CN116421670A - Camellia oleifera shell polyphenol extract and preparation method and application thereof - Google Patents
Camellia oleifera shell polyphenol extract and preparation method and application thereof Download PDFInfo
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Classifications
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- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
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- A61K36/82—Theaceae (Tea family), e.g. camellia
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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
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- A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
- A61K47/36—Polysaccharides; Derivatives thereof, e.g. gums, starch, alginate, dextrin, hyaluronic acid, chitosan, inulin, agar or pectin
- A61K47/38—Cellulose; Derivatives thereof
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Abstract
The invention belongs to the technical field of extraction and application of plant active ingredients, and particularly relates to a camellia oleifera shell polyphenol extract, a preparation method and application thereof. According to the invention, the tea-oil camellia shells which are by-products of tea-oil processing are fully utilized as raw materials, the tea-oil camellia shells are fully destroyed by grinding under mild conditions, the tea-oil camellia shells are extracted by combining a microwave-assisted extraction method, and then the tea-oil camellia shells are purified by macroporous resin and nanofiltration, so that the activity of the obtained tea-oil camellia shell polyphenol extract is obviously improved on the premise of keeping higher yield, and the amylase is inhibited more. In addition, the invention can improve the comprehensive utilization rate of waste, reduce the production cost, increase the economic benefit, reduce the environmental pollution problem caused by the waste of the camellia oleifera shells, and has positive economic benefit and social significance.
Description
Technical Field
The invention belongs to the technical field of extraction and application of plant active ingredients. More particularly relates to a camellia oleifera shell polyphenol extract and a preparation method and application thereof.
Background
The oil tea fruit shells are also called oil tea cattails, account for more than 60% of the weight of a single oil tea fruit, are byproducts in the processing process of the oil tea, and are about three million tons per year. In actual production, the oil tea fruit shells are mostly abandoned and burnt, so that the oil tea fruit shells cannot be effectively utilized. However, the prior art researches show that the camellia oleifera shells are rich in active substances such as saponin, tannin, polyphenol, polysaccharide and the like, and have various physiological activities such as antioxidation, bacteriostasis, anti-inflammatory, anticancer and the like; phenolic substances such as procyanidine, hydrolyzed tannin and the like in oil tea shells have proved to have good inhibition effect on alpha-amylase and alpha-glucosidase of intestinal starch digestive enzyme, can help to realize slow digestion and absorption of starch, maintain postprandial blood sugar steady-state effect, and can prevent various diet-related chronic diseases. And the camellia oleifera shell polyphenol substances as natural products accord with the pursuit concept of people on natural healthy, safe and nontoxic medicines.
In order to improve the extraction efficiency of polyphenol in camellia oleifera shells, the prior art commonly uses technologies such as microwaves, ultrasonic waves, ultrahigh pressure and the like to carry out auxiliary solvent extraction, for example, chinese patent application CN103030710A discloses a method for recycling camellia oleifera shells, firstly, the camellia oleifera shells are subjected to microwave treatment, then are subjected to high-temperature, long-time and repeated alcohol extraction and water extraction to obtain active components of polyphenol and polysaccharide, and then auxiliary agents are added into residues to prepare biological oil and biological carbon products by pyrolysis. Although the yield of the product is higher, under the conditions of high temperature, long time and multiple extraction operations, active ingredients in the oil tea fruit shells, particularly active ingredients with poor stability such as polyphenol, are easy to change, so that the activity of the extract is reduced. Therefore, there is an urgent need to provide a method for extracting camellia oleifera shells, which improves the biological activity of the extract on the premise of maintaining a higher extraction rate.
Disclosure of Invention
The invention aims to overcome the defects and shortcomings of reduced activity of an extract caused by high temperature, long time and repeated extraction in the existing extraction method of the camellia oleifera shell polyphenol, and provides a preparation method of the camellia oleifera shell polyphenol extract, which is carried out under mild conditions and can rapidly obtain the camellia oleifera shell polyphenol extract with higher activity in high yield.
The invention aims to provide the camellia oleifera shell polyphenol extract obtained by the preparation method.
The invention further aims to provide application of the camellia oleifera shell polyphenol extract.
The above object of the present invention is achieved by the following technical scheme:
a preparation method of a camellia oleifera shell polyphenol extract specifically comprises the following steps:
s1, drying oil tea shells and crushing to obtain coarse oil tea shell powder;
s2, mixing and grinding the coarse oil tea fruit shell powder obtained in the step S1 with a composite solid auxiliary agent to obtain oil tea fruit shell fine powder;
s3, adding an ethanol solution into the oil tea fruit shell fine powder obtained in the step S2, and extracting for 60-90S under the assistance of 210-350W microwaves;
s4, centrifuging the extracting solution, collecting supernatant, removing ethanol, adjusting the pH to 4-5, and supplementing water until the volume is consistent with the volume of the ethanol solution added in the step S3;
s5, diluting the extracting solution obtained in the step S4, loading the diluted extracting solution into a macroporous resin column, washing with water to remove impurities after the adsorption is finished, eluting with ethanol solution, collecting eluent, and removing ethanol;
and S6, filtering the eluent obtained in the step S5 by using a nanofiltration membrane, and collecting filtrate.
Further, in step S1, the drying is performed until the moisture content is 5 to 8%.
Preferably, in step S1, the crushed material is sieved by a 40-80 mesh sieve.
Preferably, in step S1, the drying is hot air drying or heat pump drying, and the temperature is 40-60 ℃.
Further, in step S2, the compound solid auxiliary agent is sodium carbonate, hydroxypropyl- β -cyclodextrin, and microcrystalline cellulose.
Further, in step S2, the mass ratio of sodium carbonate, hydroxypropyl- β -cyclodextrin (HP- β -CD) and microcrystalline cellulose (MCC) is 1 to 4:0.5 to 1:1 to 2
Further, in the step S2, the addition amount of the solid auxiliary agent is 1-4% of the mass of the coarse powder of the camellia oleifera shells. Preferably, the addition amount of the solid auxiliary agent is 2-4% of the mass of the coarse powder of the camellia oleifera shell.
Preferably, in step S2, the grinding is planetary ball milling, vibratory ball milling or stirred ball milling.
Further, in step S2, the grinding time is 5 to 20 minutes.
Further, in the step S3, the volume concentration of the ethanol solution is 30% -50%.
Further, in the step S3, the mass-volume ratio of the oil tea fruit shell fine powder to the ethanol solution is 1:5-20 g/ml.
Preferably, in step S5, the dilution factor is 2 to 4 times.
Further, in step S5, the macroporous resin is HP20, LSA-21 or AB-8.
Further, in step S5, the volume concentration of the ethanol solution used for the elution is 20%, 50%, 70% and 90% in this order.
Preferably, in step S5, the flow rate of the sample is 1-2 mL/min, and the flow rate of the elution is 1-2 mL/min.
Further, in the step S6, the molecular weight cut-off of the nanofiltration membrane is 500-3000 Da.
In addition, the invention also provides the camellia oleifera shell polyphenol extract obtained by the preparation method.
Researches show that the camellia oleifera shell polyphenol extract prepared by the method has a remarkable inhibition effect on amylase, so that the application of the camellia oleifera shell polyphenol extract in preparing amylase inhibitors is also claimed. Preferably, the amylase is an alpha-amylase or an alpha-glucosidase.
The invention has the following beneficial effects:
according to the invention, the tea-oil camellia shells which are by-products of tea-oil processing are fully utilized as raw materials, the tea-oil camellia shells are fully destroyed by grinding under mild conditions, and the extraction is carried out by combining a microwave-assisted extraction method, so that the activity of the obtained tea-oil camellia shell polyphenol extract is obviously improved on the premise of keeping a higher yield, and the amylase is inhibited more. In addition, the invention can improve the comprehensive utilization rate of waste, reduce the production cost, increase the economic benefit, reduce the environmental pollution problem caused by the waste of the camellia oleifera shells, and has positive economic benefit and social significance.
Detailed Description
The present invention is further illustrated below with reference to specific examples, which are not intended to limit the invention in any way. Unless specifically stated otherwise, the reagents, methods and apparatus employed in the present invention are those conventional in the art.
Reagents and materials used in the following examples are commercially available unless otherwise specified.
Example 1 preparation method of tea-oil camellia Shell polyphenol extract
The preparation method of the camellia oleifera shell polyphenol extract specifically comprises the following steps:
s1, drying oil tea shells until the moisture content is 5%, crushing, and sieving with a 60-mesh sieve to obtain oil tea shell coarse powder;
s2, uniformly mixing the coarse oil tea fruit shell powder obtained in the step S1 and a composite solid auxiliary agent (composed of sodium carbonate, hydroxypropyl-beta-cyclodextrin and microcrystalline cellulose according to the proportion of 2:1:2) accounting for 2% of the mass of the coarse oil tea fruit shell powder, and then putting the mixture into a planetary ball mill, and grinding for 10min to obtain fine oil tea fruit shell powder;
s3, adding 50% ethanol solution with a feed-liquid ratio of 1:20 (g/ml) into the oil tea fruit shell fine powder, and extracting for 1min by using a microwave auxiliary extraction method with a microwave instrument power of 280W;
s4, centrifuging the extracting solution at 5000r/min for 10min, collecting supernatant, removing ethanol by rotary evaporation, adjusting pH to 5, and supplementing distilled water until the volume is consistent with the volume of the ethanol solution added in the step S3;
s5, loading pretreated LSA-21 macroporous resin into a glass chromatographic column, diluting the extract obtained in the step S4 by 4 times, loading the sample at a loading flow rate of 2mL/min, eluting with distilled water of 1BV to remove part of impurities after the adsorption is finished, eluting with ethanol solutions of 20%, 50%, 70% and 90% of 1BV in sequence at an eluting flow rate of 2mL/min, mixing the eluents, and removing ethanol by rotary evaporation;
s6, filtering the eluent obtained in the step S5 by using a nanofiltration membrane with a molecular weight cut-off of 3000Da, and collecting filtrate to obtain the camellia oleifera shell polyphenol extract.
Example 2 preparation method of tea-oil camellia Shell polyphenol extract
The preparation method of the camellia oleifera shell polyphenol extract specifically comprises the following steps:
s1, drying oil tea shells until the moisture content is 5%, crushing, and sieving with a 60-mesh sieve to obtain oil tea shell coarse powder;
s2, uniformly mixing the coarse oil tea fruit shell powder obtained in the step S1 and a composite solid auxiliary agent (composed of sodium carbonate, hydroxypropyl-beta-cyclodextrin and microcrystalline cellulose according to the proportion of 2:1:2) accounting for 3% of the mass of the coarse oil tea fruit shell powder, and then putting the mixture into a planetary ball mill, and grinding for 10min to obtain fine oil tea fruit shell powder;
s3, adding 50% ethanol solution with a feed-liquid ratio of 1:20 (g/ml) into the oil tea fruit shell fine powder, and extracting for 1min by using a microwave auxiliary extraction method with a microwave instrument power of 280W;
s4, centrifuging the extracting solution at 5000r/min for 10min, collecting supernatant, removing ethanol by rotary evaporation, adjusting pH to 5, and supplementing distilled water until the volume is consistent with the volume of the ethanol solution added in the step S3;
s5, loading pretreated LSA-21 macroporous resin into a glass chromatographic column, diluting the extract obtained in the step S4 by 4 times, loading the sample at a loading flow rate of 2mL/min, eluting with distilled water of 1BV to remove part of impurities after the adsorption is finished, eluting with ethanol solutions of 20%, 50%, 70% and 90% of 1BV in sequence at an eluting flow rate of 2mL/min, mixing the eluents, and removing ethanol by rotary evaporation;
s6, filtering the eluent obtained in the step S5 by using a nanofiltration membrane with a molecular weight cut-off of 3000Da, and collecting filtrate to obtain the camellia oleifera shell polyphenol extract.
Example 3 preparation method of tea-oil camellia Shell polyphenol extract
The preparation method of the camellia oleifera shell polyphenol extract specifically comprises the following steps:
s1, drying oil tea shells until the moisture content is 5%, crushing, and sieving with a 60-mesh sieve to obtain oil tea shell coarse powder;
s2, uniformly mixing the coarse oil tea fruit shell powder obtained in the step S1 and a composite solid auxiliary agent (composed of sodium carbonate, hydroxypropyl-beta-cyclodextrin and microcrystalline cellulose according to the proportion of 2:1:2) accounting for 4% of the mass of the coarse oil tea fruit shell powder, and then putting the mixture into a planetary ball mill, and grinding for 10min to obtain fine oil tea fruit shell powder;
s3, adding 50% ethanol solution with a feed-liquid ratio of 1:20 (g/ml) into the oil tea fruit shell fine powder, and extracting for 1min by using a microwave auxiliary extraction method with a microwave instrument power of 280W;
s4, centrifuging the extracting solution at 5000r/min for 10min, collecting supernatant, removing ethanol by rotary evaporation, adjusting pH to 5, and supplementing distilled water until the volume is consistent with the volume of the ethanol solution added in the step S3;
s5, loading pretreated LSA-21 macroporous resin into a glass chromatographic column, diluting the extract obtained in the step S4 by 4 times, loading the sample at a loading flow rate of 2mL/min, eluting with distilled water of 1BV to remove part of impurities after the adsorption is finished, eluting with ethanol solutions of 20%, 50%, 70% and 90% of 1BV in sequence at an eluting flow rate of 2mL/min, mixing the eluents, and removing ethanol by rotary evaporation;
s6, filtering the eluent obtained in the step S5 by using a nanofiltration membrane with a molecular weight cut-off of 3000Da, and collecting filtrate to obtain the camellia oleifera shell polyphenol extract.
Example 4 preparation method of tea-oil camellia Shell polyphenol extract
The preparation method of the camellia oleifera shell polyphenol extract specifically comprises the following steps:
s1, drying oil tea shells until the moisture content is 5%, crushing, and sieving with a 60-mesh sieve to obtain oil tea shell coarse powder;
s2, uniformly mixing the coarse oil tea fruit shell powder obtained in the step S1 and a composite solid auxiliary agent (composed of sodium carbonate, hydroxypropyl-beta-cyclodextrin and microcrystalline cellulose according to the proportion of 2:1:1) accounting for 3% of the mass of the coarse oil tea fruit shell powder, and then putting the mixture into a planetary ball mill, and grinding for 10min to obtain fine oil tea fruit shell powder;
s3, adding 50% ethanol solution with a feed-liquid ratio of 1:20 (g/ml) into the oil tea fruit shell fine powder, and extracting for 1min by using a microwave auxiliary extraction method with a microwave instrument power of 280W;
s4, centrifuging the extracting solution at 5000r/min for 10min, collecting supernatant, removing ethanol by rotary evaporation, adjusting pH to 5, and supplementing distilled water until the volume is consistent with the volume of the ethanol solution added in the step S3;
s5, loading pretreated LSA-21 macroporous resin into a glass chromatographic column, diluting the extract obtained in the step S4 by 4 times, loading the sample at a loading flow rate of 2mL/min, eluting with distilled water of 1BV to remove part of impurities after the adsorption is finished, eluting with ethanol solutions of 20%, 50%, 70% and 90% of 1BV in sequence at an eluting flow rate of 2mL/min, mixing the eluents, and removing ethanol by rotary evaporation;
s6, filtering the eluent obtained in the step S5 by using a nanofiltration membrane with a molecular weight cut-off of 3000Da, and collecting filtrate to obtain the camellia oleifera shell polyphenol extract.
Example 5 preparation method of tea-oil camellia Shell polyphenol extract
The preparation method of the camellia oleifera shell polyphenol extract specifically comprises the following steps:
s1, drying oil tea shells until the moisture content is 5%, crushing, and sieving with a 60-mesh sieve to obtain oil tea shell coarse powder;
s2, uniformly mixing the coarse oil tea fruit shell powder obtained in the step S1 and a composite solid auxiliary agent (composed of sodium carbonate, hydroxypropyl-beta-cyclodextrin and microcrystalline cellulose according to the ratio of 4:1:1) accounting for 3% of the mass of the coarse oil tea fruit shell powder, and then putting the mixture into a planetary ball mill, and grinding for 10min to obtain fine oil tea fruit shell powder;
s3, adding 50% ethanol solution with a feed-liquid ratio of 1:20 (g/ml) into the oil tea fruit shell fine powder, and extracting for 1min by using a microwave auxiliary extraction method with a microwave instrument power of 280W;
s4, centrifuging the extracting solution at 5000r/min for 10min, collecting supernatant, removing ethanol by rotary evaporation, adjusting pH to 5, and supplementing distilled water until the volume is consistent with the volume of the ethanol solution added in the step S3;
s5, loading pretreated LSA-21 macroporous resin into a glass chromatographic column, diluting the extract obtained in the step S4 by 4 times, loading the sample at a loading flow rate of 2mL/min, eluting with distilled water of 1BV to remove part of impurities after the adsorption is finished, eluting with ethanol solutions of 20%, 50%, 70% and 90% of 1BV in sequence at an eluting flow rate of 2mL/min, mixing the eluents, and removing ethanol by rotary evaporation;
s6, filtering the eluent obtained in the step S5 by using a nanofiltration membrane with a molecular weight cut-off of 3000Da, and collecting filtrate to obtain the camellia oleifera shell polyphenol extract.
Example 6 preparation method of tea-oil camellia Shell polyphenol extract
The preparation method of the camellia oleifera shell polyphenol extract specifically comprises the following steps:
s1, drying oil tea shells until the moisture content is 5%, crushing, and sieving with a 60-mesh sieve to obtain oil tea shell coarse powder;
s2, uniformly mixing the coarse oil tea fruit shell powder obtained in the step S1 and a composite solid auxiliary agent (composed of sodium carbonate, hydroxypropyl-beta-cyclodextrin and microcrystalline cellulose according to the proportion of 2:1:2) accounting for 3% of the mass of the coarse oil tea fruit shell powder, and then putting the mixture into a planetary ball mill, and grinding for 10min to obtain fine oil tea fruit shell powder;
s3, adding 50% ethanol solution with a feed-liquid ratio of 1:20 (g/ml) into the oil tea fruit shell fine powder, and extracting for 1min by using a microwave auxiliary extraction method with a microwave instrument power of 210W;
s4, centrifuging the extracting solution at 5000r/min for 10min, collecting supernatant, removing ethanol by rotary evaporation, adjusting pH to 5, and supplementing distilled water until the volume is consistent with the volume of the ethanol solution added in the step S3;
s5, loading pretreated LSA-21 macroporous resin into a glass chromatographic column, diluting the extract obtained in the step S4 by 4 times, loading the sample at a loading flow rate of 2mL/min, eluting with distilled water of 1BV to remove part of impurities after the adsorption is finished, eluting with ethanol solutions of 20%, 50%, 70% and 90% of 1BV in sequence at an eluting flow rate of 2mL/min, mixing the eluents, and removing ethanol by rotary evaporation;
s6, filtering the eluent obtained in the step S5 by using a nanofiltration membrane with a molecular weight cut-off of 3000Da, and collecting filtrate to obtain the camellia oleifera shell polyphenol extract.
Example 7 preparation method of tea-oil camellia Shell polyphenol extract
The preparation method of the camellia oleifera shell polyphenol extract specifically comprises the following steps:
s1, drying oil tea shells until the moisture content is 5%, crushing, and sieving with a 60-mesh sieve to obtain oil tea shell coarse powder;
s2, uniformly mixing the coarse oil tea fruit shell powder obtained in the step S1 and a composite solid auxiliary agent (composed of sodium carbonate, hydroxypropyl-beta-cyclodextrin and microcrystalline cellulose according to the proportion of 2:1:2) accounting for 3% of the mass of the coarse oil tea fruit shell powder, and then putting the mixture into a planetary ball mill, and grinding for 10min to obtain fine oil tea fruit shell powder;
s3, adding 50% ethanol solution with a feed-liquid ratio of 1:20 (g/ml) into the oil tea fruit shell fine powder, and extracting for 1min by using a microwave auxiliary extraction method with a microwave instrument power of 350W;
s4, centrifuging the extracting solution at 5000r/min for 10min, collecting supernatant, removing ethanol by rotary evaporation, adjusting pH to 5, and supplementing distilled water until the volume is consistent with the volume of the ethanol solution added in the step S3;
s5, loading pretreated LSA-21 macroporous resin into a glass chromatographic column, diluting the extract obtained in the step S4 by 4 times, loading the sample at a loading flow rate of 2mL/min, eluting with distilled water of 1BV to remove part of impurities after the adsorption is finished, eluting with ethanol solutions of 20%, 50%, 70% and 90% of 1BV in sequence at an eluting flow rate of 2mL/min, mixing the eluents, and removing ethanol by rotary evaporation;
s6, filtering the eluent obtained in the step S5 by using a nanofiltration membrane with a molecular weight cut-off of 3000Da, and collecting filtrate to obtain the camellia oleifera shell polyphenol extract.
Example 8 preparation method of tea-oil camellia Shell polyphenol extract
The preparation method of the camellia oleifera shell polyphenol extract specifically comprises the following steps:
s1, drying oil tea shells until the moisture content is 5%, crushing, and sieving with a 60-mesh sieve to obtain oil tea shell coarse powder;
s2, uniformly mixing the coarse oil tea fruit shell powder obtained in the step S1 and a composite solid auxiliary agent (composed of sodium carbonate, hydroxypropyl-beta-cyclodextrin and microcrystalline cellulose according to the proportion of 2:1:2) accounting for 3% of the mass of the coarse oil tea fruit shell powder, and then putting the mixture into a planetary ball mill, and grinding for 10min to obtain fine oil tea fruit shell powder;
s3, adding 50% ethanol solution with a feed-liquid ratio of 1:20 (g/ml) into the oil tea fruit shell fine powder, and extracting for 1.5min by using a microwave auxiliary extraction method with a microwave instrument power of 280W;
s4, centrifuging the extracting solution at 5000r/min for 10min, collecting supernatant, removing ethanol by rotary evaporation, adjusting pH to 5, and supplementing distilled water until the volume is consistent with the volume of the ethanol solution added in the step S3;
s5, loading pretreated LSA-21 macroporous resin into a glass chromatographic column, diluting the extract obtained in the step S4 by 4 times, loading the sample at a loading flow rate of 2mL/min, eluting with distilled water of 1BV to remove part of impurities after the adsorption is finished, eluting with ethanol solutions of 20%, 50%, 70% and 90% of 1BV in sequence at an eluting flow rate of 2mL/min, mixing the eluents, and removing ethanol by rotary evaporation;
s6, filtering the eluent obtained in the step S5 by using a nanofiltration membrane with a molecular weight cut-off of 3000Da, and collecting filtrate to obtain the camellia oleifera shell polyphenol extract.
Comparative example 1 preparation method of tea-oil camellia husk polyphenol extract
The preparation method of the camellia oleifera shell polyphenol extract specifically comprises the following steps:
s1, drying oil tea shells until the moisture content is 5%, crushing, and sieving with a 60-mesh sieve to obtain oil tea shell coarse powder;
s2, placing the coarse oil tea fruit shell powder obtained in the step S1 into a planetary ball mill, and grinding for 10min to obtain oil tea fruit shell fine powder;
s3, adding 50% ethanol solution with a feed-liquid ratio of 1:20 (g/ml) into the oil tea fruit shell fine powder, and extracting for 1min by using a microwave auxiliary extraction method with a microwave instrument power of 280W;
s4, centrifuging the extracting solution at 5000r/min for 10min, collecting supernatant, removing ethanol by rotary evaporation, adjusting pH to 5, and supplementing distilled water until the volume is consistent with the volume of the ethanol solution added in the step S3;
s5, loading pretreated LSA-21 macroporous resin into a glass chromatographic column, diluting the extract obtained in the step S4 by 4 times, loading the sample at a loading flow rate of 2mL/min, eluting with distilled water of 1BV to remove part of impurities after the adsorption is finished, eluting with ethanol solutions of 20%, 50%, 70% and 90% of 1BV in sequence at an eluting flow rate of 2mL/min, mixing the eluents, and removing ethanol by rotary evaporation;
s6, filtering the eluent obtained in the step S5 by using a nanofiltration membrane with a molecular weight cut-off of 3000Da, and collecting filtrate to obtain the camellia oleifera shell polyphenol extract.
Comparative example 2 preparation method of tea-oil camellia husk polyphenol extract
The preparation method of the camellia oleifera shell polyphenol extract specifically comprises the following steps:
s1, drying oil tea shells until the moisture content is 5%, crushing, and sieving with a 60-mesh sieve to obtain oil tea shell coarse powder;
s2, uniformly mixing the coarse oil tea fruit shell powder obtained in the step S1 and a composite solid auxiliary agent (composed of sodium carbonate, hydroxypropyl-beta-cyclodextrin and microcrystalline cellulose according to the proportion of 2:1:2) accounting for 1% of the mass of the coarse oil tea fruit shell powder, and then putting the mixture into a planetary ball mill, and grinding for 10min to obtain fine oil tea fruit shell powder;
s3, adding 50% ethanol solution with a feed-liquid ratio of 1:20 (g/ml) into the oil tea fruit shell fine powder, and extracting for 1min by using a microwave auxiliary extraction method with a microwave instrument power of 280W;
s4, centrifuging the extracting solution at 5000r/min for 10min, collecting supernatant, removing ethanol by rotary evaporation, adjusting pH to 5, and supplementing distilled water until the volume is consistent with the volume of the ethanol solution added in the step S3;
s5, loading pretreated LSA-21 macroporous resin into a glass chromatographic column, diluting the extract obtained in the step S4 by 4 times, loading the sample at a loading flow rate of 2mL/min, eluting with distilled water of 1BV to remove part of impurities after the adsorption is finished, eluting with ethanol solutions of 20%, 50%, 70% and 90% of 1BV in sequence at an eluting flow rate of 2mL/min, mixing the eluents, and removing ethanol by rotary evaporation;
s6, filtering the eluent obtained in the step S5 by using a nanofiltration membrane with a molecular weight cut-off of 3000Da, and collecting filtrate to obtain the camellia oleifera shell polyphenol extract.
Comparative example 3 preparation method of tea-oil camellia husk polyphenol extract
The preparation method of the camellia oleifera shell polyphenol extract specifically comprises the following steps:
s1, drying oil tea shells until the moisture content is 5%, crushing, and sieving with a 60-mesh sieve to obtain oil tea shell coarse powder;
s2, uniformly mixing the coarse oil tea fruit shell powder obtained in the step S1 with sodium carbonate accounting for 3% of the mass of the coarse oil tea fruit shell powder, and then putting the mixture into a planetary ball mill for grinding for 10min to obtain fine oil tea fruit shell powder;
s3, adding 50% ethanol solution with a feed-liquid ratio of 1:20 (g/ml) into the oil tea fruit shell fine powder, and extracting for 1min by using a microwave auxiliary extraction method with a microwave instrument power of 280W;
s4, centrifuging the extracting solution at 5000r/min for 10min, collecting supernatant, removing ethanol by rotary evaporation, adjusting pH to 5, and supplementing distilled water until the volume is consistent with the volume of the ethanol solution added in the step S3;
s5, loading pretreated LSA-21 macroporous resin into a glass chromatographic column, diluting the extract obtained in the step S4 by 4 times, loading the sample at a loading flow rate of 2mL/min, eluting with distilled water of 1BV to remove part of impurities after the adsorption is finished, eluting with ethanol solutions of 20%, 50%, 70% and 90% of 1BV in sequence at an eluting flow rate of 2mL/min, mixing the eluents, and removing ethanol by rotary evaporation;
s6, filtering the eluent obtained in the step S5 by using a nanofiltration membrane with a molecular weight cut-off of 3000Da, and collecting filtrate to obtain the camellia oleifera shell polyphenol extract.
Comparative example 4 preparation method of tea-oil camellia husk polyphenol extract
The preparation method of the camellia oleifera shell polyphenol extract specifically comprises the following steps:
s1, drying oil tea shells until the moisture content is 5%, crushing, and sieving with a 60-mesh sieve to obtain oil tea shell coarse powder;
s2, uniformly mixing the coarse oil tea fruit shell powder obtained in the step S1 with hydroxypropyl-beta-cyclodextrin accounting for 3% of the mass of the coarse oil tea fruit shell powder, and then putting the mixture into a planetary ball mill for grinding for 10min to obtain fine oil tea fruit shell powder;
s3, adding 50% ethanol solution with a feed-liquid ratio of 1:20 (g/ml) into the oil tea fruit shell fine powder, and extracting for 1min by using a microwave auxiliary extraction method with a microwave instrument power of 280W;
s4, centrifuging the extracting solution at 5000r/min for 10min, collecting supernatant, removing ethanol by rotary evaporation, adjusting pH to 5, and supplementing distilled water until the volume is consistent with the volume of the ethanol solution added in the step S3;
s5, loading pretreated LSA-21 macroporous resin into a glass chromatographic column, diluting the extract obtained in the step S4 by 4 times, loading the sample at a loading flow rate of 2mL/min, eluting with distilled water of 1BV to remove part of impurities after the adsorption is finished, eluting with ethanol solutions of 20%, 50%, 70% and 90% of 1BV in sequence at an eluting flow rate of 2mL/min, mixing the eluents, and removing ethanol by rotary evaporation;
s6, filtering the eluent obtained in the step S5 by using a nanofiltration membrane with a molecular weight cut-off of 3000Da, and collecting filtrate to obtain the camellia oleifera shell polyphenol extract.
Comparative example 5 preparation method of tea-oil camellia husk polyphenol extract
The preparation method of the camellia oleifera shell polyphenol extract specifically comprises the following steps:
s1, drying oil tea shells until the moisture content is 5%, crushing, and sieving with a 60-mesh sieve to obtain oil tea shell coarse powder;
s2, uniformly mixing the coarse oil tea fruit shell powder obtained in the step S1 and microcrystalline cellulose accounting for 3% of the mass of the coarse oil tea fruit shell powder, and then putting the mixture into a planetary ball mill for grinding for 10min to obtain fine oil tea fruit shell powder;
s3, adding 50% ethanol solution with a feed-liquid ratio of 1:20 (g/ml) into the oil tea fruit shell fine powder, and extracting for 1min by using a microwave auxiliary extraction method with a microwave instrument power of 280W;
s4, centrifuging the extracting solution at 5000r/min for 10min, collecting supernatant, removing ethanol by rotary evaporation, adjusting pH to 5, and supplementing distilled water until the volume is consistent with the volume of the ethanol solution added in the step S3;
s5, loading pretreated LSA-21 macroporous resin into a glass chromatographic column, diluting the extract obtained in the step S4 by 4 times, loading the sample at a loading flow rate of 2mL/min, eluting with distilled water of 1BV to remove part of impurities after the adsorption is finished, eluting with ethanol solutions of 20%, 50%, 70% and 90% of 1BV in sequence at an eluting flow rate of 2mL/min, mixing the eluents, and removing ethanol by rotary evaporation;
s6, filtering the eluent obtained in the step S5 by using a nanofiltration membrane with a molecular weight cut-off of 3000Da, and collecting filtrate to obtain the camellia oleifera shell polyphenol extract.
Comparative example 6 preparation method of tea-oil camellia husk polyphenol extract
The preparation method of the camellia oleifera shell polyphenol extract specifically comprises the following steps:
s1, drying oil tea shells until the moisture content is 5%, crushing, and sieving with a 60-mesh sieve to obtain oil tea shell coarse powder;
s2, uniformly mixing the coarse oil tea fruit shell powder obtained in the step S1 and a composite solid auxiliary agent (composed of sodium carbonate, hydroxypropyl-beta-cyclodextrin and microcrystalline cellulose according to the proportion of 2:1:2) accounting for 3% of the mass of the coarse oil tea fruit shell powder, and then putting the mixture into a planetary ball mill, and grinding for 10min to obtain fine oil tea fruit shell powder;
s3, adding 50% ethanol solution with a feed-liquid ratio of 1:20 (g/ml) into the oil tea fruit shell fine powder, and extracting for 50min at 70 ℃;
s4, centrifuging the extracting solution at 5000r/min for 10min, collecting supernatant, removing ethanol by rotary evaporation, adjusting pH to 5, and supplementing distilled water until the volume is consistent with the volume of the ethanol solution added in the step S3;
s5, loading pretreated LSA-21 macroporous resin into a glass chromatographic column, diluting the extract obtained in the step S4 by 4 times, loading the sample at a loading flow rate of 2mL/min, eluting with distilled water of 1BV to remove part of impurities after the adsorption is finished, eluting with ethanol solutions of 20%, 50%, 70% and 90% of 1BV in sequence at an eluting flow rate of 2mL/min, mixing the eluents, and removing ethanol by rotary evaporation;
s6, filtering the eluent obtained in the step S5 by using a nanofiltration membrane with a molecular weight cut-off of 3000Da, and collecting filtrate to obtain the camellia oleifera shell polyphenol extract.
Comparative example 7 preparation method of tea-oil camellia husk polyphenol extract
The preparation method of the camellia oleifera shell polyphenol extract specifically comprises the following steps:
s1, drying oil tea shells until the moisture content is 5%, crushing, and sieving with a 60-mesh sieve to obtain oil tea shell coarse powder;
s2, uniformly mixing the coarse oil tea fruit shell powder obtained in the step S1 and a composite solid auxiliary agent (composed of sodium carbonate, hydroxypropyl-beta-cyclodextrin and microcrystalline cellulose according to the proportion of 2:1:2) accounting for 3% of the mass of the coarse oil tea fruit shell powder, and then putting the mixture into a planetary ball mill, and grinding for 10min to obtain fine oil tea fruit shell powder;
s3, adding 50% ethanol solution with a feed-liquid ratio of 1:20 (g/ml) into the oil tea fruit shell fine powder, and extracting for 1min by using a microwave auxiliary extraction method with a microwave instrument power of 420W;
s4, centrifuging the extracting solution at 5000r/min for 10min, collecting supernatant, removing ethanol by rotary evaporation, adjusting pH to 5, and supplementing distilled water until the volume is consistent with the volume of the ethanol solution added in the step S3;
s5, loading pretreated LSA-21 macroporous resin into a glass chromatographic column, diluting the extract obtained in the step S4 by 4 times, loading the sample at a loading flow rate of 2mL/min, eluting with distilled water of 1BV to remove part of impurities after the adsorption is finished, eluting with ethanol solutions of 20%, 50%, 70% and 90% of 1BV in sequence at an eluting flow rate of 2mL/min, mixing the eluents, and removing ethanol by rotary evaporation;
s6, filtering the eluent obtained in the step S5 by using a nanofiltration membrane with a molecular weight cut-off of 3000Da, and collecting filtrate to obtain the camellia oleifera shell polyphenol extract.
Comparative example 8 preparation method of tea-oil camellia husk polyphenol extract
The preparation method of the camellia oleifera shell polyphenol extract specifically comprises the following steps:
s1, drying oil tea shells until the moisture content is 5%, crushing, and sieving with a 60-mesh sieve to obtain oil tea shell coarse powder;
s2, uniformly mixing the coarse oil tea fruit shell powder obtained in the step S1 and a composite solid auxiliary agent (composed of sodium carbonate, hydroxypropyl-beta-cyclodextrin and microcrystalline cellulose according to the proportion of 2:1:2) accounting for 3% of the mass of the coarse oil tea fruit shell powder, and then putting the mixture into a planetary ball mill, and grinding for 10min to obtain fine oil tea fruit shell powder;
s3, adding 50% ethanol solution with a feed-liquid ratio of 1:20 (g/ml) into the oil tea fruit shell fine powder, and extracting for 0.5min by using a microwave auxiliary extraction method with a microwave instrument power of 280W;
s4, centrifuging the extracting solution at 5000r/min for 10min, collecting supernatant, removing ethanol by rotary evaporation, adjusting pH to 5, and supplementing distilled water until the volume is consistent with the volume of the ethanol solution added in the step S3;
s5, loading pretreated LSA-21 macroporous resin into a glass chromatographic column, diluting the extract obtained in the step S4 by 4 times, loading the sample at a loading flow rate of 2mL/min, eluting with distilled water of 1BV to remove part of impurities after the adsorption is finished, eluting with ethanol solutions of 20%, 50%, 70% and 90% of 1BV in sequence at an eluting flow rate of 2mL/min, mixing the eluents, and removing ethanol by rotary evaporation;
s6, filtering the eluent obtained in the step S5 by using a nanofiltration membrane with a molecular weight cut-off of 3000Da, and collecting filtrate to obtain the camellia oleifera shell polyphenol extract.
Comparative example 9 preparation method of tea-oil camellia husk polyphenol extract
The preparation method of the camellia oleifera shell polyphenol extract specifically comprises the following steps:
s1, drying oil tea shells until the moisture content is 5%, crushing, and sieving with a 60-mesh sieve to obtain oil tea shell coarse powder;
s2, uniformly mixing the coarse oil tea fruit shell powder obtained in the step S1 and a composite solid auxiliary agent (composed of sodium carbonate, hydroxypropyl-beta-cyclodextrin and microcrystalline cellulose according to the proportion of 2:1:2) accounting for 3% of the mass of the coarse oil tea fruit shell powder, and then putting the mixture into a planetary ball mill, and grinding for 10min to obtain fine oil tea fruit shell powder;
s3, adding 50% ethanol solution with a feed-liquid ratio of 1:20 (g/ml) into the oil tea fruit shell fine powder, and extracting for 2min by using a microwave auxiliary extraction method with a microwave instrument power of 280W;
s4, centrifuging the extracting solution at 5000r/min for 10min, collecting supernatant, removing ethanol by rotary evaporation, adjusting pH to 5, and supplementing distilled water until the volume is consistent with the volume of the ethanol solution added in the step S3;
s5, loading pretreated LSA-21 macroporous resin into a glass chromatographic column, diluting the extract obtained in the step S4 by 4 times, loading the sample at a loading flow rate of 2mL/min, eluting with distilled water of 1BV to remove part of impurities after the adsorption is finished, eluting with ethanol solutions of 20%, 50%, 70% and 90% of 1BV in sequence at an eluting flow rate of 2mL/min, mixing the eluents, and removing ethanol by rotary evaporation;
s6, filtering the eluent obtained in the step S5 by using a nanofiltration membrane with a molecular weight cut-off of 3000Da, and collecting filtrate to obtain the camellia oleifera shell polyphenol extract.
Comparative example 10 preparation method of tea-oil camellia husk polyphenol extract
The preparation method of the camellia oleifera shell polyphenol extract specifically comprises the following steps:
s1, drying oil tea shells until the moisture content is 5%, crushing, and sieving with a 60-mesh sieve to obtain oil tea shell coarse powder;
s2, uniformly mixing the coarse oil tea fruit shell powder obtained in the step S1 and a composite solid auxiliary agent (composed of sodium carbonate, hydroxypropyl-beta-cyclodextrin and microcrystalline cellulose according to the proportion of 2:1:2) accounting for 3% of the mass of the coarse oil tea fruit shell powder, and then putting the mixture into a planetary ball mill, and grinding for 10min to obtain fine oil tea fruit shell powder;
s3, adding 50% ethanol solution with a feed-liquid ratio of 1:20 (g/ml) into the oil tea fruit shell fine powder, and extracting for 2min by using a microwave auxiliary extraction method with a microwave instrument power of 280W;
s4, centrifuging the extracting solution at 5000r/min for 10min, collecting supernatant, removing ethanol by rotary evaporation, adjusting pH to 5, and supplementing distilled water until the volume is consistent with the volume of the ethanol solution added in the step S3;
s5, diluting the extracting solution obtained in the step S4 until the polyphenol concentration of the extracting solution is consistent with that of the extracting solution obtained in the step S4 in the embodiment 2;
s6, filtering the eluent obtained in the step S5 by using a nanofiltration membrane with a molecular weight cut-off of 3000Da, and collecting filtrate to obtain the camellia oleifera shell polyphenol extract.
Experimental examples influence of different extraction conditions on extraction amount and extract activity
The extraction amount, the alpha-amylase inhibition rate and the alpha-glucosidase inhibition rate of the polyphenol extracts of the oil tea fruit shells obtained in the examples and the comparative examples are measured, and the specific methods are as follows:
1. measurement of the amount of extraction:
the Folin-Ciocalteu method was used to determine the polyphenol content. Accurately weighing 10mg of gallic acid, dissolving with water, transferring to a 100mL brown volumetric flask for constant volume, and obtaining 0.1mg/mL mother liquor. And respectively sucking 0, 1, 2, 3, 4 and 5mL of mother solution into a 10mL brown volumetric flask, and fixing the volume to obtain gallic acid standard solutions with concentrations of 0.01, 0.02, 0.03, 0.04 and 0.05 mg/mL. Adding 2mL (0.1N) Fu Lin Fen reagent into 1mL standard solution, mixing, standing for 5min, and adding 4mL 7.5% Na 2 CO 3 Constant volume to 10mL, standing at normal temperature in the dark for 1h, and measuring absorbance at 765 nm. And drawing a standard curve by taking the concentration c of the gallic acid standard sample as an abscissa and the absorbance as an ordinate to obtain a regression equation: y=15.05x-0.007 (R 2 ＝0.999)。
The polyphenol extract solution of the camellia oleifera shells is fixed to a certain volume, 1mL of the solution is sucked into a colorimetric tube, the operation is repeated, the polyphenol content in a sample is calculated according to the following equation, and the result is expressed as mg/g.
Wherein c represents the measured polyphenol concentration, mg/mL; v is the volume of the polyphenol extract solution of the camellia oleifera shells, and mL; m is dry weight of oil tea fruit shell and g.
2. Determination of the inhibition of alpha-amylase:
0.2mL of the camellia oleifera shell polyphenol extract solution and 0.2mL of 0.4mg/mL of alpha-amylase solution (prepared by buffer solution with pH of 6.8) are placed in a colorimetric tube, placed in a constant-temperature water bath at 37 ℃ for 10min, added with 0.2mL of 1% starch solution, reacted for 10min, added with 0.2mL of DNS reagent, and heated in a boiling water bath for 8min. After cooling, the volume was set to 10mL and absorbance was measured at 540 nm. The inhibition rate is calculated according to the following formula:
wherein A is i Represents the absorbance value of the polyphenol extract solution of the camellia oleifera shells, A 0 Represents the absorbance value measured by replacing the polyphenol extract solution of the camellia oleifera shells with an equal volume of phosphoric acid buffer solution, A t Representative of absorbance values measured with the bottom tube of tea-oil camellia husk polyphenol extract solution using phosphate buffer solution instead of alpha-amylase solution.
3. Determination of the inhibition ratio of α -glucosidase:
adding 0.1mL of oil tea fruit shell extract solution and 0.2mL of 0.2U/mL alpha-glucosidase (prepared from buffer solution with pH of 6.8) into a test tube with 0.9mL of phosphate buffer solution, standing in a constant temperature water bath at 37deg.C for 10min, adding 0.2mL of 2.5mmol of pNPG solution, reacting for 30min, and adding 0.8mL of 0.2mol/L Na 2 CO 3 After standing for 15min, the absorbance was measured at 405nm and the inhibition was calculated according to the following formula:
wherein A is i Represents the absorbance value of the polyphenol extract solution of the camellia oleifera shells, A 0 Represents the absorbance value measured by replacing the polyphenol extract solution of the camellia oleifera shells with an equal volume of phosphoric acid buffer solution, A t Representative of absorbance values measured with the bottom tube of tea-oil camellia husk polyphenol extract solution using phosphate buffer solution instead of alpha-amylase solution.
The effect of the type and amount of solid auxiliary on the extraction amount and the activity of the extract is shown in Table 1.
TABLE 1 influence of the amount of solid auxiliary on the extraction yield and the activity of the extract
From Table 1, it is known that pretreatment of the raw materials with 2% -4% of the composite solid auxiliary agent, 3% of sodium carbonate, 3% of hydroxypropyl-beta-cyclodextrin and 3% of microcrystalline cellulose can effectively increase the extraction amount of the camellia oleifera shell polyphenol, thereby improving the inhibition capability of the camellia oleifera shell polyphenol on alpha-amylase and alpha-glucosidase.
The extraction amount and activity of the extract of the fruit shell treated by the 1% composite solid auxiliary agent are not obviously changed, when the addition amount of the composite solid auxiliary agent is increased from 1% to 4%, the extraction amount and activity of the extract are in a growing trend, and when the addition amount reaches 3%, the extraction amount and activity are both maximum and then basically unchanged.
When the addition amount of the solid auxiliary agent is 3%, the polyphenol extraction amount of the solid auxiliary agent shows a consistent trend in the capability of inhibiting two enzymes: the composite solid auxiliary agent (composed of sodium carbonate, hydroxypropyl-beta-cyclodextrin and microcrystalline cellulose according to different proportions) is more than hydroxypropyl-beta-cyclodextrin is more than sodium carbonate is more than microcrystalline cellulose. Sodium carbonate can react with polyphenol rich in phenolic hydroxyl groups to form salt, and the activities of alpha-amylase and alpha-glucosidase are easily influenced by the phenolic hydroxyl groups of the polyphenol structure in the environment, so that a certain solid alkali auxiliary agent is beneficial to the dissolution of polyphenol substances, and the capacity of inhibiting the alpha-amylase and the alpha-glucosidase of a polyphenol extract can be improved. The hydroxypropyl-beta-cyclodextrin and microcrystalline cellulose can solubilize polyphenols and increase extraction yield. The extract obtained from the oil tea fruit shell treated by the composite solid auxiliary agent prepared by mixing the three solid auxiliary agents according to a certain proportion has higher polyphenol extraction quantity, and has stronger alpha-amylase and alpha-glucosidase inhibiting capability, so that a synergistic effect relationship exists among three substances.
The effect of microwave extraction conditions on the amount of extraction and the activity of the extract is shown in Table 2.
TABLE 2 influence of microwave extraction conditions on the extraction yield and extract Activity
As can be seen from Table 2, the extraction amount of polyphenol obtained by extracting oil tea fruit shells by 210W microwaves for 1min is similar to that obtained by extracting oil tea shells by a 70 ℃ leaching method for 50min, which shows that the microwave extraction method can effectively shorten the extraction time and obtain polyphenol with the extraction amount similar to that of the traditional leaching method. And further, the determination of the alpha-amylase inhibition rate and the alpha-glucosidase inhibition rate shows that the inhibition activity difference of the camellia oleifera shell polyphenol extract obtained by the two methods is larger: the extraction method has long action time and sufficient extraction, but the long-time thermal environment easily causes macromolecular polyphenol to be decomposed into micromolecular phenolic substances, so that the phenolic compounds with strong inhibition effect are converted into phenolic compounds with weak inhibition effect, compared with the phenolic compounds with weak inhibition effect, the phenolic substances extracted by the microwave extraction method possibly have higher content of substances for inhibiting the activities of alpha-amylase and alpha-glucosidase, so the extraction method is similar to the microwave extraction method in polyphenol content, but the enzyme inhibition activity is inferior to that of the microwave extraction method.
In addition, in a certain time, as the microwave power (210W-350W) is increased, the system is heated up quickly, the solid-liquid diffusion speed is also high, the cell rupture is increased, the extraction amount of the shell polyphenol is gradually increased, and the maximum extraction amount is reached at about 350W; however, when the microwave power is increased to 420W, the local temperature may be too high, and the polyphenols may be easily decomposed. The oil tea fruit shell polyphenol extract with higher extraction quantity has stronger activity on the oil tea fruit shell polyphenol extracted under the power condition of 280W instead of the highest inhibition activity on alpha-amylase and alpha-glucosidase; it is possible that the microwave power is increased from 280W to 350W, and more components are dissolved out of the phenols in the shell, but the thermal effect caused by the high power also causes the conversion of the phenol compound with strong inhibition to the phenol compound with weak inhibition.
With the increase of the microwave time (0.5 min-2 min), the extraction amount of the shell polyphenol is gradually increased, the inhibition rate of the alpha-amylase and the alpha-glucosidase is gradually increased, and the maximum value is obtained at 1min; when the microwave time is further prolonged, the extraction amount and the inhibition activity of the shell polyphenol are reduced. The extraction amount of the shell polyphenol is lower than that of the polyphenol extract obtained by the leaching method when microwaves are respectively acted for 0.5min and 2min, but the enzyme inhibition activity is equivalent.
The effect of macroporous resin purification on the amount extracted and the activity of the extract is shown in Table 3.
TABLE 3 Effect of macroporous resin purification on extraction yield and extract Activity
The results in Table 3 show that macromolecular substances such as polysaccharide, protein and the like in the camellia oleifera shell polyphenol extract obtained in the step S4 can be effectively removed through macroporous resin purification, and the blocking of nanofiltration membranes is effectively avoided, so that more active polyphenols are enriched, and the enzyme inhibition activity is obviously improved.
The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.
Claims (10)
1. The preparation method of the camellia oleifera shell polyphenol extract is characterized by comprising the following steps of:
s1, drying oil tea shells and crushing to obtain coarse oil tea shell powder;
s2, mixing and grinding the coarse oil tea fruit shell powder obtained in the step S1 with a composite solid auxiliary agent to obtain oil tea fruit shell fine powder;
s3, adding an ethanol solution into the oil tea fruit shell fine powder obtained in the step S2, and extracting for 60-90S under the assistance of 210-350W microwaves;
s4, centrifuging the extracting solution, collecting supernatant, removing ethanol, adjusting the pH to 4-5, and supplementing water until the volume is consistent with the volume of the ethanol solution added in the step S3;
s5, diluting the extracting solution obtained in the step S4, loading the diluted extracting solution into a macroporous resin column, washing with water to remove impurities after the adsorption is finished, eluting with ethanol solution, collecting eluent, and removing ethanol;
and S6, filtering the eluent obtained in the step S5 by using a nanofiltration membrane, and collecting filtrate.
2. The method according to claim 1, wherein in step S2, the complex solid auxiliary agent is sodium carbonate, hydroxypropyl- β -cyclodextrin, or microcrystalline cellulose.
3. The preparation method according to claim 2, wherein the mass ratio of the sodium carbonate, the hydroxypropyl-beta-cyclodextrin and the microcrystalline cellulose is 1-4: 0.5 to 1:1 to 2.
4. The preparation method of claim 1, wherein in the step S2, the total addition amount of the composite solid auxiliary agent is 1% -4% of the mass of the coarse powder of the camellia oleifera shell.
5. The method according to claim 1, wherein in the step S3, the volume concentration of the ethanol solution is 30% to 50%.
6. The process according to claim 1, wherein in step S5, the macroporous resin is HP20, LSA-21 or AB-8.
7. The method according to claim 1, wherein the volume concentration of the ethanol solution in step S5 is 20%, 50%, 70% and 90% in this order.
8. The method according to claim 1, wherein in step S6, the nanofiltration membrane has a molecular weight cut-off of 500-3000 Da.
9. The camellia oleifera shell polyphenol extract obtained by the preparation method of any one of claims 1 to 8.
10. Use of the camellia oleifera shell polyphenol extract of claim 9 for the preparation of amylase inhibitors.
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