CN112057409B

CN112057409B - Sheep placenta extracting solution with anti-aging function and preparation method and application thereof

Info

Publication number: CN112057409B
Application number: CN202010988954.3A
Authority: CN
Inventors: 张向飞; 胡忠国; 王翔; 王洁; 朱熠; 樊红柳; 肖静
Original assignee: CHENGDU QINGKE BIO-TECHNOLOGY CO LTD
Current assignee: CHENGDU QINGKE BIO-TECHNOLOGY CO LTD
Priority date: 2020-09-18
Filing date: 2020-09-18
Publication date: 2022-09-06
Anticipated expiration: 2040-09-18
Also published as: CN112057409A

Abstract

The invention discloses a sheep placenta extract with an anti-aging function, a preparation method and an application thereof, wherein the preparation method comprises the following steps: s1, taking sheep placenta in an embryo stage, separating cotyledons, removing a fetal membrane, washing the cotyledons with normal saline, and then cutting the cotyledons into blocks; s2, soaking the cotyledon blocks in a peroxyacetic acid solution, and then cleaning with purified water; s3, adding a taurine solution into the cotyledon blocks, and crushing and homogenizing to obtain a slurry; s4, oscillating and centrifuging the slurry, and respectively collecting supernatant and precipitate; s5, adding taurine into the precipitate, performing negative pressure cavitation extraction, centrifuging, and collecting supernatant; s6, mixing the supernatants, adjusting the pH, oscillating, centrifuging and collecting the supernatants; s7, adjusting the pH value of the supernatant, then performing complement inactivation, adjusting the pH value, centrifuging, and collecting the supernatant; s8, carrying out ultrafiltration concentration on the supernatant, and carrying out filtration sterilization to obtain the product. The extract disclosed by the invention is applied to cosmetics, has the effects of moisturizing, resisting wrinkles, delaying senescence and enhancing the skin immune function, and has a wide market prospect.

Description

Sheep placenta extracting solution with anti-aging function and preparation method and application thereof

Technical Field

The invention belongs to the technical field of extract preparation, and particularly relates to a sheep placenta extracting solution with an anti-aging function, and a preparation method and application thereof.

Background

Aging is a necessary stage in the process of life activities for any organism. With the age, people are affected by various factors inside and outside the body, and the body must have the aging process of the morphological structure and physiological function of the whole body. The skin, as the organ of the body most easily exposed to the environment, plays a role in protecting, feeling, regulating body temperature, excreting, and immunizing the body, and is one of the most obvious organs in aging of the body. The conclusion that ultraviolet rays cause skin photoaging has been widely accepted in recent years. Ultraviolet rays are classified into short, medium, and long wavelength ultraviolet rays according to wavelength. Excessive ultraviolet radiation can cause skin inflammatory infiltration, induce delayed anaphylaxis, abnormal DNA synthesis, decreased hyaluronic acid content and fibroblast damage.

Skin anti-aging has become a hot spot of research on medicines and skin care cosmetics, and a plurality of products which can delay skin aging appear in the market, wherein the products comprise some cosmetics added with sheep placenta or sheep embryo/sheep placenta extract, and the sheep placenta or sheep embryo/sheep placenta extract is considered to be capable of effectively fading wrinkles, enhancing skin elasticity and delaying skin aging.

In the existing sheep placenta extraction method, technological means of enzymolysis and high-temperature sterilization are mostly adopted in the technological process, and natural active substances in the placenta can be damaged by enzymolysis and high temperature, so that the types and the content of the natural active substances are difficult to guarantee, the quality of sheep placenta extract is not favorable to guarantee, and the extraction efficiency is not high. In the prior art, the anti-aging effect of the sheep embryo/sheep placenta extracting solution is too general, and bioactive components with the anti-aging effect are not fully extracted.

Disclosure of Invention

The invention aims to: aiming at the defects in the prior art, the sheep placenta extract with the anti-aging function and the preparation method and the application thereof are provided.

The technical scheme adopted by the invention is as follows:

a method for preparing sheep placenta extract with antiaging effect comprises the following steps:

s1, taking sheep placenta in an embryo stage, separating cotyledons, removing a fetal membrane, washing the cotyledons with normal saline, and then cutting the cotyledons into blocks;

s2, soaking the cotyledon blocks obtained in the step S1 in a peroxyacetic acid solution for 10-15min, and then cleaning with purified water;

s3, adding a taurine solution into the cotyledon blocks treated in the step S2, and crushing and homogenizing to obtain a slurry;

s4, oscillating the slurry obtained in the step S3 at 30-50 ℃, then centrifuging for 10-15min, and respectively collecting supernatant and precipitate;

s5, adding taurine into the precipitate obtained in the step S4, performing negative pressure cavitation extraction, centrifuging for 10-15min, and collecting supernatant;

s6, mixing the supernatants obtained in S4 and S5, adjusting the pH to 5.0, oscillating at 30-50 ℃, centrifuging for 10-15min, and collecting the supernatant;

s7, adjusting the pH value of the supernatant obtained in the step S6 to 6.3-6.4, then performing complement inactivation, adjusting the pH value to 7.0, placing at 4 ℃ for centrifugation, and collecting the supernatant;

s8, carrying out ultrafiltration concentration on the supernatant obtained in the step S7 until the protein concentration is 2.0-2.5mg/mL, and carrying out filtration sterilization to obtain the protein.

Further, the method comprises the following steps:

s2, soaking the cotyledon blocks obtained in the step S1 in a peroxyacetic acid solution for 10min, and then cleaning with purified water;

s3, adding a taurine solution into the cotyledon blocks treated in the step S2, and crushing and homogenizing to obtain slurry;

s4, oscillating the slurry obtained in S3 at 40 ℃, 100-;

s5, adding taurine into the precipitate obtained in the step S4, performing negative pressure cavitation extraction, centrifuging at 6000rpm of 5000-;

s6, mixing the supernatants obtained in S4 and S5, adjusting the pH to 5.0, oscillating at 40 ℃, centrifuging at 10000rpm for 10min, and collecting the supernatant;

s7, adjusting the pH value of the supernatant obtained in the step S6 to 6.3-6.4, then carrying out complement inactivation, then adjusting the pH value to 7.0, placing the mixture at 4 ℃ and centrifuging the mixture at 10000rpm, and collecting the supernatant;

Furthermore, the normal saline in S1 contains 90-110u/mL kanamycin; preferably 100 u/mL.

Further, the concentration of the peracetic acid solution in S2 is 0.05 to 0.2 vt%; preferably 0.1 vt%.

Further, the concentration of the taurine solution in each of S3 and S5 is 0.5-2 wt%, preferably 1 wt%; the ratio of the cotyledon blocks to the taurine solution is 1g:2-4mL, preferably 1g:3 mL; the ratio of the precipitate to the tauric acid solution is 1g:2-4mL, preferably 1g:3 mL.

Further, in S3, the homogenization rotation speed is 10000-.

Further, the negative pressure cavitation extraction conditions in S5 are: the temperature is 20-55 ℃, the extraction pressure is-0.01 MPa to-0.1 MPa, and the extraction time is 20-50 min.

Further, the complement inactivation in S7 specifically comprises: placing in 60 deg.C water bath for 10-15 min.

Further, in S8, a 3-5KD ultrafiltration membrane package is adopted for ultrafiltration concentration; the cells were sterilized by filtration through a 0.22 μm filter.

The sheep placenta extract with the function of resisting aging is prepared by the method.

The sheep placenta extract with antiaging effect can be used for preparing cosmetics.

In summary, due to the adoption of the technical scheme, the invention has the beneficial effects that:

1. according to the invention, fresh sheep placenta is used as a raw material, taurine is used as an extraction solvent, and the extraction and cavitation extraction are combined through twice extraction without adopting high temperature and enzymolysis, so that effective components are fully reserved and extracted, and the sheep placenta extracting solution with the remarkable anti-aging function is prepared;

2. in the invention, bioactive substances obtained by the first homogeneous leaching are limited, intracellular active ingredients are further released through negative pressure cavitation extraction of the precipitate, the active ingredients are fully extracted, partial substances are precipitated after the PH is adjusted, and effective active substances in the precipitate are fully extracted again through the second leaching; taurine is non-essential amino acid, has the functions of nourishing skin and strengthening the activity of cortical cells, and can be used as an anti-aging and anti-wrinkle agent by combining the anti-oxygen property of the taurine; in addition, the protein has stable chemical properties, is acidic amino acid, has the characteristic of existing in a zwitterion form, is beneficial to extracting various proteins in tissues, can protect the activity of the proteins so that the proteins are not easy to damage, and has higher extraction rate;

3. after the sheep placenta extracting solution is co-cultured with cells damaged by UVA irradiation, the number of living cells is obviously increased, so that compared with the extracting solution obtained by the comparative example method, the extracting solution obtained by the method has an obvious protection effect on human umbilical cord mesenchymal stem cells damaged by ultraviolet, the damaged cells are activated, the skin problem caused by excessive ultraviolet irradiation is repaired, the aging is delayed by repairing the cell damage, and the anti-aging effect is obvious;

4. the sheep placenta extract has strong oxidation resistance, can remove free radicals, can lighten aging signs, can lighten wrinkles, can improve the underlying structure of skin, can promote cell proliferation, can enhance the elasticity of the skin, and can delay skin aging;

5. the sheep placenta extract is applied to cosmetics, has the effects of moisturizing, resisting wrinkles, delaying senescence and enhancing the skin immunity, and has wide market prospect.

Drawings

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.

FIG. 1 is a morphological diagram of example cells;

FIG. 2 is a morphology chart of comparative example cells.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the detailed description and specific examples, while indicating the preferred embodiment of the invention, are intended for purposes of illustration only and are not intended to limit the scope of the invention. The components of embodiments of the present invention generally described and illustrated in the figures herein may be arranged and designed in a wide variety of different configurations.

Thus, the following detailed description of the embodiments of the present invention, presented in the figures, is not intended to limit the scope of the invention, as claimed, but is merely representative of selected embodiments of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments of the present invention without making any creative effort, shall fall within the protection scope of the present invention.

It is noted that relational terms such as "first" and "second," and the like, may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other identical elements in the process, method, article, or apparatus that comprises the element.

The features and properties of the present invention are described in further detail below with reference to examples.

Example 1

The preferred embodiment of the invention provides a preparation method of sheep placenta extract with an anti-aging function, which comprises the following specific steps:

1. cleaning and shearing: separating out cotyledon from placenta Caprae Seu Ovis at embryo stage, discarding fetal membrane, washing cotyledon with 100u/mL kanamycin-containing normal saline for 5 times until blood is completely removed, and cutting to about 1cm ³ The tissue blocks of size are ready for use.

2. Virus inactivation: soaking in 0.1 vt% peroxyacetic acid solution for 10min, and repeatedly washing with purified water for 3 times to remove peroxyacetic acid residue.

3. Crushing and homogenizing: according to the mass volume ratio of 1: 3, adding pre-cooled 1 wt% taurine solution into the soaked cotyledon tissue blocks, crushing and homogenizing at 15000rpm for 2 times, each time for 3 min.

4. Leaching and centrifuging: placing the homogenized sheep placenta serous fluid in a constant-temperature shaking water bath at 40 ℃, shaking and leaching at 120rpm for 3h, finishing leaching, centrifuging at 5000rpm for 10min, and collecting supernatant; adding 1 wt% taurine solution into the precipitate, shaking uniformly, extracting by negative pressure cavitation at 20-55 deg.C under-0.01-0.03 MPa for 25min, centrifuging the extractive solution for 10min, and discarding the precipitate.

And 5, pH adjustment: the supernatants were combined and the pH adjusted to 5.0 with 3M citric acid solution.

6. And (3) centrifuging for the second leaching: placing the supernatant with adjusted pH in a constant temperature shaking water bath at 40 deg.C, shaking and leaching at 120rpm for 3h, centrifuging at 4 deg.C at 10000rpm for 10min, collecting supernatant, and discarding precipitate.

7. Complement inactivation: adjusting pH to 6.3-6.4 with 20 wt% potassium carbonate solution, treating in 60 deg.C water bath for 10min for complement inactivation, and adjusting pH of the supernatant to 7.0 with 20 wt% potassium carbonate solution.

8. Centrifuging: centrifuging at 10000rpm at 4 deg.C for 10min to collect supernatant, and discarding precipitate.

9. And (3) ultrafiltration concentration: performing ultrafiltration concentration on the filtered and sterilized extracting solution by using an ultrafiltration membrane package with 3KD, and concentrating until the protein concentration is 2.4mg/mL to obtain a concentrated solution; filtering and sterilizing by using a 0.22 mu m filter, and freezing and storing.

Example 2

1. cleaning and shearing: separating cotyledon from placenta caprae seu ovis at embryo stage, removing fetal membrane, washing cotyledon with 90u/mL kanamycin normal saline for 3 times until blood is removed, and cutting cotyledon to about 1cm ³ The tissue blocks of size are ready for use.

2. Virus inactivation: soaking in 0.08 vt% peroxyacetic acid solution for 10min, and repeatedly washing with purified water for 3 times to remove peroxyacetic acid residue.

3. Crushing and homogenizing: according to the mass volume ratio of 1: 2.5 adding pre-cooled 1 wt% taurine solution into the soaked cotyledon tissue block, crushing and homogenizing at 15000rpm for 2 times, each time for 3 min.

6. Centrifuging for the second leaching: placing the supernatant with adjusted pH in a constant temperature shaking water bath at 40 deg.C, shaking and leaching at 120rpm for 3h, centrifuging at 4 deg.C at 10000rpm for 10min, collecting supernatant, and discarding precipitate.

8. Centrifuging: centrifuging at 4 deg.C at 10000rpm for 10min, collecting supernatant, and discarding precipitate.

9. And (3) ultrafiltration concentration: performing ultrafiltration concentration on the filtered and sterilized extracting solution by using an ultrafiltration membrane package with 3KD, and concentrating until the protein concentration is 2.3mg/mL to obtain a concentrated solution; filtering and sterilizing by using a 0.22 mu m filter, and freezing and storing.

Example 3

1. cleaning and shearing: separating cotyledon from placenta caprae seu ovis at embryo stage, discarding fetal membrane, washing cotyledon with 110u/mL kanamycin-containing normal saline for 4 times until blood is completely removed, and cutting to about 1cm ³ The tissue blocks of size are ready for use.

2. Virus inactivation: soaking in 0.12 vt% peroxyacetic acid solution for 10min, and repeatedly washing with purified water for 3 times to remove peroxyacetic acid residue.

3. Crushing and homogenizing: according to the mass volume ratio of 1: 3.2 adding pre-cooled 1 wt% taurine solution into the soaked cotyledon tissue blocks, crushing and homogenizing at 15000rpm for 2 times, each time for 3 min.

4. Leaching and centrifuging: placing the homogenized sheep placenta serous fluid in a constant-temperature shaking water bath at 40 ℃, shaking and leaching at 120rpm for 3h, finishing leaching, centrifuging at 5000rpm for 10min, and collecting supernatant; adding 1 wt% taurine solution into the precipitate, shaking uniformly, extracting by negative pressure cavitation at 20-55 deg.C under-0.01-0.03 MPa for 25min, centrifuging the extractive solution for 10min, and removing the precipitate.

6. And (3) centrifuging for the second leaching: placing the supernatant with the adjusted pH into a constant-temperature shaking water bath kettle at 40 ℃, shaking and leaching at 120rpm for 3h, finishing leaching, centrifuging at 10000rpm for 10min at 4 ℃, collecting the supernatant, and discarding the precipitate.

9. And (3) ultrafiltration concentration: performing ultrafiltration concentration on the filtered and sterilized extracting solution by using an ultrafiltration membrane package with 3KD, and concentrating until the protein concentration is 2.2mg/mL to obtain a concentrated solution; the cells were sterilized by filtration through a 0.22 μm filter and frozen.

Comparative example

1. Cleaning and shearing: separating cotyledon from placenta caprae seu ovis at embryo stage, removing fetal membrane, washing cotyledon with 100u/mL kanamycin normal saline for 3-5 times until blood is removed, and cutting cotyledon to about 1cm ³ The tissue blocks of size are ready for use.

2. Virus inactivation: soaking in 0.1 wt% peroxyacetic acid solution for 10min, and repeatedly washing with purified water for 3 times to remove peroxyacetic acid residue.

3. Crushing and homogenizing: according to the mass volume ratio of 1: 3, adding pre-cooled 1 wt% taurine solution into the soaked tissue blocks for crushing and homogenizing at 15000rpm for 2 times each for 3 min.

4. Leaching and centrifuging: placing the homogenized placenta caprae seu ovis serous fluid in a constant temperature shaking water bath kettle at 40 deg.C, shaking and leaching at 120rpm for 3h, finishing leaching, centrifuging at 5000rpm for 10min, collecting supernatant, and discarding precipitate.

And 5, pH adjustment: the supernatant was adjusted to pH 5.0 with 3M citric acid solution.

9. And (3) ultrafiltration concentration: ultrafiltering and concentrating the filtrate with ultrafiltration membrane of 3KD to obtain concentrated solution with protein concentration of 2.0-2.5 mg/mL; filtering and sterilizing by using a 0.22 mu m filter, and freezing and storing.

Experimental example 1

Establishing an in-vitro aging damage model of human Umbilical Cord Mesenchymal Stem Cells (UCMSCs), and evaluating the anti-aging function of the sheep embryo or sheep placenta extract prepared in the embodiment 1 (and the comparative example) of the invention.

1. Cell inoculation:

a) collecting 1T 75 flask to obtain cells with cell generation no more than 5, diluting 50mL cell suspension with culture medium to obtain cell concentration of 5 × 10 ⁴ Cell seeding was performed on 4 sterile 6-well plates per mL.

b) 2mL of cell suspension was added per well, with a cell count of 1X 10 ⁵ And (4) incubating for 24 h.

2. Pre-incubation of the test substance for 2 h:

a) and (3) preparing a test object: sucking 1mL of extracting solution stock solution as T1, fully dissolving in 1mL of PBS to obtain a concentration T2, taking T21 mL, diluting for 4 times by a constant dilution method, wherein the dilution solution is PBS, and the dilution multiple is 2, and obtaining 4 test substances with different concentrations. Adding 4mL of complete medium (DL + 10% FBS) respectively to obtain the target test substance.

b) Adding a test substance: discard old medium, add different concentrations of test substance, 2mL per well, blank control and injury control plus corresponding equal volume of 80% (DL + 10% FBS) + 20% (PBS); after the addition, the mixture was gently mixed, transferred to 37 ℃ with 5% CO ₂ The incubator of (2) for cultivation.

As shown in table 1 below:

TABLE 1 culture systems

3. UVA irradiation:

a) irradiation conditions: at a ratio of 10-15J/cm ² The intensity of (2) is designed into irradiation condition parameters.

b) Pre-incubating the test object for 2h, removing the old culture medium, adding 2mL of PBS solution, cleaning, removing, adding 1mL of PBS solution, slightly mixing, and irradiating; the blank control was operated identically except that no irradiation was performed.

4. Incubation of the test substance for 48 h:

after irradiation, discarding PBS, adding the prepared culture medium into each hole, slightly mixing after adding, transferring to 37 ℃, and 5% CO ₂ The incubator of (2) for cultivation.

5. Data acquisition:

a) and (3) morphology observation: after the test object is dried for 24h and 48h, the cell morphology of each group is observed every day, photographed and stored.

The results of the examples are shown in fig. 1, and it can be seen from the figure that the negative control group has normal cell morphology, the number of damaged cells is significantly reduced, the cells are aged, the experimental group has normal cell morphology, and the number of the experimental group is larger than that of the negative control group.

The results of the comparative example group are shown in fig. 2, and it can be seen from the figure that the negative control group has normal cell morphology, the number of damaged cells is significantly reduced, the cells are aged, the number of experimental cells is significantly reduced, and the cells are aged.

b) And (3) detecting the activity of the cells: 48h after the test object is dried, the cells are digested and the viable cells are counted. The results are shown in table 2 below:

TABLE 2 cell Activity Table

The result shows that the extracting solution of the method has obvious protective effect on the human umbilical cord mesenchymal stem cells damaged by ultraviolet; the extract of the comparative example does not protect the human umbilical cord mesenchymal stem cells damaged by ultraviolet rays.

Experimental example 2

The extract obtained in example 1 was subjected to an antioxidant test, and a DPPH radical scavenging experiment, a hydroxyl radical scavenging experiment, and a superoxide anion scavenging experiment were performed, respectively.

DPPH free radical scavenging experiment

Principle of experiment

1, 1-diphenyl-2-trinitrophenylhydrazine (DPPH for short) is a stable long-life free radical, and the ethanol solution of the free radical is dark purple and has strong absorption near 517 nm. In the presence of free radical scavengers, the light absorption of the DPPH ethanol solution is reduced due to its one-electron pairing. The degree of discoloration of the DPPH ethanol solution is linear with the number of electrons it receives, and thus the ability of the test sample to scavenge free radicals, i.e., the magnitude of antioxidant activity, can be evaluated.

Experimental methods

Standard Curve preparation

Dissolving vitamin E in 95% ethanol to dilute: the test system was verified by using a series of concentration gradients of 0.08mg/mL, 0.04mg/mL, 0.02mg/mL, and 0.01 mg/mL.

Sample application requirements

TABLE 3 sample addition requirements

Referring to Table 1, a 15mL centrifuge tube was used to set up the sample tube (T) and sample background (T) ₀ ) DPPH tube (C) and solvent background (C) ₀ ) For each sample concentration to be tested, 3 parallel tubes are required for each sample tube (T), and 3 parallel tubes are required for DPPH tube (C). 1mL of the same concentration of sample solution was added to each of the sample tube (T) and sample background (T0). In all test tubes (T, T) ₀ 、C、C ₀ ) Supplementing solvent, using water for water-soluble sample, using 95% ethanol for oil-soluble sample, complementing 3mL, and mixing. 1mL of DPPH ethanol solution was added to the sample tubes (T) and DPPH tube (C), the sample background (T) ₀ ) And solvent background (C) ₀ ) Replace with 95% ethanol, shake gently, and keep standing at room temperature in dark for 5 minutes. Each reaction solution was transferred to a 1cm cuvette and absorbance was measured at 517 nm.

Calculation of results

Calculating DPPH free radical clearance rate:

t-average value of 3 times of light absorption value of the sample tube, namely the light absorption value of the solution after the sample reacts with DPPH;

T ₀ -sample background absorbance;

c-average value of light absorption values of the DPPH tube for 3 times, namely the light absorption value of DPPH solution when no sample is added;

C ₀ -background absorbance of solvent.

TABLE 4 DPPH radical scavenging results table

Hydroxy radical scavenging ability detection experiment

Principle of experiment

Using Fe ²⁺ /H ₂ O ₂ Method for measuring Hydroxyl Radical Scavenging Ability (HRSA), phenanthroline-Fe ²⁺ Has strong absorption at 536nm, OH can react with phenanthrene-Fe ²⁺ Oxidation to phenanthrene-Fe ³⁺ The absorption at 536nm is reduced. The antioxidant has effects of scavenging and neutralizing OH, and can inhibit the oxidation process. FN represents o-diazaphenanthrene, and the reaction formula is as follows: FeSO ⁴⁺ 3FN→[Fe(FN) ₃ ]SO ₄ 。

Detection step

Negative control: 0.5mL of 1.0mmol/L o-diazaphenanthrene ethanol solution, 0.5mL of deionized water, 1.0mL of 0.15mol/L PBS with pH of 7.4 and 0.5mL of 0.75mmol/L FeSO are added in sequence ₄ Finally, 0.5mL of deionized water is added, the mixture is put into a water bath kettle at 37 ℃ for constant temperature 60min, and after the reaction is fully carried out, the absorbance A of the mixture is measured at 536nm ₀ 。

Positive control: sequentially adding 0.5mL of 1.0mmol/L o-diazophenanthrene ethanol solution, 0.5mL of deionized water, 1.0mL of 0.15mol/L PBS with pH of 7.4 and0.5mL of 0.75mmol/L FeSO ₄ Finally, 0.5mL of 0.01% (v/v) H was added ₂ O ₂ Placing into a 37 deg.C water bath kettle, maintaining the temperature for 60min, reacting completely, and measuring absorbance A at 536nm _i 。

Sample detection: 0.5mL of 1.0mmol/L orthophenanthrene ethanol solution, 0.5mL of sample solution, 1.0mL of 0.15mol/L PBS with pH of 7.4 and 0.5mL of 0.75mmol/L FeSO are added in sequence ₄ Finally, 0.5mL of 0.01% (v/v) H was added ₂ O ₂ Placing into a 37 deg.C water bath kettle, maintaining the temperature for 60min, reacting completely, and measuring absorbance A at 536nm _j 。

Samples were loaded as in Table 5 below

Calculation of results

The hydroxyl radical (. OH) clearance was calculated by the following formula.

TABLE 6 hydroxyl radical scavenging results

Superoxide anion radical scavenging Capacity test

Principle of experiment

Under the alkaline condition, pyrogallol can be quickly oxidized to release superoxide anions (during autoxidation), a colored intermediate product is generated (the reaction just begins to turn yellow green, and turns yellow after a few minutes), the absorption is generated at the wavelength of 320nm, the absorbance is increased along with the change of the color, and the absorbance value and the reaction time form a good linear relationship. When the antioxidant exists, the autoxidation rate of the antioxidant is inhibited, so that the absorbance value is reduced, and the autoxidation rate of the pyrogallol is reduced.

Procedure for the preparation of the

Pyrogallol autooxidation rate (a 0): taking 2.9mLA solution, carrying out water bath at 25 ℃ for 30min, quickly adding 0.1mL B solution with constant temperature of 25 ℃, shaking uniformly, reading the absorbance value A at 320nm every 30s, continuously measuring for 3min, and drawing by using A as a time (taking A as a vertical coordinate and time as a horizontal coordinate), wherein the slope is the self-oxidation rate A0 of the pyrogallol.

And (3) sample determination: taking 0.1mL of sample determination solution, adding 2.8mLA solution, carrying out water bath at 25 ℃ for 30min, quickly adding 0.1mL of solution B with constant temperature of 25 ℃, shaking up, reading absorbance values A at 320nm every 30s, continuously determining for 3min, and drawing a graph by using A AS time (taking A AS an ordinate and time AS an abscissa), wherein the slope is the pyrogallol oxidation rate AS of the sample.

Calculation of results

Superoxide anion radical scavenging (SRSA) was calculated according to the following equation.

TABLE 7 superoxide anion scavenging results table

Time	A0	Sheep placenta extractive solution
			30	0.458	0.402
60	0.475	0.407
			90	0.492	0.41
120	0.51	0.413
			150	0.527	0.415
180	0.543	0.417
			A0	0.0006
As		0.0001
			Clearance rate%		83.33333333

As can be seen from the above results, the extract obtained by the method of the present invention has strong antioxidant property.

The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principle of the present invention are intended to be included within the scope of the present invention.

Claims

1. A preparation method of sheep placenta extract with an anti-aging function is characterized by comprising the following steps:

s5, adding a taurine solution into the precipitate obtained in the step S4, performing negative pressure cavitation extraction, centrifuging for 10-15min, and collecting a supernatant;

s7, adjusting the pH value of the supernatant obtained in the step S6 to 6.3-6.4, then carrying out complement inactivation, adjusting the pH value to 7.0, placing at 4 ℃ for centrifugation, and collecting the supernatant;

2. The method for preparing the extract solution from sheep placenta with anti-aging function of claim 1, wherein the physiological saline solution of S1 contains kanamycin at a concentration of 90-110 u/mL.

3. The method for preparing the sheep placenta extract with anti-aging function according to claim 1, wherein the concentration of the peroxyacetic acid solution in S2 is 0.05-0.2 vt%.

4. The method for preparing sheep placenta extract with antiaging effect as claimed in claim 1, wherein the concentration of taurine solution in S3 and S5 is 0.5-2 wt%; the ratio of the cotyledon blocks to the taurine solution is 1g:2-4 mL; the ratio of the precipitate to the taurine solution was 1g:2-4 mL.

5. The method for preparing extractive solution of placenta caprae seu ovis with antiaging function as claimed in claim 1, wherein the homogenizing rotation speed in S3 is 10000-20000rpm, and homogenizing is performed for 2-3 times, each time for 3 min.

6. The method for preparing the sheep placenta extract with the anti-aging function according to claim 1, wherein the negative pressure cavitation extraction conditions in the S5 are as follows: the temperature is 20-55 ℃, the extraction pressure is-0.01 MPa-0.1 MPa, and the extraction time is 20-50 min.

7. The method for preparing the sheep placenta extract with anti-aging function according to claim 1, wherein the complement inactivation in S7 specifically comprises: placing in 60 deg.C water bath for 10-15 min.

8. The method for preparing the sheep placenta extract with the anti-aging function according to claim 1, wherein the step of S8 is performed by ultrafiltration concentration with a 3-5KD ultrafiltration membrane; the bacteria were removed by filtration using a 0.22 μm filter.

9. The sheep placenta extractive solution with antiaging effect prepared by the method of any one of claims 1-8.

10. Use of the extractive solution of sheep placenta of claim 9 for preparing cosmetics.