Method for extracting flavone from exocarpium citri rubrum and application of flavone
Through pre-degreasing of petroleum ether and two-stage ethanol reflux extraction, combined with polar extraction methods of ethyl acetate and n-butanol, the low extraction rate caused by the influence of pigment and waxes and polar differences in traditional flavonoid extraction is solved, and the flavonoid compounds in orange red are efficiently extracted, which is suitable for applications in functional food, health products and medicine.
Patent Information
- Application Number
- CN202510594999.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-05-09
- Publication Date
- 2025-06-06
AI Technical Summary
In traditional flavonoid extraction methods, pigments and waxes in orange red coexist with flavonoids, which affects the extraction rate, and the difference in polarity leads to the loss of some flavonoids and the total extraction rate is reduced.
Petroleum ether was pre-degreased to remove pigments and waxes, combined with two-stage ethanol reflux extraction, and polar extraction was performed using ethyl acetate and n-butanol respectively to obtain low-polar and high-polar flavonoid compounds.
The total extraction rate of flavonoids is improved, and the effective extraction of high-polar and low-polar flavonoids is ensured. The method is simple to operate and stable in process, which is suitable for industrial production needs.
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Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of extracting medicine and food, and particularly relates to a method for extracting flavonoids from tangerine peel and application thereof. Background Art
[0002] As a traditional medicinal and edible plant, the dried peel of tangerine peel is widely used in traditional Chinese medicine for cough and phlegm relief, anti-inflammatory and antibacterial effects. Modern research shows that tangerine peel is rich in flavonoids such as naringin, hesperidin and neohesperidin, which have multiple biological activities such as antioxidant, anti-inflammatory, anti-tumor and immune regulation. With the development of the health industry, flavonoids have attracted much attention in the development of functional foods, health products and anti-tumor drugs due to their natural origin and low toxicity.
[0003] Traditional flavonoid extraction mostly uses single solvent extraction or reflux extraction, but fat-soluble impurities such as pigments and waxes in orange peel coexist with flavonoids, which will affect the extraction rate of flavonoids. Moreover, flavonoids have different polarities, and extraction with only a single solvent can easily lead to the loss of high-polarity or low-polarity flavonoids, resulting in a decrease in the overall extraction rate.
[0004] Based on the above problems, the inventors used petroleum ether pre-degreasing to remove pigments and waxes, combined with two-stage ethanol reflux extraction, which helps to improve the dissolution rate of flavonoids. Then, according to the polarity difference, ethyl acetate and n-butanol were used to separate and extract low-polarity and high-polarity flavonoid compounds, which helped to improve the total flavonoid extraction rate, and can provide support for promoting the application of tangerine peel medicinal and edible products in functional foods, health products and pharmaceutical fields.
[0005] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention and should not be regarded as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art. Summary of the invention
[0006] The purpose of the present invention is to provide a method for extracting flavonoids from tangerine peel and its application, which can provide support for promoting the application of tangerine peel medicine and food homologous products in the fields of functional foods, health products and medicine.
[0007] To achieve the above object, the present invention provides the following technical solutions:
[0008] A method for extracting flavonoids from tangerine peel comprises the following steps:
[0009] S1. Petroleum ether degreasing
[0010] The tangerine peel was crushed and sieved, petroleum ether was added to remove the fat-soluble pigment, refluxed in a water bath at 80°C for 2 hours, and the petroleum ether was removed by suction to leave the filter residue;
[0011] S2. Ethanol reflux extraction
[0012] Add 70% ethanol by volume to the residue, reflux in a 70°C water bath for 2 hours, collect the filtrate, and keep the residue; repeat the operation twice and combine the two filtrates;
[0013] S3. Concentration under reduced pressure
[0014] The filtrate is concentrated under reduced pressure to a primary paste, ethanol with a volume concentration of 80% is added to the primary paste, mixed and allowed to stand at 4°C for 24 hours, the supernatant is taken and the ethanol is recovered under reduced pressure at 50-60°C to obtain an extract;
[0015] S4. Polar extraction
[0016] The extract was suspended with purified water in a volume ratio of 1:1, and extracted three times with ethyl acetate and n-butanol respectively. The extracts of each phase were combined, and the solvent was recovered under reduced pressure to obtain ethyl acetate phase extract and n-butanol phase extract, respectively. The extracts were dried to powder and stored at -20°C.
[0017] Furthermore, in step S1, the tangerine peel is crushed and passed through a 50-mesh sieve; the mass volume ratio of the tangerine peel to petroleum ether is 1:10.
[0018] Furthermore, in step S2, the ratio of the filter residue to the ethanol solution is 1:10.
[0019] Furthermore, in step S3, the mass volume ratio of the initial paste and ethanol is 1:2.
[0020] The present invention also provides the use of the ethyl acetate phase extract or the n-butanol phase extract in preparing an in vitro antioxidant active drug.
[0021] The invention also provides application of the ethyl acetate phase extract or the n-butanol phase extract in preparing anti-tumor drugs.
[0022] The present invention also provides the use of the ethyl acetate phase extract or the n-butanol phase extract in preparing a drug for inhibiting lung cancer.
[0023] Compared with the prior art, the present invention has the following beneficial effects:
[0024] (1) The present invention crushes the tangerine peel and then pre-degreases it with petroleum ether to remove pigments and wax, combines it with two-stage ethanol reflux extraction, and then extracts it with ethyl acetate and n-butanol respectively to obtain tangerine peel ethyl acetate phase flavonoid extract and tangerine peel n-butanol phase flavonoid extract, and the extraction rates are 0.1% and 0.5% respectively. The method is simple to operate and has a stable process. It can effectively control the quality of the tangerine peel flavonoid extract and can meet the needs of industrial production.
[0025] (2) The scavenging rates of the flavonoid extracts of ethyl acetate phase and n-butanol phase of tangerine peel of the present invention for DPPH at low concentrations are close to that of vitamin C, indicating that they have a strong DPPH scavenging rate.
[0026] (3) The flavonoid extracts of ethyl acetate phase and n-butanol phase of tangerine peel of the present invention can effectively inhibit the in vitro proliferation of lung cancer 549 cells and reduce the mitochondrial membrane potential of the cells. Therefore, the flavonoid compounds obtained from tangerine peel of the present invention have strong biological activity, provide an important raw material basis for the further development of natural antioxidants and anti-tumor drugs, and are expected to promote the wide application of medicinal and edible products in the fields of functional foods, health products and medicine, with high economic and social benefits. BRIEF DESCRIPTION OF THE DRAWINGS
[0027] Figure 1 To draw a rutin standard curve with absorbance as the ordinate and rutin concentration as the abscissa;
[0028] Figure 2 The scavenging ability of flavonoid extracts from ethyl acetate and n-butanol phases of orange peel on DPPH free radicals;
[0029] Figure 3 The effects of ethyl acetate phase extract and n-butanol phase extract of orange peel on the morphology of A549 cells;
[0030] Figure 4 MTT was used to detect the effect of ethyl acetate phase extract of orange peel on the proliferation of A549 cells;
[0031] Figure 5 MTT was used to detect the effect of n-butanol phase extract of orange peel on the proliferation of A549 cells;
[0032] Figure 6 To observe the red and green fluorescence of A549 cells using a fluorescence microscope;
[0033] Figure 7 It is the ratio of red fluorescence intensity to green fluorescence intensity of A549 cells.
[0034] Description of the main drawings;
[0035] Figure 3 Middle, picture magnification: 10×20;
[0036] Figure 4 Medium, ***P < 0.001, ****P < 0.0001;
[0037] Figure 5 Medium, ****P < 0.0001;
[0038] Figure 7***P<0.001, ****P<0.0001. DETAILED DESCRIPTION
[0039] The following is a clear and complete description of the technical solution of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by technicians in this field without creative work are within the scope of protection of the present invention.
[0040] 1. Materials and Methods
[0041] 1.1 Extraction of total flavonoids from tangerine peel
[0042] S1. Petroleum ether degreasing
[0043] Crush the tangerine peel through a 50-mesh sieve, take 100 g of tangerine peel powder, add petroleum ether at a mass volume ratio of 1:10 to remove fat-soluble pigments, mix well and transfer to a round-bottom flask, reflux in a water bath at 80°C for 2 h, filter to remove petroleum ether, and leave a residue;
[0044] S2. Ethanol reflux extraction
[0045] Add 70% ethanol by volume to the filter residue at a solid-liquid ratio of 1:10, reflux in a water bath at 70°C for 2 hours, collect the filtrate, and keep the filter residue; add 70% ethanol by volume to the filter residue at a solid-liquid ratio of 1:10 again, reflux in a water bath at 70°C for 2 hours, collect the filtrate, and then combine the two filtrates;
[0046] S3. Concentration under reduced pressure
[0047] The filtrate was concentrated to a primary paste by a rotary evaporator under reduced pressure. At room temperature, 80% ethanol was added to the primary paste at a mass volume ratio of 1:2. After stirring and mixing, the mixture was allowed to stand at 4°C for 24 hours. The supernatant was taken to remove water-soluble impurities such as polysaccharides and proteins. The supernatant was decompressed at 60°C to recover ethanol to obtain an extract.
[0048] S4. Polar extraction
[0049] The extract was suspended with purified water in a volume ratio of 1:1, and extracted three times with ethyl acetate and n-butanol respectively. The extracts of each phase were combined respectively, and the extracts were reduced in pressure on a rotary evaporator to recover the solvent to obtain the ethyl acetate phase extract of tangerine peel and the n-butanol phase extract of tangerine peel, respectively, which were freeze-dried into powder in a vacuum freeze dryer and stored at -20°C.
[0050] 1.2 Drawing the standard curve of rutin
[0051] Accurately weigh 20 mg of rutin standard, dissolve it completely in 60% ethanol, transfer it into a 50 mL volumetric flask, adjust the volume, shake well, and obtain a 0.4 mg / mL rutin standard solution. Accurately pipette 2, 4, 6, 8, 10, and 12 mL of the above standard solution into a 50 mL volumetric flask, add 1 mL of 5% NaNO solution, shake well, and let stand for 6 minutes; then add 10% Al(NO 3 ) 3 1 mL of solution, shake well, let stand for 6 min; add 4.0 mL of 5% NaOH solution, add 60% ethanol to make up to volume, shake well, let stand for 10 min. Measure the absorbance at a wavelength of 510 nm, and draw a rutin standard curve with rutin concentration as the horizontal axis and absorbance value (A) as the vertical axis.
[0052] 1.3 Quantitative determination of flavonoids from orange peel extract
[0053] Weigh 20 mg of the aforementioned ethyl acetate phase extract of orange peel and the n-butanol phase extract of orange peel respectively into a beaker, add 60% ethanol to dissolve, and transfer to a 50 mL volumetric flask to make up the volume and shake well. Prepare the ethyl acetate phase extract solution of orange peel and the n-butanol phase extract solution of orange peel respectively according to the steps of preparing the above rutin standard solution, measure the absorbance value (A) at a wavelength of 510 nm, substitute it into the above linear regression equation, calculate the flavonoid concentration in the ethyl acetate phase extract solution of orange peel and the n-butanol phase extract solution of orange peel respectively, and calculate the flavonoid extraction rate.
[0054] Flavonoid extraction rate (%) = (C × V × N) / m × 100%
[0055] Where, C--the concentration of flavonoids in tangerine peel extract (mg / mL);
[0056] V--volume of tangerine peel extract solution (mL);
[0057] N--Dilution multiple of tangerine peel extract;
[0058] m--Mass of tangerine peel powder used for extraction (mg).
[0059] 1.4 In vitro antioxidant activity of flavonoid extracts from orange peel
[0060] 95% ethanol was used to prepare 0.5 mg / mL vitamin C solution, ethyl acetate phase extract solution of orange red and n-butanol phase extract solution of orange red, and then diluted to 1.0, 2.0, 4.0, 8.0, 16.0 and 32.0 μg / mL, respectively. 95% ethanol was used to prepare 0.25 mM DPPH (1,1-diphenyl-2-trinitrophenylhydrazine reagent) solution.
[0061] Take 2 mL of the above vitamin C solution, ethyl acetate phase extract solution of orange peel, and n-butanol phase extract solution of orange peel respectively, mix them evenly with 2 mL of DPPH solution in a test tube, and keep them at room temperature and away from light for 30 minutes. Use 95% ethanol solution as blank control, and measure the absorbance values A1 (sample + DPPH), A2 (sample + anhydrous ethanol), and A0 (DPPH + anhydrous ethanol) at 517 nm, and repeat 6 times. Calculate the DPPH clearance rate.
[0062] DPPH removal rate (%) = [1-(A 1 -A 2 ) / A 0 ]×100%.
[0063] 1.5 Inhibitory effect of tangerine peel flavonoids extract on lung cancer A549 cells
[0064] The inhibitory effect of tangerine peel flavonoids extract on lung cancer A549 cells was detected by MTT method. The cells were seeded in 96-well plates, and 200 μL of 8×10 3 DMEM culture medium of cells was placed in a cell culture incubator for culture. When the cell fusion rate reached about 70%, sterile ethyl acetate phase extract solutions of 0, 0.4, 0.8, 1.2 and 1.6 mg / mL of orange peel were added in sequence, and sterile n-butanol phase extract solutions of 0, 0.8, 1.6 and 2.4 mg / mL of orange peel were added in sequence. Six wells were set for each concentration, and incubated for 24, 48 and 72 hours. 20 μL MTT solution (concentration of 5 mg / mL) was added to each well, and incubated for 4 hours. The supernatant was removed, and 100 μL dimethyl sulfoxide was added to each well. The wells were placed on a shaker for low-speed shaking for 10 minutes. The absorbance value (A) of each well was detected at a wavelength of 570 nm using an enzyme marker, and the absorbance value was used for cell activity analysis.
[0065] 1.6 Effect of tangerine peel flavonoids extract on mitochondrial membrane potential of lung cancer A549 cells
[0066] A549 cells were seeded in 6-well plates and 2 mL of 2 × 10 5 The 6-well plate was placed in DMEM medium containing 5% CO at 37°C. 2Incubate in a 37°C incubator. When the cell confluence reaches 70%, add sterile orange peel n-butanol phase extract solution and orange peel ethyl acetate phase extract solution to make the final concentrations 0.8 mg / mL and 1.6 mg / mL, respectively, and continue incubation for 24 hours. Aspirate the culture medium, wash 1-2 times with PBS buffer (0.01M, pH7.2-7.4), add 1 mL of cell culture medium and 1 mL of JC-1 staining working solution in turn, place on a shaker to mix thoroughly for 2 minutes, then place in a cell culture incubator at 37°C for 20 minutes, aspirate the supernatant, wash twice with JC-1 staining buffer, add 2 mL of cell culture medium, observe under an inverted fluorescence microscope, take pictures and record fluorescence images, and use Image J software to calculate the ratio of red fluorescence intensity to green fluorescence intensity to reflect the changes in mitochondrial membrane potential.
[0067] 1.7 Statistical methods
[0068] SPSS26.0 software was used for statistical analysis of the data: the experimental data were first analyzed by Shapiro-Wilk test to see if the data conformed to normal distribution, and the variance homogeneity was tested. When the data conformed to normal distribution, the data were expressed as mean ± standard deviation. The data between the two groups were compared by two independent sample t test, and the data between multiple groups were compared by one-way analysis of variance. P < 0.001 indicated that the difference was statistically significant, and P < 0.0001 indicated that the difference was extremely statistically significant.
[0069] 2. Results and Analysis
[0070] 2.1 Rutin standard curve
[0071] See Figure 1 .
[0072] Depend on Figure 1 It can be seen that the standard curve of rutin is: y=11.68x-0.0019, R 2 =0.9997.
[0073] 2.2 Quantitative determination of flavonoids from orange peel extract
[0074] The extract obtained by reflux extraction of 100g of tangerine peel powder was extracted with ethyl acetate and n-butanol in turn, and then vacuum freeze-dried to obtain 1.30g of tangerine peel ethyl acetate phase extract and 6.20g of tangerine peel n-butanol phase extract respectively; the flavonoid content of tangerine peel ethyl acetate phase was calculated to be 96.95mg, and the flavonoid extraction rate from the ethyl acetate phase was 0.1%; the flavonoid content of tangerine peel n-butanol phase was 494.33mg, and the flavonoid extraction rate from the n-butanol phase was 0.5%.
[0075] 2.3 In vitro antioxidant activity of flavonoids from orange peel extract
[0076] Results Figure 2 .
[0077] Depend on Figure 2 It can be seen that within the concentration range of 0-32 μg / mL, the scavenging ability of the ethyl acetate phase extract and the n-butanol phase flavonoid extract of orange peel on DPPH free radicals increases with the increase of concentration, and the scavenging rate of both on DPPH is similar to that of vitamin C in a lower concentration range. When the concentration of the ethyl acetate phase and the n-butanol phase of orange peel is 32 μg / mL, the scavenging rates of DPPH free radicals are 73.66% and 84.36%, respectively. This shows that the ethyl acetate phase and n-butanol phase flavonoid extracts of orange peel have strong in vitro antioxidant capacity.
[0078] 2.4 Inhibitory effect of tangerine peel flavonoids extract on lung cancer A549 cells
[0079] Results Figure 3-5 .
[0080] The flavonoids extracts from ethyl acetate phase and n-butanol phase of tangerine peel were treated on A549 cells for 24 hours. Figure 3 As shown. Under the microscope, it can be observed that the cells are wrinkled and dull, and some cells become slender spindle-shaped; while the cells in the control group are intact and bright.
[0081] Depend on Figure 4 and Figure 5 It can be seen that the flavonoid extracts from the ethyl acetate phase and the n-butanol phase of orange peel can significantly inhibit the proliferation of lung cancer A549 cells, and the inhibitory effect increases with the increase of their concentrations.
[0082] 2.5 Effect of tangerine peel flavonoids extract on mitochondrial membrane potential of lung cancer A549 cells
[0083] The JC-1 probe was used to detect changes in mitochondrial membrane potential. When JC-1 is a polymer, it produces red fluorescence, but when JC-1 is a monomer, it produces green fluorescence. The stronger the red fluorescence, the higher the mitochondrial membrane potential, and the weaker the red fluorescence, the lower the mitochondrial membrane potential. Therefore, using Image J software to process and calculate the ratio of red fluorescence intensity to green fluorescence intensity can reflect changes in mitochondrial membrane potential.
[0084] Results Figure 6 and Figure 7 .
[0085] Depend on Figure 6 It can be seen that the red fluorescence intensity of the untreated control group cells is stronger than that of the groups treated with the orange-butanol phase extract and the ethyl acetate phase extract.
[0086] Depend on Figure 7It can be seen that the ratio of red fluorescence to green fluorescence of A549 cells treated with the n-butanol phase extract and the ethyl acetate phase extract of Tangerine peel was significantly lower than that of the control group, indicating that the mitochondrial membrane potential of A549 cells was significantly reduced after being treated with the flavonoid extract of Tangerine peel. The decrease in mitochondrial membrane potential can be used as an early judgment of cell apoptosis.
[0087] In summary, the present invention crushes the tangerine peel and then pre-degreases it with petroleum ether to remove pigments and wax, combines it with two-stage ethanol reflux extraction, and then extracts it with ethyl acetate and n-butanol respectively to obtain the tangerine peel ethyl acetate phase flavonoid extract and the tangerine peel n-butanol phase flavonoid extract, and the extraction rates are 0.1% and 0.5% respectively. The method is simple to operate and has a stable process, can effectively control the quality of the tangerine peel flavonoid extract, and can meet the needs of industrial production.
[0088] In addition, it was found through experiments that the flavonoid extract of ethyl acetate phase of tangerine peel and the flavonoid extract of n-butanol phase of tangerine peel of the present invention have a strong DPPH scavenging rate, and the DPPH scavenging rate under low concentration is close to that of vitamin C. In addition, the flavonoid extract of ethyl acetate phase of tangerine peel and the flavonoid extract of n-butanol phase of tangerine peel of the present invention can effectively inhibit the in vitro proliferation ability of lung cancer 549 cells and reduce the mitochondrial membrane potential of the cells. Therefore, the flavonoid compounds obtained from tangerine peel of the present invention have strong biological activity, provide an important raw material basis for further development of natural antioxidants and anti-tumor drugs, and are expected to promote the wide application of medicinal and edible products in the fields of functional foods, health products and medicine, with high economic and social benefits.
[0089] The foregoing description of specific exemplary embodiments of the present invention is for the purpose of illustration and demonstration. These descriptions are not intended to limit the present invention to the precise form disclosed, and it is clear that many changes and variations can be made based on the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present invention and its practical application, so that those skilled in the art can realize and utilize various different exemplary embodiments of the present invention and various different selections and changes. The scope of the present invention is intended to be limited by the claims and their equivalents.
Claims
1. A method for extracting flavonoids from orange peel, characterized in that: The following steps are involved: S1. Petroleum ether degreasing The tangerine peel was crushed and sieved, petroleum ether was added to remove the fat-soluble pigment, refluxed in a water bath at 80°C for 2 hours, and the petroleum ether was removed by suction to leave the filter residue; S2. Ethanol reflux extraction Add 70% ethanol by volume to the residue, reflux in a 70°C water bath for 2 hours, collect the filtrate, and keep the residue; repeat the operation twice and combine the two filtrates; S3. Concentration under reduced pressure The filtrate is concentrated under reduced pressure to a primary paste, 80% ethanol by volume is added to the primary paste, mixed and allowed to stand at 4°C for 24 hours, the supernatant is taken and the ethanol is recovered under reduced pressure at 50-60°C to obtain an extract; S4. Polar extraction The extract was suspended with purified water in a volume ratio of 1:1, and extracted three times with ethyl acetate and n-butanol respectively. The extracts of each phase were combined, and the solvent was recovered under reduced pressure to obtain ethyl acetate phase extract and n-butanol phase extract, respectively. The extracts were dried to powder and stored at -20°C.
2. The method for extracting flavonoids from tangerine peel according to claim 1, characterized in that: In step S1, the tangerine peel is crushed and passed through a 50-mesh sieve; the mass volume ratio of the tangerine peel to petroleum ether is 1:
10.
3. The method for extracting flavonoids from tangerine peel according to claim 1, characterized in that: In step S2, the ratio of the filter residue to the ethanol solution is 1:
10.
4. The method for extracting flavonoids from tangerine peel according to claim 1, characterized in that: In step S3, the mass volume ratio of the initial paste to ethanol is 1:
2.
5. Use of the ethyl acetate phase extract or the n-butanol phase extract according to any one of claims 1 to 4 in the preparation of an in vitro antioxidant active drug.
6. Use of the ethyl acetate phase extract or the n-butanol phase extract according to any one of claims 1 to 4 in the preparation of anti-tumor drugs.
7. Use of the ethyl acetate phase extract or the n-butanol phase extract according to any one of claims 1 to 4 in the preparation of drugs for inhibiting lung cancer.