "响应面分析法优化离子液体双水相提取香樟叶中总黄酮的工艺条件", 《现代食品科技》 * |
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