A kind of okra extract and its extraction method and use

Abstract

The application provides a kind of okra extract and its extraction method and use.The extraction method includes the following steps: S1, okra calyx is first subjected to crushing treatment, then subjected to extraction treatment, to obtain okra flower residue;S2, the okra flower residue is sequentially subjected to fermentation treatment, enzymatic hydrolysis treatment and filtration treatment, to obtain the okra extract;The strain used in the fermentation treatment is selected from at least one of saccharomyces cerevisiae, bacillus subtilis and lipomyces starkeyi;Wherein, the enzyme used in the enzymatic hydrolysis treatment is selected from one of alkaline protease, cellulase and pectinase.The extraction method of the application extracts active ingredients from okra flower residue, and the obtained okra extract has a high yield and a high polypeptide content.

Description

Technical Field

[0001] This application relates to the technical field of extracting active ingredients from the calyx of Hibiscus mutabilis, specifically to an extract of Hibiscus mutabilis, its extraction method, and its uses. Background Technology

[0002] Hibiscus mutabilis L. is a plant belonging to the genus Hibiscus in the Malvaceae family. It has a slightly pungent taste and neutral properties, and enters the lung and liver meridians. It possesses the effects of clearing heat and detoxifying, reducing swelling and draining pus, and cooling the blood and stopping bleeding. It is mainly used to treat carbuncles, boils, shingles, red and swollen eyes, and traumatic injuries. The flowers, leaves, and roots of Hibiscus mutabilis can all be used medicinally. Among them, the flowers are rich in flavonoids, such as quercetin, kaempferol, guavaside, crassamol, isoquercitrin, rutin, quercetin xanthoside, hyperoside, quercetin-3-O-moribiglycoside, cyanidin, and cyanidin diglycoside.

[0003] Currently, conventional methods for extracting active ingredients from the calyx of Hibiscus mutabilis include supercritical fluid extraction, microwave extraction, and alcohol extraction (e.g., ethanol, 1,3-butanediol). However, the Hibiscus mutabilis flower residue obtained by supercritical fluid extraction, microwave extraction, or alcohol extraction (e.g., ethanol, 1,3-butanediol) still contains a certain amount of active ingredients. Summary of the Invention

[0004] In view of this, this application provides a hibiscus extract, its extraction method, and its uses. The extraction method of this application extracts the active ingredients from hibiscus flower residue, and the resulting hibiscus extract has a high yield and a high peptide content.

[0005] In a first aspect, this application provides a method for extracting an extract from Hibiscus mutabilis, the extraction method comprising the following steps:

[0006] Step S1: The calyx of the hibiscus flower is first crushed and then extracted to obtain hibiscus flower residue;

[0007] The hibiscus flower residue obtained in steps S2 and S1 is subjected to fermentation and enzymatic hydrolysis in sequence to obtain hibiscus extract; the strains used in the fermentation are selected from at least one of Saccharomyces cerevisiae, Bacillus subtilis and Saccharomyces davidii; the enzymes used in the enzymatic hydrolysis are selected from one of alkaline protease, cellulase and pectinase.

[0008] In some embodiments, in step S1, the calyx of the Hibiscus mutabilis is selected from at least one of dried calyx and fresh calyx of Hibiscus mutabilis. Further, in step S1, the calyx of the Hibiscus mutabilis is selected from dried calyx of Hibiscus mutabilis.

[0009] In some embodiments, in step S1, the extraction process is selected from at least one of supercritical fluid extraction, ethanol extraction, and 1,3-butanediol extraction.

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

[0011] In some embodiments, the supercritical fluid extraction process is selected from supercritical carbon dioxide fluid extraction processes.

[0012] In some embodiments, the supercritical fluid extraction process is performed under the following conditions in the extraction section: extraction temperature is (50-60)℃ (e.g., 50℃, 51℃, 52℃, 53℃, 54℃, 55℃, 56℃, 57℃, 58℃, 59℃, or 60℃, etc.), and extraction pressure is (20-40) MPa (e.g., 20 MPa, 21 MPa, 22 MPa, 23 MPa, 24 MPa, 25 MPa, 26 MPa, 27 MPa, 28 MPa, 29 MPa, 30 MPa, 31 MPa, 32 MPa, 33 MPa, 34 MPa, 35 MPa, 36 MPa, 37 MPa, 38 MPa, 39 MPa, or 40 MPa, etc.).

[0013] In some embodiments, the extraction temperature is (53-57)°C. Further, the extraction temperature is 55°C.

[0014] In some embodiments, the extraction pressure is (25-35) MPa. Further, the extraction pressure is 30 MPa.

[0015] In some embodiments, the supercritical fluid extraction process has an extraction time of 2 to 5 hours in the extraction section (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 supercritical fluid extraction process has an extraction time of 3 hours in the extraction section.

[0016] In some embodiments, the analytical method in the analytical section of the supercritical fluid extraction process is selected from the variable temperature and pressure method.

[0017] In some embodiments, the supercritical fluid extraction process is performed under the following conditions in the analytical section: Separation I pressure is (19–21) MPa (e.g., 19 MPa, 19.5 MPa, 20 MPa, 20.5 MPa, or 21 MPa, etc.), and 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.), and Separation II temperature is (48–52) °C (e.g., 48 °C, 49 °C, 50 °C, 51 °C, or 52 °C, etc.). Further, the supercritical fluid extraction process is performed under the following conditions in the analytical section: Separation I pressure is 20 MPa, and Separation I temperature is 60 °C; Separation II pressure is 5 MPa, and Separation II temperature is 50 °C.

[0018] In some embodiments, the fermentation temperature in step S2 is (25-35)℃; for example, 25℃, 26℃, 27℃, 28℃, 29℃, 30℃, 31℃, 32℃, 33℃, 34℃, or 35℃. Further, in step S2, the fermentation temperature is 28℃.

[0019] In some embodiments, in step S2, the microbial strain used in the fermentation process is selected from one of Saccharomyces cerevisiae, Bacillus subtilis, and Saccharomyces davidii.

[0020] In some embodiments, in step S2, the microbial strain used for fermentation is selected from *Saccharomyces cerevisiae*. Further, the strain number of the *Saccharomyces cerevisiae* is CICC 32883.

[0021] In some embodiments, in step S2, the bacterial strain used for the fermentation treatment is selected from Bacillus subtilis. Further, the strain number of the Bacillus subtilis is CICC 24713.

[0022] In some embodiments, in step S2, the microbial strain used for fermentation is selected from *Saccharomyces davidii*. Further, the strain number of *Saccharomyces davidii* is 2.2291.

[0023] In some embodiments, in step S2, the enzyme used in the enzymatic hydrolysis is selected from alkaline proteases.

[0024] In some embodiments, the alkaline protease operates under the following conditions: a temperature of (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 a pH value of 10.5–11.5 (e.g., 10.5, 10.6, 10.7, 10.8, 10.9, 11.0, 11.1, 11.2, 11.3, 11.4, or 11.5, etc.). Further, the alkaline protease operates under the following conditions: a temperature of 50°C and a pH value of 11.0.

[0025] In some embodiments, in step S2, the enzyme used in the enzymatic hydrolysis is selected from cellulase.

[0026] In some embodiments, the cellulase operates under the following conditions: a temperature of (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 a pH of 4.3–5.3 (e.g., 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, or 5.3, etc.). Further, the alkaline protease operates under the following conditions: a temperature of 50°C and a pH of 4.8.

[0027] In some embodiments, in step S2, the enzyme used in the enzymatic hydrolysis is selected from pectinase.

[0028] In some embodiments, the operating conditions of the pectinase are: a temperature of (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 a pH value of 3.5-4.5 (e.g., 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, or 4.5, etc.). Further, the operating conditions of the pectinase are: a temperature of 50°C and a pH value of 4.0.

[0029] In a second aspect of this application, this application provides a hibiscus extract, which is obtained by the extraction method described in the first aspect of this application.

[0030] In a third aspect of this application, this application provides the use of the hibiscus extract described in the first aspect of this application in the preparation of products having antioxidant, and / or antibacterial, and / or elastase-inhibiting, and / or integrin α2β1 expression-promoting properties.

[0031] This application has the following beneficial effects:

[0032] First, this application employs a combination of fermentation and enzymatic hydrolysis to extract active ingredients from Hibiscus mutabilis flower residue, resulting in a high yield and high polypeptide content in the extracted Hibiscus mutabilis extract. In particular, the combination of supercritical CO2 fluid extraction, Bacillus subtilis fermentation, and alkaline protease hydrolysis significantly improves both the yield and polypeptide content of the Hibiscus mutabilis extract when extracting active ingredients from the calyx.

[0033] Secondly, this application uses a combination of "supercritical fluid extraction followed by fermentation and enzymatic hydrolysis" to extract active ingredients from the calyx of Hibiscus mutabilis. The resulting Hibiscus mutabilis extract exhibits high inhibition rates against Malassezia, Propionibacterium acnes, elastase activity, COX-2, DPPH free radical scavenging, and integrated α2β1 expression. In particular, it significantly enhances the DPPH free radical scavenging and COX-2 inhibition rates of the Hibiscus mutabilis extract. Detailed Implementation

[0034] This application discloses an extract of Hibiscus mutabilis, its extraction method, and its uses. Those skilled in the art can refer to this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this application. The methods and applications of this application have been described through preferred embodiments. Those skilled in the art can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this application to realize and apply the technology of this application.

[0035] To make the objectives, technical solutions, and advantages of this application clearer, the implementation schemes of this application will be further described in detail below with reference to the embodiments.

[0036] Unless otherwise stated, the strain number of Saccharomyces cerevisiae used in this application is CICC 32883, and it was purchased from the China Industrial Microbial Culture Collection Center.

[0037] Unless otherwise stated, the strain number of Bacillus subtilis used in this application is CICC24713, and it was purchased from the China Industrial Microbial Culture Collection Center.

[0038] Unless otherwise stated, the strain number of Lipomyces starkeyi used in this application is 2.2291, and it was purchased from the China General Microbiological Culture Collection Center.

[0039] Examples 1-5: The extraction method of Hibiscus mutabilis extract includes steps S1 and S2.

[0040] The specific operation process of step S1 in Examples 1-5 is as follows: The dried calyxes of Hibiscus mutabilis are pulverized into powder using a pulverizer. 25g of the powdered dried calyxes are placed in an extraction vessel for supercritical fluid extraction (extraction conditions: extraction temperature 55℃, extraction pressure 30MPa, extraction time 3 hours; analysis conditions: variable temperature and pressure method, separation I pressure 20MPa, separation I temperature 60℃, separation II pressure 5MPa, separation II temperature 50℃). The extracted paste-like solid and caprylic / capric triglyceride (CAS No. 65381-09-1, abbreviated as GTCC) are compounded at a weight ratio of 1:20 to obtain the supercritical extract of Hibiscus mutabilis. The residue in the extraction vessel is filtered using a Buchner funnel to obtain Hibiscus mutabilis flower residue. The obtained Hibiscus mutabilis flower residue is divided into five equal portions, and each of the five portions is then subjected to step S2 in Examples 1-5.

[0041] The specific procedure for step S2 in Example 1 is as follows: Place one portion of Hibiscus mutabilis flower residue in an Erlenmeyer flask (250 mL), then add yeast extract peptone glucose medium (5 g, also known as YPD medium or YEPD medium) and purified water (100 mL), and sterilize by sonication for 20 minutes; after cooling to 28°C, inoculate with Bacillus subtilis (inoculation amount 2 wt%, inoculum concentration after inoculation is 1 × 10⁻⁶). 6 Fermentation was carried out (CFU / mL) under the following conditions: temperature 28℃, rotation speed 200 r / min, fermentation time 4 days. After fermentation, the temperature was first raised to 50℃ and the pH was adjusted to 11.0. Alkaline protease (2wt%) was added and enzymatically hydrolyzed for 2 hours at 50℃ and pH 11.0. Then the temperature was raised to 90℃ and held at 90℃ for 15 minutes. After enzymatic hydrolysis, the mixture was cooled to room temperature and then subjected to coarse filtration through 300-mesh gauze, fine filtration through a membrane separation system (the membrane used was SPPM-C-10 with a flux of 6.0 L / h), concentration under reduced pressure to a normal volume, nanofiltration dialysis for 48 hours (dialysis bag specification 200 Da), and spray drying to obtain the Hibiscus mutabilis extract.

[0042] The specific procedure for step S2 in Example 2 is as follows: Place one portion of Hibiscus mutabilis flower residue in an Erlenmeyer flask (250 mL), then add yeast extract peptone glucose medium (5 g, also known as YPD medium or YEPD medium) and purified water (100 mL), and sterilize by ultrasonication for 20 minutes; after cooling to 28°C, inoculate with Saccharomyces cerevisiae (inoculation amount 2 wt%, inoculum concentration after inoculation is 1 × 10⁻⁶). 6Fermentation was carried out (CFU / mL) under the following conditions: temperature 28℃, rotation speed 200 r / min, fermentation time 4 days. After fermentation, the temperature was first raised to 50℃ and the pH was adjusted to 11.0. Alkaline protease (2wt%) was added at 50℃ and pH 11.0 and enzymatic hydrolysis was carried out for 2 hours. Then the temperature was raised to 90℃ and held at 90℃ for 15 minutes. After enzymatic hydrolysis, the mixture was cooled to room temperature and then subjected to coarse filtration through 300-mesh gauze, fine filtration through a membrane separation system (the membrane used was SPPM-C-10 with a membrane flux of 6.0 L / h), concentration under reduced pressure to a normal volume, nanofiltration dialysis for 48 hours (dialysis bag specification 200 Da), and spray drying to obtain Hibiscus mutabilis extract.

[0043] The specific operation process of step S2 in Example 3 is as follows: Place one portion of Hibiscus mutabilis flower residue in a conical flask (250 mL), then add yeast extract peptone glucose medium (5 g, also known as YPD medium or YEPD medium) and purified water (100 mL), and sterilize by ultrasonication for 20 minutes; after cooling to 28°C, inoculate with *Saccharomyces cerevisiae* (inoculation amount 2 wt%, post-inoculation concentration 1 × 10⁻⁶). 6 Fermentation was carried out (CFU / mL) under the following conditions: temperature 28℃, rotation speed 200 r / min, fermentation time 4 days. After fermentation, the temperature was first raised to 50℃ and the pH was adjusted to 11.0. Alkaline protease (2wt%) was added and enzymatically hydrolyzed for 2 hours at 50℃ and pH 11.0. Then the temperature was raised to 90℃ and held at 90℃ for 15 minutes. After enzymatic hydrolysis, the mixture was cooled to room temperature and then subjected to coarse filtration through 300-mesh gauze, fine filtration through a membrane separation system (the membrane used was SPPM-C-10 with a flux of 6.0 L / h), concentration under reduced pressure to a normal volume, nanofiltration dialysis for 48 hours (dialysis bag specification 200 Da), and spray drying to obtain the Hibiscus mutabilis extract.

[0044] The specific procedure for step S2 in Example 4 is as follows: Place one portion of Hibiscus mutabilis flower residue in an Erlenmeyer flask (250 mL), then add yeast extract peptone glucose medium (5 g, also known as YPD medium or YEPD medium) and purified water (100 mL), and sterilize by sonication for 20 minutes; after cooling to 28°C, inoculate with Bacillus subtilis (inoculation amount 2 wt%, inoculum concentration after inoculation is 1 × 10⁻⁶). 6Fermentation was carried out (CFU / mL) under the following conditions: temperature 28℃, rotation speed 200 r / min, fermentation time 4 days. After fermentation, the temperature was first raised to 50℃ and the pH was adjusted to 4.8. Cellulase (2wt%) was added at 50℃ and pH 4.8 and enzymatic hydrolysis was performed for 2 hours. Then the temperature was raised to 90℃ and held at 90℃ for 15 minutes. After enzymatic hydrolysis, the mixture was cooled to room temperature and then subjected to coarse filtration through 300-mesh gauze, fine filtration through a membrane separation system (the membrane used was SPPM-C-10 with a flux of 6.0 L / h), concentration under reduced pressure to a normal volume, nanofiltration dialysis for 48 hours (dialysis bag specification 200 Da), and spray drying to obtain Hibiscus mutabilis extract.

[0045] The specific procedure for step S2 in Example 5 is as follows: Place a portion of Hibiscus mutabilis flower residue in an Erlenmeyer flask (250 mL), then add yeast extract peptone glucose medium (5 g, also known as YPD medium or YEPD medium) and purified water (100 mL), and sterilize by sonication for 20 minutes; after cooling to 28°C, inoculate with Bacillus subtilis (inoculation amount 2 wt%, inoculum concentration after inoculation is 1 × 10⁻⁶). 6 Fermentation was carried out (CFU / mL) under the following conditions: temperature 28℃, rotation speed 200 r / min, fermentation time 4 days. After fermentation, the temperature was first raised to 50℃ and the pH was adjusted to 4.0. Pectinase (2wt%) was added at 50℃ and pH 4.0 and enzymatic hydrolysis was performed for 2 hours. Then the temperature was raised to 90℃ and held at 90℃ for 15 minutes. After enzymatic hydrolysis, the mixture was cooled to room temperature and then subjected to coarse filtration through 300-mesh gauze, fine filtration through a membrane separation system (the membrane used was SPPM-C-10 with a flux of 6.0 L / h), concentration under reduced pressure to a normal volume, nanofiltration dialysis for 48 hours (dialysis bag specification 200 Da), and spray drying to obtain Hibiscus mutabilis extract.

[0046] Example 6: The extraction method of Hibiscus mutabilis extract includes steps S1 and S2.

[0047] Step S1: Crush the calyx of Hibiscus mutabilis into powder using a pulverizer. Mix the powdered calyx (5g) with 100g of a 75% ethanol aqueous solution to obtain a mixture. Then, sonicate the mixture at 100W for 2 hours to obtain an extract. Immediately afterward, filter the extract using a Buchner funnel to obtain a filtrate and a residue (i.e., Hibiscus mutabilis flower residue). Ethanol is evaporated from the filtrate at 45°C using a rotary evaporator to obtain a crude ethanol extract of Hibiscus mutabilis flowers. Add purified water to the crude ethanol extract of Hibiscus mutabilis flowers to 70% of the volume of the mixture, and then add 1,3-butanediol to 100% of the volume of the mixture. Afterward, filter the mixture using a Buchner funnel to obtain a supercritical extract of Hibiscus mutabilis.

[0048] Step S2 is the same as in Example 1.

[0049] Example 7: The extraction method of Hibiscus mutabilis extract includes steps S1 and S2.

[0050] Step S1: Use a grinder to grind the calyx of Hibiscus mutabilis into powder. Mix the powdered calyx (5g) with 100g of 30% 1,3-butanediol aqueous solution and reflux for 2.5 hours. After that, after the reaction solution is cooled to room temperature, filter it through a Buchner funnel to obtain the filtrate (i.e., Hibiscus mutabilis supercritical extract) and the filter residue (i.e., Hibiscus mutabilis flower residue).

[0051] Step S2 is the same as in Example 1.

[0052] The weight of the calyx of the Hibiscus mutabilis flower is denoted as W1 (g), the weight of the Hibiscus mutabilis extract is denoted as W2 (g), and the yield of the Hibiscus mutabilis extract is denoted as y (wt%). The formula for calculating the yield of the Hibiscus mutabilis extract is as follows:

[0053] Table 1. Condition selection and yield of Hibiscus mutabilis extract in Examples 1-7:

[0054]

[0055] Test Example 1: Determination of total flavonoid content in supercritical extract of Hibiscus mutabilis:

[0056] In this test example, the method for detecting the total flavonoid content specifically includes the following steps:

[0057] Step S1-1, Construction of the standard curve:

[0058] Using rutin (CAS No. 153-18-4) as a standard, weigh 0.1 g of rutin and place it in a 100 mL volumetric flask. First, add 75% ethanol aqueous solution to dissolve the standard, and then add 75% ethanol aqueous solution to make up to 100 mL to obtain a rutin standard solution with a concentration of 1.0 g / L.

[0059] Pipettes of 0.0 mL, 2.0 mL, 4.0 mL, 6.0 mL, 8.0 mL, and 10.0 mL of 1.0 g / L rutin standard solution were placed into 10 mL volumetric flasks and diluted to 10 mL with 75% ethanol aqueous solution to obtain rutin standard solutions with gradient concentrations of 0.0 mg / mL, 0.2 mg / mL, 0.4 mg / mL, 0.6 mg / mL, 0.8 mg / mL, and 1.0 mg / mL.

[0060] Pipe 1 mL of rutin standard solutions of varying concentrations into a 10 mL volumetric flask. First, add 4 mL of deionized water and 0.3 mL of 5% NaNO2 aqueous solution. After 5 minutes, add 0.3 mL of 10% Al(NO3)3 aqueous solution. After 6 minutes, add 2 mL of 1 mol / L NaOH aqueous solution and 2.4 mL of deionized water. After 10 minutes, measure the absorbance (A) at 510 nm using UV-Vis spectrophotometry. Plot a standard curve with absorbance A on the ordinate and the concentration of the standard solutions on the abscissa.

[0061] Step S1-2, Determination of total flavonoid content in supercritical extract of Hibiscus mutabilis:

[0062] Take 1 mL of supercritical fluid extract of Hibiscus mutabilis and place it in a test tube. Add 4 mL of deionized water and 0.3 mL of 5% NaNO2 aqueous solution. After 5 minutes, add 0.3 mL of 10% Al(NO3)3 aqueous solution. After 6 minutes, add 2 mL of 1 mol / L NaOH aqueous solution and 2.4 mL of deionized water. After 10 minutes, measure the absorbance at 510 nm using UV-Vis spectrophotometry. Calculate the total flavonoid content in the supercritical fluid extract of Hibiscus mutabilis based on the standard curve.

[0063] Table 2. Results of total flavonoid content in supercritical extract of Hibiscus mutabilis:

[0064] Supercritical extract of Hibiscus mutabilis Total flavonoid content, mg / mL Examples 1-5 4.04±0.007 Example 6 2.32±0.002 Example 7 1.66±0.009

[0065] As can be seen from Table 2, the total flavonoid content of the supercritical extract of Hibiscus mutabilis obtained by supercritical carbon dioxide extraction in this application is significantly higher than that of the supercritical extract of Hibiscus mutabilis obtained by ethanol extraction and 1,3-butanediol extraction.

[0066] Test Example 2: Determination of polypeptide content in Hibiscus mutabilis extract:

[0067] In this test example, the peptide content was detected using the biuret reagent method. The specific steps of the peptide content detection method are as follows:

[0068] Step S2-1: Preparation of bovine serum albumin standard curve:

[0069] Using bovine serum albumin (CAS No. 9048-46-8, abbreviated as BAS) as a standard, a BAS aqueous solution with a concentration of 10 mg / mL was prepared. 0 μL, 50 μL, 100 μL, 150 μL, 200 μL, and 250 μL of the 10 mg / mL BAS aqueous solution were respectively placed into microcentrifuge tubes (EP tubes) and water was added to a final volume of 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.

[0070] Add 2 mL of biuret reagent to an EP tube containing BAS standard solution and react at room temperature for 30 minutes. Then, measure the absorbance (A) at 540 nm using UV-Vis spectrophotometry. Plot a standard curve with absorbance A on the ordinate and the concentration of the standard solution on the abscissa.

[0071] The biuret reagent comprises reagent A and reagent B. Reagent A: a homogenous mixture of CuSO4 (0.15g, CAS No. 7758-98-7), sodium potassium tartrate (0.6g, CAS No. 304-59-6), and distilled water (50mL); Reagent B: a 10% (w / w) NaOH aqueous solution (30mL). The biuret reagent is prepared by thoroughly mixing reagents A and B, and should be prepared immediately before use.

[0072] Step S2-2, Determination of polypeptide content in Hibiscus mutabilis extract:

[0073] Add 1.0 mL of 10% trichloroacetic acid aqueous solution to 5 mg of Hibiscus mutabilis extract, centrifuge, collect 0.5 mL of the supernatant, add 2 mL of biuret reagent, and react at room temperature for 30 minutes. Then, measure the absorbance at 540 nm using UV-Vis spectrophotometry. Calculate the polypeptide content in the Hibiscus mutabilis extract based on the standard curve.

[0074] Table 3. Results of polypeptide content detection in Hibiscus mutabilis extract:

[0075] Hibiscus extract Peptide content, wt% Example 1 44.25±1.64 Example 2 35.49±0.54 Example 3 30.28±0.73 Example 4 20.85±0.70 Example 5 28.21±1.10 Example 6 26.82±1.59 Example 7 25.17±0.14

[0076] As can be seen from Table 3, the extract of Hibiscus mutabilis obtained by this application through "supercritical fluid extraction followed by fermentation and enzymatic hydrolysis" has a high polypeptide content.

[0077] A comparison of Examples 1, 6, and 7 reveals that the difference lies in the extraction method used in step S1. Example 1 uses supercritical fluid extraction, Example 6 uses ethanol extraction, and Example 7 uses 1,3-butanediol extraction. Based on the polypeptide content in the Hibiscus mutabilis extracts from Examples 1, 6, and 7, it is clear that compared to "first ethanol extraction, then fermentation and enzymatic hydrolysis" and "first 1,3-butanediol extraction, then fermentation and enzymatic hydrolysis," the method described in this application, "first supercritical fluid extraction, then fermentation and enzymatic hydrolysis," significantly increases the polypeptide content in the Hibiscus mutabilis extract.

[0078] A comparison of Examples 1-3 reveals that the difference lies in the bacterial strains used in step S2 of the fermentation process. Example 1 used *Bacillus subtilis*, Example 2 used *Saccharomyces cerevisiae*, and Example 3 used *Saccharomyces davidii*. The polypeptide content in the extracts of *Hibiscus mutabilis* from Examples 1-3 indicates that *Bacillus subtilis* is superior to *Saccharomyces davidii*, which in turn is superior to *Saccharomyces cerevisiae*.

[0079] A comparison of Examples 1, 4, and 5 reveals that the difference lies in the enzymes used in step S2 for enzymatic hydrolysis. Specifically, Example 1 uses alkaline protease, Example 4 uses cellulase, and Example 5 uses pectinase. The polypeptide content in the Hibiscus mutabilis extracts from Examples 1, 4, and 5 indicates that alkaline protease is superior to pectinase, which in turn is superior to cellulase.

[0080] As can be seen from Tables 2 and 3, the combination of "supercritical fluid extraction followed by fermentation and enzymatic hydrolysis" used in this application can balance the total flavonoid content in the supercritical extract of Hibiscus mutabilis and the polypeptide content in the extract, and can significantly increase the total flavonoid content in the supercritical extract of Hibiscus mutabilis and the polypeptide content in the extract.

[0081] Test Example 3 determined the p-2,2-biphenyl-1-picrylhydrazyl (CAS No. 1898-66-4, abbreviated as DPPH) free radical in the sample. Sweep rate:

[0082] In this test example, the determination of DPPH free radical scavenging rate specifically includes the following steps:

[0083] Step S3-1: Prepare DPPH ethanol solution: Dissolve 20 mg of DPPH in anhydrous ethanol and dilute to a final volume of 250 mL in a volumetric flask with anhydrous ethanol to obtain a DPPH ethanol solution with a concentration of 0.08 mg / mL. Store at 0–4°C protected from light. Prepare fresh and use immediately. Effective within 4 hours.

[0084] Step S3-2: Prepare sample solutions of different test concentrations: Using anhydrous ethanol as a solvent, prepare sample solutions of different test concentrations (specifically 2.5wt%, 5wt%, and 10wt%).

[0085] Step S3-3: Prepare positive control solution: Use vitamin C (CAS No. 50-81-7) as a positive control. Prepare a positive control solution with anhydrous ethanol to a concentration of 1 mg / mL. Store at 0-4℃ protected from light and use immediately after preparation.

[0086] Steps S3-4: Prepare three EP tubes, labeled A, B, and C respectively. Add reagents to tubes A, B, and C according to Table 4; then, incubate at room temperature for 30 minutes. After the reaction is complete, measure the absorbance (OD) of the solutions in tubes A, B, and C at 517 nm using a microplate reader, and record the OD values ​​as follows. A管 OD B管 OD C管 .

[0087] Table 4. Feed-to-liquid ratios in tubes A, B, and C during the DPPH free radical scavenging experiment:

[0088] serial number DPPH ethanol solution Anhydrous ethanol Sample solution or positive control solution Total volume Pipe A 1mL —— 1mL 2mL B tube 1mL 1mL —— 2mL C-tube — 1mL 1mL 2mL

[0089] Step S3-5: Calculate the DPPH free radical scavenging rate of the sample solution or positive control solution. The formula for calculating the DPPH free radical scavenging rate is:

[0090] The DPPH radical scavenging rate of the positive control solution was 96.36%; the detection results of the DPPH radical scavenging rate of the supercritical extract of Hibiscus mutabilis and the extract of Hibiscus mutabilis are shown in Table 5.

[0091] Table 5 shows the results of the DPPH free radical scavenging rate of the samples:

[0092]

[0093] As can be seen from Table 5, the Hibiscus mutabilis extract obtained by the process of "supercritical fluid extraction followed by fermentation and enzymatic hydrolysis" in this application all have a high scavenging effect on DPPH free radicals. In particular, the Hibiscus mutabilis extract obtained by the combination of "supercritical CO2 fluid extraction, Bacillus subtilis fermentation and alkaline protease hydrolysis" has the best scavenging effect on DPPH free radicals.

[0094] Test Example 4: The inhibition rate of the sample against cyclooxygenase 2 (COX-2) was determined.

[0095] In this test case, the COX-2 inhibition rate was detected using a COX-2 enzyme-linked immunosorbent assay (ELISA) kit. The detection process included the following steps:

[0096] S4-1. Use a 96-well blackboard and set up control wells (including blank control, 100% enzyme activity control, and positive inhibitor control) and sample wells. Add the reagents to the wells of the 96-well blackboard from top to bottom as described in Table 6. After adding the sample to be tested, mix well and incubate at 37°C for 10 minutes.

[0097] Table 6. Amounts of each reagent added to the control and sample wells:

[0098]

[0099] S4-2. Add COX-2Probe (5μL) to each well.

[0100] S4-3. Add 5 μL of COX-2 Substrate working solution to each well and incubate at 37°C in the dark for 5 minutes. After incubation, measure the fluorescence intensity (RFU) using a microplate reader at an excitation wavelength of 560 nm and an 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 样品 .

[0101] S4-4. Calculate the COX-2 inhibition rate of the sample. The calculation formula is shown below:

[0102]

[0103] Table 7 shows the results of the COX-2 inhibition rate of the samples:

[0104]

[0105] As shown in Table 7, the Hibiscus mutabilis extract obtained by the process of "supercritical fluid extraction followed by fermentation and enzymatic hydrolysis" in this application exhibits an inhibitory effect on COX-2. In particular, the Hibiscus mutabilis extract obtained by using the combination of "supercritical CO2 fluid extraction, Bacillus subtilis fermentation, and alkaline protease hydrolysis" showed the best inhibitory effect on COX-2.

[0106] Test Example 5: Determining the inhibition rate of elastase activity in the sample:

[0107] In this test example, the determination of the elastase activity inhibition rate specifically includes the following steps:

[0108] Step S5-1: Prepare PBS buffer: Adjust the pH of the PBS (1X) solution to 6.5 using 1 mol / L HCl aqueous solution and 0.1 mol / L HCl aqueous solution.

[0109] Step S5-2: Prepare elastase solution: Prepare an elastase solution (derived from porcine pancreas, purchased from Shanghai Maclean Biochemical Technology Co., Ltd.) with PBS buffer to a concentration of 5 μg / mL.

[0110] Step S5-3: Prepare substrate solution: Prepare a 1 mmol / L substrate solution using Tris-HCl buffer (0.1 mol / L, pH = 6.5). The substrate is N-succinyl-alanine-alanine-alanine-p-nitroaniline (CAS No. 52299-14-6), purchased from Sigma-Aldrich (Shanghai) Trading Co., Ltd.

[0111] Step S5-4: Prepare sample solution: Prepare a 2wt% sample solution using Tris-HCl buffer solution (0.1mol / L, pH=6.5).

[0112] Step S5-5: Using a 96-well plate and setting up wells A, B, and C, add the reagents to wells A, B, and C of the 96-well plate in the order listed in Table 8 from top to bottom; then incubate for 60 minutes. After incubation, measure the absorbance (OD) at 405 nm using a microplate reader and record it as OD. A孔 OD B孔 OD C孔 .

[0113] Table 8. Amounts of each reagent added to wells A, B, and C:

[0114]

[0115] Steps S5-6: Calculate the elastase activity inhibition rate. The calculation formula is shown below:

[0116]

[0117] Table 9 shows the results of the detection of the inhibition rate of elastase activity in the samples:

[0118]

[0119] As shown in Table 9, the Hibiscus mutabilis extract obtained by the combination of "supercritical CO2 fluid extraction, Bacillus subtilis fermentation, and alkaline protease hydrolysis" in this application exhibits a high inhibition rate against elastase. Therefore, the Hibiscus mutabilis extract of this application has a certain anti-wrinkle and firming effect on the skin, and this effect is superior to that of the supercritical Hibiscus mutabilis extract.

[0120] Test Example 6: Determining the inhibition rate of the sample against Propionibacterium acnes.

[0121] In this test case, the procedure for the Propionibacterium acnes antibacterial experiment specifically includes the following steps:

[0122] Step S6-1: 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. For the test wells, add 100 μL of diluted bacterial suspension and 100 μL of sample solution; for the control wells, add 100 μL of vesicular culture medium and 100 μL of sample solution; for the blank control wells, add 200 μL of vesicular culture medium; for the negative control wells, add 100 μL of diluted bacterial suspension and vesicular culture medium; for the positive control wells, add 100 μL of diluted bacterial suspension and 100 μL of a 200 μg / mL solution of penicillin sodium (PBS buffer). Perform three replicates for each well.

[0123] In this test example, PBS buffer was used as the solvent to prepare sample solutions of different test concentrations (specifically 0.1wt%, 0.5wt%, 1wt%, 3wt%, and 5wt%).

[0124] Step S6-2: Place the 96-well bacterial culture plate in a 2.5L anaerobic jar and incubate at room temperature for 24 hours; then, measure the absorbance (OD) at 600nm and record 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 .

[0125] Step S6-3: Calculate the Propionibacterium acnes inhibition rate. The formula for calculating the Propionibacterium acnes inhibition rate is as follows:

[0126]

[0127] Table 10 Results of Propionibacterium acnes inhibition rate:

[0128]

[0129]

[0130] As can be seen from Table 10, the Hibiscus mutabilis extract obtained by the combination of "supercritical CO2 fluid extraction, Bacillus subtilis fermentation and alkaline protease hydrolysis" in this application has a high inhibition rate against Propionibacterium acnes, and the effect of inhibiting Propionibacterium acnes is better than that of supercritical Hibiscus mutabilis extract.

[0131] Test Example 7 determined the inhibition rate of the sample against Malassezia:

[0132] 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. For the test wells, add 100 μL of diluted bacterial suspension and 100 μL of sample solution; for the control wells, add 100 μL of malt extract medium and 100 μL of sample solution; for the blank control wells, add 200 mL of malt extract medium; for the negative control wells, add 100 μL of diluted bacterial suspension and 100 mL of malt extract medium; for the positive control wells, add 100 μL of diluted bacterial suspension and 100 μL of 4 μg / mL ketoconazole solution (PBS buffer). Perform three replicates for each well.

[0133] In this test example, PBS buffer was used as the solvent to prepare sample solutions of different test concentrations (specifically 0.1wt%, 0.5wt%, 1wt%, 3wt%, and 5wt%).

[0134] Step S7-2: Place the 96-well bacterial culture plate in a 2.5L anaerobic jar and incubate at room temperature for 24 hours; then, measure the absorbance (OD) at 600nm and record 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 .

[0135] Step S7-3: Calculate the Malassezia inhibition rate. The formula for calculating the Malassezia inhibition rate is as follows:

[0136]

[0137] Table 11 Results of the detection of the inhibition rate of samples against Malassezia:

[0138]

[0139] As can be seen from Table 11, the Hibiscus mutabilis extract obtained by the combination of "supercritical CO2 fluid extraction, Bacillus subtilis fermentation and alkaline protease hydrolysis" in this application has a high inhibition rate against Malassezia, and the effect of inhibiting Malassezia is better than that of supercritical Hibiscus mutabilis extract.

[0140] Test Example 8 investigated the promoting effect of Hibiscus mutabilis extract on integrin α2β1:

[0141] In this test case, the research methodology specifically includes the following steps:

[0142] S8-1. Screening for safe concentrations of human skin fibroblasts (HFF):

[0143] Human skin fibroblasts at 2×10 5 Inoculate 100 μL of *Hymenoplastics* at a density of 1 / mL into 96-well plates, filling each well with sterile PBS buffer. Incubate at 37°C, 5% CO2, and then add samples after the plates adhere to the walls. After incubating the 96-well plates at 37°C, 5% CO2 for 24 hours, aspirate the culture medium and add 100 μL of *Hibiscus mutabilis* extract solution (3 replicates per concentration). For the blank control group, add 100 μL of serum-free culture medium solution; for the control group, add dimethyl sulfoxide. Then, incubate at 37°C, 5% CO2 for 24 hours. After incubation, stop the culture; add 10 μL of syncalcite (CAS No. 25126-32-3, abbreviated as CCK-8) to each well of the 96-well plate in the dark. Incubate the 96-well plates at 37°C, 5% CO2 for 1 hour. When the culture medium color changes from light pink to orange-yellow, measure the OD value at 450 nm using a microplate reader and record it as OD. 木芙蓉提取物 and OD 空白 The cell viability is calculated using the formula shown below:

[0144]

[0145] Cell activity is directly proportional to OD value, and a cell survival rate greater than 80% is considered a safe concentration for Hibiscus mutabilis extract.

[0146] Table 12 Results of cell viability detection for different concentrations of Hibiscus mutabilis extract:

[0147]

[0148] As can be seen from Table 12, the safe concentration of human skin fibroblasts (HFF) is 5 μg / mL.

[0149] S8-2, Integrin α2β1 content detection:

[0150] Three experimental groups were set up: a blank control group, a model group, and a Hibiscus mutabilis extract group. HFF cells were cultured at a rate of 2 × 10⁶ cells / year. 5HFF cells were seeded at a density of 1 mL / well in 24-well plates. When the HFF cells reached 80%–90% confluence, the culture medium was aspirated and the plates were washed twice with PBS buffer. Then, serum-free culture medium (1 mL) was added to the blank group and the model group, while 1 mL of 5 μg / mL hibiscus extract solution was added to the 24-well plates. The 24-well plates were then incubated in an incubator (37°C, 5% CO2) for 6 hours. Finally, the model group and the hibiscus extract group were stimulated with ultraviolet B radiation (UVB) and then incubated in an incubator (37°C, 5% CO2) for another 18 hours. The supernatant was then collected.

[0151] S8-3. Detect the expression level of integrin α2β1 using an ELISA kit.

[0152] Table 13 Results of integrin α2β1 expression level detection:

[0153] Group Integrating α2β1 content, ng / mL Blank control group 16.76±0.12 Model group 11.24±0.26 Hibiscus mutabilis extract group (Example 1: Hibiscus mutabilis extract) 14.55±0.07

[0154] As can be seen from Table 13, the hibiscus extract obtained by the combination of "supercritical CO2 fluid extraction, Bacillus subtilis fermentation and alkaline protease hydrolysis" in this application has a promoting effect on the secretion of integrin α2β1 after UVB damage.

[0155] The foregoing has provided a detailed description of a Hibiscus mutabilis extract, its extraction method, and its uses. Specific examples have been used to illustrate the principles and implementation methods of this application. The descriptions of the embodiments above are merely for the purpose of helping to understand the method and core ideas of this application. It should be noted that those skilled in the art can make various improvements and modifications to this application without departing from its principles, and these improvements and modifications also fall within the protection scope of the claims of this application.

Claims

1. A method for extracting Hibiscus mutabilis extract, characterized in that, The extraction method includes the following steps: Step S1: The calyx of the Hibiscus mutabilis flower is first crushed and then extracted to obtain Hibiscus mutabilis flower residue; The extraction process is selected from supercritical fluid extraction. The hibiscus flower residue obtained in steps S2 and S1 is successively subjected to fermentation and enzymatic hydrolysis to obtain the hibiscus extract. The bacterial strain used in the fermentation process is selected from Bacillus subtilis; The enzyme used in the enzymatic hydrolysis is selected from alkaline protease, and the working conditions of the alkaline protease are: temperature of 45-55℃ and pH of 10.5-11.

5.

2. The extraction method according to claim 1, characterized in that, In step S2, the fermentation temperature is 25–35°C.

3. 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.

4. The extraction method according to claim 1, characterized in that, In step S1, the supercritical fluid extraction process is performed under the following conditions in the extraction section: extraction temperature is 50-60℃, and extraction pressure is 20-40 MPa.

5. The extraction method according to claim 1, characterized in that, In step S1, the analytical method used in the analytical section of the supercritical fluid extraction process is selected from the variable temperature and pressure method.

6. The extraction method according to claim 1, characterized in that, In step S1, the conditions for the supercritical fluid extraction process in the analytical section are as follows: 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.

7. A hibiscus extract, characterized in that, The hibiscus extract is obtained by the extraction method according to any one of claims 1 to 6.

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