Extract based on roselle oil and roselle polypeptide as well as extraction method and application of extract

Abstract

The invention relates to the related technical field of extraction of active ingredients in roselle calyx, and particularly provides an extract based on roselle oil and roselle polypeptide as well as an extraction method and application of the extract. The extraction method comprises the following steps: S1, sequentially performing crushing treatment and supercritical fluid extraction treatment on roselle calyx to obtain a roselle oil extract and flower residues; s2, the roselle residues are sequentially subjected to fermentation treatment and enzymolysis treatment, and a roselle polypeptide extract is obtained; wherein a strain used in the fermentation treatment is selected from at least one of saccharomyces cerevisiae, bacillus subtilis and lipomyces starkeyi; an enzyme used in the enzymolysis treatment is selected from at least one of acid protease and compound protease; the extract comprises the roselle oil extract and the roselle polypeptide extract. The extraction method provided by the invention improves the extraction rate of active ingredients extracted from roselle calyx.

Description

Technical Field

[0001] The present application relates to the technical field related to the extraction of active ingredients from roselle calyx, and specifically to an extract based on roselle oil and roselle polypeptide, and an extraction method and use thereof. Background Art

[0002] Roselle (Hibiscus sabdarifa L.), also known as Roselle, belongs to the genus Hibiscus of the Malvaceae family and is a medicinal and edible plant. Its calyx is brightly colored and has good color quality. It is rich in active ingredients such as organic acids, anthocyanins, polyphenols, polysaccharides and flavonoids. It has antioxidant, anti-cancer and blood sugar-lowering effects, and is widely used in food, cosmetics and other fields.

[0003] At present, there are two methods for extracting active ingredients from roselle calyx: water extraction and alcohol extraction. Among them, the extract obtained by alcohol extraction has a strong antioxidant capacity, and its IC50 value for scavenging hydroxyl free radicals is 169.1μg / mL, which is better than the IC50 value of vitamin C, which is 448.5μg / mL. However, the extraction rate of active ingredients from roselle calyx needs to be improved. Summary of the invention

[0004] In view of this, the present application provides an extract based on roselle oil and roselle polypeptide, and an extraction method and use thereof. The extraction method of the present application improves the extraction rate of active ingredients from roselle calyx, and can not only extract roselle oil extract from roselle calyx, but also further extract roselle polypeptide extract from flower residue.

[0005] In a first aspect of the present application, the present application provides an extraction method based on an extract of Roselle oil and Roselle polypeptide, the extraction method comprising the following steps:

[0006] Step S1, the roselle calyx is subjected to a crushing treatment and a supercritical fluid extraction treatment in sequence to obtain a roselle oil extract and flower residue;

[0007] Step S2, the flower residue is subjected to fermentation treatment and enzymolysis treatment in sequence to obtain a Roselle polypeptide extract; wherein the strain used in the fermentation treatment is selected from at least one of Saccharomyces cerevisiae, Bacillus subtilis and Lipomyces staryi; the enzyme used in the enzymolysis treatment is selected from at least one of an acidic protease and a composite protease;

[0008] The extract comprises the roselle oil extract and the roselle polypeptide extract.

[0009] In some embodiments, in step S1, the roselle calyx is selected from at least one of a dried roselle calyx and a fresh roselle calyx. Further, in step S1, the roselle calyx is selected from a dried roselle calyx.

[0010] In some embodiments, in step S1, the supercritical fluid extraction process is selected from supercritical carbon dioxide fluid extraction process.

[0011] In some embodiments, in step S1, the supercritical fluid extraction treatment is carried out under the following conditions in the extraction section: the extraction temperature is (50-60)°C (for example, 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C or 60°C, etc.), and the extraction pressure is (20-40)Mpa (for example, 20Mpa, 21Mpa, 22Mpa, 23Mpa, 24Mpa, 25Mpa, 26Mpa, 27Mpa, 28Mpa, 29Mpa, 30Mpa, 31Mpa, 32Mpa, 33Mpa, 34Mpa, 35Mpa, 36Mpa, 37Mpa, 38Mpa, 39Mpa or 40Mpa, etc.).

[0012] In some embodiments, the extraction temperature is (53-57)°C. Further, the extraction temperature is 55°C.

[0013] In some embodiments, the extraction pressure is (25-35) Mpa. Further, the extraction pressure is 30 Mpa.

[0014] In some embodiments, in step S1, the extraction time of the supercritical fluid extraction treatment in the extraction section is 2 to 5 hours (e.g., 2 hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours or 5 hours, etc.). Further, in step S1, the extraction time of the supercritical fluid extraction treatment in the extraction section is 3 hours.

[0015] In some embodiments, in step S1, the supercritical fluid extraction treatment is performed by a resolution method in the resolution section selected from a variable temperature and pressure method;

[0016] And / or, the conditions of the supercritical fluid extraction treatment in the analytical section are: separation I pressure is (19-21) MPa (e.g., 19 MPa, 19.5 MPa, 20 MPa, 20.5 MPa or 21 MPa, etc.), separation I temperature is (58-62) ° C (e.g., 58 ° C, 59 ° C, 60 ° C, 61 ° C or 62 ° C, etc.); separation II pressure is (4-6) MPa (e.g., 4 MPa, 4.5 MPa, 5 MPa, 5.5 MPa or 6 MPa, etc.), separation II temperature is (48-52) ° C (e.g., 48 ° C, 49 ° C, 50 ° C, 51 ° C or 52 ° C, etc.). Further, in step S1, the conditions of the supercritical fluid extraction treatment in the analytical section are: separation I pressure is 20 MPa, separation I temperature is 60 ° C; separation II pressure is 5 MPa, separation II temperature is 50 ° C.

[0017] In some embodiments, in step S2, the fermentation temperature is (25-35)°C; for example, 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C, 34°C or 35°C. Further, in step S2, the fermentation temperature is 28°C.

[0018] In some embodiments, in step S2, the strain used in the fermentation treatment is selected from one of Saccharomyces cerevisiae, Bacillus subtilis and Lipozyme Starchus. Further, in step S2, the strain used in the fermentation treatment is selected from Saccharomyces cerevisiae or Lipozyme Starchus.

[0019] In some embodiments, in step S2, the strain used for the fermentation treatment is selected from Saccharomyces cerevisiae. Further, the strain number of the Saccharomyces cerevisiae is CICC 32883.

[0020] In some embodiments, in step S2, the strain used for the fermentation treatment is selected from Bacillus subtilis. Further, the strain number of the Bacillus subtilis is CICC 24713.

[0021] In some embodiments, in step S2, the strain used for the fermentation treatment is selected from Lipomyces staryi. Further, the strain number of Lipomyces staryi is 2.2291.

[0022] In some embodiments, in step S2, the enzymatic hydrolysis treatment includes a first enzymatic hydrolysis treatment and a second enzymatic hydrolysis treatment performed sequentially; the enzyme used in the first enzymatic hydrolysis treatment and the enzyme used in the second enzymatic hydrolysis treatment are each independently selected from an acidic protease or a composite protease, and the enzyme used in the first enzymatic hydrolysis treatment and the enzyme used in the second enzymatic hydrolysis treatment are different.

[0023] In some embodiments, the enzyme used in the first enzymatic treatment is selected from acidic proteases, and the enzyme used in the second enzymatic treatment is selected from composite proteases.

[0024] In some embodiments, the working conditions of the acidic protease are: the temperature is (45-55)°C (e.g., 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, 51°C, 52°C, 53°C, 54°C or 55°C, etc.), and the pH value is 2.0-4.0 (e.g., 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, etc.). Further, the working conditions of the acidic protease are: the temperature is 50°C and the pH value is 3.0.

[0025] In some embodiments, the working conditions of the composite protease are: temperature of (50-60)°C (e.g., 50°C, 51°C, 52°C, 53°C, 54°C, 55°C, 56°C, 57°C, 58°C, 59°C or 60°C, etc.), pH of 8.0-9.0 (e.g., 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9 or 9.0, etc.). Further, the working conditions of the composite protease are: temperature of 55°C, pH of 8.0.

[0026] In the second aspect of the present application, the present application provides an extract based on Roselle oil and Roselle polypeptide, wherein the extract is obtained by the extraction method described in the first aspect of the present application.

[0027] In the second aspect of the present application, the present application provides a use of the extract described in the first aspect of the present application in preparing a product having antioxidant, and / or anti-inflammatory, and / or antibacterial, and / or inhibiting elastase activity, and / or promoting integrin α2β1 expression.

[0028] In some embodiments, the anti-inflammatory activity includes anti-COX-2 induced inflammation.

[0029] In some embodiments, the antibacterial agents include anti-Propionibacterium acnes, anti-Malassezia, anti-Fusobacterium nucleatum, and anti-Streptococcus mutans.

[0030] In some embodiments, the product can be a scalp care product with oil control and dandruff removal functions, an oral care product with antibacterial and anti-inflammatory functions, and a skin care product with anti-wrinkle, firming, and keratin conditioning effects. Among them, the roselle oil extract is mainly used to prepare a scalp care product with oil control and dandruff removal functions and an oral care product with antibacterial and anti-inflammatory functions. The roselle polypeptide extract is mainly used to prepare a skin care product with anti-wrinkle, firming, and keratin conditioning effects. The present application has the following beneficial effects:

[0031] First, the extraction method of the present application improves the extraction rate of active ingredients from the roselle calyx, and can not only extract the roselle oil extract from the roselle calyx, but also further extract the roselle polypeptide extract from the flower residue.

[0032] Second, the present application adopts the combination of "first undergoing supercritical fluid extraction treatment, then undergoing fermentation treatment and enzymatic hydrolysis treatment", which can take into account the total flavonoids content and proanthocyanidins content in the Roselle oil extract, as well as the polypeptide content and proanthocyanidins content in the Roselle polypeptide extract, and can significantly increase the total flavonoids content and proanthocyanidins content in the Roselle oil extract, as well as the polypeptide content and proanthocyanidins content in the Roselle polypeptide extract.

[0033] Third, the enzymatic treatment of the present application using a combination of "acidic protease and composite protease" can increase the content of polypeptides and proanthocyanidins in the Roselle polypeptide extract. Furthermore, the order of using the composite protease and the acidic protease can also affect the content of polypeptides and proanthocyanidins in the Roselle polypeptide extract; among them, "using acidic protease first and then composite protease" in the enzymatic treatment can significantly increase the content of polypeptides and proanthocyanidins in the Roselle polypeptide extract. DETAILED DESCRIPTION

[0034] The present application discloses an extract based on Roselle oil and Roselle polypeptide and its extraction method and use. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar replacements and modifications are obvious to those skilled in the art, and they are all deemed to be included in this application. The method and application of this application have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application described herein without departing from the content, spirit and scope of this application to realize and apply the technology of this application.

[0035] In order to make the purpose, technical solutions and advantages of the present application clearer, the implementation scheme of the present application will be further described in detail below in conjunction with examples.

[0036] Unless otherwise specified, the Saccharomyces cerevisiae used in this application has a strain number of CICC 32883, which was purchased from the China Industrial Microbiological Culture Collection Center.

[0037] Unless otherwise specified, the strain number of Bacillus subtilis used in this application is CICC24713, which was purchased from China Industrial Microbiological Culture Collection Center.

[0038] Unless otherwise specified, the strain number of Lipomyces starkeyi used in the present application is 2.2291, which was purchased from China General Microbiological Culture Collection Administration Center.

[0039] Examples 1 to 6: wherein the extraction method of the extract based on Roselle oil and Roselle polypeptide comprises step S1 and step S2.

[0040] In Examples 1 to 6, the operation process of step S1 is specifically as follows: using a pulverizer to crush the dried roselle calyx into powdered dried roselle calyx, taking the powdered dried roselle calyx (30 g) and placing it in an extraction kettle for supercritical fluid extraction treatment (extraction section conditions: extraction temperature 55°C, extraction pressure 30 MPa, extraction time 3 hours; analysis section conditions: variable temperature and pressure method, separation I pressure 20 MPa, separation I temperature 60°C, separation II pressure 5 MPa, separation II temperature 50°C); wherein, the paste solid obtained by extraction and caprylic acid capric acid triglyceride (CAS No. 65381-09-1, referred to as GTCC) are compounded in a weight ratio of 1:20 to obtain a roselle oil extract; using a Buchner funnel to filter the remaining material in the extraction kettle to obtain flower residues. wherein, the obtained flower residues are divided into six portions, and the six portions of flower residues are respectively subjected to step S2 in Examples 1 to 6.

[0041] In Example 1, the operation process of step S2 is specifically as follows: a portion of flower residue is placed in a conical flask (250 mL), and then yeast extract peptone glucose medium (5 g, also known as YPD medium or YEPD medium) and purified water (100 mL) are added, and ultrasonic sterilization is performed for 20 minutes; after cooling to 28°C, Bacillus subtilis (inoculation amount 2wt%, after inoculation, the strain concentration is 1×10 6 CFU / mL), fermentation (conditions: temperature 28°C, rotation speed 200r / min, fermentation time 4 days). After the fermentation, acid protease (2wt%) was added for enzymolysis at a temperature of 50°C and a pH value of 3.0 for 2 hours; then the temperature was raised to 90°C and kept at 90°C for 15 minutes. After the enzymolysis was completed, after cooling to room temperature, coarse filtration (300 mesh gauze filtration) and fine filtration (SPPM-C-10 membrane separation) were performed in sequence, and then spray drying was performed to obtain the roselle polypeptide extract.

[0042] In Example 2, the operation process of step S2 is specifically as follows: a portion of flower residue is placed in a conical flask (250 mL), and then yeast extract peptone glucose medium (5 g, also known as YPD medium or YEPD medium) and purified water (100 mL) are added, and ultrasonic sterilization is performed for 20 minutes; after cooling to 28°C, Stark oil yeast (inoculation amount 2wt%, after inoculation, the strain concentration is 1×10 6 CFU / mL), fermentation (conditions: temperature 28°C, rotation speed 200r / min, fermentation time 4 days). After the fermentation, acid protease (2wt%) was added for enzymolysis at a temperature of 50°C and a pH value of 3.0 for 2 hours; then the temperature was raised to 90°C and kept at 90°C for 15 minutes. After the enzymolysis was completed, after cooling to room temperature, coarse filtration (300 mesh gauze filtration) and fine filtration (SPPM-C-10 membrane separation) were performed in sequence, and then spray drying was performed to obtain the roselle polypeptide extract.

[0043] In Example 3, the operation process of step S2 is specifically as follows: a portion of flower residue is placed in a conical flask (250 mL), and then yeast extract peptone glucose medium (5 g, also known as YPD medium or YEPD medium) and purified water (100 mL) are added, and ultrasonic sterilization is performed for 20 minutes; after cooling to 28°C, brewer's yeast (inoculation amount 2wt%, after inoculation, the strain concentration is 1×10 6 CFU / mL), fermentation (conditions: temperature 28°C, rotation speed 200r / min, fermentation time 4 days). After the fermentation is completed, acid protease (2wt%) is added for enzymolysis for 2 hours at a temperature of 50°C and a pH value of 3.0; the temperature is then raised to 90°C and kept at 90°C for 15 minutes; after the enzymolysis is completed, it is cooled to room temperature, coarse filtration (300 mesh gauze filtration) and fine filtration (SPPM-C-10 membrane separation) are performed in sequence, and then spray drying is performed to obtain the roselle polypeptide extract.

[0044] In Example 4, the operation process of step S2 is specifically as follows: a portion of flower residue is placed in a conical flask (250 mL), and then yeast extract peptone glucose medium (5 g, also known as YPD medium or YEPD medium) and purified water (100 mL) are added, and ultrasonic sterilization is performed for 20 minutes; after cooling to 28°C, brewer's yeast (inoculation amount 2wt%, strain concentration after inoculation is 1×10 6 CFU / mL), fermentation (conditions: temperature 28°C, rotation speed 200r / min, fermentation time 4 days). After the fermentation is completed, the temperature is first raised to 55°C and the pH value is adjusted to 7.5, and a composite protease (2wt%) is added for enzymolysis for 2 hours at a temperature of 55°C and a pH value of 7.5; then the temperature is raised to 90°C and kept at 90°C for 15 minutes. After the enzymolysis is completed, after cooling to room temperature, coarse filtration (300 mesh gauze filtration) and fine filtration (SPPM-C-10 membrane separation) are performed in sequence, and then spray drying is performed to obtain the Roselle polypeptide extract.

[0045] In Example 5, the operation process of step S2 is as follows: a portion of flower residue is placed in a conical flask (250 mL), and then yeast extract peptone glucose medium (5 g, also known as YPD medium or YEPD medium) and purified water (100 mL) are added, and ultrasonic sterilization is performed for 20 minutes; after cooling to 28°C, brewer's yeast (inoculation amount 2wt%, strain concentration after inoculation is 1×10 6CFU / mL), fermentation (conditions: temperature 28°C, rotation speed 200r / min, fermentation time 4 days). After the fermentation is completed, acid protease (2wt%) is added for enzymolysis at a temperature of 50°C and a pH value of 3.0 for 2 hours; then the temperature is raised to 55°C and the pH value is adjusted to 7.5, and composite protease (2wt%) is added for enzymolysis at a temperature of 55°C and a pH value of 7.5 for 2 hours; finally, the temperature is raised to 90°C and kept at 90°C for 15 minutes; after the enzymolysis is completed, after cooling to room temperature, coarse filtration (300 mesh gauze filtration) and fine filtration (SPPM-C-10 membrane separation) are performed in sequence, and then spray drying is performed to obtain the roselle polypeptide extract.

[0046] In Example 6, the operation process of step S2 is specifically as follows: a portion of flower residue is placed in a conical flask (250 mL), and then yeast extract peptone glucose medium (5 g, also known as YPD medium or YEPD medium) and purified water (100 mL) are added, and ultrasonic sterilization is performed for 20 minutes; after cooling to 28°C, brewer's yeast (inoculation amount 2wt%, after inoculation, the strain concentration is 1×10 6 CFU / mL), fermentation (conditions: temperature 28°C, rotation speed 200r / min, fermentation time 4 days). After the fermentation is completed, the temperature is first raised to 55°C and the pH value is adjusted to 7.5, and a composite protease (2wt%) is added for enzymolysis for 2 hours at a temperature of 55°C and a pH value of 7.5; then the temperature is lowered to 50°C and the pH value is adjusted to 7.0, and an acid protease (2wt%) is added for enzymolysis for 2 hours at a temperature of 50°C and a pH value of 3.0; finally, the temperature is raised to 90°C and kept at 90°C for 15 minutes; after the enzymolysis is completed, after cooling to room temperature, coarse filtration (300 mesh gauze filtration) and fine filtration (SPPM-C-10 membrane separation) are performed in sequence, and then spray drying is performed to obtain a roselle polypeptide extract.

[0047] Comparative Example 1: In this comparative example, the method for extracting the Roselle polypeptide extract comprises the following steps:

[0048] The dried roselle calyx was crushed into powder by a grinder, and the powdered dried roselle calyx (5 g) was placed in a conical flask (250 mL), and then yeast extract peptone glucose medium (5 g, also known as YPD medium or YEPD medium) and purified water (100 mL) were added, and ultrasonic sterilization was performed for 20 minutes; after cooling to 28° C., Saccharomyces cerevisiae was inoculated (inoculation amount 2wt%, strain concentration after inoculation was 1×10 6CFU / mL), fermentation (conditions: temperature 28°C, rotation speed 200r / min, fermentation time 4 days). After the fermentation is completed, acid protease (2wt%) is added for enzymolysis at a temperature of 50°C and a pH value of 3.0 for 2 hours; then the temperature is raised to 55°C and the pH value is adjusted to 7.5, and composite protease (2wt%) is added for enzymolysis at a temperature of 55°C and a pH value of 7.5 for 2 hours; finally, the temperature is raised to 90°C and kept at 90°C for 15 minutes. After the enzymolysis is completed, after cooling to room temperature, coarse filtration (300 mesh gauze filtration) and fine filtration (SPPM-C-10 membrane separation) are performed in sequence, and then spray drying is performed to obtain the roselle polypeptide extract.

[0049] Comparative Example 2: In this comparative example, the extraction method based on the extract of Roselle oil and Roselle polypeptide specifically includes the following steps:

[0050] Step S1, using a grinder to grind the dried roselle calyx into powdered dried roselle calyx, mixing the powdered dried roselle calyx (5 g) with an ethanol aqueous solution (100 g) with a volume concentration of 75% to obtain a mixed solution; then ultrasonicating for 2 hours at a power of 100 W to obtain an extract; then, immediately filtering the extract with a Buchner funnel to obtain a filtrate and a filter residue (i.e., flower residue); wherein, using a rotary evaporator to evaporate the ethanol in the filtrate at 45° C. to obtain a crude roselle ethanol extract; firstly adding purified water to the crude roselle ethanol extract to 70% of the volume of the above-mentioned mixed solution, and then adding 1,3-butanediol to 100% of the volume of the above-mentioned mixed solution, and then filtering through a Buchner funnel to obtain a roselle oil extract.

[0051] Step S2: Same as in Example 1.

[0052] Comparative Example 3: In this comparative example, the extraction method based on the extract of Roselle oil and Roselle polypeptide specifically includes the following steps:

[0053] Step S1, using a grinder to grind the dried roselle calyx into powdered dried roselle calyx, mixing the powdered dried roselle calyx (5 g) and a 1,3-butanediol aqueous solution (100 g) having a volume concentration of 30%, and then reflux reacting for 2.5 hours; after that, the reaction solution is cooled to room temperature and filtered through a Buchner funnel to obtain a filtrate (i.e., Roselle oil extract) and a filter residue (i.e., flower residue).

[0054] Step S2: Same as in Example 1.

[0055] Table 1 Condition selection of Examples 1 to 6 and Comparative Examples 1 to 2:

[0056]

[0057] Test Example 1 Determination of the content of total flavonoids in a sample:

[0058] In this test example, the method for detecting the total flavonoid content specifically includes the following steps:

[0059] Step S1-1, preparation of standard curve:

[0060] Using rutin (CAS No. 153-18-4) as a standard, weigh rutin (0.1 g) and place it in a 100 mL volumetric flask, first add 75% ethanol aqueous solution to dissolve the standard, then add 75% ethanol aqueous solution to make the volume up to 100 mL to obtain a rutin standard solution with a concentration of 1.0 g / L.

[0061] Respectively pipette 0.0mL, 2.0mL, 4.0mL, 6.0mL, 8.0mL, and 10.0mL of 1.0g / L rutin standard solution into a 10mL volumetric flask, and dilute to 10mL with 75% ethanol aqueous solution to obtain rutin standard solutions with gradient concentrations of 0.0mg / mL, 0.2mg / mL, 0.4mg / mL, 0.6mg / mL, 0.8mg / mL, and 1.0mg / mL.

[0062] Take the gradient concentration of rutin standard solution (1mL) and place it in a 10mL volumetric flask, first add deionized water (4mL), 5% NaNO2 aqueous solution (0.3mL) by weight concentration in sequence; after 5 minutes, add 10% Al(NO3)3 aqueous solution (0.3mL) by weight concentration; after 6 minutes of reaction, add 1mol / L NaOH aqueous solution (2mL) and deionized water (2.4mL) by weight concentration in sequence; after 10 minutes, measure the absorbance (A) at 510nm using UV-visible spectrophotometry. Draw a standard curve with absorbance A as the ordinate and the concentration of the standard solution as the abscissa.

[0063] Step S1-2, determination of total flavonoid content in the sample:

[0064] Take a sample (1mL) and place it in a test tube, add deionized water (4mL) and a 5% NaNO2 aqueous solution (0.3mL) by weight; after 5 minutes, add a 10% Al(NO3)3 aqueous solution (0.3mL) by weight; after 6 minutes of reaction, add a 1mol / L NaOH aqueous solution (2mL) and deionized water (2.4mL) by weight; after 10 minutes, use UV-visible spectrophotometry to measure the absorbance at 510nm. According to the standard curve, calculate the content of total flavonoids in the sample.

[0065] Table 2 Test results of total flavonoids content in samples:

[0066]

[0067] It can be seen from Table 2 that the total flavonoids content in the Roselle oil extract obtained by supercritical carbon dioxide fluid extraction in the present application is significantly higher than that in the Roselle oil extract extracted by ethanol extraction and the Roselle oil extract extracted by 1,3-butanediol extraction.

[0068] Test Example 2 Determination of the content of polypeptides in samples:

[0069] In this test example, the method for detecting the polypeptide content is the biuret reagent method, which specifically includes the following steps:

[0070] Step S2-1, preparation of bovine serum albumin standard curve:

[0071] Take bovine serum albumin (CAS No. 9048-46-8, BAS for short) as the standard substance and prepare a BAS aqueous solution with a concentration of 10 mg / mL; take 0 μL, 50 μL, 100 μL, 150 μL, 200 μL, and 250 μL of the BAS aqueous solution with a concentration of 10 mg / mL and place them in microcentrifuge tubes (EP tubes) and add water to 0.5 mL to obtain BAS standard solutions with gradient concentrations of 0.0 mg / mL, 1.0 mg / mL, 2.0 mg / mL, 3.0 mg / mL, 4.0 mg / mL, and 5.0 mg / mL.

[0072] Add biuret reagent (2 mL) to the EP tube containing the BAS standard solution and react at room temperature for 30 minutes; then, measure the absorbance (A) at 540 nm using UV-visible spectrophotometry. Draw a standard curve with absorbance A as the ordinate and the concentration of the standard solution as the abscissa.

[0073] The biuret reagent includes reagent A and reagent B. Reagent A: CuSO4 (0.15 g, CAS No. 7758-98-7), potassium sodium tartrate (0.6 g, CAS No. 304-59-6) and distilled water (50 mL) are mixed evenly; reagent B: NaOH aqueous solution (30 mL) with a weight percentage concentration of 10%. Reagent A and reagent B are mixed evenly to obtain the biuret reagent, and the biuret reagent is prepared before use.

[0074] Step S2-2, determination of polypeptide content in the sample:

[0075] A 10% trichloroacetic acid aqueous solution (1.0 mL) was added to the sample (5 mg), centrifuged, and the supernatant (0.5 mL) was taken and a biuret reagent (2 mL) was added thereto, and reacted at room temperature for 30 minutes; then, the absorbance was measured at 540 nm using UV-visible spectrophotometry. The polypeptide content (wt%) in the sample was calculated based on the standard curve.

[0076] Table 3 Test results of peptide content in samples:

[0077]

[0078]

[0079] The calculation formula for the sample yield is: Wherein, y represents the yield of the sample (unit: wt%), the weight of the roselle calyx is recorded as W1 (unit: g), and the weight of the sample is recorded as W2 (unit: g).

[0080] It can be seen from Table 3 that the Hibiscus sabdariffa polypeptide extract obtained by the present application "first undergoing supercritical fluid extraction treatment, then undergoing fermentation treatment and enzymatic hydrolysis treatment" has a higher polypeptide content.

[0081] By comparing Example 3 and Comparative Example 1, it can be seen that the difference between Example 1 and Comparative Example 1 is whether or not the supercritical fluid extraction treatment is performed in step S1, wherein Example 1 is subjected to supercritical fluid treatment, and Comparative Example 1 is not subjected to supercritical fluid treatment. From the polypeptide content in the Roselle polypeptide extract of Example 1 and Comparative Example 1, it can be seen that, compared with directly undergoing fermentation treatment and enzymolysis treatment, "first undergoing supercritical fluid extraction treatment, then undergoing fermentation treatment and enzymolysis treatment" can significantly increase the polypeptide content in the Roselle polypeptide extract.

[0082] By comparing Example 3 with Comparative Examples 2 to 3, it can be seen that the difference between Example 3 and Comparative Examples 2 to 3 is that the extraction method used in step S1 is different, wherein Example 3 is a supercritical fluid extraction treatment, Comparative Example 2 is an ethanol extraction treatment, and Comparative Example 3 is a 1,3-butanediol extraction treatment. From the content of polypeptides in the Roselle polypeptide extracts of Example 3 and Comparative Examples 2 to 3, it can be seen that compared with "first undergoing ethanol extraction treatment, then fermentation treatment and enzymolysis treatment" and "first undergoing 1,3-butanediol extraction treatment, then fermentation treatment and enzymolysis treatment", the present application "first undergoing supercritical fluid extraction treatment, then fermentation treatment and enzymolysis treatment" can significantly increase the content of polypeptides in the Roselle polypeptide extract.

[0083] By comparing Examples 1 to 3, it can be seen that the only difference between Examples 1 to 3 is that the strains used for fermentation are different, wherein the strain used for fermentation in Example 1 is Bacillus subtilis, the strain used for fermentation in Example 2 is Lipozyme Starchus, and the strain used for fermentation in Example 3 is Saccharomyces cerevisiae. From the polypeptide content in the Roselle polypeptide extracts of Examples 1 to 3, it can be seen that Saccharomyces cerevisiae is better than Lipozyme Starchus and better than Bacillus subtilis.

[0084] By comparing Examples 3 to 6, it can be seen that the only difference between Examples 3 to 6 is that the enzymes used for the enzymatic hydrolysis treatment are different, wherein the enzyme used in Example 3 is an acidic protease, the enzyme used in Example 4 is a composite protease, and the enzymes used in Examples 5 and 6 are both acidic protease and composite protease. From the polypeptide content in the Roselle polypeptide extracts of Examples 3 to 6, it can be seen that the combination of "acidic protease and composite protease" used in the enzymatic hydrolysis treatment can increase the polypeptide content in the Roselle polypeptide extract.

[0085] Further, the difference between Examples 5 and 6 is that the order of using the acidic protease and the composite protease used in the enzymatic hydrolysis treatment is different, wherein the order of using the enzymes used in the enzymatic hydrolysis treatment of Example 5 is acidic protease and composite protease, and the order of using the enzymes used in the enzymatic hydrolysis treatment of Example 6 is composite protease and acidic protease. It can be seen from the polypeptide content in the Roselle polypeptide extract of Examples 5 and 6 that the order of using the composite protease and the acidic protease can affect the polypeptide content in the Roselle polypeptide extract; wherein, "using the acidic protease first and then the composite protease" in the enzymatic hydrolysis treatment can significantly increase the polypeptide content in the Roselle polypeptide extract.

[0086] Test Example 3 Determination of the content of proanthocyanidins in the sample:

[0087] In this test example, the content of proanthocyanidins in the sample was determined according to DB12 / T 885-2019 "Determination of proanthocyanidins in plant extracts - UV / visible spectrophotometry".

[0088] Table 4 Test results of proanthocyanidin content in samples:

[0089]

[0090] It can be seen from Table 4 that the Roselle oil extract and the Roselle polypeptide extract obtained by "first undergoing supercritical fluid extraction treatment, and then undergoing fermentation treatment and enzymatic hydrolysis treatment" in the present application both have a high content of proanthocyanidins.

[0091] By comparing Example 3 with Comparative Examples 2 to 3, it can be seen that the difference between Example 3 and Comparative Examples 2 to 3 is that the extraction method used in step S1 is different, wherein Example 1 is a supercritical fluid extraction treatment, Comparative Example 2 is an ethanol extraction treatment, and Comparative Example 3 is a 1,3-butanediol extraction treatment. From the content of proanthocyanidins in the primary extraction of roselle and the polypeptide extract of Roselle in Example 3 and Comparative Examples 2 to 3, it can be seen that compared with "first undergoing ethanol extraction treatment, then fermentation treatment and enzymolysis treatment" and "first undergoing 1,3-butanediol extraction treatment, then fermentation treatment and enzymolysis treatment", "first undergoing supercritical fluid extraction treatment, then fermentation treatment and enzymolysis treatment" can significantly increase the content of proanthocyanidins in the primary extraction of roselle and the polypeptide extract of Roselle at the same time.

[0092] By comparing Example 3 and Comparative Example 1, it can be seen that the difference between Example 1 and Comparative Example 1 is whether or not the supercritical fluid extraction treatment in step S1 is performed, wherein Example 1 is subjected to supercritical fluid treatment, and Comparative Example 1 is not subjected to supercritical fluid treatment. From the content of proanthocyanidins in the Roselle polypeptide extract in Example 1 and Comparative Example 1, it can be seen that, compared with directly undergoing fermentation treatment and enzymolysis treatment, "first undergoing supercritical fluid extraction treatment, then undergoing fermentation treatment and enzymolysis treatment" can significantly increase the content of proanthocyanidins in the Roselle polypeptide extract.

[0093] By comparing Examples 1 to 3, it can be seen that the only difference between Examples 1 to 3 is that the strains used for fermentation are different, wherein the strain used for fermentation in Example 1 is Bacillus subtilis, the strain used for fermentation in Example 2 is Lipozyme Starchus, and the strain used for fermentation in Example 3 is Saccharomyces cerevisiae. From the proanthocyanidin content in the Roselle polypeptide extracts of Examples 1 to 3, it can be seen that Saccharomyces cerevisiae is better than Lipozyme Starchus and better than Bacillus subtilis.

[0094] By comparing Examples 3 to 6, it can be seen that the only difference between Examples 3 to 6 is that the enzymes used for the enzymatic hydrolysis treatment are different, wherein the enzyme used in Example 3 is an acidic protease, the enzyme used in Example 4 is a composite protease, and the enzymes used in Examples 5 and 6 are both acidic protease and composite protease. From the proanthocyanidin content in the Roselle polypeptide extracts of Examples 3 to 6, it can be seen that the combination of "acidic protease and composite protease" used in the enzymatic hydrolysis treatment can increase the proanthocyanidin content in the Roselle polypeptide extract.

[0095] Further, the difference between Examples 5 and 6 is that the order of using the acidic protease and the composite protease used in the enzymatic hydrolysis treatment is different, wherein the order of using the enzymes used in the enzymatic hydrolysis treatment of Example 5 is acidic protease and composite protease, and the order of using the enzymes used in the enzymatic hydrolysis treatment of Example 6 is composite protease and acidic protease. It can be seen from the proanthocyanidin content in the Roselle polypeptide extract of Examples 5 and 6 that the order of using the composite protease and the acidic protease can affect the proanthocyanidin content in the Roselle polypeptide extract; wherein, "using the acidic protease first and then the composite protease" in the enzymatic hydrolysis treatment can significantly increase the proanthocyanidin content in the Roselle polypeptide extract.

[0096] From Tables 2 to 4, it can be seen that the present application adopts the combination of "first undergoing supercritical fluid extraction treatment, then undergoing fermentation treatment and enzymatic hydrolysis treatment", which can take into account the total flavonoids content and proanthocyanidins content in the Roselle oil extract, as well as the polypeptide content and proanthocyanidins content in the Roselle polypeptide extract, and can significantly increase the total flavonoids content and proanthocyanidins content in the Roselle oil extract, as well as the polypeptide content and proanthocyanidins content in the Roselle polypeptide extract.

[0097] The enzymatic hydrolysis treatment of the present application uses a combination of "acidic protease and composite protease" to increase the content of polypeptides and proanthocyanidins in the Roselle polypeptide extract. Furthermore, the order of using the composite protease and the acidic protease can also affect the content of polypeptides and proanthocyanidins in the Roselle polypeptide extract; among them, "using acidic protease first and then composite protease" in the enzymatic hydrolysis treatment can significantly increase the content of polypeptides and proanthocyanidins in the Roselle polypeptide extract.

[0098] Test Example 4 Determination of the free radical of 2,2-biphenyl-1-picrylhydrazyl (CAS No. 1898-66-4, referred to as DPPH) in the sample Clearance rate:

[0099] In this test example, the determination process of DPPH free radical scavenging rate specifically includes the following steps:

[0100] Step S4-1, prepare DPPH ethanol solution: dissolve DPPH (20 mg) in anhydrous ethanol and dilute to volume in a 250 mL volumetric flask with anhydrous ethanol to obtain a DPPH ethanol solution with a concentration of 0.08 mg / mL. Store at 0-4°C in the dark, use immediately after preparation, and it is effective within 4 hours.

[0101] Step S4-2, preparing sample solutions with different test concentrations: using anhydrous ethanol as a solvent, preparing the sample into sample solutions with different test concentrations (specifically, the test concentrations are 2.5wt%, 5wt%, and 10wt%).

[0102] Step S4-3, prepare a positive control solution: use vitamin C (CAS No. 50-81-7) as a positive control, use anhydrous ethanol as a solvent, and prepare a positive control solution with a concentration of 1 mg / mL of vitamin C; store at 0-4° C. away from light and use it immediately after preparation.

[0103] Step S4-4, prepare three EP tubes and record them as tube A, tube B, and tube C. Add reagents to tube A, tube B, and tube C respectively according to Table 5; then, react at room temperature for 30 minutes. After the reaction is completed, use an ELISA reader to measure the absorbance (OD) of the solution in tube A, tube B, and tube C at 517nm and record them as OD A管 OD B管 OD C管 .

[0104] Table 5: Material-liquid ratio in tube A, tube B and tube C in DPPH free radical scavenging experiment:

[0105] serial number DPPH ethanol solution Anhydrous ethanol Sample solution or positive control solution Total volume A-tube 1mL —— 1mL 2mL B tube 1mL 1mL —— 2mL C-tube — 1mL 1mL 2mL

[0106] Step S4-5, calculating the scavenging rate of the sample solution or the positive control solution on the DPPH free radical, the calculation formula of the scavenging rate of the DPPH free radical is:

[0107] Among them, the DPPH free radical scavenging rate of the positive control solution is 96.36%; the test results of the DPPH free radical scavenging rate of the sample solution are shown in Table 6.

[0108] Table 6 Test results of DPPH free radical scavenging rate of samples:

[0109]

[0110]

[0111] As can be seen from Table 6, the Roselle oil extract and the Roselle polypeptide extract obtained by "first undergoing supercritical fluid extraction treatment, then fermentation treatment and enzymatic hydrolysis treatment" in the present application both have a high DPPH free radical scavenging rate. In particular, the Roselle polypeptide extract obtained by the combination of "supercritical CO2 fluid extraction treatment, brewer's yeast fermentation treatment, acid protease enzymatic hydrolysis treatment and composite protease enzymatic hydrolysis treatment" has the best DPPH free radical scavenging rate.

[0112] Test Example 5 Determination of the inhibition rate of the sample on cyclooxygenase 2 (COX-2):

[0113] In this test example, a COX-2 enzyme linked immunosorbent assay (ELISA) detection kit is used to detect the COX-2 inhibition rate. The detection process includes the following steps:

[0114] S5-1. Use a 96-well black board and set up control wells (including blank control, 100% enzyme activity control, positive inhibitor control) and sample wells, and add each reagent to the wells of the 96-well black board from top to bottom in sequence according to Table 6; after adding the sample, mix well and incubate at 37°C for 10 minutes.

[0115] Table 7 Amount of each reagent added to the control well and sample well:

[0116]

[0117] S5-2. Add COX-2 Probe (5 μL) to each well.

[0118] S5-3, add COX-2 substrate working solution (5 μL) to each well, incubate at 37°C in the dark for 5 minutes; after the incubation, measure the fluorescence intensity (RFU) with a microplate reader under the conditions of excitation wavelength of 560 nm and emission wavelength of 590 nm, and record it as RFU 空白对照 、RFU 100％酶活性对照 、RFU 阳性抑制剂对照 、RFU 样本 .

[0119] S5-4. Calculate the COX-2 inhibition rate of the sample, where the calculation formula is:

[0120]

[0121] Table 8 Test results of COX-2 inhibition rate of samples:

[0122]

[0123] As can be seen from Table 8, the Roselle oil extract and the Roselle polypeptide extract obtained by "first undergoing supercritical fluid extraction treatment, then undergoing fermentation treatment and enzymatic hydrolysis treatment" in the present application both have a high COX-2 inhibition rate. In particular, the Roselle polypeptide extract obtained by the combination of "supercritical CO2 fluid extraction treatment, brewer's yeast fermentation treatment, acid protease enzymatic hydrolysis treatment and composite protease enzymatic hydrolysis treatment" has the best inhibition rate on COX-2.

[0124] Test Example 6 Determination of the activity inhibition rate of the sample on elastase:

[0125] In this test example, the determination process of the elastase activity inhibition rate specifically includes the following steps:

[0126] Step S6-1, preparing PBS buffer: adjusting the pH value of PBS (1X) solution to 6.5 with 1 mol / L HCl aqueous solution and 0.1 mol / L HCl aqueous solution.

[0127] Step S6-2, preparing an elastase solution: using PBS buffer, elastase (derived from porcine pancreas, purchased from Shanghai MacLean Biochemical Technology Co., Ltd.) was prepared into an elastase solution with a concentration of 5 μg / mL.

[0128] Step S6-3, preparing substrate solution: using Tris-HCl buffer solution (0.1 mol / L, pH=6.5) to prepare substrate solution with a concentration of 1 mmol / L, wherein the substrate is N-succinyl-alanine-alanine-alanine-p-nitroaniline (CAS No. 52299-14-6), purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.

[0129] Step S6-4, preparing a sample solution: using a Tris-HCl buffer solution (0.1 mol / L, pH=6.5) to prepare a sample solution with a concentration of 2 wt%.

[0130] Step S6-5, using a 96-well plate and setting wells A, B and C, respectively, adding each reagent to wells A, B and C of the 96-well plate from top to bottom according to Table 9; then, incubating for 60 minutes. After the incubation is completed, the absorbance (OD) is measured at 405nm using an ELISA reader and recorded as OD A孔 OD B孔 OD C孔 .

[0131] Table 9 The amount of each reagent added to well A, well B and well C:

[0132] Reagents A hole B hole C hole Elastase solution 25μL 25μL 25μL Sample solution 50μL 50μL — Tris-HCl buffer (0.1 mol / L, pH = 6.5) — 15μL 50μL Substrate solution 15μL — 15μL Buffer 10μL 10μL 10μL

[0133] Step S6-6, calculating the inhibition rate of elastase activity, the calculation formula is as follows:

[0134]

[0135] Table 10 Test results of the inhibition rate of samples on elastase activity:

[0136]

[0137] As can be seen from Table 10, the Roselle oil extract and the Roselle polypeptide extract obtained by the combination of "supercritical CO2 fluid extraction treatment, brewer's yeast fermentation treatment, acid protease enzymatic treatment and composite protease enzymatic treatment" in the present application both have a high activity inhibition rate against elastase. It can be seen that the Roselle oil extract and the Roselle polypeptide extract of the present application both have a certain anti-wrinkle and firming effect on the skin.

[0138] Test Example 7: Determination of the antibacterial rate of the sample against Propionibacterium acnes:

[0139] In this test example, the operation process of the Propionibacterium acnes antibacterial experiment specifically includes the following steps:

[0140] Step S7-1, use a sterile 96-well bacterial culture plate, and set up test wells, control wells, blank control wells, negative control wells and positive control wells. Among them, add diluted bacterial suspension (100μL) and sample solution (100μL) to the test well; add blister culture medium (100μL) and sample solution (100μL) to the control well; add blister culture medium (200μL) to the blank control well; add diluted bacterial suspension (100μL) and blister culture medium (100μL) to the negative control well; add diluted bacterial suspension (100μL) and a solution of sodium penicillin at a concentration of 200μg / mL (100μL, the solvent is PBS buffer) to the positive control well. Make three replicate wells for each.

[0141] In this test example, PBS buffer was used as a solvent to prepare sample solutions with different test concentrations (specifically, the test concentrations were 0.5 wt %, 1 wt %, 3 wt % and 5 wt %).

[0142] Step S7-2, placing the 96-well bacterial culture plate in a 2.5L anaerobic tank and culturing at room temperature for 24 hours; then, measuring the absorbance (OD) at 600nm, and recording it as OD 试验孔 OD 对照孔 OD 空白对照孔 OD 阴性对照孔 OD 阳性对照孔 .

[0143] Step S7-3, calculating the inhibition rate of Propionibacterium acnes. The calculation formula of the inhibition rate of Propionibacterium acnes is as follows:

[0144]

[0145] Table 11 Test results of the inhibition rate of Propionibacterium acnes of samples:

[0146]

[0147] It can be seen from Table 11 that the Roselle oil extract and Roselle polypeptide extract obtained by the combination of "supercritical CO2 fluid extraction treatment, brewer's yeast fermentation treatment, acid protease enzymatic hydrolysis treatment and composite protease enzymatic hydrolysis treatment" adopted in the present application have a high active inhibition rate against Propionibacterium acnes.

[0148] Test Example 8: Determination of the antibacterial rate of samples against Malassezia:

[0149] Step S8-1, use a sterile 96-well bacterial culture plate, and set up test wells, control wells, blank control wells, negative control wells and positive control wells. Among them, add diluted bacterial suspension (100 μL) and sample solution (100 μL) to the test well; add malt juice medium (100 μL) and sample solution (100 μL) to the control well; add malt juice medium (200 mL) to the blank control well; add diluted bacterial suspension (100 μL) and malt juice medium (100 mL) to the negative control well; add diluted bacterial suspension (100 μL) and ketoconazole solution (100 μL, solvent is PBS buffer) at a concentration of 4 μg / mL to the positive control well. Make 3 duplicate wells for each.

[0150] In this test example, PBS buffer was used as a solvent to prepare sample solutions with different test concentrations (specifically, the test concentrations were 0.5 wt %, 1 wt %, 3 wt % and 5 wt %).

[0151] Step S8-2, placing the 96-well bacterial culture plate in a 2.5L anaerobic tank and culturing at room temperature for 24 hours; then, measuring the absorbance (OD) at 600nm, and recording it as OD 试验孔 OD 对照孔 OD 空白对照孔 OD 阴性对照孔 OD 阳性对照孔 .

[0152] Step S8-3, calculating the Malassezia inhibition rate, the calculation formula of the Malassezia inhibition rate is as follows:

[0153]

[0154] Table 12 Test results of the inhibition rate of samples on Malassezia:

[0155]

[0156] As can be seen from Table 12, the Roselle oil extract and Roselle polypeptide extract obtained by the combination of "supercritical CO2 fluid extraction treatment, brewer's yeast fermentation treatment, acid protease enzymatic hydrolysis treatment and composite protease enzymatic hydrolysis treatment" adopted in the present application both have a high active inhibition rate against Malassezia.

[0157] Test Example 9: Determination of the antibacterial rate of the sample against Fusobacterium nucleatum and Streptococcus mutans:

[0158] Step S9-1, use a sterile 96-well bacterial culture plate, and set up test wells, control wells, blank control wells, negative control wells and positive control wells. Among them, add diluted bacterial suspension (100 μL) and sample solution (100 μL) to the test well; add malt juice medium (100 μL) and sample solution (100 μL) to the control well; add malt juice medium (200 mL) to the blank control well; add diluted bacterial suspension (100 μL) and malt juice medium (100 mL) to the negative control well; add diluted bacterial suspension (100 μL) and sodium penicillin solution (100 μL, solvent is PBS buffer) with a concentration of 4 μg / mL to the positive control well. Make 3 duplicate wells for each.

[0159] In this test example, PBS buffer was used as a solvent to prepare sample solutions with different test concentrations (specifically, the test concentrations were 1.25wt%, 2.5wt%, 5wt% and 10wt%).

[0160] Step S9-2, placing the 96-well bacterial culture plate in a 2.5L anaerobic jar and culturing at room temperature for 24 hours; then, measuring the absorbance (OD) at 600nm, and recording it as OD 试验孔 OD 对照孔 OD 空白对照孔 OD 阴性对照孔 OD阳性对照孔 .

[0161] Step S9-3, calculating the inhibition rate of Fusobacterium nucleatum and Streptococcus mutans, the calculation formula is as follows:

[0162]

[0163] Table 13 Test results of the inhibition rate of samples on Fusobacterium nucleatum:

[0164]

[0165] Table 14 Test results of the inhibition rate of samples on Streptococcus mutans:

[0166]

[0167] It can be seen from Tables 13 and 14 that the Roselle oil extract obtained by the combination of "supercritical CO2 fluid extraction treatment, brewer's yeast fermentation treatment, acid protease enzymatic hydrolysis treatment and composite protease enzymatic hydrolysis treatment" adopted in the present application has a high active inhibition rate against Fusobacterium nucleatum and Streptococcus mutans.

[0168] Test Example 10 Study on the promoting effect of samples on integrin α2β1:

[0169] In this test case, the research method specifically includes the following steps:

[0170] S10-1. Safe concentration screening of human skin fibroblasts (HFF):

[0171] Human skin fibroblasts were cultured at 2×10 5 The cells were inoculated at a density of 100 μL / mL in a 96-well plate, and 100 μL was added to each well. The edge wells were filled with sterile PBS buffer. After incubation in an incubator (37°C, 5% CO2), the samples were attached to the wall. After the 96-well plate was incubated in an incubator (37°C, 5% CO2) for 24 hours, the culture medium was aspirated, and 100 μL of the sample solution was added (3 replicates for each concentration). 100 μL of serum-free culture medium solution was added to the blank group, and dimethyl sulfoxide was added to the control group; then, the plate was placed in an incubator (37°C, 5% CO2) and incubated for 24 hours. After the incubation, the culture was terminated; 10 μL of sincalide (CAS No. 25126-32-3, referred to as CCK-8) was added to each well of the 96-well plate in the dark; after the 96-well plate was incubated in an incubator (37°C, 5% CO2) for 1 hour, the color of the culture medium changed from light pink to orange-yellow. At this time, the OD value was measured at a wavelength of 450nm using an enzyme reader and recorded as OD 样本 and OD 空白 .

[0172] The cell survival rate was calculated using the following formula: Among them, cell activity is proportional to the OD value, and when the cell survival rate is greater than 80%, it is the safe concentration of the sample.

[0173] In this test, the sample solution uses PBS buffer as a solvent to prepare sample solutions with different test concentrations (specifically, the test concentrations are 1.25 μg / mL, 2.5 μg / mL, 5 μg / mL, and 10 μg / mL).

[0174] Table 15 Test results of cell viability of sample solutions with different test concentrations:

[0175]

[0176] As can be seen from Table 15, the safe concentration for human skin fibroblasts (HFF) is 5 μg / mL.

[0177] S10-2, integrin α2β1 content detection:

[0178] Three experimental groups were set up, namely blank control group, model group and sample group. HFF cells were cultured at 2×10 5 The cells were inoculated in a 24-well plate at a density of 1 mL / well. When the HFF cell fusion reached 80% to 90%, the culture medium was removed and the cells were washed twice with PBS buffer. After that, serum-free culture medium (1 mL) was added to the blank group and the model group, and sample solution (1 mL) with a concentration of 5 μg / mL was added to the sample group. After that, the 24-well plate was placed in an incubator (37°C, 5% CO2) for 6 hours. Finally, the model group and the sample group were stimulated by ultraviolet radiation b (UVB) and placed in an incubator (37°C, 5% CO2) for 18 hours, and the supernatant was collected.

[0179] S10-3. Use ELISA kit to detect the expression level of integrin α2β1.

[0180] Table 16 Detection results of the expression level of integrin α2β1:

[0181] Group Integrated α2β1 content, ng / mL Blank control group 16.76±0.12 Model Group 11.24±0.26 Sample group (Example 5 Roselle polypeptide extract) 14.31±0.11

[0182] As can be seen from Table 16, the Hibiscus sabdariffa polypeptide extract obtained by the combination of "supercritical CO2 fluid extraction treatment, brewer's yeast fermentation treatment, acid protease enzymatic treatment and composite protease enzymatic treatment" adopted in the present application has a promoting effect on the secretion of integrin α2β1 after UVB damage.

[0183] The above is a detailed introduction to an extract based on Roselle oil and Roselle polypeptide provided by the present application, as well as its extraction method and use. This article uses specific examples to illustrate the principles and implementation methods of the present application. The description of the above embodiments is only used to help understand the method and core idea of ​​the present application. It should be pointed out that for those skilled in the art, without departing from the principles of the present application, several improvements and modifications can be made to the present application, and these improvements and modifications also fall within the scope of protection of the claims of the present application.

Claims

1. A method for extracting an extract based on Roselle oil and Roselle polypeptide, characterized in that: The extraction method comprises the following steps: Step S1, the roselle calyx is subjected to a crushing treatment and a supercritical fluid extraction treatment in sequence to obtain a roselle oil extract and flower residue; Step S2, the flower residue is subjected to fermentation treatment and enzymolysis treatment in sequence to obtain a Roselle polypeptide extract; wherein the strain used in the fermentation treatment is selected from at least one of Saccharomyces cerevisiae, Bacillus subtilis and Lipomyces staryi; the enzyme used in the enzymolysis treatment is selected from at least one of an acidic protease and a composite protease; The extract comprises the Roselle oil extract and the Roselle polypeptide extract.

2. The extraction method according to claim 1, characterized in that In step S1, the supercritical fluid extraction process is selected from supercritical carbon dioxide fluid extraction process; And / or, the conditions of the supercritical fluid extraction treatment in the extraction section are: extraction temperature is (50-60)°C, and extraction pressure is (20-40)Mpa.

3. The extraction method according to claim 1, characterized in that In step S1, the supercritical fluid extraction treatment is performed by a analytical method in the analytical section selected from a variable temperature and pressure method; And / or, the conditions of the supercritical fluid extraction treatment in the analytical section are: separation I pressure is (19-21) MPa, separation I temperature is (58-62)°C; separation II pressure is (4-6) MPa, separation II temperature is (48-52)°C.

4. The extraction method according to claim 1, characterized in that In step S2, the temperature of the fermentation treatment is (25-35)°C.

5. The extraction method according to claim 1 or 4, characterized in that In step S2, the bacterial species used for the fermentation treatment is selected from one of Saccharomyces cerevisiae, Bacillus subtilis and Lipomyces staryi.

6. The extraction method according to claim 1, characterized in that In step S2, the enzymatic hydrolysis treatment includes a first enzymatic hydrolysis treatment and a second enzymatic hydrolysis treatment performed sequentially; the enzyme used in the first enzymatic hydrolysis treatment and the enzyme used in the second enzymatic hydrolysis treatment are each independently selected from an acidic protease or a composite protease, and the enzyme used in the first enzymatic hydrolysis treatment and the enzyme used in the second enzymatic hydrolysis treatment are different.

7. The extraction method according to claim 6, characterized in that The enzyme used in the first enzymatic treatment is selected from acidic proteases, and the enzyme used in the second enzymatic treatment is selected from composite proteases.

8. The extraction method according to claim 5 or 6, characterized in that The working conditions of the acidic protease are: temperature of (45-55)°C and pH of 2.0-4.0; And / or, the working conditions of the composite protease are: temperature of (50-60)°C and pH of 8.0-9.

0.

9. An extract based on Roselle oil and Roselle polypeptide, characterized in that: The extract is obtained by the extraction method according to any one of claims 1 to 8.

10. Use of the extract according to claim 9 in preparing a product having anti-free radical oxidation, and / or anti-inflammatory, and / or antibacterial, and / or inhibiting elastase activity, and / or promoting integrin α2β1 expression; Optionally, the anti-inflammatory includes anti-inflammation caused by cyclooxygenase 2; Optionally, the antibacterial agents include anti-Propionibacterium acnes, anti-Malassezia, anti-Fusobacterium nucleatum and anti-Streptococcus mutans.

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