CN116870061A - Peach blossom extract and preparation method and application thereof - Google Patents
Peach blossom extract and preparation method and application thereof Download PDFInfo
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- CN116870061A CN116870061A CN202310981219.3A CN202310981219A CN116870061A CN 116870061 A CN116870061 A CN 116870061A CN 202310981219 A CN202310981219 A CN 202310981219A CN 116870061 A CN116870061 A CN 116870061A
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Classifications
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- 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
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/73—Rosaceae (Rose family), e.g. strawberry, chokeberry, blackberry, pear or firethorn
- A61K36/736—Prunus, e.g. plum, cherry, peach, apricot or almond
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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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- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
- A61K8/9783—Angiosperms [Magnoliophyta]
- A61K8/9789—Magnoliopsida [dicotyledons]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/08—Anti-ageing preparations
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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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
- A61K2236/10—Preparation or pretreatment of starting material
- A61K2236/19—Preparation or pretreatment of starting material involving fermentation using yeast, bacteria or both; enzymatic treatment
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- A—HUMAN NECESSITIES
- 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
- A61K2236/30—Extraction of the material
- A61K2236/33—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones
- A61K2236/331—Extraction of the material involving extraction with hydrophilic solvents, e.g. lower alcohols, esters or ketones using water, e.g. cold water, infusion, tea, steam distillation or decoction
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- A—HUMAN NECESSITIES
- 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
- A61K2236/53—Liquid-solid separation, e.g. centrifugation, sedimentation or crystallization
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Abstract
The invention belongs to the technical field of deep processing of peach blossom, and particularly relates to a peach blossom extract and a preparation method and application thereof. According to the preparation method, a specific protein mixed enzyme is adopted to hydrolyze a precipitated product after alcohol precipitation on the basis of traditional water extraction and alcohol precipitation, and then an ultrafiltration membrane with a specific aperture is adopted to carry out fractional ultrafiltration on the enzymolysis product, so that more active effective components are obtained through enrichment, the anti-inflammatory/soothing effect and the tightening effect of the obtained peach blossom extract are obviously improved, and the preparation method has a wide application prospect in the preparation fields of medicines, foods or cosmetics.
Description
Technical Field
The invention belongs to the technical field of deep processing of peach blossom. More particularly, relates to a peach blossom extract and a preparation method and application thereof.
Background
The peach flower is flower of peach tree of Rosaceae, can be used as Chinese medicinal material after being picked and dried in spring, has effects of purgation, diuresis and detumescence, and is mainly used for treating edema, ascites and constipation.
Chinese patent application CN108014191A discloses a peach flower extract obtained by carrying out ultrasonic extraction on peach flowers by using water or a hydrophilic solvent and an aqueous solution thereof, concentrating under reduced pressure and evaporating to dryness, wherein the peach flower extract has obvious inhibition effect on hyaluronidase activity and shows that the peach flower extract can inhibit the hyaluronidase activity so as to relieve some inflammation associated with degradation of hyaluronic acid by preventing the degradation of hyaluronic acid, however, the invention has no relevant experimental data to directly indicate that the extracted peach flower extract can relieve inflammation symptoms, whether the effect can reach the expected value of people or not has clear data can be judged by referring, the extract is obtained by a simple solvent extraction mode, the active ingredients obtained by different extraction modes are different, and the peach flower extract extracted by different extraction modes can have better efficacy or even different efficacy. Therefore, development of different extraction processes of peach flower extracts and development of performance effects of peach flower extracts extracted by specific processes have important research significance and application value.
Disclosure of Invention
The invention aims to overcome the defects and shortcomings that the prior art is not deep enough in the performance research of the independent peach blossom extract, and no direct data can indicate that the peach blossom extract can reduce inflammation symptoms and has other performance effects, and provides a preparation method of the peach blossom extract.
The invention aims to provide the peach blossom extract prepared by the preparation method.
It is another object of the present invention to provide the use of peach blossom extract in the preparation of a pharmaceutical, food or cosmetic product.
The above object of the present invention is achieved by the following technical scheme:
the invention provides a preparation method of a peach blossom extract, which comprises the following steps:
s1, adding water into peach blossom, heating, reflux-extracting, concentrating, adding ethanol, mixing uniformly, fully standing, and filtering to obtain a precipitate;
s2, adding water into the precipitate obtained in the step S1 for full dissolution, adding papain and neutral protease for full enzymolysis, inactivating the enzyme after the enzymolysis is finished, and concentrating the enzymolysis liquid to obtain an enzymolysis product;
s3, treating the enzymolysis product obtained in the step S2 by using nonpolar or weak polar resin, eluting with water to obtain eluent, concentrating the eluent, performing primary ultrafiltration by using a 9.5-10.5 kDa ultrafiltration membrane, performing secondary ultrafiltration on the obtained filtrate by using a 6.5-7.5 kDa ultrafiltration membrane, and concentrating the trapped fluid to obtain the peach blossom extract.
The inventors found in earlier studies that the selection of enzymes in the enzymatic hydrolysis step of the invention was critical. The anti-inflammatory/soothing effects of the peach flower extract obtained by enzymatic hydrolysis with different enzymes are different. The inventor creatively discovers in a large number of experiments that compared with peach flower extracts prepared by papain alone, neutral protease alone or bromelain alone or other mixed enzymes, the peach flower extracts prepared by adopting the mixed enzymes consisting of papain and neutral protease as enzymes in the enzymolysis step synergistically improve the enzymolysis efficiency, better remove protein and improve the yield of polysaccharide, and jointly improve the anti-inflammatory/soothing effect of the prepared peach flower extracts.
The inventor finds that after elution by macroporous resin, the peach blossom extract prepared under the ultrafiltration parameter condition can be enriched to obtain more active effective components, and the anti-inflammatory/soothing effect and the tightening effect of the peach blossom extract are further improved, so that the prepared peach blossom extract has excellent anti-inflammatory/soothing effect and tightening effect.
Preferably, in the step S2, the weight ratio of the papain to the neutral protease is 1-3:1-3.
More preferably, the weight ratio of the papain to the neutral protease is 1-2:1-2.
Most preferably, the weight ratio of papain to neutral protease is 1:1.
Preferably, in the step S2, the ratio of the total weight of the papain and the neutral protease to the weight of the water is 3-6:100.
More preferably, in step S2, the ratio of the total weight of papain and neutral protease to the weight of water is 5:100.
Preferably, in step S3, the first ultrafiltration is performed with an ultrafiltration membrane of 9.5-10 kDa.
More preferably, in step S3, the first ultrafiltration is performed using a 10kDa ultrafiltration membrane.
Preferably, in the step S3, the ultrafiltration membrane of 6.5-7 kDa is used for the second ultrafiltration.
More preferably, in step S3, the second ultrafiltration is performed using a 7kDa ultrafiltration membrane.
Further, in step S3, the treatment with the nonpolar or weakly polar resin is a resin column treatment filled with the nonpolar or weakly polar resin.
Preferably, in step S3, the nonpolar or weakly polar resin is AB-8 or D101 macroporous resin.
Preferably, in step S3, the weight of the nonpolar or weakly polar resin is 5 to 15 times that of the enzymatic hydrolysis product.
More preferably, in step S3, the weight of the nonpolar or weakly polar resin is 10 times the weight of the enzymatic hydrolysate.
Preferably, in step S3, the eluting with water is performed using 5 to 10 column volumes of water.
More preferably, in step S3, the eluting with water is performed using 8 column volumes of water.
Preferably, in the steps S1 to S3, the temperature of the concentration is 50 to 70 ℃.
Preferably, in the steps S1 to S3, the concentration is performed until the solid mass fraction is 25% to 40%.
Further, in step S1, the ethanol is added so that the final volume concentration of the ethanol is 75% -85%.
Preferably, in step S1, the ethanol is added such that the final volume concentration of ethanol is 80%.
Preferably, in the step S1, the weight ratio of the peach blossom to the water is 1:15-25.
More preferably, in step S1, the weight ratio of peach blossom to water is 1:20.
Preferably, in the step S2, the weight ratio of the sediment to the water is 1:5-8.
More preferably, in step S2, the weight ratio of the precipitate to water is 1:6.
The invention also protects the peach blossom extract prepared by the preparation method.
Further, the peach blossom extract also has the effect of promoting the expression of the Eln1 gene.
Further, the Eln1 gene is zebra fish Eln1 gene.
The invention also provides application of the peach blossom extract in preparation of medicines, foods or cosmetics.
Further, the peach blossom extract is applied to the preparation of cosmetics with a tightening effect.
Further, the use of the peach blossom extract in the preparation of cosmetics having anti-inflammatory/soothing effects.
The invention has the following beneficial effects: the invention provides a brand-new preparation method of peach blossom extract, which is characterized in that a specific protein mixed enzyme is adopted to hydrolyze a precipitated product after alcohol precipitation on the basis of traditional water extraction and alcohol precipitation, and then an ultrafiltration membrane with a specific aperture is adopted to carry out graded ultrafiltration on the enzymolysis product, so that more active ingredients are obtained through enrichment, the anti-inflammatory/soothing effect and the tightening effect of the obtained peach blossom extract are obviously improved, and the preparation method has wide application prospects in the fields of preparation of medicines, foods or cosmetics.
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 of peach blossom extract
S1, taking peach blossom, heating the peach blossom with water of which the weight is 20 times that of the peach blossom to 100 ℃ for reflux extraction for 1.5 hours to obtain peach blossom water extract, and concentrating the peach blossom water extract until the solid mass fraction is 30% to obtain water concentrate;
s2, adding ethanol with the volume concentration of 95% into the water extraction concentrate to ensure that the final volume concentration of the ethanol is 80%, standing for 12 hours, and filtering to obtain a precipitate;
s3, mixing the precipitate with water of which the weight is 6 times that of the precipitate, then adding enzyme for enzymolysis, heating to 100 ℃ after the enzymolysis is finished, maintaining for 10min to inactivate the enzyme, concentrating and drying the enzymolysis liquid to obtain an enzymolysis product; wherein the weight ratio of enzyme to water is 5:100; the enzyme is a mixed enzyme consisting of papain (20 ten thousand U/g) and neutral protease (20 ten thousand U/g) in a weight ratio of 1:1;
s4, loading the enzymolysis product on an AB-8 type macroporous resin column (the weight dosage of macroporous resin in the macroporous resin column is 10 times of the weight of the enzymolysis product), eluting with water with the volume of 8 times of the column volume to obtain macroporous resin eluent, and concentrating the macroporous resin eluent until the solid mass fraction is 30%, thus obtaining elution concentrate;
s5, ultrafiltering the eluting concentrated solution obtained in the step S4 by using a 10kDa ultrafiltration membrane to obtain trapped fluid A and filtrate A; then ultrafiltering the filtrate A with 7kDa ultrafilter membrane to obtain trapped fluid B and filtrate B; concentrating and drying the trapped fluid B to obtain the peach blossom extract.
Example 2 preparation of peach blossom extract
The main difference from example 1 is that in step S5, an ultrafiltration membrane of 10.5kDa is used followed by an ultrafiltration membrane of 7.5 kDa.
Other steps and parameters refer to example 1.
Example 3 preparation of peach blossom extract
The main difference from example 1 is that in step S5, an ultrafiltration membrane of 9.5kDa is used followed by an ultrafiltration membrane of 6.5 kDa.
Other steps and parameters refer to example 1.
Comparative example 1 preparation of peach blossom extract
S1, taking peach blossom, heating the peach blossom with water of which the weight is 20 times that of the peach blossom to 100 ℃ for reflux extraction for 1.5 hours to obtain peach blossom water extract, and concentrating the peach blossom water extract until the solid mass fraction is 30% to obtain water concentrate;
s2, adding ethanol with the volume concentration of 95% into the water extraction concentrate to ensure that the final volume concentration of the ethanol is 80%, standing for 12 hours, and filtering to obtain a precipitate;
s3, mixing the precipitate with water of which the weight is 6 times that of the precipitate, then adding enzyme for enzymolysis, heating to 100 ℃ after the enzymolysis is finished, maintaining for 10min to inactivate the enzyme, concentrating and drying the enzymolysis liquid to obtain an enzymolysis product; wherein the weight ratio of enzyme to water is 5:100; the enzyme is papain (20 ten thousand U/g);
s4, loading the enzymolysis product on an AB-8 type macroporous resin column (the weight of macroporous resin in the macroporous resin column is 10 times of that of the enzymolysis product), and eluting with water with the volume of 8 times of that of the column to obtain macroporous resin eluent; concentrating and drying the macroporous resin eluent to obtain the peach blossom extract.
The difference from example 1 is that in step S3, papain is used for enzymolysis; no ultrafiltration was performed.
Comparative example 2 preparation of peach blossom extract
S1, taking peach blossom, heating the peach blossom with water of which the weight is 20 times that of the peach blossom to 100 ℃ for reflux extraction for 1.5 hours to obtain peach blossom water extract, and concentrating the peach blossom water extract until the solid mass fraction is 30% to obtain water concentrate;
s2, adding ethanol with the volume concentration of 95% into the water extraction concentrate to ensure that the final volume concentration of the ethanol is 80%, standing for 12 hours, and filtering to obtain a precipitate;
s3, mixing the precipitate with water of which the weight is 6 times that of the precipitate, then adding enzyme for enzymolysis, heating to 100 ℃ after the enzymolysis is finished, maintaining for 10min to inactivate the enzyme, concentrating and drying the enzymolysis liquid to obtain an enzymolysis product; wherein the weight ratio of enzyme to water is 5:100; the enzyme is bromelain (20 ten thousand U/g);
s4, loading the enzymolysis product on an AB-8 type macroporous resin column (the weight of macroporous resin in the macroporous resin column is 10 times of that of the enzymolysis product), and eluting with water with the volume of 8 times of that of the column to obtain macroporous resin eluent; concentrating and drying the macroporous resin eluent to obtain the peach blossom extract.
The difference from example 1 is that in step S3, bromelain is used for the enzymatic hydrolysis; no ultrafiltration was performed.
Comparative example 3 preparation of peach blossom extract
S1, taking peach blossom, heating the peach blossom with water of which the weight is 20 times that of the peach blossom to 100 ℃ for reflux extraction for 1.5 hours to obtain peach blossom water extract, and concentrating the peach blossom water extract until the solid mass fraction is 30% to obtain water concentrate;
s2, adding ethanol with the volume concentration of 95% into the water extraction concentrate to ensure that the final volume concentration of the ethanol is 80%, standing for 12 hours, and filtering to obtain a precipitate;
s3, mixing the precipitate with water of which the weight is 6 times that of the precipitate, then adding enzyme for enzymolysis, heating to 100 ℃ after the enzymolysis is finished, maintaining for 10min to inactivate the enzyme, concentrating and drying the enzymolysis liquid to obtain an enzymolysis product; wherein the weight ratio of enzyme to water is 5:100; the enzyme is neutral protease (20 ten thousand U/g);
s4, loading the enzymolysis product on an AB-8 type macroporous resin column (the weight of macroporous resin in the macroporous resin column is 10 times of that of the enzymolysis product), and eluting with water with the volume of 8 times of that of the column to obtain macroporous resin eluent; concentrating and drying the macroporous resin eluent to obtain the peach blossom extract.
The difference from example 1 is that in step S3, enzymolysis is performed using neutral protease; no ultrafiltration was performed.
Comparative example 4 preparation of peach blossom extract
S1, taking peach blossom, heating the peach blossom with water of which the weight is 20 times that of the peach blossom to 100 ℃ for reflux extraction for 1.5 hours to obtain peach blossom water extract, and concentrating the peach blossom water extract until the solid mass fraction is 30% to obtain water concentrate;
s2, adding ethanol with the volume concentration of 95% into the water extraction concentrate to ensure that the final volume concentration of the ethanol is 80%, standing for 12 hours, and filtering to obtain a precipitate;
s3, mixing the precipitate with water of which the weight is 6 times that of the precipitate, then adding enzyme for enzymolysis, heating to 100 ℃ after the enzymolysis is finished, maintaining for 10min to inactivate the enzyme, concentrating and drying the enzymolysis liquid to obtain an enzymolysis product; wherein the weight ratio of enzyme to water is 5:100; the enzyme is a mixed enzyme consisting of papain (20 ten thousand U/g) and neutral protease (20 ten thousand U/g) in a weight ratio of 1:1;
s4, loading the enzymolysis product on an AB-8 type macroporous resin column (the weight of macroporous resin in the macroporous resin column is 10 times of that of the enzymolysis product), and eluting with water with the volume of 8 times of that of the column to obtain macroporous resin eluent; concentrating and drying the macroporous resin eluent to obtain the peach blossom extract.
The difference from example 1 is that no ultrafiltration was performed.
Comparative example 5 preparation of peach blossom extract
S1, taking peach blossom, heating the peach blossom with water of which the weight is 20 times that of the peach blossom to 100 ℃ for reflux extraction for 1.5 hours to obtain peach blossom water extract, and concentrating the peach blossom water extract until the solid mass fraction is 30% to obtain water concentrate;
s2, adding ethanol with the volume concentration of 95% into the water extraction concentrate to ensure that the final volume concentration of the ethanol is 80%, standing for 12 hours, and filtering to obtain a precipitate;
s3, mixing the precipitate with water of which the weight is 6 times that of the precipitate, then adding enzyme for enzymolysis, heating to 100 ℃ after the enzymolysis is finished, maintaining for 10min to inactivate the enzyme, concentrating and drying the enzymolysis liquid to obtain an enzymolysis product; wherein the weight ratio of enzyme to water is 5:100; the enzyme is a mixed enzyme consisting of papain (20 ten thousand U/g) and bromelain (20 ten thousand U/g) in a weight ratio of 1:1;
s4, loading the enzymolysis product on an AB-8 type macroporous resin column (the weight of macroporous resin in the macroporous resin column is 10 times of that of the enzymolysis product), and eluting with water with the volume of 8 times of that of the column to obtain macroporous resin eluent; concentrating and drying the macroporous resin eluent to obtain the peach blossom extract.
The difference from example 1 is that in step S3, papain and bromelain are used as the mixed enzymes; no ultrafiltration was performed.
Comparative example 6 preparation of peach blossom extract
S1, taking peach blossom, heating the peach blossom with water of which the weight is 20 times that of the peach blossom to 100 ℃ for reflux extraction for 1.5 hours to obtain peach blossom water extract, and concentrating the peach blossom water extract until the solid mass fraction is 30% to obtain water concentrate;
s2, adding ethanol with the volume concentration of 95% into the water extraction concentrate to ensure that the final volume concentration of the ethanol is 80%, standing for 12 hours, and filtering to obtain a precipitate;
s3, mixing the precipitate with water of which the weight is 6 times that of the precipitate, then adding enzyme for enzymolysis, heating to 100 ℃ after the enzymolysis is finished, maintaining for 10min to inactivate the enzyme, concentrating and drying the enzymolysis liquid to obtain an enzymolysis product; wherein the weight ratio of enzyme to water is 5:100; the enzyme is a mixed enzyme consisting of bromelain (20 ten thousand U/g) and neutral protease in a weight ratio of 1:1;
s4, loading the enzymolysis product on an AB-8 type macroporous resin column (the weight of macroporous resin in the macroporous resin column is 10 times of that of the enzymolysis product), and eluting with water with the volume of 8 times of that of the column to obtain macroporous resin eluent; concentrating and drying the macroporous resin eluent to obtain the peach blossom extract.
The difference from example 1 is that in step S3, the mixed enzymes used are neutral protease and bromelain; no ultrafiltration was performed.
Comparative example 7 preparation of peach blossom extract
And (3) taking peach flowers, then heating the peach flowers to 100 ℃ by 20 times of water, carrying out reflux extraction for 1.5 hours to obtain peach flower water extract, concentrating and drying the peach flower water extract to obtain the peach flower water extract.
Comparative example 8 preparation of peach blossom extract
S1, taking peach blossom, heating the peach blossom with water of which the weight is 20 times that of the peach blossom to 100 ℃ for reflux extraction for 1.5 hours to obtain peach blossom water extract, and concentrating the peach blossom water extract until the solid mass fraction is 30% to obtain water concentrate;
s2, adding ethanol with the volume concentration of 95% into the water extraction concentrate to ensure that the final volume concentration of the ethanol is 80%, standing for 12 hours, and filtering to obtain a precipitate;
s3, mixing the precipitate with water of which the weight is 6 times that of the precipitate, then adding enzyme for enzymolysis, heating to 100 ℃ after the enzymolysis is finished, maintaining for 10min to inactivate the enzyme, concentrating and drying the enzymolysis liquid to obtain an enzymolysis product; wherein the weight ratio of enzyme to water is 5:100; the enzyme is a mixed enzyme consisting of papain (20 ten thousand U/g) and neutral protease (20 ten thousand U/g) in a weight ratio of 1:1;
s4, loading the enzymolysis product on an AB-8 type macroporous resin column (the weight dosage of macroporous resin in the macroporous resin column is 10 times of the weight of the enzymolysis product), eluting with water with the volume of 8 times of the column volume to obtain macroporous resin eluent, and concentrating the macroporous resin eluent until the solid mass fraction is 30%, thus obtaining elution concentrate;
s5, ultrafiltering the eluting concentrated solution obtained in the step S4 by using a 20kDa ultrafiltration membrane to obtain trapped fluid A and filtrate A; then ultrafiltering the filtrate A with a 15kDa ultrafiltration membrane to obtain trapped fluid B and filtrate B; concentrating and drying the trapped fluid B to obtain the peach blossom extract.
The difference from example 1 is that in step S4, ultrafiltration treatment is performed by using an ultrafiltration membrane of 20kDa followed by an ultrafiltration membrane of 15 kDa.
Comparative example 9 preparation of peach blossom extract
S1, taking peach blossom, heating the peach blossom with water of which the weight is 20 times that of the peach blossom to 100 ℃ for reflux extraction for 1.5 hours to obtain peach blossom water extract, and concentrating the peach blossom water extract until the solid mass fraction is 30% to obtain water concentrate;
s2, adding ethanol with the volume concentration of 95% into the water extraction concentrate to ensure that the final volume concentration of the ethanol is 80%, standing for 12 hours, and filtering to obtain a precipitate;
s3, mixing the precipitate with 6 times of water, adding enzyme for enzymolysis, and heating after the enzymolysis is finished
Inactivating enzyme at 100deg.C for 10min, concentrating and drying the enzymatic hydrolysate to obtain enzymatic hydrolysate; wherein the weight ratio of enzyme to water is 5:100; the enzyme is a mixed enzyme consisting of papain (20 ten thousand U/g) and neutral protease (20 ten thousand U/g) in a weight ratio of 1:1;
s4, loading the enzymolysis product on an AB-8 type macroporous resin column (the weight dosage of macroporous resin in the macroporous resin column is 10 times of the weight of the enzymolysis product), eluting with water with the volume of 8 times of the column volume to obtain macroporous resin eluent, and concentrating the macroporous resin eluent until the solid mass fraction is 30%, thus obtaining elution concentrate;
s5, ultrafiltering the eluting concentrated solution obtained in the step S4 by using a 15kDa ultrafiltration membrane to obtain trapped fluid A and filtrate A; then ultrafiltering the filtrate A with 10kDa ultrafilter membrane to obtain trapped fluid B and filtrate B; concentrating and drying the trapped fluid B to obtain the peach blossom extract.
The difference from example 1 is that in step S4, ultrafiltration treatment is carried out by using an ultrafiltration membrane of 15kDa followed by an ultrafiltration membrane of 10 kDa.
Comparative example 10 preparation of peach blossom extract
S1, taking peach blossom, heating the peach blossom with water of which the weight is 20 times that of the peach blossom to 100 ℃ for reflux extraction for 1.5 hours to obtain peach blossom water extract, and concentrating the peach blossom water extract until the solid mass fraction is 30% to obtain water concentrate;
s2, adding ethanol with the volume concentration of 95% into the water extraction concentrate to ensure that the final volume concentration of the ethanol is 80%, standing for 12 hours, and filtering to obtain a precipitate;
s3, mixing the precipitate with 6 times of water, adding enzyme for enzymolysis, and heating after the enzymolysis is finished
Inactivating enzyme at 100deg.C for 10min, concentrating and drying the enzymatic hydrolysate to obtain enzymatic hydrolysate; wherein the weight ratio of enzyme to water is 5:100; the enzyme is a mixed enzyme consisting of papain (20 ten thousand U/g) and neutral protease (20 ten thousand U/g) in a weight ratio of 1:1;
s4, loading the enzymolysis product on an AB-8 type macroporous resin column (the weight dosage of macroporous resin in the macroporous resin column is 10 times of the weight of the enzymolysis product), eluting with water with the volume of 8 times of the column volume to obtain macroporous resin eluent, and concentrating the macroporous resin eluent until the solid mass fraction is 30%, thus obtaining elution concentrate;
s5, ultrafiltering the eluting concentrated solution obtained in the step S4 by using an ultrafiltration membrane of 8kDa to obtain trapped fluid A and filtrate A; then ultrafiltering the filtrate A with 5kDa ultrafilter membrane to obtain trapped fluid B and filtrate B; concentrating and drying the trapped fluid B to obtain the peach blossom extract.
The difference from example 1 is that in step S4, the ultrafiltration treatment is performed by using an ultrafiltration membrane of 8kDa followed by an ultrafiltration membrane of 5 kDa.
Experimental example 1 experiment of anti-inflammatory and soothing Effect of peach blossom extract
1. Experimental method
Selecting normal Tg (corola: EGFP) transgenic zebra fish of 3dpf (days post fertilization), pretreating 1h with 1.00mg/mL sample to be tested, taking standard dilution water as a blank control group, cutting 50% of 3dpf zebra fish tail fins by a surgical knife under a stereoscopic microscope, placing the cut zebra fish tail fins in a 6-hole cell culture plate, adding 14 pieces/hole into each hole of the test group to obtain the sample to be tested, taking zebra fish system culture water as the blank control group, and continuously incubating in an incubator for 6h. After 6h incubation, 3dpf zebra fish is anesthetized by 0.02% of tricaine, macrophage and neutrophil aggregation condition at the wound of the zebra fish tail fin is observed under a fluorescence microscope, photographing is carried out, the cell number is counted, and the average aggregation number of inflammatory cells of each group of zebra fish at the wound is calculated.
Wherein the samples to be tested are the peach blossom extracts prepared in comparative examples 1-10 and example 1, respectively, and the test results are shown in Table 1.
2. Experimental results
TABLE 1 anti-inflammatory and soothing test results of peach blossom extract of the invention
| Group of | Average number of inflammatory cells accumulated at the wound site |
| Example 1 | 6.8 |
| Blank control group | 16.7 |
| Comparative example 1 | 14.3 |
| Comparative example 2 | 15.0 |
| Comparative example 3 | 14.8 |
| Comparative example 4 | 10.7 |
| Comparative example 5 | 14.6 |
| Comparative example 6 | 14.9 |
| Comparative example 7 | 16.0 |
| Comparative example 8 | 16.7 |
| Comparative example 9 | 15.3 |
| Comparative example 10 | 11.5 |
As can be seen from table 1, comparing the data of the blank control and comparative examples 1 to 7, only the peach blossom extract prepared by using the mixed enzyme composed of papain and neutral protease in the enzymolysis step has significantly improved anti-inflammatory/soothing effect; the peach flower extract obtained by water extraction or the peach flower extract prepared by mixed enzymes consisting of other enzymes or single enzymolysis has weak anti-inflammatory/soothing effect and cannot produce synergistic anti-inflammatory/soothing effect.
From the data of comparative examples 8 to 10, it is known that the improvement effect of anti-inflammatory/soothing effect is weaker than that of the peach blossom extract prepared in example 1, which indicates that in the ultrafiltration step of the present invention, the selection of the ultrafiltration condition is critical, the ultrafiltration is performed by using a 10kDa ultrafiltration membrane, then the ultrafiltration is performed by using a 7kDa ultrafiltration membrane, and the anti-inflammatory/soothing effect of the peach blossom extract prepared by taking the retentate obtained after ultrafiltration by the 7kDa ultrafiltration membrane is remarkably improved. The peach blossom extracts prepared in example 2 and example 3 achieve substantially the same effect as in example 1.
Experimental example 2 experiment of the Tight Effect of peach blossom extract
1. Experimental method
Collecting wild type zebra fish juvenile fish with a length of 4dpf (four days after fertilization), placing the juvenile fish in a 6-well plate, adding 5mL of sample solution with different concentrations into each well for treatment, treating the 20 juvenile fish with standard dilution water in the last well as a blank control group, respectively collecting all juvenile fish after 72 hours of treatment and washing the juvenile fish for 2 times by using sterilized double distilled water respectively, then respectively transferring the juvenile fish into labeled sterilized 1.5mL EP pipes, extracting zebra fish RNA, then carrying out reverse transcription on the RNA into cDNA, and detecting the change of an Eln1 gene in mRNA level on a fluorescent quantitative PCR instrument.
The samples tested are the peach blossom extract prepared in example 1 and the peach blossom extracts prepared in comparative examples 4 and 7-9, and the mass concentration is 0.5mg/mL; wherein the positive control drug is 0.5mg/mL dendrobium polysaccharide, and the test result is shown in Table 2.
2. Experimental results
TABLE 2 influence on the relative expression level of the Eln1 Gene of zebra fish
| Relative expression level of Eln1 Gene | |
| Example 1 | 2.99±0.28 |
| Blank control group | 1.00±0.15 |
| Positive control group | 1.91±0.10 |
| Comparative example 4 | 1.78±0.11 |
| Comparative example 7 | 1.65±0.17 |
| Comparative example 8 | 1.44±0.13 |
| Comparative example 9 | 1.31±0.15 |
As can be seen from table 2, compared with the blank control group, the peach blossom extract prepared in comparative example 4 improves the relative expression level of the zebra fish Eln1 gene, which indicates that the peach blossom extract obtained by enzymolysis with papain and neutral protease has the effect of promoting Eln1 gene expression, shows a certain tightening effect, and the obtained peach blossom extract (i.e. example 1) is subjected to ultrafiltration under specific parameters on the basis of comparative example 4, so that the relative expression level of the zebra fish Eln1 gene is further greatly improved, and the relative expression level of the zebra fish Eln1 gene is higher than that of the positive control dendrobium polysaccharide, which indicates that the papain and neutral protease are adopted for enzymolysis together, and further more tightening effective components can be enriched under specific ultrafiltration conditions, so that the relative expression level of the zebra fish Eln1 gene is remarkably improved, and the relative expression level of the peach blossom extract is better than that of the positive control dendrobium polysaccharide.
Compared with the peach blossom extract prepared in the embodiment 1, the data of the comparative examples 7-9 show that the relative expression level of the Eln1 gene of the zebra fish cannot be obviously improved, which indicates that in the ultrafiltration step of the invention, the selection of the ultrafiltration condition is critical, the ultrafiltration is performed by using an ultrafiltration membrane of 9.5-10.5 kDa, the ultrafiltration is performed by using an ultrafiltration membrane of 6.5-7.5 kDa, and the tightening effect of the peach blossom extract prepared from the retentate obtained by ultrafiltration of the ultrafiltration membrane of 6.5-7.5 kDa can be obviously improved. The peach blossom extracts prepared in example 2 and example 3 achieve substantially the same effect as in example 1.
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 peach blossom extract is characterized by comprising the following steps:
s1, adding water into peach blossom, heating, reflux-extracting, concentrating, adding ethanol, mixing uniformly, fully standing, and filtering to obtain a precipitate;
s2, adding water into the precipitate obtained in the step S1 for full dissolution, adding papain and neutral protease for full enzymolysis, inactivating the enzyme after the enzymolysis is finished, and concentrating the enzymolysis liquid to obtain an enzymolysis product;
s3, treating the enzymolysis product obtained in the step S2 by using nonpolar or weak polar resin, eluting with water to obtain eluent, concentrating the eluent, performing primary ultrafiltration by using a 9.5-10.5 kDa ultrafiltration membrane, performing secondary ultrafiltration on the obtained filtrate by using a 6.5-7.5 kDa ultrafiltration membrane, and concentrating the trapped fluid to obtain the peach blossom extract.
2. The preparation method according to claim 1, wherein in the step S2, the weight ratio of the papain to the neutral protease is 1-3:1-3.
3. The preparation method according to claim 2, wherein the weight ratio of the papain to the neutral protease is 1-2:1-2.
4. The method according to claim 1, wherein in step S3, the first ultrafiltration is performed using an ultrafiltration membrane of 9.5kDa to 10 kDa.
5. The method according to claim 1, wherein in the step S3, the ultrafiltration membrane of 6.5-7 kDa is used for the second ultrafiltration.
6. The method according to claim 1, wherein in step S3, the nonpolar or weakly polar resin is an AB-8 or D101 type macroporous resin.
7. The method according to claim 1, wherein the concentration temperature in steps S1 to S3 is 50 to 70 ℃.
8. The method according to claim 1, wherein in the steps S1 to S3, the concentration is performed so that the solid mass fraction is 25% to 40%.
9. The peach blossom extract prepared by the preparation method of any one of claims 1 to 8.
10. Use of the peach blossom extract of claim 9 for the preparation of a medicament, food or cosmetic.
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