CN111437311A - Method for extracting flavonoids from pharbitis seed peels - Google Patents
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Abstract
The invention relates to the technical field of ugly citrus extracts, and discloses a method for extracting flavonoids from ugly citrus peel, which is used for separating and extracting the flavonoids from the ugly citrus peel by a method combining ethanol extraction and ultrasonic-microwave assistance and has high extraction efficiency. On the basis, by adopting a means of applying impact load after mixing and freezing the ugly peel and water, the cell integrity of the ugly peel is damaged by utilizing the brittleness improvement of the ugly peel and the brittleness failure of ice crystals under the condition of large load and quick application, the extraction of flavonoids substances by ethanol extraction and ultrasonic-microwave auxiliary combination is promoted, and meanwhile, the purification of the flavonoids substances is realized by adopting the characteristic of the reaction of the flavonoids compounds and metal salts, and the activity is improved.
Description
Technical Field
The invention relates to the technical field of ugly citrus extracts, in particular to a method for extracting flavonoids from ugly citrus peel.
Background
The bioflavonoid, polysaccharide and alkaloid are three natural products of plant origin and widely exist in plants in the nature. The flavonoids compounds are prepared by connecting 2 benzene rings with phenolic hydroxyl groups through a central three-carbon atom, and have the effects of resisting oxidation, resisting cancer, inhibiting lipase, resisting bacteria, diminishing inflammation and the like. At present, flavonoid natural products such as resveratrol and ellagic acid appear in the market, and are used for health care, immunity enhancement, cancer resistance and the like.
Citrus is one of the most important economic crops in the world and is the second economic fruit crop in China. The production of a large amount of byproducts such as peels and peel residues is accompanied in the orange processing process, for example, the peels after the canned oranges are processed account for about 30-40% of the fruit weight, the juiced orange residues account for about 50% of the wet weight, and the current accumulated production amount of the peels/peel residues of the oranges in the whole country can reach 500 million tons, so that how to improve the utilization efficiency of the peel residues of the oranges is a great problem facing the orange processing industry in the country. The citrus peel is rich in functional substances such as flavonoid, phenolic acid, essential oil, carotenoid and the like, and the content of the functional substances is higher than that of the pulp. The method for separating and obtaining flavonoid substances from the citrus peel and peel residues is an important direction for improving the comprehensive utilization efficiency of the citrus peel.
The ugu citrus is a kind of citrus, introduced from japan, and has been planted in large quantities in Zhejiang, Chongqing, Yunnan, Jiangxi, Hunan, and the like. The ugly oranges have the characteristic of high-sugar crisp meat, the sugar content reaches 13-14 degrees, the maximum sugar content can reach more than 16 degrees, the taste sweetness is moderate, the ugly oranges contain less than 1% of acid during maturation, and the ugly oranges are well loved by consumers. The ugly oranges are also accompanied by a large amount of skin dregs generated in the processing process, and how to extract flavonoid compounds from the skins of the ugly oranges has important significance for further improving the economic value of the ugly oranges.
Chinese patent 201811609330.5, the patent name "a citrus peel extract and its extraction method and application", discloses a method for obtaining a citrus peel extract containing polyphenol and flavonoid compounds by using Nehel navel orange peel as a raw material and performing ethanol extraction, reflux extraction and rotary evaporation, then petroleum ether extraction and ethyl acetate extraction and purification, and the method is applied to the field of food preservation due to the characteristics of oxidation resistance and antibiosis, but the method has low efficiency of obtaining the citrus peel extract by repeatedly extracting with ethanol as a solvent.
Disclosure of Invention
In order to efficiently extract flavonoids from the peel of the ugly citrus, the invention aims to provide a method for extracting flavonoids from the peel of the ugly citrus, which adopts a method combining solvent extraction and ultrasound-microwave to improve the extraction efficiency of the flavonoids.
The invention provides the following technical scheme:
a method for extracting flavonoids from ugly citrus peel comprises the following steps:
(1) cleaning peel of fresh ugly oranges, drying, crushing and sieving to obtain ugly orange peel powder;
(2) dispersing the ugly peel powder into an ethanol water solution, and applying microwave and ultrasonic combined extraction;
(3) centrifuging and separating supernatant after extraction, performing rotary evaporation to obtain an organic paste, and performing freeze drying to obtain crude extract powder;
(4) purifying and separating the crude extract powder to obtain flavonoids.
The extraction method provided by the invention comprises the steps of drying and crushing the peel of the ugly oranges to obtain the ugly orange peel powder, dispersing the ugly orange peel powder into ethanol, and extracting flavonoids from the ugly orange peel powder by adopting a mode of combined action of ultrasound and microwave. Wherein ultrasonic cavitation forms local hot spots in ethanol and micro-jet impacts the surface of the ugly citrus peel to form cavitation corrosion, which causes the damage of the microstructure of ugly citrus peel cells and accelerates the dissolution and diffusion of components in the cells in the ethanol; the microwave breaks plant cells under the action of a microwave field in an instantaneous penetration heating mode, and the microwave is cooperated with the ultrasonic action, so that the extraction of flavonoids is enhanced.
As an improvement of the method, the drying process of the peel in the step (1) comprises the following steps: mixing peel of the ugly oranges with water according to a mass ratio of 1: 0.3-0.7, then placing the mixture in a low-temperature environment box at the temperature of-80 to-40 ℃ for freezing for 12-14 hours, then applying an impact load of 20-40 MPa to the peel for stamping, wherein the acceleration of the impact load is 8-10 g, and quickly moving the peel of the ugly oranges into a freeze drying device for freeze drying and dehydration. The ugly citrus peel contains a large amount of fiber structures, the cell wall structure is thick, the general crushing process is usually the macro peel crushing, but the cell structure is still kept complete on the microstructure, and the subsequent ultrasonic-microwave-ethanol combined extraction is not facilitated. Therefore, the pericarp is mixed with water and then frozen at low temperature, the water is frozen into ice on the surface of the pericarp and after being immersed into pericarp cells, the pericarp is made crisp, particularly the brittleness of the fibrous structure of the pericarp is improved, then the pericarp is impacted at low temperature, ice crystals on the surface of the pericarp and in the pericarp are converted into brittleness and broken by the larger load impact force and the faster load application speed, so that the cell structure of the pericarp is expanded, the integrity of the cells is damaged, and then the moisture is removed by adopting a freeze drying mode, so that the extraction of the flavonoid compound can be greatly improved.
As an improvement of the method, in the step (2), the ultrasonic treatment temperature is 60-65 ℃, the ultrasonic power is 300-500W, the ultrasonic frequency is 30-40 KHz, the microwave power is 200-250W, the microwave frequency is 500-600 MHz, and the treatment time is 20-30 min. The microwave power should be controlled during the extraction process, and the excessive microwave power can easily cause solution explosion boiling and liquid spraying, resulting in flavone loss.
As an improvement of the method, the feed-liquid ratio of the ugly orange peel powder and the ethanol water solution in the step (2) is 1: 15-22, and the concentration of the ethanol water solution is 55-65 wt%.
As an improvement of the method, the rotating speed of the rotary evaporation in the step (3) is 40-60 rpm/min, and the temperature is 30-50 ℃.
As an improvement of the method of the invention, the purification process of the step (4) comprises the following steps: preparing the crude extract into water solution, adding zinc chloride solution, adjusting pH value until precipitation is completely generated, centrifuging, collecting precipitate, dissolving with ethanol water solution, adding ethylenediamine tetraacetic acid, vacuum drying to obtain solid, dissolving with methanol, centrifuging, and vacuum drying supernatant to obtain purified flavonoids. The flavonoids have large pi conjugated bonds and can perform complex reaction with metal ions to form complex precipitates. The characteristic of the flavonoid is utilized to separate the flavonoid, and the rest crude components are left in the aqueous dispersion because of the non-reaction. Then dissociating by adopting ethylenediamine tetraacetic acid, dissolving and separating by using methanol, and drying in vacuum to obtain the purified flavonoid substance. In the prior art, calcium chloride is used for separating flavonoids, but the inventor finds that zinc chloride has a better separation effect on flavonoids in the orange peel. Meanwhile, the inventor intends to show that a small amount of plant polyphenol has the capability of complexing with metal ions, so that the flavonoid substance purified by the method is still a flavonoid substance containing a small amount of plant polyphenol impurities, and further purification and separation can be realized by means of liquid phase and the like.
As an improvement of the method, the concentration of the aqueous solution of the crude extract is 0.1-0.25 g/L, the concentration of the zinc chloride solution is 5-8 mmol/L, the pH value is 8-9, and the concentration of the ethanol aqueous solution is 20-30 wt%.
The improvement of the method is characterized in that the molar ratio of the ethylene diamine tetraacetic acid to the zinc chloride is 1-3: 1, and the retention time is 3-5 hours.
The invention has the following beneficial effects:
compared with the prior art, the preparation method disclosed by the invention has the advantages that the flavonoid substances are separated and extracted from the peel of the pharbitis seed by a method combining ethanol extraction and ultrasonic-microwave assistance, and the extraction efficiency is high. On the basis, a means of applying impact load after mixing and freezing the ugly peel and water is adopted, and the cell integrity of the ugly peel is damaged by using the brittleness improvement of the ugly peel and the brittleness failure of ice crystals under the conditions of large load and quick application, so that the extraction of flavonoids substances by ethanol extraction and ultrasonic-microwave auxiliary combination is promoted. Meanwhile, the characteristic of reaction of the flavonoid compound and the metal salt is adopted to realize the purification of the flavonoid substance and improve the activity.
Detailed Description
The following further describes the embodiments of the present invention.
The starting materials used in the present invention are commercially available or commonly used in the art, unless otherwise specified, and the methods in the following examples are conventional in the art, unless otherwise specified.
As the content of flavonoids is influenced by the growth cycle, the semen pharbitidis which is picked in the same batch in 12 months and 1 day in 2019 is adopted for testing.
Example 1
A method for extracting flavonoids from ugly citrus peel comprises the following steps:
(1) cleaning peel of fresh ugly citrus, freeze-drying at-80 deg.C, pulverizing, sieving with 200 mesh sieve to obtain ugly citrus peel powder, and storing at-80 deg.C;
(2) dispersing the pharbitis seed peel powder into an ethanol water solution to obtain a mixed system, wherein the material-liquid ratio of the pharbitis seed peel powder to the ethanol water solution is 1:20, the concentration of the ethanol water solution is 60 wt%, ultrasonic and microwave combined extraction is applied to the mixed system, the ultrasonic treatment temperature is 62 ℃, the ultrasonic power is 500W, the ultrasonic frequency is 40KHz, the microwave power is 200W, the microwave frequency is 600MHz, the microwave treatment time is 30s every 5min, and the treatment time is 30 min;
(3) centrifuging the mixed system after extraction, retaining the supernatant and performing rotary evaporation at 30 ℃ to obtain a crude extract, wherein the rotary evaporation rate is 45 rpm/min;
(4) filtering and purifying the crude extract with 0.25 μm microfiltration membrane, freeze drying to obtain flavonoids, separating by liquid phase, and calculating the peak area of four standard substances including hesperidin, rutin, Sichuan hesperetin, and hesperetin to be 19.42g/kg semen Pharbitidis peel dry powder. The reason why the hesperidin, the rutin, the Sichuan hesperetin and the hesperetin are adopted as the standard substances is that the inventor finds that four main substances, namely the hesperidin, the rutin, the Sichuan hesperetin and the hesperetin, account for more than 50% of the total amount in the research on flavonoid substances in the pharbitis seed peels picked in different periods, and the rest peak areas are all calculated as flavonoid substances; the following examples are the same.
Example 2
A method for extracting flavonoids from ugly peel is different from the method in the example 1 in that in the step (2): the feed-liquid ratio of the ugly peel powder to the ethanol aqueous solution is 1:22, the concentration of the ethanol aqueous solution is 55 wt%, ultrasonic and microwave combined extraction is applied to a mixed system, the temperature of ultrasonic treatment is 65 ℃, the ultrasonic power is 300W, the ultrasonic frequency is 30KHz, the microwave power is 250W, the microwave frequency is 500MHz, the microwave treatment time is 30s every 5min, the treatment time is 20min, and the content is 18.95g/kg of ugly peel dry powder.
Example 3
A method for extracting flavonoids from ugly citrus peel is different from the method in the example 1 in that in the step (2): the feed-liquid ratio of the ugly peel powder to the ethanol aqueous solution is 1:15, the concentration of the ethanol aqueous solution is 65 wt%, ultrasonic and microwave combined extraction is applied to a mixed system, the temperature of ultrasonic treatment is 60 ℃, the ultrasonic power is 400W, the ultrasonic frequency is 35KHz, the microwave power is 200W, the microwave frequency is 550MHz, the microwave treatment time is 30s every 5min, the treatment time is 25min, and 19.02g/kg of ugly peel dry powder is calculated.
Example 4
A method for extracting flavonoids from ugly peel is different from the method in example 1 in that the drying process in the step (1) is as follows: mixing peel of the ugly oranges with water according to the mass ratio of 1:0.5, then placing the mixture in a low-temperature environment box at the temperature of minus 80 ℃ for freezing for 12 hours, then applying an impact load of 30MPa to the peel to punch the peel of the ugly oranges, wherein the acceleration of the impact load is 10g, and then quickly moving the peel of the ugly oranges into a freeze drying device for freeze drying and dehydration. The final extraction flavone amount is 26.02g/kg of ugly orange peel dry powder.
Example 5
A method for extracting flavonoids from ugly peel is different from the method in example 1 in that the drying process in the step (1) is as follows: mixing peel of the ugly oranges with water according to the mass ratio of 1:0.3, then placing the mixture in a low-temperature environment box at the temperature of-70 ℃ for freezing for 12 hours, then applying an impact load of 40MPa to the peel to punch the peel of the ugly oranges, wherein the acceleration of the impact load is 9.5g, and then quickly moving the peel of the ugly oranges into a freeze-drying device for freeze-drying and dehydration. Finally extracting the dry powder of the ugly citrus peel with the flavone amount of 25.68 g/kg.
Example 6
A method for extracting flavonoids from ugly peel is different from the method in example 1 in that the drying process in the step (1) is as follows: mixing peel of the ugly oranges with water according to the mass ratio of 1:0.7, then placing the mixture in a low-temperature environment box at the temperature of minus 40 ℃ for freezing for 14 hours, then applying an impact load of 20MPa to the peel to punch the peel of the ugly oranges, wherein the acceleration of the impact load is 8g, and then quickly moving the peel of the ugly oranges into a freeze drying device for freeze drying and dehydration. The final extraction flavone amount is 25.33g/kg of ugly citrus peel dry powder.
Comparative example 1
A method for extracting flavonoids from ugly peel is different from the method in example 1 in that the drying process in the step (1) is as follows: mixing peel of semen Pharbitidis and water at a mass ratio of 1:0.5, freezing in a low-temperature environment box at-80 deg.C for 12 hr, and freeze-drying in a freeze-drying device. The final extraction flavone amount is 19.14g/kg of ugly citrus peel dry powder.
Comparative example 2
A method for extracting flavonoids from ugly peel is different from the method in example 1 in that the drying process in the step (1) is as follows: freezing peel of the ugly oranges in a low-temperature environment box at the temperature of-80 ℃ for 12 hours, then applying an impact load of 30MPa to the peel to punch the peel of the ugly oranges, wherein the acceleration of the impact load is 10g, and then quickly moving the peel of the ugly oranges into a freeze-drying device for freeze-drying and dehydration. The final extraction amount was 19.55g/kg of ugly citrus peel dry powder.
Example 7
A method for extracting flavonoids from pharbitis seed peels is different from the embodiment 1 in that the separation and purification process of the step (4) is that the crude extract is prepared into an aqueous solution with the concentration of 0.1 g/L, the pH value is adjusted to 8, then 5 mmol/L zinc chloride solution is dripped into the aqueous solution until the precipitate is completely generated, centrifugal separation is carried out after the precipitate is completely generated, the precipitate is taken out and dissolved by 20 wt% of ethanol aqueous solution, ethylene diamine tetraacetic acid is added into the ethanol aqueous solution for keeping for 3 hours, the molar ratio of the ethylene diamine tetraacetic acid to the zinc chloride is 1:1, then vacuum drying is carried out to obtain a solid, then methanol is dissolved and centrifugal separation is carried out, and the supernatant is vacuum dried to obtain purified flavonoids, and the final extraction amount is 13.46g/kg pharbitis seed peel dry powder.
Example 8
A method for extracting flavonoids from pharbitis seed peels is different from the embodiment 1 in that the separation and purification process of the step (4) is that a crude extract is prepared into an aqueous solution with the concentration of 0.2 g/L, the pH value is adjusted to 8, then 6 mmol/L zinc chloride solution is dripped into the aqueous solution until precipitation is completed, centrifugal separation is carried out after the precipitation is completely generated, the precipitate is taken out and dissolved by 25 wt% of ethanol aqueous solution, ethylene diamine tetraacetic acid is added into the ethanol aqueous solution for keeping for 4 hours, the molar ratio of the ethylene diamine tetraacetic acid to the zinc chloride is 1:2, then vacuum drying is carried out to obtain a solid, then methanol is dissolved and centrifugal separation is carried out, and the supernatant is vacuum dried to obtain purified flavonoids, wherein the final extraction amount is 13.22g/kg pharbitis seed peel dry powder.
Example 9
A method for extracting flavonoids from pharbitis seed peels is different from the embodiment 1 in that the separation and purification process of the step (4) is that a crude extract is prepared into an aqueous solution with the concentration of 0.25 g/L, the pH value is adjusted to 9, then 8 mmol/L zinc chloride solution is dripped into the aqueous solution until precipitation is completed, centrifugal separation is carried out after the precipitation is completely generated, the precipitate is taken out and dissolved by 30 wt% of ethanol aqueous solution, ethylene diamine tetraacetic acid is added into the ethanol aqueous solution for keeping for 5 hours, the molar ratio of the ethylene diamine tetraacetic acid to the zinc chloride is 1:3, then vacuum drying is carried out to obtain a solid, then methanol is dissolved and centrifugal separation is carried out, and the supernatant is vacuum dried to obtain purified flavonoids, wherein the final extraction amount is 13.68g/kg pharbitis seed peel dry powder.
Example 10
A method for extracting flavonoids from ugly citrus peel comprises the following steps:
(1) cleaning peel of fresh ugly oranges, drying, crushing, sieving with a 200-mesh sieve to obtain ugly orange peel powder, and storing in an environment at-80 ℃ for later use, wherein the drying process comprises the following steps: mixing peel of the ugly oranges with water according to a mass ratio of 1:0.5, then placing the mixture in a low-temperature environment box at the temperature of minus 80 ℃ for freezing for 12 hours, then applying an impact load of 30MPa to the peel to punch the peel of the ugly oranges, wherein the acceleration of the impact load is 10g, and then quickly moving the peel of the ugly oranges into a freeze drying device for freeze drying and dehydration;
(2) dispersing the pharbitis seed peel powder into an ethanol water solution to obtain a mixed system, wherein the material-liquid ratio of the pharbitis seed peel powder to the ethanol water solution is 1:20, the concentration of the ethanol water solution is 60 wt%, ultrasonic and microwave combined extraction is applied to the mixed system, the ultrasonic treatment temperature is 62 ℃, the ultrasonic power is 500W, the ultrasonic frequency is 40KHz, the microwave power is 200W, the microwave frequency is 600MHz, the microwave treatment time is 30s every 5min, and the treatment time is 30 min;
(3) centrifuging the mixed system after extraction, retaining the supernatant and performing rotary evaporation at 30 ℃ to obtain a crude extract, wherein the rotary evaporation rate is 45 rpm/min;
(4) separating and purifying the crude extract by preparing the crude extract into an aqueous solution with the concentration of 0.1 g/L, adjusting the pH value to 8, then dropwise adding a 5 mmol/L zinc chloride solution, centrifugally separating after complete precipitation, dissolving the precipitate with 20 wt% of an ethanol aqueous solution, adding ethylene diamine tetraacetic acid, keeping for 3 hours, keeping the molar ratio of the ethylene diamine tetraacetic acid to the zinc chloride at 1:1, then carrying out vacuum drying to obtain a solid matter, dissolving the methanol, centrifugally separating, and carrying out vacuum drying on a supernatant to obtain a purified flavonoid substance, wherein the extraction amount is 17.82g/kg of the dried pharbitis orange peel powder.
Example 11
A method for extracting flavonoids from ugly citrus peel comprises the following steps:
(1) cleaning peel of fresh ugly oranges, drying, crushing, sieving with a 200-mesh sieve to obtain ugly orange peel powder, and storing in an environment at-80 ℃ for later use, wherein the drying process comprises the following steps: mixing peel of the ugly oranges with water according to a mass ratio of 1:0.3, then placing the mixture in a low-temperature environment box at the temperature of-70 ℃ for freezing for 12 hours, then applying an impact load of 40MPa to the peel to punch the peel of the ugly oranges, wherein the acceleration of the impact load is 9.5g, and then quickly moving the peel of the ugly oranges into a freeze-drying device for freeze-drying and dehydration;
(2) dispersing the pharbitis seed peel powder into an ethanol water solution to obtain a mixed system, wherein the material-liquid ratio of the pharbitis seed peel powder to the ethanol water solution is 1:20, the concentration of the ethanol water solution is 60 wt%, ultrasonic and microwave combined extraction is applied to the mixed system, the ultrasonic treatment temperature is 62 ℃, the ultrasonic power is 500W, the ultrasonic frequency is 40KHz, the microwave power is 200W, the microwave frequency is 600MHz, the microwave treatment time is 30s every 5min, and the treatment time is 30 min;
(3) centrifuging the mixed system after extraction, retaining the supernatant and performing rotary evaporation at 30 ℃ to obtain a crude extract, wherein the rotary evaporation rate is 45 rpm/min;
(4) separating and purifying the crude extract by preparing the crude extract into an aqueous solution with the concentration of 0.2 g/L, adjusting the pH value to 8, then dropwise adding 6 mmol/L zinc chloride solution, carrying out centrifugal separation after complete precipitation, dissolving the precipitate with 25 wt% of ethanol aqueous solution, adding ethylene diamine tetraacetic acid, keeping for 4 hours, wherein the molar ratio of the ethylene diamine tetraacetic acid to the zinc chloride is 1:2, then carrying out vacuum drying to obtain a solid matter, dissolving the methanol, carrying out centrifugal separation, and carrying out vacuum drying on the supernatant to obtain a purified flavonoid substance, wherein the extraction amount is 17.26g/kg of the dried pharbitis orange peel powder.
Example 12
A method for extracting flavonoids from ugly citrus peel comprises the following steps:
(1) cleaning peel of fresh ugly oranges, drying, crushing, sieving with a 200-mesh sieve to obtain ugly orange peel powder, and storing in an environment at-80 ℃ for later use, wherein the drying process comprises the following steps: mixing peel of the ugly oranges with water according to a mass ratio of 1:0.7, then placing the mixture in a low-temperature environment box at the temperature of minus 40 ℃ for freezing for 14 hours, then applying an impact load of 20MPa to the peel to punch the peel of the ugly oranges, wherein the acceleration of the impact load is 8g, and then quickly moving the peel of the ugly oranges into a freeze drying device for freeze drying and dehydration;
(2) dispersing the pharbitis seed peel powder into an ethanol water solution to obtain a mixed system, wherein the material-liquid ratio of the pharbitis seed peel powder to the ethanol water solution is 1:20, the concentration of the ethanol water solution is 60 wt%, ultrasonic and microwave combined extraction is applied to the mixed system, the ultrasonic treatment temperature is 62 ℃, the ultrasonic power is 500W, the ultrasonic frequency is 40KHz, the microwave power is 200W, the microwave frequency is 600MHz, the microwave treatment time is 30s every 5min, and the treatment time is 30 min;
(3) centrifuging the mixed system after extraction, retaining the supernatant and performing rotary evaporation at 30 ℃ to obtain a crude extract, wherein the rotary evaporation rate is 45 rpm/min;
(4) separating and purifying the crude extract by preparing the crude extract into an aqueous solution with the concentration of 0.25 g/L, adjusting the pH value to 9, then dropwise adding 8 mmol/L zinc chloride solution, carrying out centrifugal separation after complete precipitation, dissolving the precipitate with 30 wt% of ethanol aqueous solution, adding ethylenediamine tetraacetic acid, keeping for 5 hours, wherein the molar ratio of the ethylenediamine tetraacetic acid to the zinc chloride is 1:3, then carrying out vacuum drying to obtain a solid matter, carrying out centrifugal separation after methanol dissolution, and carrying out vacuum drying on the supernatant to obtain a purified flavonoid substance, wherein the extraction amount is 16.88g/kg of the dried pharbitis orange peel powder.
ABTS free radical scavenging test
(1) Preparing ABTS working solution, namely mixing 7 mmol/L ABTS of 5m L and 140 mmol/L potassium persulfate solution of 88 mu L, standing for 16h at room temperature in a dark condition, and finally forming ABTS stock solution, wherein the ABTS stock solution is stable at room temperature in the dark condition, is diluted into the working solution by absolute ethyl alcohol before use, and requires the absorbance at 734nm to be 0.70 +/-0.05;
(2) preparing 100, 200, 300, 400, 500, 600, 700, 800, 900 and 1000 mu mol/L Trolox standard substance by using absolute ethyl alcohol, taking 10 small test tubes with the length of 15m L, adding ABTS working solution with the length of 2m L, respectively adding 100, 200, 300, 400, 500, 600, 700, 800, 900 and 1000 mu mol/L Trolox standard substance with the length of 40u L, uniformly oscillating, reacting in water bath at 30 ℃ for 6min, measuring the A value (734nm), paralleling for 3 times, taking the average value to calculate, taking the concentration (mu mol/L) of Trolox as the horizontal coordinate, and obtaining the clearance rate ([ A mol/L)0-A]A 100%) as ordinate, drawing a standard curve;
(3) determination of samples the extracts of the samples obtained in each example were prepared with absolute ethanol at a concentration of 800ppm, ABTS working solution 2m L was added with sample extract of 800ppm of 40u L, uniformly shaken, reacted in a water bath at 30 ℃ for 6min, measured for A value (734nm) in parallel 3 times, and averaged to calculate the antioxidant activity equivalent TEAC, i.e. the free radical scavenging capacity of 1g of flavonoids of each example corresponds to the mg of Trolox scavenging capacity, the results are shown in Table 1.
TABLE 1 TEAC (mg/g) of flavonoids in examples
Claims (8)
1. A method for extracting flavonoids from ugly citrus peel is characterized by comprising the following steps:
(1) cleaning peel of fresh ugly oranges, drying, crushing and sieving to obtain ugly orange peel powder;
(2) dispersing the ugly peel powder into an ethanol water solution, and applying microwave and ultrasonic combined extraction;
(3) centrifuging and separating supernatant after extraction, performing rotary evaporation to obtain an organic paste, and performing freeze drying to obtain crude extract powder;
(4) purifying and separating the crude extract powder to obtain flavonoids.
2. The method for extracting flavonoids from ugly citrus peel according to claim 1, wherein the drying process of the peel in the step (1) comprises: mixing peel of the ugly oranges with water according to a mass ratio of 1: 0.3-0.7, then placing the mixture in a low-temperature environment box at the temperature of-80 to-40 ℃ for freezing for 12-14 hours, then applying an impact load of 20-40 MPa to the peel for stamping, wherein the acceleration of the impact load is 8-10 g, and quickly moving the peel of the ugly oranges into a freeze drying device for freeze drying and dehydration.
3. The method for extracting flavonoids from pharbitis seed peels as claimed in claim 1, wherein the ultrasonic treatment temperature in the step (2) is 60-65 ℃, the ultrasonic power is 300-500W, the ultrasonic frequency is 30-40 KHz, the microwave power is 200-250W, the microwave frequency is 500-600 MHz, and the treatment time is 20-30 min.
4. The method for extracting flavonoids from pharbitis seed peels as claimed in claim 1 or 3, wherein the feed-liquid ratio of the pharbitis seed peel powder and the ethanol water solution in the step (2) is 1: 15-22, and the concentration of the ethanol water solution is 55-65 wt%.
5. The method for extracting flavonoids from pharbitis seed peels as claimed in claim 1, wherein the rotation speed of the rotary evaporation in the step (3) is 40-60 rpm/min, and the temperature is 30-50 ℃.
6. The method for extracting flavonoids from ugly citrus peel according to claim 1, wherein the purification process of the step (4) is as follows: preparing the crude extract into water solution, adding zinc chloride solution, adjusting pH value until precipitation is completely generated, centrifuging, collecting precipitate, dissolving with ethanol water solution, adding ethylenediamine tetraacetic acid, vacuum drying to obtain solid, dissolving with methanol, centrifuging, and vacuum drying supernatant to obtain purified flavonoids.
7. The method for extracting flavonoids from pharbitis seed peels as claimed in claim 6, wherein the concentration of the aqueous solution of the crude extract is 0.1-0.25 g/L, the concentration of the zinc chloride solution is 5-8 mmol/L, the pH value is 8-9, and the concentration of the aqueous solution of ethanol is 20-30 wt%.
8. The method for extracting flavonoids from pharbitis seed peels as claimed in claim 6, wherein the molar ratio of the ethylenediamine tetraacetic acid to the zinc chloride is 1-3: 1, and the retention time is 3-5 hours.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113209177A (en) * | 2021-05-12 | 2021-08-06 | 浙江工商大学 | Method for extracting flavonoid compounds in citrus peel with assistance of ultrasonic waves |
| CN114916672A (en) * | 2022-06-21 | 2022-08-19 | 成都农业科技中心 | Method for extracting citrus flavonoid |
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| CN104926767A (en) * | 2014-03-18 | 2015-09-23 | 五邑大学 | Method of purification preparation of citrus flavones by adopting macroporous adsorption resin |
| CN107619422A (en) * | 2017-10-27 | 2018-01-23 | 北京理工大学 | A kind of method for preparing the O glucuronides of high-purity gossypitrin 8 |
| CN108693127A (en) * | 2018-03-12 | 2018-10-23 | 湖北大学知行学院 | A method of extracting flavones by raw material of wild elephant skin mandarin orange |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104926767A (en) * | 2014-03-18 | 2015-09-23 | 五邑大学 | Method of purification preparation of citrus flavones by adopting macroporous adsorption resin |
| CN107619422A (en) * | 2017-10-27 | 2018-01-23 | 北京理工大学 | A kind of method for preparing the O glucuronides of high-purity gossypitrin 8 |
| CN108693127A (en) * | 2018-03-12 | 2018-10-23 | 湖北大学知行学院 | A method of extracting flavones by raw material of wild elephant skin mandarin orange |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113209177A (en) * | 2021-05-12 | 2021-08-06 | 浙江工商大学 | Method for extracting flavonoid compounds in citrus peel with assistance of ultrasonic waves |
| CN113209177B (en) * | 2021-05-12 | 2022-06-28 | 浙江工商大学 | Method for extracting flavonoid compounds in citrus peels with assistance of ultrasonic waves |
| CN114916672A (en) * | 2022-06-21 | 2022-08-19 | 成都农业科技中心 | Method for extracting citrus flavonoid |
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