Cottonrose hibiscus extract as well as extraction method and application thereof

Abstract

The invention provides a cottonrose hibiscus extract as well as an extraction method and application thereof. The extraction method comprises the following steps: S1, performing crushing treatment on hibiscus mutabilis calyx, and then performing extraction treatment to obtain hibiscus mutabilis flower residues; s2, sequentially performing fermentation treatment, enzymolysis treatment and filtration treatment on the hibiscus mutabilis flower residues to obtain the hibiscus mutabilis extract, a strain used in the fermentation treatment is selected from at least one of saccharomyces cerevisiae, bacillus subtilis and lipomyces starkeyi; wherein an enzyme used in the enzymolysis treatment is selected from one of alkaline protease, cellulase and pectinase. According to the extraction method disclosed by the invention, active ingredients are extracted from the hibiscus mutabilis flower residues, and the obtained hibiscus mutabilis extract has relatively high yield and relatively high polypeptide content.

Description

Technical Field

[0001] The present application relates to the technical field related to the extraction of active ingredients from Hibiscus mutabilis calyx, and specifically to a Hibiscus mutabilis extract and an extraction method and use thereof. Background Art

[0002] Hibiscus mutabilis L. is a plant of the genus Hibiscus in the Malvaceae family. It has a slightly pungent taste and is neutral in nature. It enters the lung and liver meridians and has the effects of clearing away heat and detoxifying, reducing swelling and draining pus, and cooling blood and stopping bleeding. It is mainly used to treat carbuncle, swelling, snake venom, burns, red and swollen eyes, and traumatic injuries. The flowers, leaves, and roots of Hibiscus mutabilis can all be used as medicinal parts. Among them, Hibiscus mutabilis flowers are rich in flavonoids, such as quercetin, kaempferol, guajava glycoside, praline glycoside, isoquercetin, rutin, quercetin, hyperoside, quercetin-3-O-sambubioside, cyanidin, and cyanidin.

[0003] At present, the conventional methods for extracting active ingredients from Hibiscus mutabilis calyx include supercritical fluid extraction, microwave extraction, alcohol extraction (such as ethanol, 1,3-butanediol, etc.), etc. However, the Hibiscus mutabilis flower residue obtained by extracting Hibiscus mutabilis calyx through supercritical fluid extraction, microwave extraction or alcohol extraction (such as ethanol, 1,3-butanediol, etc.) still contains a certain amount of active ingredients. Summary of the invention

[0004] In view of this, the present application provides a hibiscus extract and an extraction method and use thereof. The extraction method of the present application extracts active ingredients from hibiscus flower residues, and the obtained hibiscus extract has a high yield and a high polypeptide content in the hibiscus extract.

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

[0006] Step S1, the calyx of Hibiscus mutabilis is firstly subjected to a crushing treatment and then subjected to an extraction treatment to obtain a Hibiscus mutabilis flower residue;

[0007] The hibiscus flower residue obtained in step S2 and step S1 is subjected to fermentation treatment and enzymolysis treatment in sequence to obtain the hibiscus extract; the strain used in the fermentation treatment is selected from at least one of saccharomyces cerevisiae, bacillus subtilis and staryi yeast; the enzyme used in the enzymolysis treatment is selected from one of alkaline protease, cellulase and pectinase.

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

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

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

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

[0012] In some embodiments, the conditions of the supercritical fluid extraction treatment in the extraction section are: 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.).

[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 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.

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

[0017] In some embodiments, 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, 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.

[0018] 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.

[0019] In some embodiments, in step S2, the bacterial species used for the fermentation treatment is selected from one of Saccharomyces cerevisiae, Bacillus subtilis and Lipomyces staryi.

[0020] 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.

[0021] 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.

[0022] 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.

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

[0024] In some embodiments, the working conditions of the alkaline protease are: 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.), pH 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 working conditions of the alkaline protease are: temperature of 50°C, pH of 11.0.

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

[0026] In some embodiments, the working conditions of the cellulase are: 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.), 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 working conditions of the alkaline protease are: temperature of 50°C, pH of 4.8.

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

[0028] In some embodiments, the working conditions of the pectinase are: 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 pH 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 working conditions of the pectinase are: temperature of 50°C, and pH of 4.0.

[0029] In a second aspect of the present application, the present application provides a Hibiscus mutabilis extract, wherein the Hibiscus mutabilis extract is obtained by the extraction method described in the first aspect of the present application.

[0030] In the third aspect of the present application, the present application provides a use of the Hibiscus mutabilis extract described in the first aspect of the present application in the preparation of a product having antioxidant and / or antibacterial effects, and / or inhibiting elastase activity, and / or promoting integrin α2β1 expression.

[0031] This application has the following beneficial effects:

[0032] First, the present application adopts a combination of "fermentation treatment and enzymatic hydrolysis treatment" to extract active ingredients from Hibiscus mutabilis flower residues, and the obtained Hibiscus mutabilis extract has a high yield and a high polypeptide content in the Hibiscus mutabilis extract. In particular, the combination of "supercritical CO2 fluid extraction treatment, Bacillus subtilis fermentation treatment and alkaline protease enzymatic hydrolysis treatment" is used to extract active ingredients from Hibiscus mutabilis calyx, so that the yield of Hibiscus mutabilis extract and the polypeptide content in Hibiscus mutabilis extract are significantly improved.

[0033] Second, the present application adopts the combination of "first supercritical fluid extraction treatment, then fermentation treatment and enzymatic hydrolysis treatment" to extract the active ingredients in the calyx of Hibiscus mutabilis. The obtained Hibiscus mutabilis extract has a high Malassezia inhibition rate, Propionibacterium acnes inhibition rate, elastase activity inhibition rate, COX-2 inhibition rate, DPPH free radical scavenging rate and integrated α2β1 expression level; in particular, the DPPH free radical scavenging rate and COX-2 inhibition rate of the Hibiscus mutabilis extract are significantly improved. DETAILED DESCRIPTION

[0034] The present application discloses a Hibiscus mutabilis extract 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 methods and applications of this application have been described through preferred embodiments, and relevant personnel 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 implement 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] Embodiments 1 to 5: wherein the method for extracting the Hibiscus mutabilis extract comprises step S1 and step S2.

[0040] The operation process of step S1 in Examples 1 to 5 is specifically as follows: using a pulverizer to crush the dried calyx of Hibiscus mutabilis into powdered dried calyx of Hibiscus mutabilis, taking the powdered dried calyx of Hibiscus mutabilis (25g) and placing it in an extraction kettle for supercritical fluid extraction treatment (extraction section conditions: extraction temperature 55°C, extraction pressure 30Mpa, extraction time 3 hours; analysis section conditions: variable temperature and pressure method, separation I pressure 20Mpa, separation I temperature 60°C, separation II pressure 5Mpa, 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 supercritical extract of Hibiscus mutabilis; using a Buchner funnel to filter the remaining material in the extraction kettle to obtain Hibiscus mutabilis flower residue. wherein, the obtained Hibiscus mutabilis flower residue is divided into five parts, and the five parts of Hibiscus mutabilis flower residue are respectively subjected to step S2 in Examples 1 to 5.

[0041] The operation process of step S2 in Example 1 is as follows: a portion of Hibiscus mutabilis 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 is completed, the temperature is first raised to 50°C and the pH value is adjusted to 11.0, alkaline protease (2wt%) is added and enzymolysis is performed for 2 hours under the conditions of temperature 50°C and pH value 11.0; 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, it is sequentially subjected to 300 mesh gauze coarse filtration, membrane separation system fine filtration (the membrane model used for membrane separation is SPPM-C-10, and the membrane flux is 6.0L / h), reduced pressure concentration to a general volume, nanofiltration dialysis purification for 48h (dialysis bag specification is 200Da), and spray drying to obtain Hibiscus mutabilis extract.

[0042] The operation process of step S2 in Example 2 is as follows: a portion of Hibiscus mutabilis 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, Saccharomyces cerevisiae is inoculated (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, the temperature is first raised to 50°C and the pH value is adjusted to 11.0, alkaline protease (2wt%) is added under the conditions of temperature 50°C and pH value 11.0 and enzymolysis for 2 hours; 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, it is sequentially subjected to 300 mesh gauze coarse filtration, membrane separation system fine filtration (the membrane model used for membrane separation is SPPM-C-10, and the membrane flux is 6.0L / h), reduced pressure concentration to a general volume, nanofiltration dialysis purification for 48h (dialysis bag specification is 200Da), and spray drying to obtain Hibiscus mutabilis extract.

[0043] The operation process of step S2 in Example 3 is as follows: a portion of Hibiscus mutabilis 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 yeast (inoculation amount 2wt%, strain concentration after inoculation 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 50°C and the pH value is adjusted to 11.0, alkaline protease (2wt%) is added and enzymolysis is performed for 2 hours under the conditions of temperature 50°C and pH value 11.0; 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, it is sequentially subjected to 300 mesh gauze coarse filtration, membrane separation system fine filtration (the membrane model used for membrane separation is SPPM-C-10, and the membrane flux is 6.0L / h), reduced pressure concentration to a general volume, nanofiltration dialysis purification for 48h (dialysis bag specification is 200Da), and spray drying to obtain Hibiscus mutabilis extract.

[0044] The operation process of step S2 in Example 4 is as follows: a portion of Hibiscus mutabilis 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 6CFU / 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 50°C and the pH value is adjusted to 4.8, and cellulase (2wt%) is added under the conditions of temperature 50°C and pH 4.8 and enzymolysis for 2 hours; 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, it is sequentially subjected to 300 mesh gauze coarse filtration, membrane separation system fine filtration (the membrane model used for membrane separation is SPPM-C-10, and the membrane flux is 6.0L / h), reduced pressure concentration to a general volume, nanofiltration dialysis purification for 48h (dialysis bag specification is 200Da), and spray drying to obtain Hibiscus mutabilis extract.

[0045] The operation process of step S2 in Example 5 is as follows: a portion of Hibiscus mutabilis 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 is completed, the temperature is first raised to 50°C and the pH value is adjusted to 4.0, pectinase (2wt%) is added under the conditions of temperature 50°C and pH 4.0 and enzymolysis for 2 hours; 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, it is sequentially subjected to 300 mesh gauze coarse filtration, membrane separation system fine filtration (the membrane model used for membrane separation is SPPM-C-10, and the membrane flux is 6.0L / h), reduced pressure concentration to a general volume, nanofiltration dialysis purification for 48h (dialysis bag specification is 200Da), and spray drying to obtain Hibiscus mutabilis extract.

[0046] Embodiment 6: The method for extracting Hibiscus mutabilis extract comprises step S1 and step S2.

[0047] Step S1, using a grinder to grind Hibiscus mutabilis calyx into powdered Hibiscus mutabilis calyx, mixing the powdered Hibiscus mutabilis calyx (5 g) and 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., Hibiscus mutabilis flower residue); wherein, using a rotary evaporator to evaporate the ethanol in the filtrate at 45° C. to obtain a Hibiscus mutabilis flower ethanol crude extract; firstly adding purified water to the Hibiscus mutabilis flower ethanol crude 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 Hibiscus mutabilis supercritical extract.

[0048] Step S2: Same as in Example 1.

[0049] Embodiment 7: The method for extracting Hibiscus mutabilis extract comprises step S1 and step S2.

[0050] Step S1, using a grinder to grind the hibiscus calyx into powdered hibiscus flower, mixing the powdered hibiscus calyx (5g) and a 1,3-butanediol aqueous solution (100g) with a volume concentration of 30%, and then reflux reacting for 2.5 hours; after that, after the reaction liquid is cooled to room temperature, it is filtered through a Buchner funnel to obtain a filtrate (i.e., hibiscus supercritical extract) and a filter residue (i.e., hibiscus flower residue).

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

[0052] The weight of the hibiscus calyx is recorded as W1 (unit: g), the weight of the hibiscus extract is recorded as W2 (unit: g), and the yield of the hibiscus extract is recorded as y (unit: wt%). The calculation formula for the yield of the hibiscus extract is:

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

[0054]

[0055] Test Example 1 Determination of the content of total flavonoids 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, preparation of standard curve:

[0058] 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.

[0059] 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.

[0060] 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.

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

[0062] Take the supercritical extract of Hibiscus mutabilis (1mL) and place it in a test tube, add deionized water (4mL), a 5% NaNO2 aqueous solution (0.3mL) with a weight percentage concentration; after 5 minutes, add a 10% Al(NO3)3 aqueous solution (0.3mL) with a weight percentage concentration; after 6 minutes of reaction, add a 1mol / L NaOH aqueous solution (2mL) and deionized water (2.4mL) in sequence; after 10 minutes, measure the absorbance at 510nm using UV-visible spectrophotometry. According to the standard curve, calculate the content of total flavonoids in the supercritical extract of Hibiscus mutabilis.

[0063] Table 2 Test results of total flavonoids content in supercritical extract of Hibiscus mutabilis:

[0064] Hibiscus mutabilis supercritical extract Total flavonoids content, mg / mL Embodiments 1 to 5 4.04±0.007 Example 6 2.32±0.002 Example 7 1.66±0.009

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

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

[0067] In this test example, the method for detecting the polypeptide content is the biuret reagent method. The method for detecting the polypeptide content specifically includes the following steps:

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

[0069] 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.

[0070] 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.

[0071] 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.

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

[0073] A 10% by weight trichloroacetic acid aqueous solution (1.0 mL) was added to the Hibiscus mutabilis extract (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 by UV-visible spectrophotometry. The polypeptide content in the Hibiscus mutabilis extract was calculated based on the standard curve.

[0074] Table 3 Detection results of polypeptide content in Hibiscus mutabilis extract:

[0075] Hibiscus mutabilis 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] It can be seen from Table 3 that the hibiscus extract obtained by "first undergoing supercritical fluid extraction treatment, then undergoing fermentation treatment and enzymatic hydrolysis treatment" has a higher polypeptide content.

[0077] By comparing Examples 1, 6 and 7, it can be seen that the difference between Examples 1, 6 and 7 is that the extraction treatment methods used in step S1 are different, wherein Example 1 is a supercritical fluid extraction treatment, Example 6 is an ethanol extraction treatment, and Example 7 is a 1,3-butanediol extraction treatment. From the polypeptide content in the hibiscus extracts in Examples 1, 6 and 7, 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 polypeptide content in the hibiscus extract.

[0078] By comparing Examples 1 to 3, it can be seen that the difference between Examples 1 to 3 is that the strains used for fermentation in step S2 are different, among which Example 1 is Bacillus subtilis, Example 2 is Saccharomyces cerevisiae, and Example 3 is Lipozyme Starchus. From the content of polypeptides in the hibiscus extracts of Examples 1 to 3, it can be seen that Bacillus subtilis is better than Lipozyme Starchus and better than Saccharomyces cerevisiae.

[0079] By comparing Examples 1, 4 and 5, it can be seen that the difference between Examples 1, 4 and 5 is that the enzymes used for enzymatic treatment in step S2 are different, wherein Example 1 is alkaline protease, Example 4 is cellulase, and Example 5 is pectinase. From the polypeptide content in the hibiscus extracts of Examples 1, 4 and 5, it can be seen that alkaline protease is better than pectinase and better than cellulase.

[0080] From Table 2 and Table 3, it can be seen that the present application adopts the combination of "first supercritical fluid extraction treatment, then fermentation treatment and enzymatic hydrolysis treatment", which can take into account the total flavonoids content in the Hibiscus mutabilis supercritical extract and the polypeptide content in the Hibiscus mutabilis extract, and can significantly increase the total flavonoids content in the Hibiscus mutabilis supercritical extract and the polypeptide content in the Hibiscus mutabilis extract.

[0081] Test Example 3 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:

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

[0083] Step S3-1, 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 away from light, use immediately after preparation, and it is effective within 4 hours.

[0084] Step S3-2, 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%).

[0085] Step S3-3, prepare a positive control solution: use vitamin C (CAS No. 50-81-7) as a positive control, and use anhydrous ethanol to prepare a positive control solution with a concentration of 1 mg / mL of vitamin C; store it at 0-4° C. away from light and use it immediately after preparation.

[0086] Step S3-4, 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 4; 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 .

[0087] Table 4: Material-liquid ratio in tube A, tube B and tube C in DPPH free radical scavenging experiment:

[0088] 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

[0089] Step S3-5, calculating the DPPH free radical scavenging rate of the sample solution or the positive control solution, the calculation formula of the DPPH free radical scavenging rate is:

[0090] 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 rates of the Hibiscus mutabilis supercritical extract and the Hibiscus mutabilis extract are shown in Table 5.

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

[0092]

[0093] It can be seen from Table 5 that the hibiscus extracts obtained by the present application "first undergoing supercritical fluid extraction treatment, then undergoing fermentation treatment and enzymatic hydrolysis treatment" all have a high scavenging effect on DPPH free radicals, especially the hibiscus extracts obtained by the combination of "supercritical CO2 fluid extraction treatment, Bacillus subtilis fermentation treatment and alkaline protease enzymatic hydrolysis treatment" have the best scavenging effect on DPPH free radicals.

[0094] Test Example 4: Determination of the inhibition rate of the sample on cyclooxygenase 2 (COX-2):

[0095] 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:

[0096] S4-1. 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 to be tested, mix well and incubate at 37°C for 10 minutes.

[0097] Table 6: Amount of each reagent added to the control well and sample well:

[0098]

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

[0100] S4-3, 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 样品 .

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

[0102]

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

[0104]

[0105] As can be seen from Table 7, the hibiscus extract obtained by "first undergoing supercritical fluid extraction treatment, then undergoing fermentation treatment and enzymatic hydrolysis treatment" in the present application has an inhibitory effect on COX-2. In particular, the hibiscus extract obtained by using the combination of "supercritical CO2 fluid extraction treatment, Bacillus subtilis fermentation treatment and alkaline protease enzymatic hydrolysis treatment" has the best inhibitory effect on COX-2.

[0106] Test Example 5: Determination of the inhibition rate of sample activity on elastase:

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

[0108] Step S5-1, 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.

[0109] Step S5-2, 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.

[0110] Step S5-3, 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.

[0111] Step S5-4, 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%.

[0112] Step S5-5, 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 8; 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 .

[0113] Table 8 The amount of each reagent added in well A, well B and well C:

[0114]

[0115] Step S5-6, calculating the elastase activity inhibition rate, the calculation formula is as follows:

[0116]

[0117] Table 9 Test results of the inhibition rate of samples on elastase activity:

[0118]

[0119] As can be seen from Table 9, the hibiscus extract obtained by the combination of "supercritical CO2 fluid extraction treatment, Bacillus subtilis fermentation treatment and alkaline protease enzymatic treatment" in this application has a high inhibition rate on elastase. It can be seen that the hibiscus extract of this application has a certain anti-wrinkle and firming effect on the skin, and the anti-wrinkle and firming effect is better than that of the supercritical hibiscus extract.

[0120] Test Example 6: Determination of the antibacterial rate of the sample against Propionibacterium acnes:

[0121] In this test example, the operation process of 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. 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 with a concentration of 200μg / mL (100μL, the solvent is PBS buffer) to the positive control well. Make three replicate wells for each.

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

[0124] Step S6-2, 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 .

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

[0126]

[0127] Table 10 Test results of Propionibacterium acnes inhibition rate:

[0128]

[0129]

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

[0131] Test Example 7 Determination of the antibacterial rate of samples 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. 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) with a concentration of 4 μg / mL to the positive control well. Make 3 duplicate wells for each.

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

[0134] 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 .

[0135] Step S7-3, calculating the Malassezia inhibition rate, the calculation formula of the Malassezia inhibition rate is as follows:

[0136]

[0137] Table 11 Test results of the inhibition rate of samples against Malassezia:

[0138]

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

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

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

[0142] S8-1. Safe concentration screening of human skin fibroblasts (HFF):

[0143] 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 Hibiscus mutabilis extract 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, it 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 木芙蓉提取物 and OD 空白 . Calculate the cell survival rate. The calculation formula of cell survival rate is as follows:

[0144]

[0145] Among them, the cell activity is proportional to the OD value. When the cell survival rate is greater than 80%, it is the safe concentration of Hibiscus mutabilis extract.

[0146] Table 12 Test results of cell viability of different concentrations of Hibiscus mutabilis extracts:

[0147]

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

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

[0150] Three experimental groups were set up, namely blank control group, model group and Hibiscus mutabilis extract group. HFF cells were cultured at 2×10 5The 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 medium (1 mL) was added to the blank group and the model group, and 1 mL of hibiscus extract solution with a concentration of 5 μg / mL was added to the 24-well plate. After that, the 24-well plate was placed in an incubator (37°C, 5% CO2) for 6 hours. Finally, the model group and the hibiscus extract 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.

[0151] S8-3. Use ELISA kit to detect the expression level of integrin α2β1.

[0152] Table 13 Detection results of the expression level of integrin α2β1:

[0153] Group Integrated 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 treatment, Bacillus subtilis fermentation treatment and alkaline protease enzymatic treatment" adopted in the present application has a promoting effect on the secretion of integrin α2β1 after UVB damage.

[0155] The above is a detailed introduction to a Hibiscus mutabilis extract and its extraction method and use provided by the present application. 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 ideas 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 Hibiscus mutabilis extract, characterized in that: The extraction method comprises the following steps: Step S1, the calyx of Hibiscus mutabilis is firstly subjected to a crushing treatment and then subjected to an extraction treatment to obtain Hibiscus mutabilis flower residue; The hibiscus flower residue obtained in step S2 and step S1 is subjected to fermentation treatment and enzymolysis treatment in sequence to obtain the hibiscus extract; the strain used in the fermentation treatment is selected from at least one of saccharomyces cerevisiae, bacillus subtilis and staryi yeast; the enzyme used in the enzymolysis treatment is selected from one of alkaline protease, cellulase and pectinase.

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

3. The extraction method according to claim 1 or 2, 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.

4. The extraction method according to claim 3, characterized in that In step S2, the bacterial species used in the fermentation treatment is selected from Bacillus subtilis.

5. The extraction method according to claim 1, characterized in that The working conditions of the alkaline protease are: temperature of (45-55)°C and pH of 10.5-11.5; And / or, the working conditions of the cellulase are: temperature of (45-55)°C, pH of 4.3-5.3; And / or, the working conditions of the pectinase are: temperature of (45-55)°C and pH of 3.5-4.

5.

6. The extraction method according to claim 1 or 5, characterized in that In step S2, the enzyme used in the enzymatic treatment is selected from alkaline protease.

7. The extraction method according to claim 1, characterized in that In step S1, the extraction treatment is selected from at least one of supercritical fluid extraction treatment, ethanol extraction treatment and 1,3-butanediol extraction treatment.

8. The extraction method according to claim 7, characterized in that In step S1, the extraction process is selected from supercritical fluid extraction process; Optionally, the supercritical fluid extraction process is selected from supercritical carbon dioxide fluid extraction process; Optionally, 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. Optionally, the analytical method of the supercritical fluid extraction treatment in the analytical section is selected from a variable temperature and pressure method; Optionally, the supercritical fluid extraction treatment conditions 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.

9. A hibiscus extract, characterized in that The hibiscus mutabilis extract is obtained by the extraction method according to any one of claims 1 to 8.

10. Use of the Hibiscus mutabilis extract according to claim 9 in preparing a product having antioxidant and / or antibacterial effects, and / or inhibiting elastase activity, and / or promoting integrin α2β1 expression.

Images