Method for extracting fisetin from propolis and prepared propolis extract

Abstract

The invention discloses a method for extracting fisetin from propolis and a prepared propolis extract, the method comprises the following steps: (1) loading: loading a powdery propolis raw material into an extraction kettle, and sealing; (2) impurity removal: heating and pressurizing to enable the carbon dioxide to reach a supercritical state, and removing lipid components in the propolis; (3) extraction: adding absolute ethyl alcohol as an entrainer; in a supercritical state, a mixture of carbon dioxide and absolute ethyl alcohol is used for extracting fisetin in propolis; (4) separation: reducing pressure and cooling to recover the supercritical carbon dioxide to a gaseous state so as to separate the extract from the solvent; (5) concentration: collecting and concentrating the extract, and recovering ethanol; and (6) drying: performing vacuum drying to obtain the propolis extract. According to the method, the fisetin is extracted from the propolis by adopting a supercritical extraction technology, the content and the yield of the fisetin in the product can be remarkably improved, the content of the fisetin in the product can reach 70%, and the yield is greater than 80%.

Description

Technical Field

[0001] The invention relates to the technical field of extraction of active ingredients of natural products, and in particular to a method for extracting fisetin from propolis and the prepared propolis extract. Background Art

[0002] Propolis is a kind of colloid substance that is collected by bees from the buds and bark of long-gum plants, mixed with the secretions of the bees' tongue glands and wax glands, and synthesized by repeated metabolism by bees. Propolis is a reddish brown to greenish brown powder, or resinous block, with fragrance. When heated, wax is separated and can be dispersed in water. It also acts as a surfactant.

[0003] Propolis extract is a yellow-brown or dark-brown viscous substance secreted by the Chinese honey bee or Italian honey bee of the family Apidae. Its main components include flavonoids and terpenes, which have antibacterial, anti-inflammatory, antiviral, and immune-enhancing effects. The main active ingredients of propolis extract are flavonoids, including chrgsin, kaempferol, galangin, quercetin, fisetin, etc., as well as 10-hydroxy-α-decenoic acid, terpenes, HAD, royal jelly acid, organic acids, amino acids, enzymes, vitamins, inorganic salts, etc.

[0004] Fisetin, also known as fisetin, fisetin, butetin, and fisetin acid, CAS number: 528-48-3, is a polymer pharmaceutical raw material, yellow needle-shaped crystals, derived from Rhus succedanea L, Cotinus coggygria, etc., of the Anacardiaceae family, with a melting point of 330°C (decomposition), soluble in ethanol, acetone and acetic acid, and almost insoluble in water, ether, benzene, chloroform and petroleum ether. A team of Japanese and American researchers found through animal experiments that fisetin in vegetables and fruits helps improve memory.

[0005] High temperature ethanol extraction is the traditional method for extracting fisetin. Using the traditional alcohol extraction resin purification method, the content of fisetin extracted from the Chinese medicinal material Cotinus coggygria can only reach about 10%, and the best yield is only 3.64%. Summary of the invention

[0006] The technical problem to be solved by the present invention is to overcome the technical defects of the background technology and provide a method for extracting fisetin from propolis and a prepared propolis extract. The present invention uses supercritical extraction technology to extract fisetin from propolis, which can significantly increase the content and yield of fisetin in the product, wherein the content of fisetin in the product can reach 70% and the yield is greater than 80%.

[0007] The technical solution adopted by the present invention to solve the above technical problems is as follows:

[0008] A method for extracting fisetin from propolis comprises the following steps:

[0009] (1) Loading: Load the powdered propolis raw material into the extraction kettle and seal it;

[0010] (2) Impurity removal: heating and pressurizing to make carbon dioxide reach a supercritical state to remove the lipid components in propolis. The specific conditions are: extraction pressure 35-36 MPa, extraction temperature 30-40°C, and extraction time 10-12 h;

[0011] (3) Extraction: adding anhydrous ethanol as an entrainer, the mass ratio of anhydrous ethanol to supercritical carbon dioxide is 0.3-0.35:1; under supercritical conditions, the mixture of carbon dioxide and anhydrous ethanol extracts fisetin in propolis;

[0012] (4) Separation: After the extraction is completed, the pressure is reduced and the temperature is lowered to restore the supercritical carbon dioxide to a gaseous state, thereby separating the extract from the solvent. The separated extract contains fisetin;

[0013] (5) Concentration: collecting and concentrating the separated extract to recover ethanol;

[0014] (6) Drying: Vacuum drying to obtain propolis extract.

[0015] Preferably, in step (1), the particle size of the powdered propolis raw material is 10 to 20 meshes.

[0016] Preferably, in step (3), the extraction pressure is 30-35 MPa, the extraction temperature is 40-45° C., and the extraction time is 2.5-3.5 h.

[0017] Preferably, in step (4), the reduced pressure is reduced to room temperature and pressure.

[0018] Preferably, in step (5), the concentration pressure is -0.09 to -0.07 MPa, and the temperature is 70 to 80°C.

[0019] Preferably, in step (6), the vacuum drying pressure is -0.10 to -0.08 MPa, and the temperature is 75 to 85°C.

[0020] A propolis extract is prepared by using the method for extracting fisetin from propolis as described above.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] (1) The supercritical extraction technology used in the present invention is an efficient and environmentally friendly method for extracting the effective ingredients of propolis;

[0023] (2) The present invention can obtain higher purity fisetin by optimizing the extraction conditions and properly treating the extracted product, while reducing the solvent residue in the product;

[0024] (3) The equipment of the present invention is easy to operate, can significantly increase the content of the product, and the production method is green and environmentally friendly;

[0025] (4) The present invention can reduce the use of organic solvents and improve the yield of effective ingredients. DETAILED DESCRIPTION

[0026] In order to better understand the content of the present invention, further description is given below in conjunction with specific embodiments. It should be understood that these embodiments are only used to further illustrate the present invention, and are not intended to limit the scope of the present invention. In addition, it should be understood that after reading the content described in the present invention, those skilled in the art make some non-essential changes or adjustments to the present invention, which still belong to the protection scope of the present invention.

[0027] Example 1

[0028] A method for extracting fisetin from propolis, comprising the following steps:

[0029] (1) Loading: 100 g of powdered propolis raw material (particle size of 10 mesh) is placed in an extraction kettle and sealed;

[0030] (2) Impurity removal: The extraction kettle is heated by a heating system, and the compressor pressurizes the extraction kettle to make the carbon dioxide reach a supercritical state; under the supercritical state, the carbon dioxide removes the oil and neutral lipid components in the propolis. The specific conditions are: extraction pressure 35MPa, extraction temperature 30°C, and extraction time 10h;

[0031] (3) Extraction: Anhydrous ethanol was added as an entrainer, and the mass ratio of anhydrous ethanol to supercritical carbon dioxide was 0.3:1; under the supercritical state, the mixture of carbon dioxide and anhydrous ethanol extracted fisetin in propolis, and the specific conditions were: extraction pressure 30 MPa, extraction temperature 40 ° C, and extraction time 2.5 h;

[0032] (4) Separation: After the extraction is completed, the pressure is reduced and the temperature is lowered to normal temperature and pressure, so that the supercritical carbon dioxide returns to a gaseous state, thereby separating the extract from the solvent. The separated extract contains fisetin;

[0033] (5) Concentration: drying at 70°C under -0.09 MPa, collecting and concentrating the separated extract, and recovering ethanol;

[0034] (6) Drying: vacuum drying at 75°C under -0.10 MPa to obtain 1.65 g of propolis extract with a yield of 80.05%;

[0035] (7) Analysis and testing: The product was subjected to component analysis, and the content of fisetin in the propolis extract obtained was 70.83%.

[0036] Example 2

[0037] A method for extracting fisetin from propolis, comprising the following steps:

[0038] (1) Loading: 300 g of powdered propolis raw material (particle size of 10 mesh) is placed in an extraction kettle and sealed;

[0039] (2) Impurity removal: The extraction kettle is heated by a heating system, and the compressor pressurizes the extraction kettle to make the carbon dioxide reach a supercritical state; under the supercritical state, the carbon dioxide removes the oil and neutral lipid components in the propolis. The specific conditions are: extraction pressure 36MPa, extraction temperature 40°C, and extraction time 12h;

[0040] (3) Extraction: Anhydrous ethanol was added as an entrainer, and the mass ratio of anhydrous ethanol to supercritical carbon dioxide was 0.35:1; under the supercritical state, the mixture of carbon dioxide and anhydrous ethanol extracted fisetin in propolis, and the specific conditions were: extraction pressure 35 MPa, extraction temperature 45 ° C, and extraction time 3.5 h;

[0041] (4) Separation: After the extraction is completed, the pressure is reduced and the temperature is lowered to normal temperature and pressure, so that the supercritical carbon dioxide returns to a gaseous state, thereby separating the extract from the solvent. The separated extract contains fisetin;

[0042] (5) Concentration: drying at 80°C under -0.07 MPa, collecting and concentrating the separated extract, and recovering ethanol;

[0043] (6) Drying: vacuum drying at 85°C under -0.08 MPa to obtain 4.98 g of propolis extract with a yield of 81.17%;

[0044] (7) Analysis and testing: The product was subjected to component analysis, and the content of fisetin in the propolis extract obtained was 70.65%.

[0045] Example 3

[0046] A method for extracting fisetin from propolis, comprising the following steps:

[0047] (1) Loading: 500 g of powdered propolis raw material (particle size of 10 mesh) is placed in an extraction kettle and sealed;

[0048] (2) Impurity removal: The extraction kettle is heated by a heating system, and the compressor pressurizes the extraction kettle to make the carbon dioxide reach a supercritical state; under the supercritical state, the carbon dioxide removes the oil and neutral lipid components in the propolis. The specific conditions are: extraction pressure 35.5MPa, extraction temperature 35°C, extraction time 11h;

[0049] (3) Extraction: Anhydrous ethanol was added as an entrainer, and the mass ratio of anhydrous ethanol to supercritical carbon dioxide was 0.325:1; under the supercritical state, the mixture of carbon dioxide and anhydrous ethanol extracted fisetin in propolis, and the specific conditions were: extraction pressure 32.5 MPa, extraction temperature 42.5 °C, and extraction time 3 h;

[0050] (4) Separation: After the extraction is completed, the pressure is reduced and the temperature is lowered to normal temperature and pressure, so that the supercritical carbon dioxide returns to a gaseous state, thereby separating the extract from the solvent. The separated extract contains fisetin;

[0051] (5) Concentration: drying at 75°C under -0.08 MPa, collecting and concentrating the separated extract, and recovering ethanol;

[0052] (6) Drying: vacuum drying at 80°C under -0.09 MPa to obtain 8.28 g of propolis extract with a yield of 80.39%;

[0053] (7) Analysis and testing: The product was subjected to component analysis, and the content of fisetin in the propolis extract obtained was 70.87%.

[0054] Comparative Example 1

[0055] Compared with Example 3, the only difference is that in step (2), during impurity removal, the extraction pressure is 34 MPa, and the content of fisetin in the prepared propolis extract is 55.72%, and the yield is 62.17%.

[0056] Comparative Example 2

[0057] Compared with Example 3, the only difference is that in step (2), during impurity removal, the extraction temperature is 29° C., and the content of fisetin in the prepared propolis extract is 51.09%, and the yield is 55.36%.

[0058] Comparative Example 3

[0059] Compared with Example 3, the only difference is that in step (2), when removing impurities, the extraction time is 9 hours, and the content of fisetin in the prepared propolis extract is 50.15%, and the yield is 45.91%.

[0060] Comparative Example 4

[0061] Compared with Example 3, the only difference is that in step (3), no entrainer is used during supercritical carbon dioxide extraction, and the content of fisetin in the prepared propolis extract is 12.78%, and the yield is 35.25%.

[0062] Comparative Example 5

[0063] Compared with Example 3, the only difference is that in step (3), during supercritical carbon dioxide extraction, the mass ratio of anhydrous ethanol to supercritical carbon dioxide is 0.25:1, and the content of fisetin in the prepared propolis extract is 62.43%, and the yield is 72.44%.

[0064] The content of fisetin in the propolis extracts prepared in Examples 1 to 3 and Comparative Examples 1 to 5 was determined using a liquid chromatograph (ultraviolet detector) in the following manner:

[0065] 1. Chromatographic conditions

[0066] Mobile phase: A: 0.1% phosphoric acid: B: acetonitrile = 77:23

[0067] Chromatographic column: Vensil XBP C18(2) (4.6×150mm, 5μm);

[0068] Column temperature: 35°C

[0069] Flow rate: 1.0 mL / min;

[0070] Wavelength: 368nm;

[0071] Injection volume: 10 μL.

[0072] 2. Solution Preparation

[0073] (1) Test sample solution: Take about 25 mg of the test sample, accurately weigh (1 / 100,000), place in a 100-ml volumetric flask, add about 10 ml of methanol, sonicate to dissolve, remove, cool to room temperature, add methanol to the mark, and shake well.

[0074] (2) Reference substance solution: Take appropriate amount of fisetin reference substance, add methanol, and ultrasonically dissolve to prepare reference substance stock solution. Accurately measure 2 ml of the reference substance stock solution and place it in a 10 ml volumetric flask. Add water to make up to volume to prepare mixed reference substance solution.

[0075] 3. Preparation of standard curve: Accurately aspirate the fisetin reference solution and dilute it with methanol solution to form a series of concentrations of standard mixed solutions. Repeat the injection 3 times under the above chromatographic conditions, 10 μl each time, and perform regression processing based on the peak area integral value and the average value of the peak area integral and the concentration.

[0076] 4. Content calculation formula:

[0077]

[0078] Where:

[0079] C1: test sample concentration obtained according to the standard curve;

[0080] V1: volume of the test sample dissolved;

[0081] m: sample weight (g).

[0082] The calculation formula for the yield of fisetin in the propolis extracts prepared in Examples 1 to 3 and Comparative Examples 1 to 5 is:

[0083]

[0084] Where:

[0085] y: is the overall yield of the process, %;

[0086] M1: weight of the product, in grams (g);

[0087] M0: weight of raw material, in g;

[0088] w1: mass fraction of fisetin in the test sample, %;

[0089] w0: mass fraction of fisetin in raw materials, %.

[0090] The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by a person skilled in the art within the spirit and scope of the present invention shall also fall within the protection scope of the present invention.

Claims

1. A method for extracting fisetin from propolis, characterized in that: The steps include: (1) Loading: Load the powdered propolis raw material into the extraction kettle and seal it; (2) Impurity removal: heating and pressurizing to make carbon dioxide reach a supercritical state to remove the lipid components in propolis. The specific conditions are: extraction pressure 35-36 MPa, extraction temperature 30-40°C, and extraction time 10-12 h; (3) Extraction: adding anhydrous ethanol as an entrainer, the mass ratio of anhydrous ethanol to supercritical carbon dioxide is 0.3-0.35:1; under supercritical conditions, the mixture of carbon dioxide and anhydrous ethanol extracts fisetin in propolis; (4) Separation: After the extraction is completed, the pressure is reduced and the temperature is lowered to restore the supercritical carbon dioxide to a gaseous state, thereby separating the extract from the solvent. The separated extract contains fisetin; (5) Concentration: collecting and concentrating the separated extract to recover ethanol; (6) Drying: vacuum drying to obtain propolis extract.

2. A method for extracting fisetin from propolis according to claim 1, characterized in that: In step (1), the particle size of the powdered propolis raw material is 10 to 20 meshes.

3. A method for extracting fisetin from propolis according to claim 1, characterized in that: In step (3), the extraction pressure is 30-35 MPa, the extraction temperature is 40-45° C., and the extraction time is 2.5-3.5 h.

4. A method for extracting fisetin from propolis according to claim 1, characterized in that: In step (4), the temperature is lowered to room temperature and pressure.

5. The method for extracting fisetin from propolis according to claim 1, characterized in that: In step (5), the concentration pressure is -0.09 to -0.07 MPa, and the temperature is 70 to 80°C.

6. A method for extracting fisetin from propolis as claimed in claim 1, characterized in that: In step (6), the vacuum drying pressure is -0.10 to -0.08 MPa, and the temperature is 75 to 85°C.

7. A propolis extract, characterized in that: The product is prepared by the method for extracting fisetin from propolis as described in any one of claims 1 to 6.

Images