Peony seed crude oil, refined peony seed oil, and their preparation methods and applications

Abstract

The present application discloses a crude peony seed oil, a refined peony seed oil, and their preparation methods and applications. The extraction method of the crude peony seed oil comprises the following steps: mixing peony seeds with water, mixing with a complex enzyme under the conditions of a pH of 4 to 5.5 and a temperature of 40 to 60 °C for enzymatic hydrolysis, then performing ultrasonic extraction, centrifuging, and collecting the oil phase to obtain the crude peony seed oil. The temperature of the ultrasonic extraction is 40 to 70 °C, and the time is 30 to 60 min; the mass ratio of the peony seeds to the complex enzyme is 1:(0.02 to 0.1); the complex enzyme comprises cellulase, hemicellulase, acid protease, and pectinase, and the mass ratio of the four enzymes is 1:(0.5 to 2):(0.2 to 2):(0.5 to 3). The present application enzymatically hydrolyzes peony seeds with specific types of enzymes, combines ultrasonic extraction to obtain the crude peony seed oil, and then obtains the refined peony seed oil through purification. After purification, the acid value and peroxide value of the peony seed oil are reduced, it is not easy to deteriorate, and it has an ideal beauty effect.

Description

Technical Field

[0001] This application belongs to the technical fields of plant biology and cosmetics, and particularly relates to a crude peony seed oil, a refined peony seed oil, and their preparation methods and applications. Background Art

[0002] Peony seed oil is rich in unsaturated fatty acids such as linoleic acid and linolenic acid. Among them, the content of the essential fatty acid α-linolenic acid in the human body is as high as 60%, which is 10 to 100 times that of commercially available vegetable oils. At present, peony seed oil is mainly applied in fields such as food, and its application in the cosmetics field is less, and the application fields are relatively limited.

[0003] At present, the extraction methods of peony seed oil mainly include steam distillation method, organic solvent extraction method, adsorption extraction method, and supercritical CO2 extraction method. However, there are various drawbacks in the above methods for extracting peony seed oil. For example, when using the steam distillation method, due to the too high temperature, the active ingredients will be damaged, resulting in a decrease in the yield and quality of peony seed oil; when using organic solvents for treatment, a large amount of organic solvents will be used. On the one hand, it will damage the environment and increase the cost; on the other hand, it will lead to the residue of organic solvents in the final product. When used in the cosmetics field, it will irritate the skin and cause allergies. The production cycle of the adsorption extraction method is relatively long and the production efficiency is low. The supercritical CO2 extraction method has high requirements for conditions and equipment and is not suitable for industrial production. The peony seed oil obtained by rough processing is also prone to contain impurities and is also prone to rancidity of the peony seed oil.

[0004] The enzymatic hydrolysis method has many advantages. For example, no organic solvents are used during the enzymatic hydrolysis process, reducing environmental pollution and improving the production and use safety of products. However, the technology of the aqueous enzymatic method is not yet mature. For example, some studies have shown that the aqueous enzymatic method will have some adverse effects. When extracting baicalin by the method of enzymatic hydrolysis with cellulase, baicalin will also be hydrolyzed during the enzymatic hydrolysis process, resulting in a decrease in the yield of baicalin, that is, the content of the target active ingredient is reduced. Some studies have also shown that when using the aqueous enzymatic method to extract oil, the oil yield is low and the quality of the oil is not good.

[0005] Therefore, there is an urgent need in this field to develop an enzymatic hydrolysis process suitable for the extraction of peony seed oil, which can maintain the good beauty effect of the product while increasing the yield of peony seed oil, and broaden the application of peony seed oil in the cosmetics field. Summary of the Invention

[0006] The technical problem to be solved by this application is to overcome the defects that when extracting peony seed oil by methods such as steam distillation method, organic solvent extraction method, adsorption extraction method and supercritical CO2 extraction method in the prior art, it is easy to cause low yield of peony seed oil, poor quality of the oil, organic solvent residues, environmental damage and high extraction cost, the aqueous enzymatic method is not yet mature, the extraction rate is low, or it is easy to damage active ingredients, etc., and to provide a crude peony seed oil, a refined peony seed oil, and their preparation methods and applications. This application enzymatically hydrolyzes peony seeds with specific types of enzymes, and then combines with ultrasonic extraction to obtain crude peony seed oil. The crude peony seed oil is purified to obtain refined peony seed oil. After refining, the acid value and peroxide value of the peony seed oil are reduced, it is not easy to deteriorate, has ideal beauty effects, and the preparation process is simple, has little environmental pollution and low cost. It expands the research of peony seed oil in the cosmetics field and meets the needs of consumers.

[0007] This application adopts the following technical solutions to solve the above technical problems:

[0008] This application provides a method for extracting crude peony seed oil, which specifically includes the following steps: mixing shelled and dried peony seeds with water, mixing with a complex enzyme under the conditions of a pH value of 4 to 5.5 and a temperature of 40 to 60 °C for enzymatic hydrolysis. After the enzymatic hydrolysis, ultrasonic extraction is carried out. The temperature of the ultrasonic extraction is 40 to 70 °C, the time of the ultrasonic extraction is 30 to 60 min, centrifugation is carried out, and the oil phase is collected to obtain the crude peony seed oil;

[0009] Among them, the mass ratio of the peony seeds to the complex enzyme is 1:(0.02 - 0.1); the complex enzyme includes cellulase, hemicellulase, acid protease and pectinase; the mass ratio of the cellulase, the hemicellulase, the acid protease and the pectinase is 1:(0.5 - 2):(0.2 - 2):(0.5 - 3).

[0010] In some embodiments, the mass ratio of the peony seeds to the complex enzyme is preferably 1:(0.03 - 0.1), more preferably 1:(0.045 - 0.1).

[0011] In some embodiments, the mass ratio of the cellulase, the hemicellulase, the acid protease and the pectinase is preferably 1:1:1:1.

[0012] In some embodiments, the drying conditions and methods can be conventional in the art and can generally be carried out in an oven. The drying temperature can be 50 to 60 °C.

[0013] In some embodiments, the peony seeds may also be pulverized according to the conventional operations in the art before use, and the conditions and methods of pulverization may be conventional in the art. The mesh number of the pulverized peony seeds may be 50-200 mesh, preferably 60-120 mesh, such as 100 mesh.

[0014] In some embodiments, the mass ratio of the peony seeds to the water may be 1:(2-15), preferably 1:(4-12), such as 1:6.

[0015] In some embodiments, when enzymatic hydrolysis is carried out, the pH value of the system is preferably 4.5.

[0016] In some embodiments, the enzymatic hydrolysis is carried out under stirring conditions, and the conditions and methods of stirring may be conventional in the art. The rotation speed of stirring may be 200-400 r / min, preferably 300-400 r / min.

[0017] In some embodiments, the time of enzymatic hydrolysis may be 45-75 min, preferably 60-75 min.

[0018] In some embodiments, the temperature of enzymatic hydrolysis is preferably 40-65 °C, such as 50 °C.

[0019] In some embodiments, the power of ultrasonic extraction may be 200-400 W, preferably 300 W.

[0020] In some embodiments, the temperature of ultrasonic extraction is preferably 60-70 °C.

[0021] In some embodiments, the ultrasonic frequency of ultrasonic extraction may be 30-50 KHz, preferably 40 KHz.

[0022] In some embodiments, the time of ultrasonic extraction is preferably 50-60 min.

[0023] In some embodiments, the conditions and methods of centrifugation may be the conventional conditions and methods for such operations in the art, and the main purpose is to separate the solid and liquid in the system.

[0024] In some embodiments, the rotation speed of centrifugation may be 4500-6000 r / min, preferably 5000 r / min.

[0025] In some embodiments, the time of centrifugation may be 25-40 min, preferably 30 min.

[0026] The present application also provides a crude peony seed oil, which is prepared by the method for extracting crude peony seed oil as described above.

[0027] The present application also provides a method for refining peony seed oil, which specifically includes the following steps: The crude peony seed oil as described above is subjected to degumming, deacidification and decolorization treatments to obtain refined peony seed oil.

[0028] In some embodiments, the conditions and methods for degumming can be the conventional conditions and methods for such operations in the art, and specifically include the following steps: Mix the crude peony seed oil with water, and degum under the conditions of a temperature of 50-70°C and stirring.

[0029] During the degumming process, the mass ratio of the crude peony seed oil to the water can be (10-30):1, preferably 20:1.

[0030] During the degumming process, the rotation speed of the stirring can be 150-250 r / min, preferably 200 r / min.

[0031] During the degumming process, the stirring time can be conventional in the art, generally until flocculent substances appear, preferably 1-2.5 h, more preferably 1.5 h.

[0032] During the degumming process, the degumming temperature is preferably 55°C.

[0033] In some embodiments, after the degumming operation, centrifugation and collection of the oil phase can be further included according to the conventional methods in the art. Among them, the rotation speed of the centrifugation can be the conventional rotation speed for such operations in the art, generally 4000-60000 r / min, preferably 4800 r / min. The centrifugation time can be 15-45 min, preferably 20 min.

[0034] In some embodiments, the conditions and methods for deacidification can be the conventional conditions and methods for such operations in the art, and specifically include the following steps: Mix the degummed peony seed oil with an aqueous solution of alkali, and deacidify under the conditions of a temperature of 50-60°C and stirring.

[0035] During the deacidification process, the aqueous solution of alkali can include an aqueous solution of sodium hydroxide commonly used in the art.

[0036] During the deacidification process, the concentration of the alkali in the aqueous solution of alkali can be conventional in the art, generally the volume concentration can be 5%-10%, preferably 8%.

[0037] During the deacidification process, the volume ratio of the degummed peony seed oil to the aqueous solution of alkali can be (200-500):1, such as 390:1.

[0038] During the deacidification process, the rotation speed of the stirring can be conventional in the art, generally 150-250 r / min, preferably 200 r / min.

[0039] During the deacidification process, the stirring time can be conventional in the art. When saponin particles appear in the system, the stirring speed is slowed down to promote colloid flocculation, and when the saponin and the oil layer are clearly separated, the stirring is stopped; the stirring time is preferably 1 to 3 h.

[0040] In some embodiments, after the deacidification operation, centrifugation and the operation of collecting the oil phase can be further included according to the convention in the art. Among them, the rotation speed of the centrifugation can be the conventional rotation speed for such operations in the art, generally 4000 to 6000 r / min, preferably 4800 r / min. The centrifugation time can be 15 to 30 min, preferably 20 min.

[0041] In some embodiments, the conditions and methods for decolorization can be the conventional conditions and methods for such operations in the art, specifically including the following steps: mixing the deacidified peony seed oil and the decolorizing agent.

[0042] During the decolorization process, the decolorizing agent can be a decolorizing agent that is conventionally used in the art and does not chemically react with peony seed oil and can adsorb colored substances and metals in the system. For example, it can include activated carbon.

[0043] During the decolorization process, the mass ratio of the deacidified peony seed oil to the decolorizing agent can be conventional in the art, generally (10 - 30):1, such as 20:1.

[0044] During the decolorization process, the mixing time can be conventional in the art, generally 30 to 60 min, preferably 45 min.

[0045] During the decolorization process, the mixing temperature can be conventional in the art, generally 40 to 60 °C, preferably 50 °C.

[0046] In some embodiments, after the decolorization operation, centrifugation and / or filtration operations can be further included. According to the convention in the art, the purpose of the centrifugation and the filtration is to remove the decolorizing agent.

[0047] Among them, the rotation speed of the centrifugation can be the conventional rotation speed for such operations in the art, generally 4000 to 6000 r / min, preferably 5000 r / min. The centrifugation time can be 15 to 30 min, preferably 20 min.

[0048] Among them, the pore size of the filter membrane for filtration can be conventional in the art, generally 0.22 to 0.8 μm, preferably 0.45 μm.

[0049] This application also provides a refined peony seed oil, which is prepared by the above-mentioned refining method of peony seed oil.

[0050] The present application also provides an application of the refined peony seed oil as described above as a raw material in the preparation of skin external agents.

[0051] Based on common knowledge in the art, the above preferred conditions can be combined arbitrarily to obtain various preferred examples of the present application.

[0052] The reagents and raw materials used in the present application are all commercially available.

[0053] The positive and progressive effects of the present application are as follows: The present application enzymatically hydrolyzes peony seeds with specific types of enzymes, and then combines with ultrasonic extraction to obtain crude peony seed oil. The crude peony seed oil is purified to obtain refined peony seed oil. After purification, the acid value and peroxide value of the peony seed oil are reduced, it is not easy to deteriorate, has ideal beauty effects, and the preparation process is simple, with little environmental pollution and low cost. It expands the research of peony seed oil in the cosmetic field and meets the needs of consumers. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] The present application can be better understood by referring to the description given below in conjunction with the accompanying drawings. The accompanying drawings, together with the following detailed description, are included in this specification and form a part of this specification, and are used to further illustrate the preferred embodiments of the present application and explain the principles and advantages of the present application.

[0055] Wherein:

[0056] Figure 1 It is a comparison chart of the acid values of the refined peony seed oil prepared in Examples 1 to 4 and Comparative Examples 1 to 5;

[0057] Figure 2 It is a comparison chart of the peroxide values of the refined peony seed oil prepared in Examples 1 to 4 and Comparative Examples 1 to 5;

[0058] Figure 3 It is a comparison chart of the cytotoxicity of the refined peony seed oil prepared in Examples 1 to 4 and Comparative Examples 1 to 5;

[0059] Figure 4 It is a comparison chart of the in vitro post - sun repair effects of the refined peony seed oil prepared in Examples 1 to 4 and Comparative Examples 1 to 5. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0060] The present application will be further illustrated below by way of examples, but the present application is not limited to the scope of the described examples. The experimental methods without specific conditions noted in the following examples are carried out according to conventional methods and conditions, or selected according to the product specifications.

[0061] The experimental methods used in the following examples are all conventional methods unless otherwise specified.

[0062] The cellulase in the following examples was purchased from Xiasheng (Beijing) Biotechnology Development Co., Ltd., and the product number was CEL-01;

[0063] The hemicellulase in the following examples was purchased from Xiasheng (Beijing) Biotechnology Development Co., Ltd., and the model was FDG-2255;

[0064] The acid protease in the following examples was purchased from Xiasheng (Beijing) Biotechnology Development Co., Ltd., and the model was FDG-2237;

[0065] The pectinase in the following examples was purchased from Xiasheng (Beijing) Biotechnology Development Co., Ltd., and the model was FFG-0658.

[0066] Example 1

[0067] (1) Shell, dry, crush and sieve Heze peony seeds. Preferably, the peony seeds with a particle size of 100 mesh are taken. 100 g of peony seeds are mixed evenly with 600 g of deionized water, the pH value of the system is adjusted to 4.5, and after heating to 50 °C, 4.5 g of a compound enzyme (the mass ratio of cellulase, hemicellulase, acid protease, and pectinase is 1:1:1:1) is added, and enzymatic hydrolysis is carried out under stirring conditions. The stirring speed is 300 r / min, the enzymatic hydrolysis time is 60 min. After the enzymatic hydrolysis is completed, ultrasonic extraction is carried out. The temperature of ultrasonic extraction is 60 °C, the power of ultrasonic extraction is 300 W, the frequency of ultrasonic extraction is 40 KHz, and the time of ultrasonic extraction is 50 min; after the ultrasonic extraction is completed, centrifugation is carried out, and centrifugation is carried out at 5000 r / min for 30 min, and the oil phase is collected to obtain crude peony seed oil;

[0068] (2) The crude peony seed oil is prepared batch by batch according to the above method. 200 g of crude peony seed oil is taken for degumming treatment. The crude peony seed oil is heated to 55 °C, 10 g of deionized water is added and stirred. The stirring speed is 200 r / min. Flocculants appear after stirring for 1.5 h. Stirring is stopped, and centrifugation is carried out. The centrifugation speed is 4800 r / min, and the centrifugation time is 15 min. After centrifugation, the oil phase is collected to obtain 195 mL of degummed peony seed oil;

[0069] (3) The degummed peony seed oil is heated to 50 °C, 0.5 mL of an aqueous sodium hydroxide solution with a volume percentage of 8% is added and stirred. The stirring speed is 200 r / min, and the stirring time is 1 h. After stirring is completed, centrifugation is carried out at 4800 r / min for 20 min. After centrifugation, the oil phase is collected to obtain 192 g of deacidified peony seed oil;

[0070] (4) Add 9.6 g of activated carbon to the deacidified peony seed oil, heat and stir for decolorization. The stirring speed is 200 r / min, the decolorization time is 45 min, and the decolorization temperature is 50 °C. After decolorization, centrifuge to remove the activated carbon. The centrifugation speed is 5000 r / min, and the centrifugation time is 30 min. After centrifugation, perform suction filtration. The pore size of the filter membrane during suction filtration is 0.45 μm. After suction filtration, refined peony seed oil is obtained.

[0071] Example 2

[0072] Compared with Example 1, the only difference is that the addition amount of the complex enzyme in step (1) is 10 g, and other condition parameters are the same as those in Example 1, and refined peony seed oil is obtained.

[0073] Example 3

[0074] Compared with Example 1, the only difference is that the addition amount of the complex enzyme in step (1) is 3 g, and other condition parameters are the same as those in Example 1, and refined peony seed oil is obtained.

[0075] Example 4

[0076] Compared with Example 1, the only difference is that the addition ratios of the four enzymes in the complex enzyme added in step (1) are different. The mass ratio of cellulase, hemicellulase, acid protease, and pectinase is 1:2:1:1, and other condition parameters are the same as those in Example 1, and refined peony seed oil is obtained.

[0077] Comparative Example 1

[0078] Compared with Example 1, the only difference is that in step (1), ultrasonic extraction treatment is not performed, and other condition parameters are the same as those in Example 1, and refined peony seed oil is obtained.

[0079] Comparative Example 2

[0080] Compared with Example 1, the only difference is that the complex enzyme added in step (1) is different. Specifically, it is a mixed enzyme of cellulase, hemicellulase, and acid protease; the addition amount of the complex enzyme is 4.5 g; among them, the mass ratio of cellulase, hemicellulase, and acid protease is 3:1:1, and other condition parameters are the same as those in Example 1, and refined peony seed oil is obtained.

[0081] Comparative Example 3

[0082] Compared with Example 1, the only difference is that the complex enzyme added in step (1) is different. Specifically, it is a mixed enzyme of cellulase, hemicellulase, and pectinase; the addition amount of the complex enzyme is 4.5 g; among them, the mass ratio of cellulase, hemicellulase, and pectinase is 1:3:1, and other condition parameters are the same as those in Example 1, and refined peony seed oil is obtained.

[0083] Comparative Example 4

[0084] Compared with Example 1, the difference is only that in step (1), only 4.5 g of cellulase is added, and other condition parameters are the same as those in Example 1, and refined peony seed oil is obtained.

[0085] Comparative Example 5

[0086] Compared with Example 1, the difference is only that the conditions for ultrasonic extraction in step (1) are different. The ultrasonic extraction time is 15 min, and the ultrasonic extraction temperature is 30 °C. Other condition parameters are the same as those in Example 1, and refined peony seed oil is obtained.

[0087] Determination of acid value in Effect Example 1

[0088] The acid value is one of the indicators to measure whether the quality of oil is excellent and whether it is fresh. The lower the acid value, the more suitable it is to be used as the base oil for cosmetics. Refer to GB5009.229-2016 to test the acid value (calculated as KOH) of the refined peony seed oil prepared in the above examples and comparative examples. The results are shown in Table 1 and Figure 1 ( Figure 1 Among them, p>0.05, ns indicates no statistical difference compared with Example 1; **p<0.01, indicating a statistical difference compared with Example 1; ***p<0.001, indicating an extremely significant statistical difference compared with Example 1).

[0089] Table 1

[0090] Number Acid value (calculated as KOH) (mg / g) Example 1 0.34±0.02 Example 2 0.37±0.01 Example 3 0.44±0.02 Example 4 0.45±0.03 Comparative Example 1 0.88±0.02 Comparative Example 2 1.18±0.04 Comparative Example 3 1.03±0.03 Comparative Example 4 0.97±0.03 Comparative Example 5 1.51±0.04

[0091] Effect Example 2: Determination of peroxide value

[0092] The peroxide value is the reaction product generated during the oxidation process of oil by itself in a certain environment. The peroxide value represents the freshness and refining degree of oil. The lower the peroxide value, the fresher the oil and the more ideal the refining degree. Refer to GB / T 5009.227-2016 to test the peroxide value of the refined peony seed oil prepared in the above examples and comparative examples. The results are shown in Table 2 and Figure 2 ( Figure 2 Among them, p>0.05, ns indicates no statistical difference compared with Example 1; *p<0.05, indicating a statistical difference compared with Example 1, ***p<0.001, indicating an extremely significant statistical difference compared with Example 1).

[0093] Table 2

[0094] Number Peroxide value (mmol / kg) Example 1 0.22±0.03 Example 2 0.27±0.02 Example 3 0.29±0.01 Example 4 0.3±0.02 Comparative Example 1 0.81±0.05 Comparative Example 2 0.64±0.03 Comparative Example 3 0.87±0.04 Comparative Example 4 0.75±0.03 Comparative Example 5 1.01±0.04

[0095] Effect Example 3: Promote cell proliferation

[0096] In this experiment, human skin fibroblasts from the Cell Bank of the Chinese Academy of Sciences were used to verify the cytotoxicity of the products prepared in the above-mentioned examples and comparative examples.

[0097] Reagents: The manufacturer of 0.25% (containing EDTA) trypsin is GIBCO, USA; the manufacturer of DMEM medium is GIBCO, USA; the manufacturer of double antibiotics is Corning, USA; the manufacturer of CCK-8 is Beijing Baierdi Biotechnology Co., Ltd.; the manufacturer of fetal bovine serum is GIBCO, USA; the manufacturer of phosphate buffer solution is Beijing Bairuiji Biotechnology Co., Ltd.

[0098] Equipment: The manufacturer of the WJ-80A-II type CO2 constant temperature incubator is Shanghai Shengke Instrument and Equipment Co., Ltd.; the manufacturer of the Sunrise microplate reader is Tecan Trading Co., Ltd.; the manufacturer of the TL80-2 type medical centrifuge is Jiangsu Tianli Medical Instrument Co., Ltd.; the manufacturer of the NUNC 96-well cell culture plate is Thermo Fisher Scientific.

[0099] 1. Experimental steps:

[0100] The products prepared in the above-mentioned examples and comparative examples were respectively diluted to a volume fraction of 10% with DMSO, and then configured into a test solution of the experimental group with a volume percentage of 0.1% using serum-free DMEM medium.

[0101] Human skin fibroblasts were cultured in DMEM medium containing 10% fetal bovine serum and 1% double antibiotics (1×10 5 U / L penicillin, 100 mg / L streptomycin). The cells grew in an incubator at 37°C and 5% CO2 saturated humidity. When the cell confluence reached more than 85%, the cells in the logarithmic growth phase were digested with 0.05% trypsin, and the digestion reaction was terminated with serum-containing DMEM. The cells were counted using a cell counting plate, and the cell suspension concentration was adjusted to 7×10 4 cells / mL. The cell suspension was inoculated into a 96-well plate at a ratio of 100 μL per well and incubated in an incubator at 37°C and 5% CO2 for 12 h. After incubation, the old culture medium was removed, and the cells were washed twice with phosphate buffer solution.

[0102] In the experimental group, 100 μL of the filtered and sterilized test solution of the experimental group with different concentrations prepared above was added to each well, and 6 replicates were made for each test solution; the control group contained cells and serum-free DMEM medium was added; the blank control group had no cells and 100 μL of PBS was added. Then it was incubated in an incubator at 37°C and 5% CO2 for 24 h. Then 10 μL of CCK-8 solution was added to each well, and it was incubated for another 3 h. The absorbance value was measured at a wavelength of 450 nm, and the cell viability of each group was calculated. The results are shown in Table 3 and Figure 3 ( Figure 3Among them, p > 0.05, ns indicates no statistical difference compared with Example 1; *p < 0.05 indicates a statistical difference compared with Example 1; ***p < 0.001 indicates an extremely significant statistical difference compared with Example 1).

[0103] The calculation formula for cell viability is as follows:

[0104] Cell viability (%) = (A experimental group - A blank control group) / (A control group - A blank control group) × 100%.

[0105] Table 3

[0106] Number Cell viability Example 1 115.76±2.82％ Example 2 112.31±3.54％ Example 3 105.53±2.85％ Example 4 104.82±3.12％ Comparative Example 1 93.94±1.58％ Comparative Example 2 95.88±1.41％ Comparative Example 3 90.3±2.00％ Comparative Example 4 92.71±1.24％ Comparative Example 5 90.7±2.85％

[0107] The results show that the refined peony seed oil prepared in Examples 1 to 4 has a growth-promoting effect on human skin fibroblasts, while the refined peony seed oil prepared in Comparative Examples 1 to 5 is slightly toxic to human skin fibroblasts.

[0108] Effect of Example 4 on post-sun repair in vitro

[0109] 1. Experimental steps:

[0110] The products prepared in the above-mentioned examples and comparative examples were respectively diluted with DMSO to a volume fraction of 10%, and then configured into a test solution of the experimental group with a volume percentage of 0.05% using serum-free DMEM medium.

[0111] HaCaT cells were cultured in DMEM medium containing 10% fetal bovine serum and 1% double antibody (1×10 5 U / L penicillin, 100 mg / L streptomycin). The cells grew in an incubator at 37°C and 5% CO2 saturated humidity. When the cell confluence reached more than 85%, the cells in the logarithmic growth phase were digested with 0.05% trypsin, and the digestion reaction was terminated with serum-containing DMEM. The cells were counted with a cell counting plate, and the cell suspension concentration was adjusted to 7×10 4 cells / mL. The cell suspension was inoculated into a 96-well plate at a ratio of 100 μL per well and incubated in an incubator at 37°C and 5% CO2 for 12 h. After incubation, the old culture medium was removed, the cells were washed twice with phosphate buffer solution, and then 100 μL of PBS solution was added. The model group and the experimental group were irradiated with UVB, and the irradiation dose was 15 mJ / cm 2, the negative control group was not irradiated; the PBS was aspirated and discarded, the model group and the negative control group were added with serum-free DMEM medium, and 100 μL of the filtered and sterilized experimental solution to be tested prepared above was added to each well in the experimental group. Six replicate wells were made for each test solution; the blank control group had no cells and 100 μL of PBS was added. Then it was incubated in an incubator at 37 °C and 5% CO2 for 24 h. Then 10 μL of CCK-8 solution was added to each well, and it was incubated for another 3 h. The absorbance value was measured at a wavelength of 450 nm, and the cell survival rate of each group was calculated. The results are shown in Table 4 and Figure 4 ( Figure 4 Among them, p > 0.05, ns indicates no statistical difference compared with the model group; *p < 0.05, indicating a statistical difference compared with the model group; ***p < 0.001, indicating an extremely significant statistical difference compared with the model group).

[0112] The calculation formula for the cell survival rate is as follows:

[0113] Cell survival rate (%) = (Aexperimental group or model group - Ablank control group) / (Anegative control group - Ablank control group) × 100%.

[0114] Table 4

[0115]

[0116]

[0117] The results show that the refined peony seed oil prepared in Examples 1 to 4 has an ideal repair effect on damaged cells, and the repair effect is significantly better than that of the refined peony seed oil prepared in Comparative Examples 1 to 5.

[0118] Effect Example 5

[0119] Calculate the yield of crude peony seed oil in the above examples and comparative examples. The results are shown in Table 5. The yield of crude peony seed oil = (crude peony seed oil / mass of peony seed powder) × 100%.

[0120] Table 5

[0121] Number Yield of crude peony seed oil Example 1 25.62％ Example 2 23.13％ Example 3 23.01％ Example 4 23.74％ Comparative Example 1 17.35％ Comparative Example 2 15.22％ Comparative Example 3 15.64％ Comparative Example 4 16.81％ Comparative Example 5 14.7％

[0122] Finally, it should be noted that in this application, the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements not only includes those elements, but also includes other elements not expressly listed, or also includes elements inherent to such process, method, article or device.

[0123] Although the present application has been disclosed above through the description of specific embodiments of the present application, it should be understood that those skilled in the art can design various modifications, improvements or equivalents to the present application within the spirit and scope of the appended solutions. These modifications, improvements or equivalents should also be considered to be included within the scope claimed by the present application.

Claims

1. A method for extracting crude peony seed oil, characterized in that, It includes the following steps: Mix the shelled and dried peony seeds with water, mix with a complex enzyme under the conditions of a pH value of 4 - 5.5 and a temperature of 40 - 60 °C, and carry out enzymatic hydrolysis. The time of the enzymatic hydrolysis is 45 - 75 min. After the enzymatic hydrolysis, carry out ultrasonic extraction. The temperature of the ultrasonic extraction is 60 - 70 °C, and the time of the ultrasonic extraction is 30 - 60 min. Then centrifuge and collect the oil phase to obtain the crude peony seed oil. Among them, the mass ratio of the peony seeds to the complex enzyme is 1:(0.02 - 0.1); the complex enzyme includes cellulase, hemicellulase, acid protease and pectinase; the mass ratio of the cellulase, the hemicellulase, the acid protease and the pectinase is 1:(0.5 - 2):(0.2 - 2):(0.5 - 3).

2. The extraction method of crude peony seed oil according to claim 1, characterized in that, The extraction method satisfies at least one of the following conditions: The mass ratio of the peony seeds to the complex enzyme is 1:(0.03 - 0.1); The mass ratio of the cellulase, the hemicellulase, the acid protease and the pectinase is 1:1:1:1; The temperature of the drying is 50 - 60 °C; Before use, the peony seeds also include a crushing operation, and the mesh number of the crushed peony seeds is 50 - 200 meshes; The mass ratio of the peony seeds to the water is 1:(2 - 15).

3. The extraction method of crude peony seed oil according to claim 2, characterized in that, The extraction method satisfies at least one of the following conditions: The mass ratio of the peony seeds to the complex enzyme is 1:(0.045 - 0.1); The mesh number of the crushed peony seeds is 60 - 120 meshes; The mass ratio of the peony seeds to the water is 1:(4 - 12).

4. The extraction method of crude peony seed oil according to claim 1 or 2 or 3, characterized in that, The extraction method satisfies at least one of the following conditions: During the enzymatic hydrolysis, the pH value of the system is 4.5; The enzymatic hydrolysis is carried out under stirring conditions, and the stirring speed is 200 - 400 r / min; The time of the enzymatic hydrolysis is 60 - 75 min; The temperature of the enzymatic hydrolysis is 40 - 65 °C; The power of the ultrasonic extraction is 200 - 400 W; The ultrasonic frequency of the ultrasonic extraction is 30 - 50 KHz; The time of the ultrasonic extraction is 50 - 60 min; The rotation speed of the centrifuge is 4500 - 6000 r / min; The time of the centrifuge is 25 - 40 min.

5. The extraction method of crude peony seed oil according to claim 4, characterized in that, The extraction method satisfies at least one of the following conditions: The enzymatic hydrolysis is carried out under stirring conditions, and the stirring speed is 300 - 400 r / min; The power of the ultrasonic extraction is 300 W; The ultrasonic frequency of the ultrasonic extraction is 40 KHz; The rotation speed of the centrifuge is 5000 r / min; The time of the centrifuge is 30 min.

6. A crude peony seed oil, characterized in that, Obtained by the extraction method of the crude peony seed oil according to any one of claims 1 - 5.

7. A refining method of peony seed oil, characterized in that, It includes the following steps: The crude peony seed oil according to claim 6 is subjected to degumming, deacidification and decolorization treatments to obtain refined peony seed oil.

8. The refining method of peony seed oil according to claim 7, characterized in that, The degumming includes the following steps: Mix the crude peony seed oil with water and carry out degumming under the conditions of a temperature of 50 - 70 °C and stirring.

9. The refining method of the peony seed oil according to claim 8, characterized in that During the degumming process, the mass ratio of the crude peony seed oil to the water is (10 - 30):1; During the degumming process, the rotation speed of the stirring is 150 - 250 r / min; During the degumming process, the stirring time is 1 - 2.5 h; During the degumming process, the degumming temperature is 55 °C; After the degumming operation, it further includes centrifugation and the operation of collecting the oil phase.

10. The refining method of peony seed oil according to claim 9, wherein During the degumming process, the mass ratio of the crude peony seed oil to the water is 20:1; The rotation speed of the centrifugation is 4000 - 60000 r / min; the centrifugation time is 15 - 45 min.

11. The refining method of peony seed oil according to claim 7, characterized in that, The deacidification includes the following steps: mixing the degummed peony seed oil with an aqueous solution of alkali, and performing deacidification under the conditions of a temperature of 50 - 60 °C and stirring.

12. The refining method of peony seed oil according to claim 11, wherein During the deacidification process, the aqueous solution of alkali includes an aqueous solution of sodium hydroxide; During the deacidification process, the volume concentration of the alkali in the aqueous solution of alkali is 5% - 10%; During the deacidification process, the volume ratio of the degummed peony seed oil to the aqueous solution of alkali is (200 - 500):1; During the deacidification process, the rotation speed of the stirring is 150 - 250 r / min; During the deacidification process, the stirring time is 1 - 3 h; After the deacidification operation, it further includes centrifugation and the operation of collecting the oil phase.

13. The refining method of peony seed oil according to claim 12, characterized in that, The rotation speed of the centrifugation is 4000 - 6000 r / min; the centrifugation time is 15 - 30 min.

14. The refining method of peony seed oil according to claim 7, characterized in that, The decolorization includes the following steps: mixing the deacidified peony seed oil with a decolorizing agent.

15. The refining method of peony seed oil according to claim 14, wherein During the decolorization process, the decolorizing agent includes activated carbon; During the decolorization process, the mass ratio of the deacidified peony seed oil to the decolorizing agent is (10 - 30):1; During the decolorization process, the mixing time is 30 - 60 min; During the decolorization process, the mixing temperature is 40 - 60 °C; After the decolorization operation, it further includes centrifugation and / or filtration operations.

16. The refining method of peony seed oil according to claim 15, characterized in that, The rotation speed of the centrifugation is 4000 - 6000 r / min; the centrifugation time is 15 - 30 min; the pore size of the filtration membrane for filtration is 0.22 - 0.8 μm.

17. A refined peony seed oil, characterized in that, Prepared by the refining method of peony seed oil according to any one of claims 7 - 16.

18. Application of a refined peony seed oil as claimed in claim 17 as a raw material in the preparation of a skin external preparation.

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