Preparation method of pyracantha fruit extract freeze-dried powder and its application in cosmetics

Abstract

The present invention provides a preparation method of freeze-dried powder of Pyracantha fruit extract and its application in cosmetics, belonging to the technical field of personal care cosmetics. The preparation steps of freeze-dried powder of Pyracantha fruit extract are as follows: 1) fresh and mature Pyracantha fruit is selected, and supercritical fluid extraction is performed to obtain a supercritical fluid extract; 2) the supercritical fluid extract is ultrasonically dissolved by petroleum ether, ethyl acetate, and n-butanol in sequence, and filtered to obtain an ethyl acetate solution and an n-butanol solution; 3) the ethyl acetate solution is solvent-recovered to obtain an ethyl acetate part; 4) the n-butanol solution is solvent-recovered to obtain an n-butanol part; 5) the ethyl acetate part is mixed with the n-butanol part, and freeze-dried to obtain freeze-dried powder of Pyracantha fruit extract. The preparation method of the present invention improves the extraction rate of total flavonoids and total polyphenols, and at the same time increases the content of total flavonoids and total polyphenols in the extract, and the obtained Pyracantha fruit extract has strong tyrosinase inhibitory activity, inhibits melanin formation to achieve whitening effect, and has anti-wrinkle and firming effects.

Description

Technical Field

[0001] The invention belongs to the technical field of personal care cosmetics, and particularly relates to a preparation method of freeze-dried powder of Pyracantha fortuneana fruit extract and application of the same in cosmetics. Background Art

[0002] Pyracantha fortuneana, also known as torch fruit, rescue food, life-saving food, red seed, etc., is the fruit of Pyracantha fortuneana of the genus Pyracantha in the Rosaceae family. The fruit, root and leaf can all be used as medicine. It is a multi-purpose flower and fruit plant with medicinal, edible and ornamental value. It was first recorded in the herbal monograph "Southern Yunnan Materia Medica" (written by Lan Mao in the Ming Dynasty). It tastes sweet and sour, has a neutral nature, can eliminate stagnation and stop dysentery, promote blood circulation and stop bleeding, and is used to treat indigestion, enteritis, dysentery, infantile malnutrition, metrorrhagia, leucorrhea, postpartum abdominal pain and other diseases. Modern research shows that Pyracantha fortuneana is rich in flavonoids, polyphenols, sugars, proteins and multiple vitamins, and has significant antioxidant, anti-tumor, anti-aging, liver and stomach protection and other multiple effects. It has great application value in the fields of medicine, food, cosmetics, etc. Chinese invention patent application CN113476356A discloses a preparation method and use of a Pyracantha fortuneana fruit extract, comprising the following steps: (1) enzymatic hydrolysis: taking Pyracantha fortuneana fruit, adding a composite enzyme for enzymatic hydrolysis, and obtaining an enzymatic hydrolysis mixture; (2) ethanol extraction: adding ethanol to the enzymatic hydrolysis mixture for extraction, filtering, and concentrating to obtain an ethanol extract; (3) macroporous adsorption resin purification: loading the ethanol extract onto a macroporous adsorption resin for purification, and collecting an analytical solution; (4) drying: concentrating and drying the analytical solution to obtain a Pyracantha fortuneana fruit extract that can effectively extract flavonoids from Pyracantha fortuneana fruit and is easy to realize industrial production, thereby obtaining a Pyracantha fortuneana fruit extract with antioxidant, brightening, and whitening effects. However, the preparation process is cumbersome, and the extraction rate of total flavonoids needs to be further improved, and the content of polyphenols in the extract is not disclosed; Chinese invention patent CN100389675C discloses a method for extracting a series of effective components of Pyracantha fruit, and the specific process is: Pyracantha fruit is squeezed after impurities are removed and cleaned to obtain Pyracantha fruit juice; the pomace after juicing is leached with an acidic low-grade alcohol aqueous solution, filtered, purified by column chromatography, and dried to obtain red pigment; the pomace after extracting red pigment is leached with ethanol or acetone, filtered, recovered solvent, extracted and purified, and dried to obtain yellow pigment; the pomace after extracting yellow pigment is hydrolyzed with hydrochloric acid, filtered, concentrated, decolorized, precipitated, purified, and dried to obtain pectin, and the remaining residual pomace is used as feed raw material. Although the above preparation method can extract a series of effective components in Pyracantha fruit, the preparation process is complicated, and acid is used in the preparation process, which causes a certain degree of damage to some components in Pyracantha fruit. Summary of the invention

[0003] The present invention aims at the problems existing in the prior art and provides a method for preparing freeze-dried powder of pyracantha fruit extract, which has the advantages of simple preparation process, high extraction efficiency for total flavonoids and total polyphenols in pyracantha fruit, high content of total flavonoids and total polyphenols in the extract, and neutral extraction solvent, thereby reducing damage to components in pyracantha fruit.

[0004] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0005] The first aspect of the present invention is to provide a method for preparing a freeze-dried powder of Pyracantha fortuneana fruit extract, the method for preparing the freeze-dried powder of Pyracantha fortuneana fruit extract comprising the following steps:

[0006] 1) Selecting fresh and ripe Pyracantha fortuneana fruit and subjecting it to supercritical fluid extraction to obtain a supercritical fluid extract;

[0007] 2) the supercritical fluid extract is ultrasonically dissolved in petroleum ether, ethyl acetate and n-butanol in sequence, and filtered to obtain an ethyl acetate solution and an n-butanol solution;

[0008] 3) recovering the solvent from the ethyl acetate solution to obtain an ethyl acetate portion;

[0009] 4) recovering the solvent from the n-butanol solution to obtain the n-butanol portion;

[0010] 5) Mix the ethyl acetate portion and the n-butanol portion, and freeze-dry to obtain freeze-dried powder of Pyracantha fortuneana fruit extract.

[0011] Preferably, the supercritical fluid extraction in step 1) is supercritical carbon dioxide fluid extraction.

[0012] Further preferably, the flow rate of the supercritical carbon dioxide fluid is 3-10 L / min, the temperature of the extraction kettle is 55-60° C., the pressure of the extraction kettle is 5-10 MPa, the pressure of the separation kettle is 5-20 MPa, the temperature of the separation kettle is 50-60° C., and the extraction time is 1-3 h.

[0013] Further preferably, the supercritical carbon dioxide fluid extraction is a two-step separation, wherein: the pressure of separation kettle I is 10-20 MPa, the temperature of separation kettle I is 50-60°C, the pressure of separation kettle II is 5-10 MPa, and the temperature of separation kettle II is 50-55°C.

[0014] Preferably, the mass of the supercritical fluid extract in step 2) and the added volume ratio of petroleum ether, ethyl acetate and n-butanol are independently selected from 1:3-6 g / mL.

[0015] Further preferably, the ratio of the mass of the supercritical fluid extract to the added volume of petroleum ether is 1:5 g / mL, and the ratio of the mass of the supercritical fluid extract to the added volume of ethyl acetate and n-butanol is 1:3 g / mL.

[0016] Preferably, in step 2), the supercritical fluid extract is ultrasonically dissolved in petroleum ether, ethyl acetate and n-butanol in sequence with power independently selected from 300-400 W, and the ultrasonic dissolution time independently selected from 3-10 min.

[0017] Further preferably, the power of ultrasonic dissolution in petroleum ether is 350 W, and the ultrasonic dissolution time is 5 min; the power of ultrasonic dissolution in ethyl acetate is 300 W, and the ultrasonic dissolution time is 6 min; the power of ultrasonic dissolution in n-butanol is 350 W, and the ultrasonic dissolution time is 3 min.

[0018] Preferably, the freeze-drying procedure in step 5) is: pre-freezing at -50 to -20°C for 5-12 hours, continuous intermittent vacuum drying for 20-60 hours, with each interruption time being 1-8 hours. When the temperature rises to 10-30°C and the vacuum degree is below 5-100 Pa, the sample is dried.

[0019] The second aspect of the present invention is to provide a use of the freeze-dried powder of Pyracantha fortuneana fruit extract prepared according to the above preparation method in the preparation of cosmetics with whitening and / or anti-wrinkle and / or firming effects.

[0020] Compared with the prior art, the present invention has the following beneficial effects: relevant medicinal ingredients in Pyracantha fortuneana fruit are extracted by supercritical fluid technology, and total flavonoids and total polyphenols in Pyracantha fortuneana fruit are enriched by removing impurities with petroleum ether and dissolving with ethyl acetate and n-butanol, thereby increasing the content of total flavonoids and total polyphenols in the extract; the freeze-dried powder of Pyracantha fortuneana fruit extract obtained by the preparation method of the present invention has strong tyrosinase inhibitory activity, inhibits melanin formation to achieve a whitening effect, and also has anti-wrinkle and firming effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 HPLC chart of freeze-dried powder of Pyracantha fortuneana fruit extract at a wavelength of 254 nm;

[0022] Figure 2 HPLC chart of freeze-dried powder of Pyracantha fortuneana fruit extract at a wavelength of 365 nm;

[0023] Figure 3 Inhibition rate of pyracantha fruit extract freeze-dried powder on tyrosinase activity;

[0024] Figure 4 Representative images of the melanin-inhibiting efficacy of freeze-dried powder of Pyracantha fortuneana fruit extract. DETAILED DESCRIPTION

[0025] Example 1

[0026] A method for preparing freeze-dried powder of Pyracantha fortuneana fruit extract, comprising the following specific steps:

[0027] 1) Select fresh ripe Pyracantha fortuneana fruit, extract it with supercritical carbon dioxide fluid, the flow rate of the supercritical carbon dioxide fluid is 5 L / min, the temperature of the extraction kettle is 55° C., the pressure of the extraction kettle is 5 MPa, the pressure of the separation kettle is 10 MPa, the temperature of the separation kettle is 50° C., and the extraction time is 2 h to obtain a supercritical fluid extract;

[0028] 2) adding petroleum ether to the supercritical fluid extract and ultrasonically dissolving it, the volume ratio of the supercritical fluid extract to petroleum ether is 1:5 g / mL, the ultrasonic power is 350 W, the ultrasonic time is 5 min, filtering to obtain petroleum ether insolubles; adding ethyl acetate to the petroleum ether insolubles and ultrasonically dissolving them, the mass ratio of the supercritical fluid extract to the added volume ratio of ethyl acetate is 1:3 g / mL, the ultrasonic power is 300 W, the ultrasonic time is 6 min, filtering to obtain ethyl acetate solution and ethyl acetate insolubles; adding n-butanol to the ethyl acetate insolubles and ultrasonically dissolving them, the mass ratio of the supercritical fluid extract to the added volume ratio of n-butanol is 1:3 g / mL, the ultrasonic power is 350 W, the ultrasonic time is 3 min, filtering to obtain n-butanol solution;

[0029] 3) recovering the solvent from the ethyl acetate solution to obtain an ethyl acetate portion;

[0030] 4) recovering the solvent from the n-butanol solution to obtain the n-butanol portion;

[0031] 5) Mix the ethyl acetate portion with the n-butanol portion, pre-freeze at -30°C for 10 hours, and dry under vacuum continuously and intermittently for 30 hours, with each interruption time being 3 hours. When the temperature rises to 15°C and the vacuum degree is 20 Pa, the freeze-dried powder of Pyracantha fortuneana fruit extract is obtained.

[0032] Example 2

[0033] A method for preparing freeze-dried powder of Pyracantha fortuneana fruit extract, comprising the following specific steps:

[0034] 1) Select fresh and ripe Pyracantha fortuneana fruit, extract it with supercritical carbon dioxide fluid, the flow rate of the supercritical carbon dioxide fluid is 5 L / min, the temperature of the extraction kettle is 55° C., the pressure of the extraction kettle is 5 MPa, separate it in two steps, the pressure of separation kettle I is 10 MPa, the temperature of separation kettle I is 60° C., the pressure of separation kettle II is 5 MPa, the temperature of separation kettle II is 55° C., and the extraction time is 3 h to obtain a supercritical fluid extract;

[0035] 2) adding petroleum ether to the supercritical fluid extract and ultrasonically dissolving it, the volume ratio of the supercritical fluid extract to petroleum ether is 1:5 g / mL, the ultrasonic power is 350 W, the ultrasonic time is 5 min, filtering to obtain petroleum ether insolubles; adding ethyl acetate to the petroleum ether insolubles and ultrasonically dissolving them, the mass ratio of the supercritical fluid extract to the added volume ratio of ethyl acetate is 1:3 g / mL, the ultrasonic power is 300 W, the ultrasonic time is 6 min, filtering to obtain ethyl acetate solution and ethyl acetate insolubles; adding n-butanol to the ethyl acetate insolubles and ultrasonically dissolving them, the mass ratio of the supercritical fluid extract to the added volume ratio of n-butanol is 1:3 g / mL, the ultrasonic power is 350 W, the ultrasonic time is 3 min, filtering to obtain n-butanol solution;

[0036] 3) recovering the solvent from the ethyl acetate solution to obtain an ethyl acetate portion;

[0037] 4) recovering the solvent from the n-butanol solution to obtain the n-butanol portion;

[0038] 5) Mix the ethyl acetate portion with the n-butanol portion, pre-freeze at -30°C for 10 hours, and dry under vacuum continuously and intermittently for 30 hours, with each interruption time being 3 hours. When the temperature rises to 15°C and the vacuum degree is 20 Pa, the freeze-dried powder of Pyracantha fortuneana fruit extract is obtained.

[0039] Example 3

[0040] A method for preparing freeze-dried powder of Pyracantha fortuneana fruit extract, comprising the following specific steps:

[0041] 1) Select fresh ripe Pyracantha fortuneana fruit, extract it with supercritical carbon dioxide fluid, the flow rate of the supercritical carbon dioxide fluid is 5 L / min, the temperature of the extraction kettle is 55° C., the pressure of the extraction kettle is 5 MPa, the pressure of the separation kettle is 10 MPa, the temperature of the separation kettle is 50° C., and the extraction time is 2 h to obtain a supercritical fluid extract;

[0042] 2) adding petroleum ether to the supercritical fluid extract and ultrasonically dissolving it, the volume ratio of the supercritical fluid extract to petroleum ether is 1:3 g / mL, the ultrasonic power is 350 W, the ultrasonic time is 5 min, filtering to obtain petroleum ether insolubles; adding ethyl acetate to the petroleum ether insolubles and ultrasonically dissolving them, the mass ratio of the supercritical fluid extract to the added volume ratio of ethyl acetate is 1:6 g / mL, the ultrasonic power is 300 W, the ultrasonic time is 6 min, filtering to obtain ethyl acetate solution and ethyl acetate insolubles; adding n-butanol to the ethyl acetate insolubles and ultrasonically dissolving them, the mass ratio of the supercritical fluid extract to the added volume ratio of n-butanol is 1:3 g / mL, the ultrasonic power is 350 W, the ultrasonic time is 3 min, filtering to obtain n-butanol solution;

[0043] 3) recovering the solvent from the ethyl acetate solution to obtain an ethyl acetate portion;

[0044] 4) recovering the solvent from the n-butanol solution to obtain the n-butanol portion;

[0045] 5) Mix the ethyl acetate portion with the n-butanol portion, pre-freeze at -30°C for 10 hours, and dry under vacuum continuously and intermittently for 30 hours, with each interruption time being 3 hours. When the temperature rises to 15°C and the vacuum degree is 20 Pa, the freeze-dried powder of Pyracantha fortuneana fruit extract is obtained.

[0046] Example 4

[0047] A method for preparing freeze-dried powder of Pyracantha fortuneana fruit extract, comprising the following specific steps:

[0048] 1) Select fresh ripe Pyracantha fortuneana fruit, extract it with supercritical carbon dioxide fluid, the flow rate of the supercritical carbon dioxide fluid is 5 L / min, the temperature of the extraction kettle is 55° C., the pressure of the extraction kettle is 5 MPa, the pressure of the separation kettle is 10 MPa, the temperature of the separation kettle is 50° C., and the extraction time is 2 h to obtain a supercritical fluid extract;

[0049] 2) adding petroleum ether to the supercritical fluid extract for ultrasonic dissolution, the volume ratio of the supercritical fluid extract to petroleum ether is 1:5 g / mL, the ultrasonic power is 350 W, the ultrasonic time is 5 min, filtering to obtain petroleum ether insolubles; adding ethyl acetate to the petroleum ether insolubles for ultrasonic dissolution, the mass ratio of the supercritical fluid extract to the added volume ratio of ethyl acetate is 1:3 g / mL, the ultrasonic power is 300 W, the ultrasonic time is 6 min, filtering to obtain ethyl acetate solution and ethyl acetate insolubles; adding n-butanol to the ethyl acetate insolubles for ultrasonic dissolution, the mass ratio of the supercritical fluid extract to the added volume ratio of n-butanol is 1:6 g / mL, the ultrasonic power is 350 W, the ultrasonic time is 3 min, filtering to obtain n-butanol solution;

[0050] 3) recovering the solvent from the ethyl acetate solution to obtain an ethyl acetate portion;

[0051] 4) recovering the solvent from the n-butanol solution to obtain the n-butanol portion;

[0052] 5) Mix the ethyl acetate portion with the n-butanol portion, pre-freeze at -30°C for 10 hours, and dry under vacuum continuously and intermittently for 30 hours, with each interruption time being 3 hours. When the temperature rises to 15°C and the vacuum degree is 20 Pa, the freeze-dried powder of Pyracantha fortuneana fruit extract is obtained.

[0053] Comparative Example 1

[0054] Fresh and ripe Pyracantha fortuneana fruit was selected, and Pyracantha fortuneana fruit extract was prepared according to Example 1 in Chinese invention patent application CN113476356A.

[0055] Comparative Example 2

[0056] A method for preparing freeze-dried powder of Pyracantha fortuneana fruit extract, comprising the following specific steps:

[0057] 1) Select fresh ripe Pyracantha fortuneana fruit, extract it with supercritical carbon dioxide fluid, the flow rate of the supercritical carbon dioxide fluid is 5 L / min, the temperature of the extraction kettle is 55° C., the pressure of the extraction kettle is 20 MPa, the pressure of the separation kettle is 30 MPa, the temperature of the separation kettle is 50° C., and the extraction time is 2 h;

[0058] 2) adding petroleum ether to the supercritical fluid extract and ultrasonically dissolving it, the volume ratio of the supercritical fluid extract to petroleum ether is 1:5 g / mL, the ultrasonic power is 350 W, the ultrasonic time is 5 min, filtering to obtain petroleum ether insolubles; adding ethyl acetate to the petroleum ether insolubles and ultrasonically dissolving them, the mass ratio of the supercritical fluid extract to the added volume ratio of ethyl acetate is 1:3 g / mL, the ultrasonic power is 300 W, the ultrasonic time is 6 min, filtering to obtain ethyl acetate solution and ethyl acetate insolubles; adding n-butanol to the ethyl acetate insolubles and ultrasonically dissolving them, the mass ratio of the supercritical fluid extract to the added volume ratio of n-butanol is 1:3 g / mL, the ultrasonic power is 350 W, the ultrasonic time is 3 min, filtering to obtain n-butanol solution;

[0059] 3) recovering the solvent from the ethyl acetate solution to obtain an ethyl acetate portion;

[0060] 4) recovering the solvent from the n-butanol solution to obtain the n-butanol portion;

[0061] 5) Mix the ethyl acetate portion with the n-butanol portion, pre-freeze at -30°C for 10 hours, and dry under vacuum continuously and intermittently for 30 hours, with each interruption time being 3 hours. When the temperature rises to 15°C and the vacuum degree is 20 Pa, the freeze-dried powder of Pyracantha fortuneana fruit extract is obtained.

[0062] Comparative Example 3

[0063] A method for preparing freeze-dried powder of Pyracantha fortuneana fruit extract, comprising the following specific steps:

[0064] 1) Select fresh and ripe Pyracantha fortuneana fruit, extract it with supercritical carbon dioxide fluid, the flow rate of supercritical carbon dioxide fluid is 5 L / min, the temperature of extraction kettle is 55° C., the pressure of extraction kettle is 5 MPa, the pressure of separation kettle is 10 MPa, the temperature of separation kettle is 50° C., and the extraction time is 2 h to obtain supercritical fluid extract;

[0065] 2) adding petroleum ether to the supercritical fluid extract and ultrasonically dissolving it, the volume ratio of the supercritical fluid extract to the petroleum ether is 1:7 g / mL, the ultrasonic power is 350 W, the ultrasonic time is 5 min, filtering to obtain the petroleum ether insoluble matter; adding ethyl acetate to the petroleum ether insoluble matter and ultrasonically dissolving it, the mass ratio of the supercritical fluid extract to the added volume ratio of ethyl acetate is 1:7 g / mL, the ultrasonic power is 300 W, the ultrasonic time is 6 min, filtering to obtain ethyl acetate solution and ethyl acetate insoluble matter; adding n-butanol to the ethyl acetate insoluble matter and ultrasonically dissolving it, the mass ratio of the supercritical fluid extract to the added volume ratio of n-butanol is 1:7 g / mL, the ultrasonic power is 350 W, the ultrasonic time is 3 min, filtering to obtain n-butanol solution;

[0066] 3) recovering the solvent from the ethyl acetate solution to obtain an ethyl acetate portion;

[0067] 4) recovering the solvent from the n-butanol solution to obtain the n-butanol portion;

[0068] 5) Mix the ethyl acetate portion with the n-butanol portion, pre-freeze at -30°C for 10 hours, and dry under vacuum continuously and intermittently for 30 hours, with each interruption time being 3 hours. When the temperature rises to 15°C and the vacuum degree is 20 Pa, the freeze-dried powder of Pyracantha fortuneana fruit extract is obtained.

[0069] Experimental Example 1 Determination of the Content of Total Flavonoids and Total Polyphenols in Freeze-dried Pyracantha Fruit Extract Powder Example 1-4, Comparative Example 1-3 I. Determination of the Content of Total Flavonoids in Freeze-dried Pyracantha Fruit Extract Powder

[0070] 1. Preparation of Reagents

[0071] (1) 5% sodium nitrite solution: Weigh 5.0 g of sodium nitrite, dissolve it in purified water, and add to a 100 mL volumetric flask. Shake well and store in a dark place.

[0072] (2) 10% aluminum nitrate solution: Weigh 10.0 g of aluminum nitrate, dissolve in purified water, and add to a 100 mL volumetric flask. Shake well and store in a dark place.

[0073] (3) 4% sodium hydroxide solution: Weigh 4.0 g of sodium hydroxide, dissolve it in purified water, add to a 100 mL volumetric flask, shake well, and store.

[0074] (4) 70% ethanol: Take 70 ml of anhydrous ethanol, add water to make up to 100 ml, and mix well.

[0075] 2. Test solution: Weigh 0.1 g of lyophilized powder and dissolve it in 10 mL of water to obtain a test solution with a concentration of 10 mg / mL.

[0076] 3. Preparation of reference solution

[0077] Accurately weigh 5 mg of rutin reference substance dried to constant weight at 120°C, place in a 50 mL brown volumetric flask, add an appropriate amount of 70% ethanol (30 ml), place in a water bath for slight heating or ultrasonic treatment to dissolve, cool, add 70% ethanol to the scale, shake well, and obtain (a solution containing 0.1 mg of anhydrous rutin per 1 mL).

[0078] 4. Preparation of Standard Curve

[0079] Accurately pipette 0mL, 1mL, 2mL, 3mL, 4mL, and 5mL of rutin standard solution into 10mL volumetric flasks, add 70% ethanol to 5mL, shake well, add 0.3mL of 5% sodium nitrite solution, mix well, and place for 6min, add 0.3mL of 10% aluminum nitrate solution, mix well, and place for 6min, then add 4mL of 4% sodium hydroxide solution, add 70% ethanol to the scale, place for 15min, use the reagent blank as reference, and immediately measure the absorbance at a wavelength of 510nm by UV-visible spectrophotometry, and draw a standard curve with absorbance as the ordinate and concentration as the abscissa. The regression equation of the rutin standard curve is y=11.443x+0.0453(R 2 =0.9993).

[0080] 5. Sample determination

[0081] According to the above method, adjust the appropriate sample concentration, take 1 mL of the test solution, place it in a 10 mL volumetric flask, add 0.3 mL of 5% sodium nitrite solution, mix, and let it stand for 6 min, add 0.3 mL of 10% aluminum nitrate solution, mix, and let it stand for 6 min, then add 4.0 mL of 4% sodium hydroxide solution, add 70% ethanol to make up to the scale, let it stand for 15 min, use the reagent blank as a reference, and measure the absorbance at a wavelength of 510 nm by UV-visible spectrophotometry, read the rutin content in the test solution from the standard curve, and calculate the content of total flavonoids in the sample.

[0082] 6. Calculation

[0083] Read the concentration of rutin in the test solution from the standard curve and calculate according to the following formula.

[0084]

[0085] Where:

[0086] C------Read the concentration of rutin in the test solution on the standard curve, mg / ml;

[0087] V------The volume of the liquid to be tested, mL.

[0088] Total flavonoid content in freeze-dried powder of Pyracantha fortuneana fruit extract:

[0089]

[0090] Total flavonoid yield (%) = total flavonoid content × test sample mass / mass of Pyracantha fortuneana fruit medicinal material added in the process × 100%

[0091] 2. Determination of total polyphenol content in freeze-dried powder of Pyracantha fortuneana fruit extract

[0092] Accurately weigh 0.01g of gallic acid reference substance, dissolve it in ultrapure water and dilute to 100mL to obtain a 0.10mg / mL standard solution. Measure 0mL, 0.1mL, 0.2mL, 0.3mL, 0.4mL and 0.5mL of 0.10mg / mL standard solution in a 10mL volumetric flask, add 5.0mL ultrapure water for dilution, then add 0.5mL of Folin phenol reagent to mix, fully react for 1min, then drop 1.5mL of 20% sodium carbonate solution into the mixed solution, dilute with ultrapure water, finally, react at 75℃ for 10min, measure the absorbance of the mixed solution at 760nm wavelength with UV-visible absorption spectrometer, and obtain the regression equation of gallic acid standard curve y=103.07x+0.0082(R 2 =0.9992). According to the above method, the appropriate sample concentration was adjusted to determine the total polyphenol content in the extract and calculate the total polyphenol content in the sample.

[0093] Calculation process:

[0094] Read the concentration of gallic acid in the test solution from the standard curve and calculate it according to the following formula:

[0095]

[0096] Where:

[0097] C------Read the concentration of gallic acid in the test solution on the standard curve, mg / mL;

[0098] V------The volume of the liquid to be tested, mL.

[0099] Total percentage of polyphenols in freeze-dried powder of Pyracantha fortuneana fruit extract:

[0100]

[0101] Total polyphenol yield (%) = total polyphenol content × test sample mass / mass of Pyracantha fruit medicinal materials added in the process × 100%

[0102] The results of the determination of the yield and content of total flavonoids and total polyphenols in the freeze-dried powder of Pyracantha fortuneana fruit extract of Examples 1-4 and Comparative Examples 1-3 are shown in Table 1.

[0103] Table 1 Contents of total flavonoids and total polyphenols in freeze-dried powder of Pyracantha fortuneana fruit extracts of Examples 1-4 and Comparative Examples 1-3

[0104]

[0105] It can be seen from the experimental results in Table 1 that the preparation method of the present invention is much higher than that of Comparative Example 1 in terms of the extraction rate of total flavonoids and total polyphenols and the content of total flavonoids and total polyphenols in the prepared extract; at the same time, it can be seen from the data results of Example 1 and Comparative Example 3 that, based on the solvent range specified in the present invention, increasing the amount of solvent does not significantly increase the extraction rate of total flavonoids and total polyphenols and the content of total flavonoids and total polyphenols in the extract.

[0106] Experimental Example 2 Identification of components in the Pyracantha fortuneana fruit extract of the present invention

[0107] Ultra high performance liquid chromatography-quadrupole-electrostatic field orbitrap mass spectrometry (UPLC-QExactive-Orbitrap-MS) was used to identify the components in the Pyracantha fortuneana fruit extract of the present invention.

[0108] Instruments: U3000 ultra-high performance liquid chromatography, Q-Exactive mass spectrometer [Thermo Fisher Scientific, USA, equipped with electrospray ionization source (ESI) and Xcalibur 3.0 data processing system.

[0109] Mass spectrometry conditions:

[0110] The ion source is ESI;

[0111] Scanning was performed in positive and negative ion modes, and the scanning method was full scan / data-dependent secondary scan (Fullscan / ddMS2);

[0112] The primary mass spectrometry resolution is Full Scan 70 000 FWHM (Full Width at Half Maximum), and the secondary mass spectrometry resolution is MS / MS 17 500 FWHM.

[0113] At the detection wavelength of 254nm and 365nm, the non-sugar components of Pyracantha fortuneana fruit extract were analyzed by HPLC-MS and data comparison, and 14 main components were confirmed. The HPLC spectrum is shown in Figure 1 , Figure 2 , the specific ingredients are shown in Table 2.

[0114] Table 2 14 main components of Pyracantha fortuneana fruit extract

[0115]

[0116] Experimental Example 3 In vitro tyrosinase activity inhibition test

[0117] 1) preparing tyrosinase solution and levodopa (L-DOPA) solution;

[0118] 2) Take a 96-well plate, add samples and tyrosinase (final concentration is 25U / mL) to the experimental wells; add PBS (PH=6.8) and tyrosinase (final concentration is 25U / mL) to the blank control group; add samples and PBS to the withering group. Place in a 37℃ constant temperature water bath for 5 minutes, then add PBS (PH=6.8) and L-DOPA (final concentration is 0.5mmol / L). Place in a 37℃ constant temperature water bath for 10 minutes, and measure the absorbance at 492nm.

[0119] 3) Calculation of enzyme activity

[0120] The enzyme activity was calculated according to the following formula:

[0121]

[0122] Wherein: A1, A2 are the absorbance values ​​of the sample, tyrosinase and L-DOPA at the beginning and end of the reaction; A3, A4 are the absorbance values ​​of the tyrosinase and L-DOPA at the beginning and end of the reaction.

[0123] Statistical analysis: All experiments were repeated at least three times according to the parallel design principle. The experimental data were expressed as mean ± standard deviation (Mean ± SD) and analyzed using T test data. Compared with the control, P < 0.05 was significant; P < 0.01 was significantly significant; P < 0.001 was very significantly significant.

[0124] The results of Examples 1-4, Comparative Examples 1-3 and α-arbutin on the inhibition rate of tyrosinase activity are shown in Figure 3 .

[0125] The inhibition rates of the pyracantha fruit extracts of Examples 1-4 and Comparative Example 3 on tyrosinase activity are all above 90%. Compared with the blank group, they can significantly inhibit the activity of tyrosinase. Moreover, the inhibitory activity of Examples 1-4 on tyrosinase is significantly higher than that of α-arbutin, and the whitening effect is good.

[0126] Experimental Example 4: Promoting effect on zebrafish type I collagen gene expression

[0127] 1. Principle

[0128] Collagen is the most abundant extracellular matrix protein in the human body, and type I collagen is the most abundant protein in the skin. The distribution of type I collagen in zebrafish is the same as that in humans and shows a high degree of conservation with humans. Their innate expression decreases significantly at 6 days after fertilization and becomes very low between 10 and 12 days after fertilization. Testing the expression of zebrafish type I collagen genes (col1a1a, col1a1b and col1a2), comparing the relative expression changes of zebrafish type I collagen genes in the treatment group and the blank control group, and calculating the promotion rate of type I collagen gene expression can evaluate the anti-wrinkle and firming effects of raw materials or products.

[0129] 2. Main Reagents

[0130] Fish embryo culture medium: 2940 mg of anhydrous calcium chloride, 1233 mg of magnesium sulfate heptahydrate, 630 mg of sodium bicarbonate, and 55 mg of potassium chloride were dissolved in 10 L of water. The pH value was 6.5-8.5. All chemicals were of analytical grade.

[0131] Acetyl hexapeptide-8 working solution: Prepare 0.8 g / L acetyl hexapeptide-8 solution with fish embryo culture medium, and prepare it fresh before use.

[0132] Pyracantha fruit extract freeze-dried powder solution: Take 0.05g of Pyracantha fruit extract freeze-dried powder and dissolve it in fish embryo culture medium, and make up to 100mL to obtain 0.05% Pyracantha fruit extract freeze-dried powder solution.

[0133] RNAlater solution: Invitrogen; AM7020, stored frozen.

[0134] TRIzol reagent: Invitrogen; 15596018, stored in refrigerator.

[0135] Isopropyl alcohol: Store frozen.

[0136] Ethanol: Weigh 37.5 mL of ethanol and dissolve it in 12.5 mL of ultrapure distilled water. Store frozen.

[0137] PrimeScript RT Reagent Kit for DNA Eraser: Takara; Cat no.RR047A

[0138] Real-time PCR kit: SYBR Premix Ex Taq (Takara; Cat no. RR420A)

[0139] 3. Testing process

[0140] Healthy zebrafish 6 days after fertilization were selected. The test required setting up a blank control group (fish embryo culture medium), a positive control group (acetyl hexapeptide-8) and a test substance treatment group (lyophilized powder solution prepared in Example 1). Blank control group: 36 zebrafish were randomly selected and evenly distributed in a 24-well plate, with 3 wells, each containing 12 zebrafish and 2.5mL fish embryo culture medium; positive control group: 36 zebrafish were randomly selected and evenly distributed in a 24-well plate, with 3 wells, each containing 12 zebrafish and 2.5mL acetyl hexapeptide-8 working solution; test substance treatment group: 36 zebrafish were randomly selected and evenly distributed in a 24-well plate, with 3 wells, each containing 12 zebrafish and 2.5mL 0.05% lyophilized powder solution of Pyracantha fruit extract. Place in a 28°C ± 1°C incubator and culture for 24h ± 1h. Twelve zebrafish from each well were collected into a 1.5 mL tube, the solution was removed, 0.5 mL RNAlater solution was added, and the tube was frozen for storage.

[0141] RNA extraction: Remove RNAlater solution and rinse with PBS solution 3 times. Place in ice bath, remove PBS solution, and add 500μL TRIzol reagent. Homogenize embryos with a pellet pestle and place in ice bath for 10 minutes. Add 100μL chloroform, vortex for 1 minute, and place in ice bath for 5 minutes. Centrifuge samples for 20 minutes, transfer the upper layer solution to a 1.5mL centrifuge tube, add 250μL isopropanol, vortex for 1 minute, place in ice bath for 10 minutes, and centrifuge samples for 20 minutes. Remove all supernatant, add 500μL ethanol, vortex for 1 minute, centrifuge samples for 5 minutes, and place in ice bath for 10 minutes. Repeat this step 3 times. Remove ethanol, centrifuge samples for 5 minutes, and air-dry samples in a fume hood with the lid open for 10 minutes. Add 10μL DNase / RNase-free ultrapure distilled water and heat at 55℃ for 15 minutes.

[0142] cDNA synthesis: Dilute the RNA sample to 1000 ng / 7 μL with DNase / RNase-free ultrapure distilled water. Synthesize RNA into cDNA using PrimerScript RT Kit with gDNAEraser (Takara; Cat no.RR047A) and store frozen.

[0143] Perform real-time RT-PCR: Prepare primer mix and real-time PCR master mix for each primer. Dilute cDNA sample 10-fold with DNase / RNase-free ultrapure distilled water. Add 9 μL PCR master mix and 1 μL diluted cDNA sample to each well of a 96-well PCR plate. Seal the 96-well PCR plate with optical film, centrifuge the plate for 5 minutes, and perform real-time PCR amplification.

[0144] Data and result calculation: Real-time PCR data were collected. Ct was used as the amplification result, and the amplification amount of β-actin gene was used as the housekeeping gene. The relative expression of each gene (col1a1a, col1a1b and col1a2) was calculated as the test result according to the following formula.

[0145]

[0146] ΔΔCt=ΔCt 空白对照组 Mean-ΔCt 受试物 ............(2)

[0147] Relative gene expression = 2 -ΔΔCt ..............................(3)

[0148]

[0149] Where:

[0150] ΔCt: the difference between the cycle number of the target gene and the cycle number of the housekeeping gene;

[0151] The number of cycles it takes for the fluorescence signal of the target gene to reach the set threshold;

[0152] The number of cycles experienced when the fluorescence signal of the housekeeping gene reaches the set threshold;

[0153] ΔΔCt: the difference between the average ΔCt of the blank control group and the ΔCt of the test substance group;

[0154] ΔCt 空白对照组 : The average value of the difference between the cycle number of the target gene in the blank control group and the cycle number of the housekeeping gene;

[0155] ΔCt 受试物 : The difference between the cycle number of the target gene and the cycle number of the housekeeping gene in the test substance group;

[0156] 2 -ΔΔCt 空白对照组 : Relative expression of target gene in blank control group;

[0157] 2 -ΔΔCt 受试物组 : Relative expression level of target gene in the test group.

[0158] The mean and standard error of each test group were calculated, and the statistical results were expressed as mean ± standard error. The data were analyzed by variance analysis using statistical software, and the relative expression of the target gene between the test group and the blank control group (or solvent control group) was tested by two-tailed T test to obtain the p value. p<0.05 indicated a significant difference.

[0159] At least 90% of the fish embryos in each test group survived after exposure, otherwise the results of the corresponding test group were invalid.

[0160] 4. Experimental Results

[0161] The specific experimental results are shown in Table 3.

[0162] Table 3 Results of the promotion effect on the expression of type I collagen gene in zebrafish

[0163]

[0164] According to the experimental results in the table, the 0.05% pyracantha fruit extract freeze-dried powder solution can significantly promote the expression of zebrafish type I collagen gene and has the effect of promoting the regeneration of type I collagen. The pyracantha fruit extract freeze-dried powder obtained by the present invention has obvious anti-wrinkle and firming effects.

[0165] Experimental Example 5: Zebrafish embryo melanin inhibition test

[0166] 1. Principle

[0167] Melanin in the human body is formed by the oxidation of tyrosine to dopa through the action of tyrosinase, which is then converted into indolequinone through oxidation and decarboxylation, and finally indolequinone is polymerized into melanin. The genes related to pigment formation in zebrafish are highly similar to those in humans in terms of sequence and regulatory mechanism. Zebrafish embryos are small and transparent, which makes it easy to take pictures under a microscope, and use software analysis to quantitatively read the melanin content level. The changes in the melanin levels of fish embryos in the treatment group and the control group are tested and compared, and the melanin inhibition rate is calculated to evaluate the melanin inhibition effect of the raw materials or products.

[0168] 2. Preparation of main reagents

[0169] Fish embryo culture medium: 2940 mg of anhydrous calcium chloride, 1233 mg of magnesium sulfate heptahydrate, 630 mg of sodium bicarbonate, and 55 mg of potassium chloride were dissolved in 10 L of water. The pH value was 6.5-8.5, and all chemicals were of analytical grade.

[0170] Phenylthiourea storage solution: weigh 15 mg of phenylthiourea and dissolve it in 10 mL of water. Store at 2℃~8℃ away from light. It is valid for half a year.

[0171] Phenylthiourea working solution: dilute the phenylthiourea stock solution 50 times with fish embryo culture medium and prepare 5 mL fresh before use.

[0172] Kojic acid solution: 2.5 g of kojic acid was dissolved in 1000 mL of water.

[0173] Methyl cellulose solution: weigh 2g-4g of methyl cellulose and dissolve it in 100mL of water.

[0174] Pyracantha fruit extract freeze-dried powder solution: Take 0.05g of Pyracantha fruit extract freeze-dried powder, dissolve it in fish embryo culture medium and make up to 100mL to obtain 0.05% Pyracantha fruit extract freeze-dried powder solution.

[0175] 3. Experimental Procedure

[0176] Healthy zebrafish embryos 6-8 hours after fertilization were selected and divided into a blank control group (fish embryo culture medium), a 100% melanin inhibition model group (phenylthiourea working solution), a positive control group (kojic acid solution) and a test substance treatment group (freeze-dried powder of Pyracantha fortuneana fruit extract prepared in Example 1). Among them, the blank control group: randomly selected 20 fish embryos to 96-well plates, each well containing one fish embryo and 0.2mL fish embryo culture medium; 100% melanin inhibition model group: randomly selected 20 fish embryos to 96-well plates, each well containing one fish embryo and 0.2mL 30mg / L phenylthiourea working solution; positive control group: randomly selected 20 fish embryos to 96-well plates, each well containing one fish embryo and 0.2mL 2.5g / L kojic acid solution; test substance treatment group: randomly selected 20 fish embryos to 96-well plates, each well containing one fish embryo and 0.2mL 0.05% pyracantha fruit extract freeze-dried powder solution. All groups were placed in a 28℃±1℃ incubator and cultured until 48h±1h after fertilization.

[0177] Randomly select at least 12 fish embryos from each group. Under a microscope (e.g., 2x eyepiece), carefully peel off the embryo shell of the unhatched fish embryo with tweezers without damaging the fish body. Cover the fish embryo with 2%-4% methylcellulose and place it with its back facing up. Then place it under a stereo microscope for photography. All fish embryos need to be photographed using the same photography parameters.

[0178] Open the photo with analysis software such as Image J, mark the head and yolk dorsal area of ​​each fish embryo, then select "Measure average intensity" in the "Measurement" column, and select the "average signal intensity" in the test results as the melanin content index.

[0179] The calculation formula of melanin inhibition rate is as follows:

[0180]

[0181] Where:

[0182] S—the average value of “mean signal intensity” of fish embryos in the test substance treatment group;

[0183] C—the average value of “average signal intensity” of fish embryos in the blank control group;

[0184] P—The average value of “mean signal intensity” of fish embryos in the 100% melanin inhibition model group.

[0185] The mean and standard error of each test group were calculated, and the statistical results were expressed as mean ± standard error. The data were analyzed by variance analysis using statistical software, and the target melanin signal intensity between the test substance treatment group and the blank control group was tested by two-tailed T test to obtain the p value. p<0.05 indicated a significant difference.

[0186] 4. Results

[0187] Test validity verification: At least 90% of the fish embryos in each test group survive after exposure, otherwise the results of the corresponding test group are invalid.

[0188] Test requirements: A 100% melanin inhibition model group must be set up for each batch of tests. It is required that each batch of fish embryos in the 100% melanin inhibition model group have no visible pigment in the measurement area (including the eyes).

[0189] Representative images are shown in Figure 4 The test results are shown in Table 4.

[0190] Table 4 Results of inhibition of melanin in zebrafish embryos

[0191]

[0192] According to the test data in the above table, it can be seen that the melanin inhibition rate of the blank control group is 0%, and the melanin inhibition rate of the Pyracantha fortuneana fruit extract added at a concentration of 0.05% is 10%, compared with the blank control group, p < 0.05, and the difference is significant. Therefore, the freeze-dried powder of the Pyracantha fortuneana fruit extract prepared by the present invention has a significant whitening effect.

[0193] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. A method for preparing freeze-dried powder of Pyracantha fortuneana fruit extract, characterized in that: The preparation method of the freeze-dried powder of Pyracantha fortuneana fruit extract comprises the following steps: 1) Select fresh and ripe Pyracantha fruit and extract it by supercritical fluid to obtain supercritical fluid extract; 2) The supercritical fluid extract is ultrasonically dissolved in petroleum ether, ethyl acetate and n-butanol in sequence, and filtered to obtain ethyl acetate solution and n-butanol solution; 3) recovering the solvent from the ethyl acetate solution to obtain an ethyl acetate portion; 4) recovering the solvent from the n-butanol solution to obtain the n-butanol portion; 5) Mix the ethyl acetate part with the n-butanol part and freeze-dry them to obtain; Wherein, the supercritical fluid extraction in step 1) is supercritical carbon dioxide fluid extraction, the flow rate of the supercritical carbon dioxide fluid is 3-10 L / min, the temperature of the extraction kettle is 55-60°C, the pressure of the extraction kettle is 5-10MPa, the pressure of the separation kettle is 5-20MPa, the temperature of the separation kettle is 50-60°C, and the extraction time is 1-3h; and the mass of the supercritical fluid extract in step 2) and the added volume ratio of petroleum ether, ethyl acetate, and n-butanol are independently selected from 1:3-6g / mL.

2. The preparation method according to claim 1, characterized in that The supercritical carbon dioxide fluid extraction is a two-step separation, wherein: the pressure of separation kettle I is 10-20MPa, the temperature of separation kettle I is 50-60°C, the pressure of separation kettle II is 5-10MPa, and the temperature of separation kettle II is 50-55°C.

3. The preparation method according to claim 1, characterized in that: The mass ratio of the supercritical fluid extract to the added volume of petroleum ether was 1:5 g / mL, and the mass ratio of the supercritical fluid extract to the added volume of ethyl acetate and n-butanol was 1:3 g / mL.

4. The preparation method according to claim 1, characterized in that: Step 2) The supercritical fluid extract is successively dissolved by ultrasonic treatment with petroleum ether, ethyl acetate and n-butanol, the power of which is independently selected from 300-400 W, and the ultrasonic dissolution time is independently selected from 3-10 min.

5. The preparation method according to claim 4, characterized in that: The power of ultrasonic dissolution in petroleum ether was 350 W, and the ultrasonic dissolution time was 5 min; the power of ultrasonic dissolution in ethyl acetate was 300 W, and the ultrasonic dissolution time was 6 min; the power of ultrasonic dissolution in n-butanol was 350 W, and the ultrasonic dissolution time was 3 min.

6. The preparation method according to claim 1, characterized in that: Step 5) The freeze-drying procedure is: pre-freezing at -50~-20℃ for 5-12h, continuous intermittent vacuum drying for 20-60h, each interruption time is 1-8h, when the temperature rises to 10-30℃ and the vacuum degree is below 5-100Pa, the sample is dried.

7. Use of the freeze-dried powder of Pyracantha fortuneana fruit extract prepared by the preparation method according to any one of claims 1 to 6 in preparing cosmetics with whitening and / or anti-wrinkle and / or firming effects.

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