CN117363678A - Preparation method of peony fresh flower extracting solution - Google Patents

Preparation method of peony fresh flower extracting solution Download PDF

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CN117363678A
CN117363678A CN202311307125.4A CN202311307125A CN117363678A CN 117363678 A CN117363678 A CN 117363678A CN 202311307125 A CN202311307125 A CN 202311307125A CN 117363678 A CN117363678 A CN 117363678A
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enzymolysis
peony
fresh flower
flower extract
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CN117363678B (en
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Shandong Payne Biotechnology Co ltd
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Beijing Feitian Peony Biotechnology Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/06—Preparation of peptides or proteins produced by the hydrolysis of a peptide bond, e.g. hydrolysate products
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P39/00—General protective or antinoxious agents
    • A61P39/06—Free radical scavengers or antioxidants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P21/00—Preparation of peptides or proteins
    • C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

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  • General Engineering & Computer Science (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Veterinary Medicine (AREA)
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  • Animal Behavior & Ethology (AREA)
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  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

The invention aims to provide a preparation method of a peony fresh flower extract, in particular to a method for preparing the peony fresh flower extract by enzymolysis, which mainly develops various peony active peptides, is purely natural and free from addition, and has the effects of resisting oxidation, whitening, moisturizing, fading spots, delaying aging, resisting bacteria and diminishing inflammation. The invention scientifically and purposely screens the types of enzymes for enzymolysis by means of modern biology, can fully decompose substances to the extent of small molecules, is favorable for absorption and utilization, and finally obtains the enzymolysis liquid with high antioxidant and high polypeptide yield and the peony active peptide with antioxidant and liver protecting effects.

Description

Preparation method of peony fresh flower extracting solution
Technical Field
The invention relates to the technical field of cosmetics, in particular to a preparation method of a peony fresh flower extract, and particularly relates to a method for preparing the peony fresh flower extract by enzymolysis.
Background
Paeoniflorin, the root bark of Paeonia suffruticosa, is used as a medicine for treating diseases such as epidemic febrile disease, hematemesis, epistaxis, night fever, early cooling, no sweat, bone steaming, amenorrhea, dysmenorrhea, carbuncle, swelling and sore, traumatic injury, etc. it is a common herb for cooling blood and removing blood stasis. Modern researches have shown that cortex moutan has antibacterial, antiinflammatory, antiallergic, antitumor, hemostatic, blood stasis dispelling, heat and toxic materials clearing away, tranquilizing, analgesic, spasmolytic, and other activities, and also has effects of promoting phagocytic function of monocytes, enhancing specific immunity, increasing weight of immune organs, and fresh flos moutan for skin care product with effects of resisting wrinkle, resisting oxidation, whitening skin, keeping moisture, removing mottle, delaying aging, resisting bacteria, and relieving inflammation.
At present, a plurality of preparation methods of peony fresh flower water are disclosed, including a supercritical CO2 fluid extraction method, and the defects of large equipment investment and complex operation are overcome; the rotary evaporation method has the defects of complex process, higher operation condition, low utilization rate and incapability of obtaining a fine product; the distillation method has the defects of high energy consumption and lower product grade. The disclosed preparation method of the peony fresh flower extract has low antioxidant activity and low utilization rate, and cannot obtain a fine product.
And the enzymolysis can fully decompose substances to the extent of small molecules, thereby being beneficial to absorption and utilization. Currently, researches on performing enzymolysis on peony exist, and CN20211107323820210914; CN20151097643920151221; although CN20141049656620140925 uses an enzymatic hydrolysis method of cellulase and pectase or cellulase, the effect of the enzymatic hydrolysis is not described. CN20141029281520140626 adopts mixed protease to obtain a peony peptide beverage; CN20141062852120141111 is a method for extracting peony polysaccharide from peony cake by biological enzyme method; CN20211145973020211202 is a process for preparing peony pistil protein powder. The invention scientifically and purposely screens the types of enzymes for enzymolysis and the enzymolysis liquid for resisting oxidation, whitening and moisturizing, fading color spots, delaying aging, resisting bacteria and diminishing inflammation through the modern biological means.
Disclosure of Invention
The invention aims to provide a preparation method of a peony fresh flower extract, in particular to a method for preparing the peony fresh flower extract by enzymolysis, which mainly develops various peony active peptides, is purely natural and free from addition, and has the effects of resisting oxidation, whitening, moisturizing, fading spots, delaying aging, resisting bacteria and diminishing inflammation.
In order to achieve the above purpose, the invention is realized by the following technical scheme:
collecting the petals of Paeonia ostii, cleaning, freezing and preserving, further crushing, sterilizing, heating and inactivating enzyme by using microwave and ultrasonic equipment, squeezing, collecting supernatant, regulating pH to 7.5-9.6 by using sodium hydroxide solution, performing preliminary enzymolysis, heating, sterilizing and inactivating enzyme after full enzymolysis, and filtering to remove enzymolysis residues and insoluble substances; and carrying out enzymolysis in stages, namely stirring each time one enzyme is added, after full enzymolysis, heating, sterilizing, inactivating enzyme, filtering to remove enzymolysis residues and insoluble substances, centrifuging, and filtering and intercepting small molecular peptides below 10000 daltons by using a filter membrane to obtain peony petal enzymolysis liquid, namely peony fresh flower extracting liquid.
The preliminary enzymolysis refers to preliminary enzymolysis by adopting lipase-containing enzyme.
The preliminary enzymolysis refers to preliminary enzymolysis by adopting enzyme containing cellulase.
The preliminary enzymolysis refers to preliminary enzymolysis by adopting enzyme containing glycosidase.
The staged enzymolysis refers to enzymolysis by adopting complex enzymes of papain, trypsin, neutral protease, alkaline protease, subtilisin and/or chymotrypsin, and retaining a plurality of glutamic acid, arginine, lysine, phenylalanine, tyrosine, cysteine and methionine as much as possible.
In order to increase the storage time of the peony petal enzymolysis liquid, bergamot and/or geranium are added into the peony petal enzymolysis liquid as natural preservative. The freezing preservation temperature is-5 to-22 ℃; the ultrasonic frequency is 50-120 kHz; the temperature is raised to 35-55 ℃ by microwaves.
The invention also aims to provide the peony fresh flower extracting solution, which is prepared by adopting the preparation method of the peony fresh flower extracting solution.
The invention also aims to provide the peony active peptide, which is prepared by vacuum drying enzymolysis liquid, sending a sample to a detection hospital for analysis and sequencing, and performing functional prediction to obtain 1 peony active peptide with the functions of resisting oxidization and protecting liver, wherein the amino acid sequence is MYECCFECKCKR.
Technical effects
The invention aims to provide a preparation method of a peony fresh flower extract, in particular to a method for preparing the peony fresh flower extract by enzymolysis, which mainly develops various peony active peptides, is purely natural and free from addition, and has the effects of resisting oxidation, whitening, moisturizing, fading spots, delaying aging, resisting bacteria and diminishing inflammation. The invention scientifically and purposefully screens the types of enzymes for enzymolysis by means of modern biology, can fully decompose substances to the extent of small molecules, is favorable for absorption and utilization, finally obtains the enzymolysis liquid with high antioxidant and high polypeptide yield, and obtains the peony active peptide with antioxidant and liver protecting effects.
Detailed Description
Embodiments of the present invention will be further described with reference to examples.
Example 1: the invention provides a preparation method of a peony fresh flower extract, which comprises the following specific preparation steps:
collecting morning danfeng peony petals, cleaning, freezing and preserving, regulating the freezing and preserving temperature to be-5 to-22 ℃, further crushing, sterilizing, heating and inactivating enzyme by utilizing microwave combined ultrasonic equipment, regulating the ultrasonic frequency to be 50-120 kHz, regulating the microwave heating to be 35-55 ℃, squeezing, collecting supernatant, regulating the pH value to be 7.5-9.6 by adopting sodium hydroxide solution, carrying out preliminary enzymolysis by adopting lipase, cellulase and glycosidase, heating, sterilizing and inactivating enzyme after full enzymolysis, and filtering to remove enzymolysis residues and insoluble matters; and carrying out enzymolysis in stages, namely adopting compound enzyme which is a combination of papain, trypsin, neutral protease, alkaline protease, subtilisin and chymotrypsin in a ratio of 1:1:1:1:1, stirring each time one enzyme is added, heating, sterilizing and inactivating enzyme after full enzymolysis, filtering to remove enzymolysis residues and insoluble substances, centrifuging, and filtering centrifugate by a filter membrane to intercept small molecular peptides below 10000 daltons to obtain peony petal enzymolysis liquid, namely peony fresh flower extract. In order to increase the storage time of the peony petal enzymolysis liquid, bergamot or/and geranium can be added into the peony petal enzymolysis liquid as a natural preservative, and the yield of the polypeptide can reach 94% by a Kjeldahl nitrogen determination method.
And (3) vacuum drying the enzymolysis liquid, sending the sample to a detection hospital for analysis and sequencing, and carrying out functional prediction to obtain the peony active peptide for resisting oxidation and protecting the liver.
The concentration of the enzymatic hydrolysate used in the medium was 10% in the experiments to verify the function of the enzymatic hydrolysate and the active peptide in the examples without specific illustration.
Comparative example 1: the steps of primary enzymolysis are removed, and the polypeptide yield reaches 18% in other steps as in example 1.
Comparative example 2: the combination of complex enzymes is replaced by a weight ratio of 1:1.2:0.8:1.2:1.2 trypsin, papain, subtilisin, alkaline 2709 protease, neutral Neutrase protease, the other steps being as described in example 1, the polypeptide yield being up to 29%. Comparative example 3: the combination of the complex enzyme is replaced by acid protease, pectase and cellulase in a ratio of 1:1:1, and the polypeptide yield reaches 24% in other steps as in the example 1.
Comparative example 4: the chymotrypsin only is reserved in the combination of the complex enzymes, and the polypeptide yield reaches 12% in other steps as in the example 1.
EXAMPLE 2 cytotoxicity
Keratinocytes (HaCat) were seeded at a density of 1×105 cells/well, inoculated in 96-well plates, cultured for 24 hours, 10% ez-Cytox was added to DMEM medium, cell viability was measured, and each group of enzymatic hydrolysate was added to the medium. The culture was carried out for 4 hours, and absorbance was measured at a wavelength of 450nm with a microplate reader, and 4 replicates were made for each group.
Cell viability (%) = (absorbance of each group/absorbance of blank group) ×100%
Results: the experimental group 1 showed cytotoxicity of 2.25%, the active peptide cytotoxicity of 2.5%, the control group 1 showed cytotoxicity of 5.5%, the control group 2 showed cytotoxicity of 3.25%, the control group 3 showed cytotoxicity of 3.75%, and the control group 4 showed cytotoxicity of 3.25%. The other groups except the control group 1 have low cytotoxicity below 5.0%, and can be used as cosmetic raw materials, and the control group 1 has complex components and is removed in subsequent experiments.
EXAMPLE 3 antioxidant Effect
Human fibroblasts were inoculated into 96-well plates containing DMEM, 1×105 cells/well, cultured for 24 hours, and each group of enzymatic hydrolysate was added to the medium. The previous medium was then replaced with serum-free DMEM medium and incubated for 24 hours. After incubation, the supernatants from each well were collected and tested for hydroxyl (.OH), superoxide anion (O2-.) and DPPH radical scavenging according to the kit instructions, the results are shown in Table 1. The hydroxyl and superoxide anion radical scavenging rates were calculated as 0% at the initial state of each group of cells, 5 replicates per group.
Table 1 hydroxyl and superoxide anion radical scavenging assay results.
Sample of OH radical scavenging Rate O2-free radical scavenging Rate DPPH radical
Experiment group 1 95.4％ 97.2％ 99.6％
Active peptide 97.6％ 98.4％ 99.8％
Comparative example 2 77.6％ 64.8％ 72.4％
Comparative example 3 65.2％ 75.4％ 79.8％
Comparative example 4 58.4％ 64.8％ 67.6％
Results: the hydroxyl and superoxide anion radical scavenging effect of the experimental group 1 is significantly better than that of the control group 2-4.
Example 4 inhibition of tyrosinase activity
The tyrosinase inhibition rate was determined using a T/SHRH 015-2018 cosmetic-tyrosinase activity inhibition test, the results of which are shown in Table 2.
Table 2 tyrosinase activity inhibition assay.
Sample of Tyrosinase activity inhibition rate
Experiment group 1 90.45％
Active peptide 97.50％
Comparative example 2 58.30％
Comparative example 3 49.12％
Comparative example 4 47.75％
Results: the hydroxyl and superoxide anion radical scavenging effect of the experimental group 1 is significantly better than that of the control group 2-4.
EXAMPLE 5 anti-autophagy
Autophagy experiments were performed on keratinocytes (HaCat) as a subject, and the number of autophagosomes within HaCat was tested as a quantification standard for autophagy to evaluate antioxidant and ultraviolet effects. Cells were transferred to 96-well plates at a density of 1×105 cells/well, and each set of enzymatic lysates was added to the medium and cultured for 24 hours. Cells were irradiated with UVB at a dose of 20mJ/cm2, and then 0.1mM H2O2 was added to the culture solution and reacted for 1 hour, and then the autophagy test agent working solution was added and reacted for 15 minutes to 1 hour, followed by washing the cells with washing solution (PBST). PBS was added and absorbance was measured at 360nm/550nm setting. The autophagy was set to 100% in the HaCaT cell blank without uv and H2O2 effects, 4 replicates per group.
Results: haCaT autophagy by UV and H2O2 reached 102.25%, experimental group 1 autophagy 125.75%, active peptide autophagy 150.25%, control 2 autophagy 106.5%, control 3 autophagy 104.75%, and control 4 autophagy 105.25%.
EXAMPLE 6 antibacterial action
The antimicrobial activity was screened using an agar disc diffusion method, and the size of the antibacterial zone of staphylococcus aureus and escherichia coli was observed, and the results are shown in table 3.
Table 3 results of the inhibition zone size measurements.
Sample of Staphylococcus aureus (mm) Coli (mm)
Experiment group 1 18.5±0.20 22.8±0.35
Active peptide 22.4±0.15 25.5±0.5
Comparative example 2 10.4±0.45 8.4±0.55
Comparative example 3 8.20±0.50 10.8±0.75
Comparative example 4 9.55±0.40 12.5±0.35
Example 7 crowd experiment:
100 subjects 20-55 years old were selected and used once in the morning and evening, and the observation period was 30 days. The total score is 5, and the score is an integer. The average scores are as follows. The results are shown in Table 4.
Table 4 results of the spread comfort, irritation, stain resistance, water smoothness scores.
Sample of Comfort of painting Irritation (irritation) Resistance to plaque Smoothness with water
Experiment group 1 4.78 0.15 4.89 4.79
Active peptide 4.83 0.10 4.95 4.87
Comparative example 2 4.87 0.37 4.34 4.15
Comparative example 3 4.89 0.21 4.49 4.27
Comparative example 4 4.77 0.17 4.42 4.36
EXAMPLE 8 liver protecting action of active peptide
Separating to obtain SD rat liver cells, culturing in DMEM suspension, adding active peptide, adding 2mmol/L H2O2 into the culture solution to cause acute liver cell damage, and detecting ALT activity, NO and MDA content, SOD activity and other indexes in the cell suspension according to the instruction of the kit. The results are shown in Table 5.
Table 5ALT, NO, SOD, MDA measurement results.
Sample of ALT(u/L) NO(umol/L) SOD(NU/ml) MDA(nmol/ml)
H2O2 injury group 170.58±4.35 30.57±0.49 31.71±0.40 4.75±0.47
Blank group 60.41±1.73 3.75±0.41 45.53±0.51 1.85±0.10
Active peptide 42.75±2.86 4.43±0.56 43.67±0.17 2.17±0.24
The above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims (10)

1. A preparation method of a peony fresh flower extract comprises the following preparation steps:
collecting the petals of Paeonia ostii, cleaning, freezing and preserving, further crushing, sterilizing, heating and inactivating enzyme by using microwave and ultrasonic equipment, squeezing, collecting supernatant, regulating pH to 7.5-9.6 by using sodium hydroxide solution, performing preliminary enzymolysis, heating, sterilizing and inactivating enzyme after full enzymolysis, and filtering to remove enzymolysis residues and insoluble substances; and carrying out enzymolysis in stages, namely stirring each time one enzyme is added, after full enzymolysis, heating, sterilizing, inactivating enzyme, filtering to remove enzymolysis residues and insoluble substances, centrifuging, and filtering and intercepting small molecular peptides below 10000 daltons by using a filter membrane to obtain peony petal enzymolysis liquid, namely peony fresh flower extracting liquid.
2. The method for preparing the peony fresh flower extract according to claim 1, which is characterized in that: the preliminary enzymolysis refers to preliminary enzymolysis by adopting lipase-containing enzyme.
3. The method for preparing the peony fresh flower extract according to claim 1, which is characterized in that: the preliminary enzymolysis refers to preliminary enzymolysis by adopting enzyme containing cellulase.
4. The method for preparing the peony fresh flower extract according to claim 1, which is characterized in that: the preliminary enzymolysis refers to preliminary enzymolysis by adopting enzyme containing glycosidase.
5. The method for preparing the peony fresh flower extract according to claim 1, which is characterized in that: the staged enzymolysis refers to enzymolysis by complex enzymes of papain, trypsin, neutral protease, alkaline protease, subtilisin and/or chymotrypsin.
6. The method for preparing the peony fresh flower extract according to claim 1, which is characterized in that: the staged enzymolysis is enzymolysis by adopting complex enzymes of papain, trypsin, neutral protease, alkaline protease, subtilisin and chymotrypsin.
7. The method for preparing the peony fresh flower extract according to claim 1, which is characterized in that: the freezing preservation temperature is-5 to-22 ℃.
8. The method for preparing the peony fresh flower extract according to claim 1, which is characterized in that: the ultrasonic frequency is 50-120 kHz; the temperature of the microwaves is raised to 35-55 ℃.
9. A peony fresh flower extracting solution is characterized in that: the method for preparing the peony fresh flower extract according to claim 1.
10. A peony active peptide, which is characterized in that: the peony active peptide with the functions of resisting oxidation and protecting liver is prepared by adopting the preparation method of the peony fresh flower extract as claimed in claim 1, and the amino acid sequence is MYECCFECKCKR.
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CN110257460A (en) * 2019-05-23 2019-09-20 世堃堂(广东)生物科技有限公司 A kind of preparation method of the tree peony protein peptides of blood pressure lowering

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Title
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