Preparation method of antioxidant repair rosemary extract as well as product and application of antioxidant repair rosemary extract

Abstract

The invention belongs to the technical field of natural plant extracts, and particularly relates to a preparation method, a product and application of an antioxidant repair rosemary extract. According to the technical scheme, snail protease and bromelain are added during rosemary leaf pretreatment, so that cell walls of rosemary leaves are fully destroyed, and release of intracellular active ingredients can be effectively promoted. According to the technical scheme, an extraction solvent A and an extraction solvent B are used, the extraction solvent A is mainly used for extracting flavonoid substances such as rutin, hesperetin and thistle flavin in the rosemary, and the extraction solvent B is mainly used for extracting terpene components such as rosmarinic acid, carnosic acid and rosmarinol in the rosemary. According to the technical scheme, the rosemary extract is obtained by mixing and extracting the extract A and the extract B, and it is found through experimental verification that the extract has remarkable effects in the aspects of oxidation resistance, soothing and skin repairing.

Description

Technical Field

[0001] The invention belongs to the technical field of natural plant extracts, and in particular relates to a preparation method of an antioxidant and repairing rosemary extract and a product and application thereof. Background Art

[0002] For a long time, people have been using synthetic antioxidants for anti-oxidation and preservation of food and oils. Studies have shown that traditional synthetic antioxidants such as butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) may be potentially toxic and harmful to the human body. In addition, synthetic antioxidants are easily lost at high temperatures. Therefore, natural antioxidants with high safety and strong antioxidant capacity are increasingly valued.

[0003] Rosemary is a herbaceous plant of the Lamiaceae family. It is a plant with high antioxidant effect. In my country, the research on rosemary started relatively late. Through research, it was found that rosemary contains carnosic acid, carnosol, rosmarinic acid, rosmarinic phenol, vanillic acid, caffeic acid, homophyllium, cyperine, naringin, chlorogenic acid, rutin, quercetin, kaempferol, epirosmarin, rosmarin, rosmarinic dialdehyde, luteolin and its derivatives, methoxyflavonoids, hesperidin, isohesperidin and other effective compounds.

[0004] At present, the main extraction methods of rosemary are reflux method, ultrasound-assisted extraction method and supercritical extraction method. However, the current research on the extraction of rosemary often focuses on the extraction of a certain active ingredient. For example, carnosic acid, carnosol, rosmarinic acid and rosmarinic phenol, but in addition to the above ingredients, there are many other phenolic components in rosemary extract, and there is a synergistic antioxidant effect between the components. Therefore, it is limited to simply judge the quality of antioxidants based on the content of a certain component. Further comprehensive research on the active chemical components and physiological functions of rosemary is needed. Summary of the invention

[0005] The present invention aims to study the influence of the extraction method on the content of beneficial substances and the antioxidant effect of rosemary extract, and to find an efficient and rapid rosemary extraction method with better antioxidant and repairing effect of the extract.

[0006] The technical solution of the present invention is specifically as follows:

[0007] A method for preparing an antioxidant and repairing rosemary extract comprises the following steps:

[0008] (1) grinding dried rosemary leaves into powder with an average particle size of 80-100 mesh, adding water to the rosemary powder to make the moisture content of the rosemary reach 20-30%, then adding snail protease and bromelain, and performing enzymolysis pretreatment at a temperature of 30-40° C. for 1-2 h;

[0009] (2) adding water to the rosemary powder after enzymatic pretreatment to make the water content reach 150-180%, soaking at room temperature for 8-10 hours, and using steam distillation to obtain rosemary essential oil-removed raw material and rosemary essential oil primary extract;

[0010] (3) adding a raw material of rosemary de-essential oil to a reaction container, adding an extraction solvent A for soaking and stirring, wherein the extraction solvent A is an ethanol-water system solution, refluxing and leaching for 20-30 min at a temperature of 60-90° C. to obtain an extract, filtering and centrifuging the extract to obtain an extract A, and the residue in the reaction container is reserved for later use;

[0011] (4) adding the residue of step (3) to the extraction solvent B, wherein the extraction solvent B is an ethyl acetate and acetone system solution, extracting for 20 to 80 minutes at an ultrasonic power of 100 to 400 W and a temperature of 40 to 50° C., centrifuging at a speed of 3000 to 5000 rpm after the extraction is completed, pouring the supernatant extract into a container, adding the extraction solvent B again to repeat the extraction, the extraction and separation times are 1 to 3 times, and filtering all the supernatant extracts to obtain an aqueous solution, and the other extracted components are fat-soluble precipitates;

[0012] (5) adjusting the pH value of the aqueous solution to neutral, and then vacuum concentrating to an extract with a liquid-to-solid ratio of 1:1.5-2 (v / w); subjecting the extract to supercritical carbon dioxide extraction and separation, placing the extract in a supercritical carbon dioxide extraction kettle, and then adding a carrier co-solvent, the separation kettle pressure is 8-10MPa, the temperature is 40-45°C, the extraction time is 1.3-1.5h, the CO2 flow rate is: 30-50L / h, to obtain extract B, and extracts A and B are combined to obtain rosemary extract.

[0013] Preferably, the amount of snail protease in step (1) is 350-400 U / g dry rosemary leaves, and the amount of bromelain is 450-600 U / g dry rosemary leaves.

[0014] Preferably, the ethanol mass fraction of the ethanol-water system solution of the extraction solvent A in step (3) is 40-70%, and the volume ratio of the rosemary essential oil removal raw material to the extraction solvent A is 1:5-10.

[0015] Preferably, in step (4), the volume ratio of ethyl acetate to acetone in the extraction solvent B is 20-30:25-20, and the volume ratio of the residue in the container to the extraction solvent B is 1:2-3.

[0016] Preferably, in step (5), calcium hydroxide is used to adjust the pH value of the solution to neutral.

[0017] Preferably, the carrier co-solvent in step (5) is one or both of anhydrous ethanol and food-grade n-hexane.

[0018] Preferably, the mass ratio of the extract to the carrier solvent in step (5) is 1:1-2.

[0019] Preferably, the primary extract of rosemary essential oil obtained in step (1) is further subjected to distillation separation to obtain rosemary essential oil and crude rosemary hydrosol.

[0020] The rosemary extract is prepared according to the method.

[0021] The rosemary extract is used as an antioxidant.

[0022] The beneficial effects of the present invention are:

[0023] 1. The technical solution used in the present invention is to add snail protease and bromelain during rosemary leaf pretreatment to fully destroy the cell wall of rosemary leaves, which can effectively promote the release of active ingredients in cells, and experimentally verify that the synergistic effect of the mixed addition of snail protease and bromelain is greater than that of using snail protease alone or bromelain alone.

[0024] 2. This technical solution uses extraction solvent A and extraction solvent B. Extraction solvent A is mainly used to extract flavonoids in rosemary, such as rutin, hesperidin, cyperin, etc. Extraction solvent B is mainly used to extract terpenoid components such as rosmarinic acid and rosmarinic phenol in rosemary, and the use of ethyl acetate and acetone in extraction solvent B can significantly improve the extraction rate of rosmarinic acid, carnosic acid, and rosmarinic phenol in rosemary. Rosemary extract is obtained by mixing extracts A and B. Experimental verification shows that the extract has significant effects in anti-oxidation, soothing, and repairing skin, and the effect is significantly better than single extraction components of rosmarinic acid or rosmarinic phenol. DETAILED DESCRIPTION

[0025] The present application is further described in detail below in conjunction with comparative examples and embodiments.

[0026] Example 1

[0027] A method for preparing an antioxidant repair rosemary extract, characterized in that it comprises the following steps:

[0028] (1) 2 kg of dried rosemary leaves were crushed into powder with an average particle size of 100 meshes, water was added to the rosemary powder to make the moisture content of the rosemary reach 30%, and then snail protease and bromelain were added, and enzymolysis pretreatment was performed at a temperature of 40° C. for 2 h, wherein the amount of the snail protease was 380 U / g of dried rosemary leaves, and the amount of the bromelain was 500 U / g of dried rosemary leaves.

[0029] (2) adding water to the rosemary powder after the enzymatic pretreatment in step (1) to make the water content reach 150%, soaking at room temperature for 8 hours, and using steam distillation to obtain a rosemary essential oil-free raw material and a rosemary essential oil primary extract;

[0030] (3) adding a raw material for removing essential oil from rosemary to a reaction container, and then adding an extraction solvent A for soaking and stirring, wherein the extraction solvent A is an ethanol-water system solution, refluxing and leaching for 25 minutes at a temperature of 82° C. to obtain an extract, filtering and centrifuging the extract to obtain an extract A, and the residue in the reaction container is reserved; the mass fraction of ethanol in the ethanol-water system solution of the extraction solvent A is 60%, and the volume ratio of the raw material for removing essential oil from rosemary to the extraction solvent A is 1:6;

[0031] (4) The residue of step (3) is further added with extraction solvent B, wherein the extraction solvent B is an ethyl acetate and acetone system solution, and extracted for 60 minutes at an ultrasonic power of 300 W and a temperature of 50° C. After the extraction is completed, the mixture is centrifuged at a speed of 5000 rpm, the supernatant extract is poured into a container, and the extraction solvent B is added again for repeated extraction. The extraction and separation times are 3 times, and all the supernatant extracts are filtered to obtain an aqueous solution. The other extracted components are fat-soluble precipitates; the volume ratio of ethyl acetate to acetone in the extraction solvent B is 25:34, and the volume of the residue in the container and the extraction solvent B is 1:2.

[0032] (5) The pH value of the aqueous solution is adjusted to neutral with calcium hydroxide, and then vacuum concentrated to an extract with a liquid-to-solid ratio of 1g:1.8mL; the extract is subjected to supercritical carbon dioxide extraction and separation, and the extract is put into a supercritical carbon dioxide extraction kettle, and the carrier solvent anhydrous ethanol is added according to the mass ratio of the extract to the carrier solvent of 1:1. The separation kettle pressure is 8MPa, the temperature is 45°C, the extraction time is 1.3h, and the CO2 flow rate is 30L / h to obtain extract B, and extracts A and B are combined to obtain rosemary extract.

[0033] Example 2

[0034] A method for preparing an antioxidant repair rosemary extract, characterized in that it comprises the following steps:

[0035] (1) 2 kg of dried rosemary leaves were crushed into powder with an average particle size of 80 meshes, water was added to the rosemary powder to make the moisture content of the rosemary reach 25%, and then snail protease and bromelain were added, and enzymolysis pretreatment was performed at 30° C. for 1 hour, wherein the amount of the snail protease was 350 U / g of dried rosemary leaves, and the amount of the bromelain was 450 U / g of dried rosemary leaves.

[0036] (2) adding water to the rosemary powder after the enzymatic pretreatment in step (1) to make the water content reach 160%, soaking at room temperature for 8 hours, and using steam distillation to obtain a rosemary essential oil-free raw material and a rosemary essential oil primary extract;

[0037] (3) adding a raw material for removing essential oil from rosemary to a reaction container, and then adding an extraction solvent A for soaking and stirring, wherein the extraction solvent A is an ethanol-water system solution, refluxing and leaching for 25 minutes at a temperature of 82° C. to obtain an extract, filtering and centrifuging the extract to obtain an extract A, and the residue in the reaction container is reserved; the mass fraction of ethanol in the ethanol-water system solution of the extraction solvent A is 40%, and the volume ratio of the raw material for removing essential oil from rosemary to the extraction solvent A is 1:8;

[0038] (4) The residue of step (3) is further added with extraction solvent B, wherein the extraction solvent B is an ethyl acetate and acetone system solution, and extracted for 40 minutes at an ultrasonic power of 400 W and a temperature of 40° C. After the extraction is completed, the mixture is centrifuged at a speed of 3000 rpm, the supernatant extract is poured into a container, and the extraction solvent B is added again for repeated extraction. The extraction and separation times are 2 times, and all the supernatant extracts are filtered to obtain an aqueous solution. The other extracted components are fat-soluble precipitates; the volume ratio of ethyl acetate to acetone in the extraction solvent B is 5:8, and the volume ratio of the residue in the container to the extraction solvent B is 1:2.

[0039] (5) The pH value of the aqueous solution is adjusted to neutral with calcium hydroxide, and then vacuum concentrated to an extract with a liquid-to-solid ratio of 1g:2mL; the extract is subjected to supercritical carbon dioxide extraction and separation, and the extract is put into a supercritical carbon dioxide extraction kettle, and the carrier solvent anhydrous ethanol is added according to the mass ratio of the extract to the carrier solvent of 1:2. The separation kettle pressure is 10MPa, the temperature is 40°C, the extraction time is 1.3h, and the CO2 flow rate is 30L / h to obtain extract B, and extracts A and B are combined to obtain rosemary extract.

[0040] Example 3

[0041] A method for preparing an antioxidant repair rosemary extract, characterized in that it comprises the following steps:

[0042] (1) 2 kg of dried rosemary leaves were crushed into powder with an average particle size of 90 meshes, water was added to the rosemary powder to make the moisture content of the rosemary reach 25%, and then snail protease and bromelain were added, and enzymolysis pretreatment was performed at 35° C. for 1.5 h, wherein the amount of the snail protease was 380 U / g of dried rosemary leaves, and the amount of the bromelain was 500 U / g of dried rosemary leaves.

[0043] (2) adding water to the rosemary powder after the enzymatic pretreatment in step (1) to make the water content reach 170%, soaking at room temperature for 8 hours, and using steam distillation to obtain a rosemary essential oil-free raw material and a rosemary essential oil primary extract;

[0044] (3) adding a raw material for removing essential oil from rosemary to a reaction container, and then adding an extraction solvent A for soaking and stirring, wherein the extraction solvent A is an ethanol-water system solution, refluxing and leaching for 30 minutes at a temperature of 70° C. to obtain an extract, filtering and centrifuging the extract to obtain an extract A, and the residue in the reaction container is reserved; the mass fraction of ethanol in the ethanol-water system solution of the extraction solvent A is 40%, and the volume ratio of the raw material for removing essential oil from rosemary to the extraction solvent A is 1:8;

[0045] (4) The residue of step (3) is further added with extraction solvent B, wherein the extraction solvent B is an ethyl acetate and acetone system solution, and extracted for 20 minutes at an ultrasonic power of 100 W and a temperature of 40° C. After the extraction is completed, the mixture is centrifuged at a speed of 5000 rpm, the supernatant extract is poured into a container, and the extraction solvent B is added again for repeated extraction. The extraction and separation times are 1 time, and all the supernatant extracts are filtered to obtain an aqueous solution. The other extracted components are fat-soluble precipitates; the volume ratio of ethyl acetate to acetone in the extraction solvent B is 20:29, and the volume of the residue in the container and the extraction solvent B is 1:2.

[0046] (5) The pH value of the aqueous solution is adjusted to neutral with calcium hydroxide, and then vacuum concentrated to an extract with a liquid-to-solid ratio of 1g:1.5mL; the extract is subjected to supercritical carbon dioxide extraction and separation, and the extract is put into a supercritical carbon dioxide extraction kettle, and a carrier solvent food grade n-hexane is added according to a mass ratio of extract to carrier solvent of 1:1. The separation kettle pressure is 8MPa, the temperature is 40°C, the extraction time is 1.4h, and the CO2 flow rate is 30L / h to obtain extract B, and extracts A and B are combined to obtain rosemary extract.

[0047] Comparative Example 1

[0048] The process technology of this embodiment is different from that of embodiment 1 in that: this embodiment does not have an enzymatic pretreatment step: step (1) is: 2 kg of dried rosemary leaves are crushed into powder with an average particle size of 100 mesh, water is added to make the water content reach 150%, soaked at room temperature for 8 hours, and steam distilled to obtain rosemary essential oil-free raw material and rosemary essential oil primary extract. The other process steps are consistent with embodiment 1.

[0049] Comparative Example 2

[0050] The process technology of this embodiment is different from that of embodiment 2 in that: this embodiment does not have an enzymatic pretreatment step, and step (1) is: 2 kg of dried rosemary leaves are crushed into powder with an average particle size of 80 mesh, water is added to the rosemary powder to make the moisture content of the rosemary reach 160%, and the mixture is soaked at room temperature for 8 hours, and steam distillation is used to obtain a rosemary essential oil-free raw material and a rosemary essential oil primary extract. The other process steps are consistent with those of embodiment 2.

[0051] Comparative Example 3

[0052] The process technology of this embodiment is different from that of embodiment 1 in that: this embodiment does not have step (3), and the rosemary de-essential oil raw material is directly added to the extraction solvent B, the extraction solvent B is an ethyl acetate and acetone system solution, and extracted for 60 minutes at an ultrasonic power of 300W and a temperature of 50°C. After the extraction is completed, the supernatant extract is centrifuged at a speed of 5000 rpm, the supernatant extract is poured into a container, and the extraction solvent B is added again for repeated extraction, and the number of extraction and separation is 3 times, all the supernatant extracts are filtered to obtain an aqueous solution, and the other extracted components are fat-soluble precipitates; the volume ratio of ethyl acetate to acetone in the extraction solvent B is 25:34, and the volume of the rosemary de-essential oil raw material and the extraction solvent B in the container is 1:2. The other process steps are consistent with those of embodiment 1 (no extraction liquid is combined).

[0053] Comparative Example 4

[0054] The process technology difference between this embodiment and embodiment 2 is that: this embodiment does not have step (3), and the rosemary de-essential oil raw material is directly added to the extraction solvent B, and the extraction solvent B is an ethyl acetate and acetone system solution. The extraction is carried out at an ultrasonic power of 400W and a temperature of 40°C for 40 minutes. After the extraction is completed, the supernatant extract is centrifuged at a speed of 3000 rpm, and the supernatant extract is poured into a container, and the extraction solvent B is added again for repeated extraction. The number of extraction and separation is 2 times, and all the supernatant extracts are filtered to obtain an aqueous solution. The other extracted components are fat-soluble precipitates; the volume ratio of ethyl acetate to acetone in the extraction solvent B is 5:8, and the volume of the rosemary de-essential oil raw material and the extraction solvent B in the container is 1:2. The other process steps are consistent with those of embodiment 1 (no extract is combined)

[0055] Comparative Example 5

[0056] The process technology of this embodiment is different from that of embodiment 3 in that: this embodiment does not have an enzymatic pretreatment step and does not have the process step (3). The specific steps are:

[0057] (1) 2 kg of dried rosemary leaves were crushed into powder with an average particle size of 90 mesh, water was added to make the water content reach 170%, and the mixture was soaked at room temperature for 8 hours, and steam distillation was performed to obtain a rosemary essential oil-removed raw material and a rosemary essential oil primary extract;

[0058] (2) Take rosemary essential oil removal raw material and add extraction solvent B, wherein the extraction solvent B is an ethyl acetate and acetone system solution, extract for 20 minutes at an ultrasonic power of 100 W and a temperature of 40° C. After the extraction is completed, centrifuge at a speed of 5000 rpm, pour the supernatant extract into a container, add the extraction solvent B again to repeat the extraction, the extraction and separation times are 1 time, and all the supernatant extracts are filtered to obtain an aqueous solution, and the other extracted components are fat-soluble precipitates; the volume ratio of ethyl acetate to acetone in the extraction solvent B is 20:29, and the volume of the residue in the container and the extraction solvent B is 1:2.

[0059] (3) The pH value of the aqueous solution is adjusted to neutral with calcium hydroxide, and then vacuum concentrated to an extract with a liquid-to-solid ratio of 1g:1.5mL; the extract is subjected to supercritical carbon dioxide extraction and separation, and the extract is placed in a supercritical carbon dioxide extraction kettle, and a carrier solvent food-grade n-hexane is added according to a mass ratio of extract to carrier solvent of 1:1. The separation kettle pressure is 8MPa, the temperature is 40°C, the extraction time is 1.4h, and the CO2 flow rate is 30L / h to obtain an extract.

[0060] (1) In vitro antioxidant effect and soothing and maintenance efficacy test

[0061] According to the 3D epidermal skin model The test was carried out according to the detection method of reactive oxygen species (ROS), nuclear transcription factor (Nrf2), keratin 10, nuclear transcription factor (NF-κB), inflammatory factor (IL-8), and inflammatory mediator (PGE2). The model used in this test is a 3D epidermal skin model Provided by Guangdong Boxi Biotechnology Co., Ltd.

[0062] Reactive oxygen species (ROS) testing

[0063] Test steps:

[0064] 1) Preparation of probe working solution: dilute the 10 mM probe solution to 20 μM probe working solution with model culture medium;

[0065] 2) UVB irradiation: According to the test grouping in Table 4, the model was transferred to a 6-well plate and randomly divided into a blank control group (BC), a negative control group (NC), a positive control group (PC1) and a sample group, with 3 replicates in each group. Except for the blank control group, the remaining groups were irradiated with UVB at a dose of 600 mJ / cm 2 ;

[0066] 3) Administration: After irradiation, 0.9 mL of KcGrowth culture medium was added to each well of the BC group and the NC group, and 0.9 mL of culture medium containing VE was added to each well of the PC1 group. The sample group evenly distributed the samples of corresponding concentrations on the surface of the model and placed it in a CO2 incubator (37°C, 5% CO2) for incubation for 24 h;

[0067] 4) Probe incubation: After the incubation, add 3.7 mL of 20 μM probe working solution to each well. Incubate in a CO2 incubator (37°C, 5% CO2) for 1 hour, shaking the well plate every 20 minutes to ensure uniform incubation of the model;

[0068] 5) Sample collection: After the incubation is completed, the model is cut and placed in a 1.5 mL centrifuge tube. 1 mL of 4% paraformaldehyde fixative is added to the tube, which is then wrapped in tin foil and sent for frozen sectioning.

[0069] 6) Washing and sealing: Wash the frozen sections with PBS for 8 minutes. After washing, use absorbent paper to cover the tissue.

[0070] Wipe the liquid clean, use a 200μL pipette tip to drop a small drop (about 20μL) of fluorescent anti-quenching mounting medium, and seal the slide with a coverslip.

[0071] 7) Take pictures: Observe with a fluorescence microscope and take fluorescent pictures.

[0072] 8) Calculation of down-regulation rate:

[0073] Table 1 Reactive oxygen species (ROS) test groups and test results

[0074]

[0075]

[0076] Note: Integrated optical density (IOD), its value reflects the content of ROS.

[0077] The above tests show that based on the 3D epidermal skin model Compared with the control group, the active oxygen (ROS) content of the rosemary extract in the sample group at a concentration of 0.5% (v / v) was significantly reduced, indicating that the sample can reduce the active oxygen (ROS) content at this concentration and has an antioxidant effect. Comparing the samples of Examples 1-3 and Comparative Examples 1-5, it can be found that the reduction rate prepared in Comparative Example 5 is 59.09%, the reduction rate of Example 1-3 is the largest, the reduction rate of Comparative Example 1-2 is second, and the reduction rate of Comparative Example 3-4 is further reduced. This shows that the technical solution disclosed in the present invention has a significant effect on the antioxidant effect of the extract.

[0078] (2) Tests of nuclear transcription factor (Nrf2), keratin 10, nuclear transcription factor (NF-κB), inflammatory factor (IL-8), and inflammatory mediator (PGE2)

[0079] Test steps:

[0080] 1) Prepare the test substance working solution according to the test groups in Table 2.

[0081] 2) UVB irradiation: According to the test grouping in Table 2, the model was randomly divided into a blank control group (BC), a negative control group (NC), a positive control group (PC1), a positive control group (PC2), a positive control group (PC3) and a sample group, with 3 replicates in each group. Except for the blank control group, the remaining groups were irradiated with UVB, and the irradiation dose was 600mJ / cm2.

[0082] 3) Administration: After irradiation, 0.9 mL of culture medium was added to each well of the BC group and the NC group, 0.9 mL of culture medium containing vitamin E was added to each well of the PC1 group, 0.9 mL of culture medium containing WY14643 was added to each well of the PC2 group, and 0.9 mL of culture medium containing dexamethasone was added to each well of the PC3 group. For the sample group, the samples of corresponding concentrations were evenly distributed on the surface of the model and incubated in a CO2 incubator (37°C, 5% CO2) for 24 h.

[0083] (Note: PC1 VE is used as a positive control for Nrf2 detection, PC2 WY14643 is used as a positive control for Kerat in10 detection, and PC3 dexamethasone is used as a positive control for IL-8, NF-κB, and PGE2 detection)

[0084] 4) After the incubation, wash the remaining test substance with sterile PBS and wipe off the remaining liquid with a sterile cotton swab.

[0085] 5) Calculation of promotion rate and inhibition rate:

[0086]

[0087] Table 2 Nuclear transcription factor (Nrf2) test groups and test results

[0088]

[0089]

[0090]

[0091] Table 3 Keratin 10 test groups and test results

[0092]

[0093]

[0094] Table 4 Nuclear transcription factor table (NF-κB) test groups and results

[0095]

[0096]

[0097] Table 5 Inflammatory factor (IL-8) test groups and results

[0098]

[0099]

[0100] Table 6 Inflammatory mediator (PGE2) test groups and results

[0101]

[0102]

[0103]

[0104] The test results in Tables 2 to 6 fully illustrate that based on the 3D epidermal skin model Compared with the control group, the content of nuclear transcription factor (NF-κB), inflammatory factor (IL-8), and inflammatory mediator (PGE2) in the rosemary extract sample at a concentration of 0.5% (v / v) was significantly reduced, indicating that the sample can reduce the content of nuclear transcription factor (NF-κB) at this concentration, inhibit the secretion of inflammatory factor (IL-8) and inflammatory mediator (PGE2), and has a significant soothing effect. Comparing the samples of Examples 1-3 and Comparative Examples 1-5, it can be found that Example 1-3 has the best effect, Comparative Example 1-2 is second, and Comparative Example 3-4 is lower. This shows that the technical solution disclosed in the present invention has a significant effect on the soothing effect of the extract.

[0105] Comparative Example 6

[0106] The difference between this embodiment and embodiment 1 is that only snail protease is used in the enzymatic pretreatment step of step 1), specifically: 2 kg of dried rosemary leaves are crushed into powder with an average particle size of 100 mesh, water is added to the rosemary powder to make the moisture content of rosemary reach 30%, and then snail protease is added, and enzymatic pretreatment is carried out at a temperature of 40° C. for 2 hours, and the amount of snail protease used is 880 U / g dried rosemary leaves.

[0107] Comparative Example 7

[0108] The difference between this embodiment and embodiment 1 is that only bromelain is used in the enzymatic pretreatment step 1), specifically: 2 kg of dried rosemary leaves are crushed into powder with an average particle size of 100 mesh, water is added to the rosemary powder to make the moisture content of rosemary reach 30%, and then snail protease is added, and enzymatic pretreatment is carried out at a temperature of 40° C. for 2 h, and the amount of bromelain used is 880 U / g dried rosemary leaves.

[0109] Comparative Example 8

[0110] The difference between this embodiment and embodiment 1 is that the extraction solvent B in this embodiment is only ethyl acetate.

[0111] Comparative Example 9

[0112] The difference between this embodiment and embodiment 1 is that the leaching solvent B in this embodiment is only acetone.

[0113] The extracts obtained by Example 1 and Comparative Examples 6-9 were tested in the same manner as described above to obtain the test data described in Table 7 (the down-regulation rates calculated in Table 1 below were all calculated based on the negative control group).

[0114] Table 7 Comparison of test data of Example 1 and Comparative Examples 6-9

[0115]

[0116]

[0117] It can be seen from the test data in Table 7 above that in the technical solution used in the present invention, regarding the enzymatic pretreatment of rosemary, the effect of the mixed addition of snail protease and bromelain is greater than that of using snail protease alone or using bromelain alone. The use of ethyl acetate and acetone in the extraction solvent B can significantly improve the extraction rate of rosmarinic acid and rosmarinic phenol in rosemary. The rosemary extract is obtained by mixing extracts A and B. It is found through experimental verification that the extract has a significant effect in anti-oxidation, soothing and repairing skin, and the effect is significantly better than the single extraction component of rosmarinic acid or rosmarinic phenol.

Claims

1. A method for preparing an antioxidant repair rosemary extract, characterized in that: The steps include: (1) grinding dried rosemary leaves into powder with an average particle size of 80-100 mesh, adding water to the rosemary powder to make the moisture content of the rosemary reach 20-30%, then adding snail protease and bromelain, and performing enzymolysis pretreatment at a temperature of 30-40° C. for 1-2 h; (2) adding water to the rosemary powder after enzymatic pretreatment to make the water content reach 150-180%, soaking at room temperature for 8-10 hours, and using steam distillation to obtain rosemary essential oil-removed raw material and rosemary essential oil primary extract; (3) adding a raw material of rosemary de-essential oil to a reaction container, adding an extraction solvent A for soaking and stirring, wherein the extraction solvent A is an ethanol-water system solution, refluxing and leaching for 20-30 min at a temperature of 60-90° C. to obtain an extract, filtering and centrifuging the extract to obtain an extract A, and the residue in the reaction container is reserved for later use; (4) adding the residue of step (3) to the extraction solvent B, wherein the extraction solvent B is an ethyl acetate and acetone system solution, extracting for 20 to 80 minutes at an ultrasonic power of 100 to 400 W and a temperature of 40 to 50° C., centrifuging at a speed of 3000 to 5000 rpm after the extraction is completed, pouring the supernatant extract into a container, adding the extraction solvent B again to repeat the extraction, the extraction and separation times are 1 to 3 times, and filtering all the supernatant extracts to obtain an aqueous solution, and the other extracted components are fat-soluble precipitates; (5) adjusting the pH value of the aqueous solution to neutral, and then vacuum concentrating to an extract with a liquid-to-solid ratio of 1:1.5-2 (v / w); subjecting the extract to supercritical carbon dioxide extraction and separation, placing the extract in a supercritical carbon dioxide extraction kettle, and then adding a carrier co-solvent, the separation kettle pressure is 8-10MPa, the temperature is 40-45°C, the extraction time is 1.3-1.5h, the CO2 flow rate is: 30-50L / h, to obtain extract B, and extracts A and B are combined to obtain rosemary extract.

2. The method for preparing an antioxidant repair rosemary extract according to claim 1, characterized in that: In step (1), the amount of snail protease is 350-400 U / g of dry rosemary leaves, and the amount of bromelain is 450-600 U / g of dry rosemary leaves.

3. The method for preparing an antioxidant repair rosemary extract according to claim 1, characterized in that: The ethanol mass fraction of the ethanol-water system solution of the extraction solvent A in step (3) is 40-70%, and the volume ratio of the rosemary essential oil removal raw material to the extraction solvent A is 1:5-10.

4. The method for preparing an antioxidant repair rosemary extract according to claim 1, characterized in that: In step (4), the volume ratio of ethyl acetate to acetone in the extraction solvent B is 20-30:25-40, and the volume ratio of the residue in the container to the extraction solvent B is 1:2-3.

5. The method for preparing an antioxidant repair rosemary extract according to claim 1, characterized in that: In step (5), the pH value of the solution is adjusted to neutral with calcium hydroxide.

6. The method for preparing an antioxidant repair rosemary extract according to claim 1, characterized in that: The carrier co-solvent in step (5) is one or both of anhydrous ethanol and food-grade n-hexane.

7. The method for preparing an antioxidant repair rosemary extract according to claim 1, characterized in that: The mass ratio of the extract to the carrier solvent in step (5) is 1:1-2.

8. The method for preparing an antioxidant and repairing rosemary extract according to claim 1, characterized in that: The primary extract of rosemary essential oil obtained in step (1) is further subjected to distillation separation to obtain rosemary essential oil and crude rosemary hydrosol.

9. The rosemary extract prepared by the method according to any one of claims 1 to 7.

10. Use of the rosemary extract according to claim 9 as an antioxidant.

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