CN111418845A - Preparation method of laver polyphenol extract and application of laver polyphenol extract - Google Patents
Preparation method of laver polyphenol extract and application of laver polyphenol extract Download PDFInfo
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- CN111418845A CN111418845A CN202010447686.4A CN202010447686A CN111418845A CN 111418845 A CN111418845 A CN 111418845A CN 202010447686 A CN202010447686 A CN 202010447686A CN 111418845 A CN111418845 A CN 111418845A
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
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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
- A61K36/02—Algae
- A61K36/04—Rhodophycota or rhodophyta (red algae), e.g. Porphyra
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/06—Antigout agents, e.g. antihyperuricemic or uricosuric agents
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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01D—SEPARATION
- B01D11/00—Solvent extraction
- B01D11/02—Solvent extraction of solids
- B01D11/0203—Solvent extraction of solids with a supercritical fluid
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- 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
- A61K2236/37—Extraction at elevated pressure or temperature, e.g. pressurized solvent extraction [PSE], supercritical carbon dioxide extraction or subcritical water extraction
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- 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
- A61K2236/50—Methods involving additional extraction steps
- A61K2236/51—Concentration or drying of the extract, e.g. Lyophilisation, freeze-drying or spray-drying
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Abstract
The invention discloses a method for preparing laver polyphenol and the application of laver polyphenol extract, which takes the laver polyphenol extract as one of active ingredients or the only active ingredient to prepare xanthine oxidase inhibitor; the preparation method of the laver polyphenol comprises the following steps: homogenizing newly picked thallus Porphyrae at low temperature, pulverizing, freeze drying to obtain thallus Porphyrae dry powder, defatting under nitrogen protection, and supercritical CO extracting2Preparing the laver polyphenol, wherein the preferable conditions for preparing the laver polyphenol are as follows: the temperature is 50 ℃, the ethanol concentration of the entrainer is 70%, the time is 2.0h, and the pressure is 30 MPa; removing organic solvent at low temperature, and freeze drying to obtain polyphenol extract; the inhibition kinetics of the laver polyphenol on the xanthine oxidase activity andthe fluorescence quenching experimental result provides reliable experimental and theoretical basis for the application of the inhibitor in the field of reducing uric acid level, and also provides a new way for high-value utilization of laver and development of an economic and safe natural xanthine oxidase inhibitor.
Description
Technical Field
The invention relates to the technical field of food biochemistry and pharmaceutical chemistry, in particular to a preparation method of a laver polyphenol extract and application of the laver polyphenol extract.
Background
Laver belongs to the family of Rhodophyta, and is called "vitamin treasure house" and contains abundant minerals and vitamins essential to human body, and has low fat content. China is a big country for laver production, and the first yield of the 21 st century leaps the world. Since the laver has high nutritional quality and good taste in the early stage, the laver can be widely used as food material, and the laver in the later stage is usually wasted due to abandoning and harvesting. In recent years, research focuses on later-stage and application, but research on analysis of nutritional vitamin components such as protein, fat, carbohydrate and the like of the laver has achieved certain results, but evaluation of chemical components of the laver is not comprehensive and thorough, physiological functions of polyphenol, flavone and other small molecular active substances are not fully researched, and researches on extraction, purification, structural analysis and biological activity of the laver are reported rarely, so that high-value application of the laver is limited.
With the improvement of the living standard of people and the change of the dietary structure, hyperuricemia (gout) becomes the fourth highest after the third highest (hypertension, hyperlipidemia and hyperglycemia), seriously threatens the health of human beings, and reports show that the patients with ventilation disease have more than 8000 ten thousands. The first method for reducing the incidence rate of hyperuricemia and gout is to reduce the serum uric acid level, xanthine oxidase can promote the formation of uric acid, and the inhibition of the activity of xanthine oxidase can reduce the rate of uric acid formation, thereby effectively reducing the formation of uric acid. At present, allopurinol, which is a typical common drug for inhibiting the production of uric acid, can effectively inhibit xanthine oxidase, but adverse reactions such as rash, pruritus, diarrhea, abdominal pain, cytopenia and the like can occur. Therefore, the development of more natural xanthine oxidase inhibitor drugs is urgently needed.
The research finds that the polyphenol of terrestrial and marine plants has better biological activity, but the application research of the polyphenol of seaweed in the aspect of enzyme inhibitor is rarely reported at present. In view of this, research and development of the application of the laver polyphenol extract as a xanthine oxidase inhibitor can provide technical and experimental theoretical bases for deep development and utilization of functional products of marine algae polyphenol and related medicines, and broaden high-value utilization of marine algae resources.
Disclosure of Invention
The invention aims to provide a preparation method of a laver polyphenol extract and application of the laver polyphenol extract, which are implemented by low-temperature pretreatment and supercritical CO treatment of raw materials2The porphyra polyphenol is extracted, an inhibition action mechanism is revealed through experimental researches such as the inhibition activity capability, the inhibition kinetics and the fluorescence quenching capability of xanthine oxidase, an experimental theoretical basis is provided for the development and the utilization of the porphyra, and meanwhile, an economical and safe xanthine oxidase inhibitor of a natural seaweed source is obtained.
In order to achieve the purpose, the technical scheme adopted by the invention for solving the technical problem is as follows:
a method for preparing laver polyphenol extract and its application as xanthine oxidase inhibitor are provided, wherein laver polyphenol extract is used as active ingredient for preparing xanthine oxidase inhibitor.
The technical scheme provided by the invention is as follows:
CO (carbon monoxide)2A supercritical extraction method of laver polyphenol comprises the following steps:
1) cleaning newly picked laver, placing the laver in ice water, homogenizing and crushing the laver at a high speed at 0-5 ℃ by a homogenizer, freeze-drying the laver, and sieving the dried laver powder with a 50-mesh sieve for later use;
2) raw material degreasing: placing the laver powder obtained in the step 1) in a reflux device with a nitrogen protection device, selecting petroleum ether for refluxing for 6 hours at 70 ℃, cooling to room temperature, performing suction filtration, washing the petroleum ether for 2 times, placing the filter cake in the reflux device with the nitrogen protection again for refluxing for 3 hours, performing suction filtration, and placing the filter cake in a vacuum drying oven for drying for later use; preferably, the boiling range of petroleum ether in step 2 is from 30 to 60 ℃.
3) Loading the laver powder obtained in the step 2) into a supercritical extraction kettle, and extracting for 0.5-2 h at the temperature of 40-70 ℃ and the volume fraction of entrainer ethanol of 50-80% under 20-40 MP.
Preferably, the extraction pressure in step three is 30 MPa.
Preferably, the extraction temperature in step three is 50 ℃.
Preferably, the extraction time in step three is 2 h.
Preferably, the volume part of the entrainer in step three is 70%.
4) Mixing the solutions of the multiple laver extracts obtained in the step 3), performing low-temperature rotary evaporation to remove the organic solvent, and performing freeze drying to obtain the laver polyphenol extract.
Preferably, the de-esterification of the raw material laver is performed under the protection of nitrogen.
Preferably, petroleum ether for de-esterification of the raw material laver has a boiling range of 30 to 60 ℃.
An application of the laver polyphenol in inhibiting xanthine oxidase activity is provided.
The invention has the following beneficial effects:
(1) the invention relates to a low-temperature pretreatment mode of raw materials and supercritical CO2The method for extracting the laver polyphenol limits various critical conditions, can reduce the loss of the laver polyphenol caused by oxidative deterioration and the like in the raw material pretreatment and extraction processes, and can obviously improve the extraction rate of the laver polyphenol and the content of the laver polyphenol in the laver polyphenol extract.
(2) The xanthine oxidase inhibitor is laver polyphenol which is an extract of laver homologous to natural food and medicine, and has the advantages of rich laver resources in China, simple preparation process of the polyphenol and easy industrialization.
(3) The dynamics of the activity of the laver polyphenol in inhibiting xanthine oxidase and fluorescence quenching experimental data provide reliable experimental theoretical basis for the development field of the food and the medicine for reducing uric acid, and provide a new way for further high-value utilization of laver resources and development and utilization of natural marine xanthine oxidase inhibitors.
(4) The half-inhibitory concentration of the laver polyphenol to xanthine oxidase is lower than that of allopurinol which is a commercially available drug selected in a positive control experiment, so that the inhibition effect is obviously better than that of allopurinol which is a sold drug in the experimental data alone.
Drawings
FIG. 1 shows the inhibitory effect of the polyphenol extract of laver on xanthine oxidase enzyme activity;
FIG. 2 is a graph showing the measurement of xanthine oxidase inhibition mechanism by the polyphenol extract of laver;
FIG. 3 is a graph showing the measurement of xanthine oxidase inhibition types and inhibition constants of the polyphenol extract of laver in accordance with the present invention;
FIG. 4 is a fluorescence spectrum of interaction between a laver polyphenol extract and xanthine oxidase according to the present invention.
Detailed Description
The present invention is further described in detail with reference to specific examples, so that those skilled in the art can implement the invention with reference to the description.
Example 1:
CO (carbon monoxide)2The supercritical extraction method of laver polyphenol comprises the following steps:
step 1) adding cleaned and newly picked laver into ice water, homogenizing and crushing at a high speed at 0-5 ℃, freeze-drying, and sieving the dried laver powder with a 50-mesh sieve for later use;
and 2) placing the laver powder obtained in the step 1 in a reflux device with a nitrogen protection device, selecting petroleum ether with a boiling range of 30-60 ℃ for refluxing for 6h at 70 ℃, cooling to room temperature, performing suction filtration, washing with the petroleum ether for 2 times, placing the filter cake in the reflux device with the nitrogen protection again for refluxing for 3h, performing suction filtration, and placing the filter cake in a vacuum drying oven for drying for later use.
Step 3) loading the treated laver powder into a supercritical extraction kettle, extracting for 1.5h at 40 ℃ under the pressure of 20MP and the volume fraction of entrainer ethanol of 60 percent,
and 4) combining the laver polyphenol extracting solution, performing low-temperature rotary evaporation to remove the organic solvent, and performing freeze drying to obtain the laver polyphenol extract.
Calculating according to gallic acid standard curve to obtain laver polyphenol extraction rate of 0.37mg/g (gallic acid equivalent).
Example 2: [ Steps 1), 2), 4) in the same manner as in example 1]
And 3) loading the treated laver powder into a supercritical extraction kettle, extracting for 2 hours at 40 ℃ under the pressure of 25MP and with 70 volume parts of entrainer ethanol.
Calculating according to gallic acid standard curve to obtain laver polyphenol extraction rate of 0.65mg/g (gallic acid equivalent).
Example 3: [ Steps 1), 2), 4) in the same manner as in example 1]
And 3) filling the treated laver powder into a supercritical extraction kettle, extracting for 2.5 hours at 40 ℃ under the pressure of 30MP and with the entrainer ethanol accounting for 80 volume percent.
Calculating according to gallic acid standard curve to obtain laver polyphenol extraction rate of 0.54mg/g (gallic acid equivalent).
Example 4: [ Steps 1), 2), 4) in the same manner as in example 1]
And 3) filling the treated laver powder into a supercritical extraction kettle, extracting for 2 hours at 50 ℃ under the pressure of 30MP and with the entrainer ethanol accounting for 60 volume percent.
Calculating according to gallic acid standard curve to obtain laver polyphenol extraction rate of 0.72mg/g (gallic acid equivalent).
Example 5: [ Steps 1), 2), 4) in the same manner as in example 1]
And 3) filling the treated laver powder into a supercritical extraction kettle, extracting for 2.5 hours at 50 ℃ under the pressure of 20MP and the volume fraction of entrainer ethanol of 70 percent.
Calculating according to gallic acid standard curve to obtain laver polyphenol extraction rate of 0.62mg/g (gallic acid equivalent).
Example 6: [ Steps 1), 2), 4) in the same manner as in example 1]
And 3) filling the treated laver powder into a supercritical extraction kettle, extracting for 1.5 hours at 50 ℃ under the pressure of 25MP and with the entrainer ethanol accounting for 80 volume percent.
Calculating according to gallic acid standard curve to obtain laver polyphenol extraction rate of 0.59mg/g (gallic acid equivalent).
Example 7: [ Steps 1), 2), 4) in the same manner as in example 1]
And 3) filling the treated laver powder into a supercritical extraction kettle, extracting for 2.5 hours at the temperature of 60 ℃ under the pressure of 25MP and with the entrainer ethanol accounting for 60 volume percent.
Calculating according to gallic acid standard curve to obtain laver polyphenol extraction rate of 0.53mg/g (gallic acid equivalent).
Example 8: [ Steps 1), 2), 4) in the same manner as in example 1]
And 3) filling the treated laver powder into a supercritical extraction kettle, extracting for 1.5 hours at the temperature of 60 ℃ under the pressure of 30MP and with the entrainer ethanol accounting for 70 volume percent.
Calculating according to gallic acid standard curve to obtain laver polyphenol extraction rate of 0.56mg/g (gallic acid equivalent).
Example 9: [ Steps 1), 2), 4) in the same manner as in example 1]
And 3) filling the treated laver powder into a supercritical extraction kettle, extracting for 1.5 hours at the temperature of 60 ℃ under the pressure of 30MP and with the entrainer ethanol accounting for 70 volume percent.
Calculating according to gallic acid standard curve to obtain laver polyphenol extraction rate of 0.73mg/g (gallic acid equivalent).
Example 10: [ Steps 1), 2), 4) in the same manner as in example 1]
And 3) filling the treated laver powder into a supercritical extraction kettle, extracting for 2 hours at 50 ℃ under the pressure of 30MP and with 70 volume parts of entrainer ethanol.
Calculating according to gallic acid standard curve to obtain laver polyphenol extraction rate of 0.79mg/g (gallic acid equivalent).
To verify the inhibitory effect of porphyra polyphenol on xanthine oxidase, the inhibitory effect of porphyra polyphenol on xanthine oxidase at different concentrations was tested by using commercial medicine allopurinol as a positive reference and xanthine as a model substrate, as specifically shown in example 11. Further discloses an inhibition mechanism of the porphyra polyphenol on xanthine oxidase, and experimental examinations are carried out by respectively utilizing enzymatic reaction kinetics and fluorescence quenching methods of small molecules and protease, specifically examples 12-14.
Example 11
The test result of the inhibition experiment of the extracted porphyra polyphenol on the xanthine oxidase activity is shown in figure 1, the inhibition rate of the porphyra polyphenol on the xanthine oxidase activity is gradually increased along with the increase of the concentration, when the polyphenol concentration is more than 0.025mg/m L, the inhibition rate tends to be in an equilibrium state, the corresponding half inhibition concentration is 0.0045mg/m L and is lower than the half inhibition concentration of allopurinol on the xanthine oxidase by 0.0245mg/m L, the inhibition activity of the porphyra polyphenol on the xanthine oxidase can be shown to be stronger than allopurinol only from the aspect of enzyme inhibition experiment data, and the in vivo activity verification is further carried out, so that the porphyra polyphenol is expected to become a potential natural xanthine oxidase inhibitor.
Example 12
Fixing the concentration of substrate xanthine, changing the concentrations of xanthine oxidase and laver polyphenol in the system, and determining the relationship between enzyme amount and enzyme activity. From fig. 2, it is known that the inhibition rates of the laver polyphenols with different concentrations and the xanthine oxidase with different concentrations are in a linear relationship and are intersected at the origin, and the inhibition rates are gradually reduced along with the increase of the polyphenol concentration, which indicates that the inhibition of the laver polyphenols on the xanthine oxidase is a reversible process.
Example 13
Under the condition that the concentration of xanthine oxidase is not changed, xanthine solutions with different concentrations are used as substrates, laver polyphenol solutions with different concentrations are added, the change of the light absorption value of a mixed system at the wavelength of 290nm is measured, the enzymatic reaction rate (V) of the xanthine oxidase is obtained, 1/V is plotted against 1/[ S ], L ineweaver-Burk double reciprocal diagram without adding or adding laver polyphenol with different concentrations is obtained, the inhibition type between the inhibition type and the inhibition type is judged, as can be seen from figure 3, the inhibition of the laver polyphenol on the xanthine oxidase is mixed inhibition, the inhibitor can be combined with both free enzyme and enzyme-substrate complex, the Ki value of the laver polyphenol on the xanthine oxidase inhibition is 0.089mg/m L, the Kis value is 0.197mg/m L, Ki < Kis shows that the binding capacity of the laver polyphenol on the xanthine oxidase is stronger than the binding capacity on the enzyme-substrate complex, and the graph shows that the xanthine oxidase only has one or one type of binding sites on the xanthine oxidase.
Example 14
In a buffer system with 0.05 mol/L pH being 7.4Tris-HCl, measuring the emission spectrum of the laver polyphenol for inhibiting xanthine oxidase in the wavelength range of 285-420 nm, as shown in figure 4, the fluorescence intensity of the xanthine oxidase is gradually reduced along with the increase of the concentration of the laver polyphenol, the peak position of the maximum fluorescence emission position generates obvious red shift, and the data obtained by combining the Stem-Volmer equation can obtain that the binding site of the laver polyphenol and the xanthine oxidase is 1, and a ground state complex is formed to cause static quenching.
Example 15
The inhibitory activity of laver polyphenol on xanthine oxidase is compared with that of rutin, quercetin, catechin and myricetin which are the polyphenols in commercial terrestrial plants, the experimental scheme is the same as that in example 11, the half inhibitory concentration IC50 of four polyphenols on xanthine oxidase is respectively 0.0193mg/m L, 0.0234mg/m L, 0.037mg/m L and 0.0217mg/m L, and the inhibitory activity of laver polyphenol on xanthine oxidase is far higher than that of rutin, quercetin, catechin and myricetin which are four terrestrial plant polyphenols.
And (4) conclusion:
in the aspect of pretreatment of experimental raw material laver, low-temperature ice water is mixed, homogenized and crushed, and freeze-dried to obtain dried laver powder. By using supercritical CO2The preparation process of the laver polyphenol is extracted and optimized, and the optimal process conditions are as follows: the extraction pressure is 30MPa, the extraction temperature is 50 ℃, 70% ethanol is used as entrainer, the extraction time is 2h, and the extraction rate of the laver polyphenol is 0.79mg/g (calculated by gallic acid equivalent) under the condition.
The inhibition of the activity of the porphyra polyphenol on enzyme belongs to reversible mixed inhibition, the binding capacity with xanthine oxidase is stronger than that with an enzyme-substrate compound, and the inhibition capacity of the porphyra polyphenol on the xanthine oxidase activity is better than that of a commercial medicament allopurinol.
3, the interaction between the laver polyphenol and the enzyme is known from the fluorescence spectrum of the interaction between the laver polyphenol and the xanthine oxidase, so that the microenvironment where the enzyme is located changes, and regular quenching is caused, the fluorescence intensity of the xanthine oxidase is gradually reduced along with the increase of the concentration of the laver polyphenol, the data obtained by combining the Stem-Volmer equation shows that the fluorescence quenching of the laver polyphenol to the xanthine oxidase belongs to static quenching, the number of the combined sites is 1, and the result is identical with the result of a dynamics double reciprocal experiment.
The above description is only a preferred embodiment of the present invention, and therefore should not be taken as limiting the scope of the invention, which is defined by the appended claims.
Claims (5)
1. A preparation method of a laver polyphenol extract is characterized by comprising the following steps:
1) and (3) pretreating the raw materials: adopting a low-temperature homogenizing, crushing and freeze-drying method:
adding cleaned and newly picked laver into ice water, homogenizing and crushing at a high speed at 0-5 ℃, freeze-drying, and sieving the dried laver powder with a 50-mesh sieve for later use;
2) and degreasing raw materials: placing the laver powder obtained in the step 1) in a reflux device with a nitrogen protection device, selecting petroleum ether for refluxing for 6 hours at 70 ℃, cooling to room temperature, performing suction filtration, washing the petroleum ether for 2 times, placing the filter cake in the reflux device with the nitrogen protection again for refluxing for 3 hours, performing suction filtration, and placing the filter cake in a vacuum drying oven for drying for later use;
3) loading the laver powder obtained in the step 2) into a supercritical extraction kettle, and extracting for 0.5-2 h at the temperature of 40-70 ℃ and the volume fraction of entrainer ethanol of 50-80% under 20-40 MP;
4) mixing the solutions of the multiple laver extracts obtained in the step 3), performing low-temperature rotary evaporation to remove the organic solvent, and performing freeze drying to obtain the laver polyphenol extract.
2. The method for preparing a polyphenol extract of laver as claimed in claim 1, wherein: : and (2) crushing the raw materials in the step 1), and performing high-speed homogeneous crushing at a low temperature of 0-5 ℃.
3. The method for preparing a polyphenol extract of laver as claimed in claim 1, wherein: the de-esterification of the raw material laver is carried out under the protection of nitrogen.
4. The method for preparing a polyphenol extract of laver as claimed in claim 1, wherein: the boiling range of petroleum ether for the de-esterification of the raw material laver is 30-60 ℃.
5. Use of a laver polyphenol extract prepared by the method as claimed in any one of claims 1 to 4, wherein: the application of the compound in inhibiting xanthine oxidase activity.
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