CN113855617A

CN113855617A - Propolis flavone refined product and preparation method and application thereof

Info

Publication number: CN113855617A
Application number: CN202111142903.XA
Authority: CN
Inventors: 柳刚
Original assignee: Hangzhou Fengqing Technology Co ltd
Current assignee: Hangzhou Fengqing Technology Co ltd
Priority date: 2021-09-28
Filing date: 2021-09-28
Publication date: 2021-12-31

Abstract

The invention provides a refined propolis flavone product and a preparation method and application thereof, the technical scheme provided by the invention is that the refined propolis flavone product is prepared by the steps of fractional extraction of sodium bicarbonate and sodium hydroxide, separation and purification, acidification treatment and macroporous adsorption resin filtration, the weight percentage of total flavone in the refined propolis flavone product is more than 90 percent calculated by rutin, and the 9 flavone monomers with the highest content are sequentially as follows: quercetin, 3, 4-dimethoxy cinnamic acid, pinobanksin, naringenin, pinocembrin, kaempferol, apigenin, chrysin, galangin; the invention adopts common alkali and acid treatment, reduces the using amount of ethanol, has no pollution to the environment, and the prepared refined propolis flavone product has definite chemical components, high total flavone content, no viscosity, golden yellow color, obvious effect on resisting skin photoaging, greatly expands the application of the refined propolis flavone product in the field of cosmetics and has higher applicability.

Description

Propolis flavone refined product and preparation method and application thereof

Technical Field

The invention relates to the research field of active ingredients in natural products, in particular to a refined propolis flavone product and a preparation method and application thereof.

Background

Propolis is an adhesive substance formed by mixing secretions such as plant resin collected by worker bees and secretions such as jaw glands and wax glands on the secretions. Researches show that flavonoid and terpene substances in the propolis have the effects of inhibiting and killing various bacteria, and also have wide biological effects of local anesthesia, tissue regeneration promotion and the like.

Propolis raw materials can not be directly eaten, and in the prior art, ethanol is mostly adopted for extracting and concentrating to obtain blocky propolis ethanol extract which is used for producing solution, granules, tablets, capsules, cosmetics and the like. However, the content of the flavonoid compounds in the propolis ethanol extract is about 24 percent, the propolis ethanol extract also contains a lot of impurities, the viscosity is strong, the color is black brown, and when the propolis ethanol extract is applied to cosmetics, the product has dark color, high viscosity, poor stability and easy precipitation, so that the developed skin care products have dark color and poor skin feel, and the application of propolis in the fields of cosmetics and the like is severely limited.

Disclosure of Invention

The invention aims to provide a refined propolis flavone product and a preparation method and application thereof, and aims to overcome the defects of dark color, high viscosity, poor stability, easiness in precipitation and the like caused by low extraction content and more impurities of the existing propolis flavone when applied to cosmetics.

In order to achieve the purpose, the invention adopts the following technical scheme:

a method for preparing refined propolis flavone product comprises the following steps:

(1): wrapping the crushed crude rubber with gauze, putting the crude rubber into a sodium bicarbonate aqueous solution for boiling, and performing heat preservation and stirring extraction to obtain filter residue and filtrate; boiling the filter residue in sodium hydroxide aqueous solution, extracting the filtrate, and mixing with the filtrate to obtain filtrate L1;

(2): adding hydrochloric acid into the filtrate L1 obtained in the step (1) until the pH value of the filtrate R1 is 4.8-5.2, standing and centrifuging to obtain a precipitate S1 and a supernatant L2; adding hydrochloric acid into the supernatant L2 until the pH value of the supernatant L2 is 3.8-4.2, standing and centrifuging to obtain a precipitate S2 and a supernatant L3; adding hydrochloric acid into the supernatant L3 until the pH value of the supernatant L3 is 2.8-3.2, standing and centrifuging to obtain a precipitate S3; mixing the precipitate S1, the precipitate S2 and the precipitate S3 to obtain a crude propolis flavone extract;

(3): adding the crude extract of the propolis flavone obtained in the step (2) into ethanol for dissolving and diluting to obtain a diluent, adjusting the pH of the diluent to 4.8-5.2, dropwise adding the diluent into macroporous adsorption resin, and standing;

(4): and (3) adding the macroporous adsorption resin obtained in the step (3) into deionized water, placing the mixture on a shaking table for elution, placing the macroporous adsorption resin in absolute ethyl alcohol, placing the mixture on the shaking table for elution, collecting eluent, eluting for three times, combining the eluents, and drying under reduced pressure to obtain the refined propolis flavone.

Preferably, in the step (1), the mass fraction of sodium bicarbonate is 5%, and the ratio of propolis in grams to 5% sodium bicarbonate aqueous solution in milliliters is 1: (8-20).

Preferably, in the step (1), the mass fraction of the sodium hydroxide is 2%, and the mass ratio of the propolis to the 2% sodium hydroxide in grams is 1: (5-10).

Preferably, in the step (1), the propolis is boiled in the sodium bicarbonate water solution for 25-35 minutes, the heat preservation temperature is 80-90 ℃, and the heat preservation stirring time is 50-70 min.

Preferably, in the step (2), the mass fraction of the hydrochloric acid is 10%, and the standing time is 20-30 hours.

Preferably, in the step (3), the mass fraction of the ethanol is 40-60%, and the density of the diluent is 0.1-0.5 g/ml.

Preferably, the sample loading amount of the diluent and the macroporous adsorption resin is 0.5-1.0 mL/g, and the standing time is 1-2 hours.

Preferably, in the step (4), the mass fraction of the absolute ethyl alcohol is 75-95%.

A propolis flavone refined substance is prepared by the preparation method of the propolis flavone refined substance, the weight percentage of total flavone in the propolis flavone refined substance is more than 90 percent based on rutin, and the 9 flavone monomers with the highest content of the propolis flavone refined substance sequentially comprise: quercetin, 3, 4-dimethoxy cinnamic acid, pinobanksin, naringenin, pinocembrin, kaempferol, apigenin, chrysin and galangin.

An application of the refined propolis flavone in cosmetics is provided.

Compared with the prior art, the invention has the beneficial effects that:

the technical scheme provides a refined propolis flavone product and a preparation method and application thereof, the preparation method adopts a method of alkali extraction and acid precipitation and macroporous adsorption resin filtration, reduces the using amount of ethanol, is environment-friendly, has high extraction and refining efficiency, basically completely extracts flavone in propolis, has high flavone content and purity and abundant varieties, and is suitable for industrial batch production.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

Fig. 1 is a liquid chromatography detection result chart provided in the sixth embodiment of the present invention.

FIG. 2 is a graph showing the ROS inhibition rate detection provided in example seven of the present invention.

FIG. 3 is a diagram of MMP-1 gene expression detection and content detection provided in example seven of the present invention.

Detailed Description

The technical solutions of the present invention will be described clearly and completely with reference to the accompanying drawings, and it should be understood that the described embodiments are some, but not all embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The embodiment of the invention provides a preparation method of a refined propolis flavone product, wherein the weight percentage of total flavone in the refined propolis flavone product prepared by the method is more than 90 percent in terms of rutin, and the 9 flavone monomers with the highest content of the refined propolis flavone product sequentially comprise the following components: quercetin, 3, 4-dimethoxy cinnamic acid, pinobanksin, naringenin, pinocembrin, kaempferol, apigenin, chrysin and galangin.

The preparation method of the refined propolis flavone product comprises the following steps:

(1): wrapping the crushed crude rubber with nylon gauze, putting the nylon gauze into a sodium bicarbonate aqueous solution with the mass fraction of 5%, boiling for 25-35 minutes, preserving heat at a temperature of more than 85 ℃, stirring and extracting for 50-70 minutes to obtain filter residue and filtrate; boiling the filter residue in a 2% sodium hydroxide aqueous solution for 25-35 minutes, keeping the temperature above 85 ℃, stirring and extracting for 50-70 minutes to obtain filter residue and filtrate, and combining the two filtrates to obtain filtrate L1;

wherein, the proportion of the crude rubber in grams and the sodium bicarbonate aqueous solution in milliliters is 1: (8-20); the proportion of the crude rubber in grams and the sodium hydroxide aqueous solution in milliliters is 1: (5-10);

(2): dropwise adding 10% hydrochloric acid into the filtrate L1 obtained in the step (1), stirring while dropwise adding until the pH of the filtrate L1 is 4.8-5.2, standing for 20-30 hours, and centrifuging to obtain a precipitate S1 and a supernatant L2; dropwise adding 10% hydrochloric acid into the supernatant L2 while stirring, keeping the pH of the supernatant L2 at 3.8-4.2, standing for 20-30 hours, and centrifuging to obtain a precipitate S2 and a supernatant L3; dropwise adding 10% hydrochloric acid into the supernatant L3 while stirring, keeping the pH of the supernatant L3 at 2.8-3.2, standing for 20-30 hours, and centrifuging to obtain a precipitate S3; mixing the precipitate S1, the precipitate S2 and the precipitate S3 to obtain a crude propolis flavone extract;

(3): adding the crude extract of the propolis flavone obtained in the step (2) into 40-60% ethanol by mass fraction for dissolving and diluting until the density of a diluent is 0.1-0.5 g/mL, adjusting the pH value of the diluent to 4.8-5.2, dropwise adding the diluent onto macroporous adsorption resin, and standing for 1-2 hours;

wherein the sample loading amount between the diluent and the macroporous resin is 0.5-1.0 mL/g (crude drug/resin amount);

(4): and (3) adding the macroporous resin subjected to sample loading in the step (3) into deionized water, placing the mixture on a shaking table for elution, removing water-soluble impurities, placing the macroporous resin in anhydrous ethanol with the mass fraction of 75-95%, placing the mixture on the shaking table for elution, performing elution for three times, collecting the eluates respectively, combining the eluates, and performing reduced pressure drying to obtain the refined propolis flavone product.

The total flavone content of the refined propolis flavone product prepared by the method is increased to over 90% from 9.62% in propolis.

The following will further describe with reference to specific examples

Example one

(1): taking 50g of crude rubber, wrapping the crushed crude rubber with nylon gauze, putting the nylon gauze into a sodium bicarbonate aqueous solution with the mass fraction of 5%, boiling for 25 minutes, keeping the temperature at 85 ℃, stirring and extracting for 50 minutes to obtain filter residue and filtrate; boiling the filter residue in 2% sodium hydroxide water solution for 25min, maintaining the temperature at 85 deg.C, stirring, extracting for 50min to obtain filter residue and filtrate, and mixing the above two filtrates to obtain filtrate L1;

wherein, the proportion of the crude rubber in grams and the sodium bicarbonate aqueous solution in milliliters is 1: 10; the proportion of the crude rubber in grams and the sodium hydroxide aqueous solution in milliliters is 1: 8;

(2): dropwise adding 10% hydrochloric acid into the filtrate L1 obtained in the step (1), stirring while dropwise adding until the pH of the filtrate L1 is 4.8, standing for 20 hours, and centrifuging to obtain a precipitate S1 and a supernatant L2; dropwise adding 10% hydrochloric acid into the supernatant L2 while stirring, keeping the pH of the supernatant L2 at 3.8, standing for 20 hours, and centrifuging to obtain a precipitate S2 and a supernatant L3; dropwise adding 10% hydrochloric acid into the supernatant L3 while stirring, keeping the pH of the supernatant L3 at 2.8, standing for 20 hours, and centrifuging to obtain a precipitate S3; mixing the precipitate S1, the precipitate S2 and the precipitate S3 to obtain a crude propolis flavone extract;

(3): adding the crude extract of the propolis flavone obtained in the step (2) into 50% ethanol by mass fraction to dissolve and dilute until the density of a diluent is 0.2g/mL, adjusting the pH value of the diluent to 4.8, dropwise adding the diluent onto macroporous adsorption resin, and standing for 1 hour;

wherein the sample loading amount between the diluent and the macroporous resin is 0.6mL/g (crude drug/resin amount);

(4): and (3) adding the macroporous resin subjected to sample loading in the step (3) into deionized water, placing the mixture on a shaking table for elution, removing water-soluble impurities, placing the macroporous resin in 75% of absolute ethyl alcohol by mass, placing the mixture on the shaking table for elution, performing elution for three times, collecting eluates respectively, combining the eluates, and performing reduced pressure drying to obtain 3.58g of the refined propolis flavone product.

Example two

(1): taking 50g of crude rubber, wrapping the crushed crude rubber with nylon gauze, putting into a sodium bicarbonate aqueous solution with the mass fraction of 5%, boiling for 30min, keeping the temperature above 85 ℃, stirring and extracting for 60min to obtain filter residue and filtrate; boiling the filter residue in 2% sodium hydroxide water solution for 30min, maintaining the temperature at 90 deg.C, stirring, extracting for 60min to obtain filter residue and filtrate, and mixing the above two filtrates to obtain filtrate L1;

wherein, the proportion of the crude rubber in grams and the sodium bicarbonate aqueous solution in milliliters is 1: 20; the proportion of the crude rubber in grams and the sodium hydroxide aqueous solution in milliliters is 1: 5;

(2): dropwise adding hydrochloric acid with the mass fraction of 10% into the filtrate L1 obtained in the step (1), stirring while dropwise adding until the pH of the filtrate L1 is 5, standing for 24 hours, and centrifuging to obtain a precipitate S1 and a supernatant L2; dropwise adding 10% hydrochloric acid into the supernatant L2 while stirring, keeping the pH of the supernatant L2 at 4, standing for 24 hours, and centrifuging to obtain a precipitate S2 and a supernatant L3; dropwise adding 10% hydrochloric acid into the supernatant L3 while stirring, keeping the pH of the supernatant L3 at 3, standing for 24 hours, and centrifuging to obtain a precipitate S3; mixing the precipitate S1, the precipitate S2 and the precipitate S3 to obtain a crude propolis flavone extract;

(3): adding the crude extract of the propolis flavone obtained in the step (2) into ethanol with the mass fraction of 60% for dissolving and diluting until the density of a diluent is 0.5g/mL, adjusting the pH value of the diluent to be 5, dropwise adding the diluent onto macroporous adsorption resin, and standing for 1.5 hours;

wherein the sample loading amount between the diluent and the macroporous resin is 0.8mL/g (crude drug/resin amount);

(4): and (3) adding the macroporous resin subjected to sample loading in the step (3) into deionized water, placing the mixture on a shaking table for elution, removing water-soluble impurities, placing the macroporous resin in absolute ethyl alcohol with the mass fraction of 95%, placing the mixture on the shaking table for elution, carrying out elution for three times, respectively collecting eluates, combining the eluates, and drying under reduced pressure to obtain 3.75g of the refined propolis flavone.

EXAMPLE III

(1): taking 50g of crude rubber, wrapping the crushed crude rubber with nylon gauze, putting into a sodium bicarbonate aqueous solution with the mass fraction of 5%, boiling for 35min, keeping the temperature at 95 ℃, stirring and extracting for 70min to obtain filter residue and filtrate; boiling the filter residue in 2% sodium hydroxide water solution for 35min, maintaining the temperature at 95 deg.C, stirring, extracting for 70min to obtain filter residue and filtrate, and mixing the above two filtrates to obtain filtrate L1;

wherein, the proportion of the crude rubber in grams and the sodium bicarbonate aqueous solution in milliliters is 1: 15; the proportion of the crude rubber in grams and the sodium hydroxide aqueous solution in milliliters is 1: 10;

(2): dropwise adding hydrochloric acid with the mass fraction of 10% into the filtrate L1 obtained in the step (1), stirring while dropwise adding until the pH of the filtrate L1 is 5.2, standing for 30 hours, and centrifuging to obtain a precipitate S1 and a supernatant L2; dropwise adding 10% hydrochloric acid into the supernatant L2 while stirring, keeping the pH of the supernatant L2 at 4.2, standing for 30 hours, and centrifuging to obtain a precipitate S2 and a supernatant L3; dropwise adding 10% hydrochloric acid into the supernatant L3 while stirring, keeping the pH of the supernatant L3 at 3.2, standing for 30 hours, and centrifuging to obtain a precipitate S3; mixing the precipitate S1, the precipitate S2 and the precipitate S3 to obtain a crude propolis flavone extract;

(3): adding the crude extract of the propolis flavone obtained in the step (2) into 50% ethanol by mass fraction to dissolve and dilute until the density of a diluent is 0.3g/mL, adjusting the pH value of the diluent to 5.2, dropwise adding the diluent onto macroporous adsorption resin, and standing for 2 hours;

wherein the sample loading amount between the diluent and the macroporous resin is 1mL/g (crude drug amount/resin amount);

(4): and (3) adding the macroporous resin subjected to sample loading in the step (3) into deionized water, placing the mixture on a shaking table for elution, removing water-soluble impurities, placing the macroporous resin in absolute ethyl alcohol with the mass fraction of 85%, placing the mixture on the shaking table for elution, carrying out elution for three times, respectively collecting eluates, combining the eluates, and drying under reduced pressure to obtain 3.69g of the refined propolis flavone product.

Example four

(1): taking 50g of crude rubber, wrapping the crushed crude rubber with nylon gauze, putting the nylon gauze into a sodium bicarbonate aqueous solution with the mass fraction of 5%, boiling for 30min, keeping the temperature above 85 ℃, stirring and extracting for 1 hour to obtain filter residue and filtrate; boiling the filter residue in 2% sodium hydroxide water solution for 30min, maintaining the temperature above 85 deg.C, stirring, extracting for 1 hr to obtain filter residue and filtrate, and mixing the above two filtrates to obtain filtrate L1;

wherein, the proportion of the crude rubber in grams and the sodium bicarbonate aqueous solution in milliliters is 1: 8; the proportion of the crude rubber in grams and the sodium hydroxide aqueous solution in milliliters is 1: 7;

(3): adding the crude extract of the propolis flavone obtained in the step (2) into ethanol with the mass fraction of 40% for dissolving and diluting until the density of a diluent is 0.1g/mL, adjusting the pH value of the diluent to be 5, dropwise adding the diluent onto macroporous adsorption resin, and standing for 1 hour;

wherein the sample loading amount between the diluent and the macroporous resin is 0.5mL/g (crude drug/resin amount);

(4): and (3) adding the macroporous resin subjected to sample loading in the step (3) into deionized water, placing the mixture on a shaking table for elution, removing water-soluble impurities, placing the macroporous resin in 80% of absolute ethyl alcohol by mass, placing the mixture on the shaking table for elution for three times, collecting eluates respectively, combining the eluates, and drying under reduced pressure to obtain 3.55g of the refined propolis flavone product.

EXAMPLE V test for measuring Total flavone content of propolis flavone refined product

(1) Drawing rutin standard curve

Precisely sucking 1mL, 2mL, 3mL, 4mL, 5mL and 6mL of rutin reference substance using solution with the density of 0.2g/L, and respectively placing the rutin reference substance using solution in a 50mL volumetric flask; adding absolute ethyl alcohol until the total volume is 15mL, sequentially adding 1mL of aluminum nitrate solution with the density of 100g/L and 1mL of potassium acetate solution with the density of 9.8g/L, shaking up, adding water to the scale, shaking up, and standing for 1 h.

Absorbance was measured at 415nm using a 1cm cuvette and a 30% ethanol solution as a blank. Taking the rutin mass (mg) in 50mL as the abscissa and the absorbance as the ordinate, drawing a standard curve or calculating according to a linear regression equation, and obtaining the linear working range of 0mg-1.2mg (50 mL).

(2) Blank test

Precisely weighing 0.1g of propolis refined product sample to be measured, placing the sample in a 50mL volumetric flask, adding absolute ethyl alcohol until the total volume is 15mL, adding water to dilute the sample to a scale, and shaking up.

(3) Determining the content of total flavonoids

Precisely weighing 0.1g of each propolis refined sample and 0.5g of crude gum in the first to fourth examples, respectively placing the propolis refined samples into a 50mL volumetric flask, adding absolute ethyl alcohol until the total volume is 15mL, sequentially adding 1mL of an aluminum nitrate solution with the density of 100g/L and 1mL of a potassium acetate solution with the density of 9.8g/L, shaking up, adding water to the scales, and shaking up. Standing for 1 h.

The absorbance of the sample solution at a wavelength of 415nm was measured using a 1cm cuvette with a blank test solution as a reference. It was found that the total flavonoids in example 92.4%, the total flavonoids in example two 94.6%, the total flavonoids in example three 95.8%, the total flavonoids in example four 91.9% and the total flavonoids in hair glue 9.86%.

TABLE 1

EXAMPLE VI test of chemical composition of propolis flavone refined product

Liquid chromatography detection conditions:

a chromatographic column: sepax HP-C18(150mm 4.6mm,5 μm) or equivalent performance chromatography columns

Mobile phase: methanol, 1% acetic acid by mass fraction

Flow rate: 1ml/min

Detection wavelength: 280nm

Column temperature: 33 deg.C

Sample introduction amount: 5 μ l

Stopping time: 135min

Post-run time: 20min

Gradient elution: the elution procedure is shown in Table 2

TABLE 2 high Performance liquid chromatography gradient elution procedure

The detection structure is shown in figure 1, the propolis flavone refined product obtained in the first embodiment, the second embodiment and the third embodiment is basically consistent with the flavonoid in the hair glue in types, and specifically comprises the following steps:

peak 1: quercetin;

peak 2: 3, 4-dimethoxycinnamic acid;

peak 3: pinobanksin;

peak 4: naringenin;

peak 5: pinocembrin;

peak 6: kaempferol;

peak 7: apigenin;

peak 8: chrysin;

peak 9: galangin.

EXAMPLE seventhly, human fibroblast-based anti-photoaging efficacy assay

The anti-aging effect test is based on an oxidation damage model of UVA irradiation fibroblasts, and the efficacy of the active substance to be tested is evaluated by analyzing the ROS inhibition rate, the expression condition of oxidation-related genes MMP-1 and the content of MMP-1. And (3) respectively dissolving the refined propolis flavone product obtained in the second embodiment in absolute ethyl alcohol and polyethylene glycol to prepare a sample with the concentration of 0.01%, and carrying out a photoaging resistance efficacy test.

(1) ROS inhibition rate detection

Digesting logarithmic phase cells, inoculating to 6-well plate, and culturing at 37 deg.C and CO₂Culturing for 24h in an incubator with the concentration of 5 percent; when the cell plating rate in the 6-hole plate reaches about 40%, the medicine is administered in groups, and each group is provided with 3 multiple holes. At a temperature of 37 ℃ and CO₂The culture was incubated for 24h in an incubator at 5%.

Directly detecting ROS activity in a blank control group; the dosage of the rest groups is 10J/cm²Irradiating the UVA, and directly detecting the activity of the ROS after the irradiation is finished; after PBS is used for washing cells, 1mL of DCFH-DA probe with the concentration of 25 MuM is added into each hole, and the cells are incubated for 45min in a cell incubator at 37 ℃; discarding the culture solution containing DCFH-DA, washing with PBS for multiple times, after trypsinizing the cells, washing the cells with PBS for 1 time, adding a certain amount of fresh PBS, and performing flow detection.

(2) MMP-1 gene expression assay

Based on the results of the cytotoxicity assays, safe concentrations were selected for MMP-1 gene expression assays. After 24h of administration, cells are collected, and the influence of the sample on the intracellular MMP-1 gene expression is detected through qRT-PCR. The specific operation steps are as follows:

digesting logarithmic phase cells, inoculating to 6-well plate, and culturing at 37 deg.C and CO₂Culturing in 5% incubator for 24 h. After the culture is finished, collecting cell culture solution for the next ELISA detection. Cells were gently washed three times with PBS, lysed using RNA isoPlus, and added per well1mL of lysate was added. Standing at room temperature for 5min to fully crack, transferring to a 1.5mL RNase-free Eppendorf tube, extracting RNA, synthesizing cDNA, and performing Elastin gene expression detection.

(3) MMP-1 content detection

Cell culture fluid was collected in 1.5mL EP tubes for MMP-1 ELISA. 1000g of the collected culture solution is centrifuged, and then supernatant fluid is absorbed and stored at the temperature of 20 ℃ below zero for MMP-1 protein ELISA detection.

The experimental results are shown in figure 2 and figure 3, compared with the unirradiated BC group, the ROS content is remarkably increased (p is less than 0.01) after UVA irradiation, and the success of modeling of the oxidative damage model is shown; compared with a UVA irradiation group, the PC group VE extremely remarkably reduces the ROS content (p is less than 0.01), and both the propolis flavone refined substance ethanol dissolving group and the polyethylene glycol dissolving group extremely remarkably reduce the ROS content (p is less than 0.01), so that the synthesis of MMP-1 is inhibited, and the propolis flavone refined substance can inhibit the degradation of extracellular matrix ECM by inhibiting the expression of MMP-1, thereby having the effect of resisting photoaging.

EXAMPLE eight eye irritation experiment

Preparing test solution:

taking 0.01 g of the refined propolis flavone product in the example 2, adding 50mL of propylene glycol to fully dissolve, adding 50mL of purified water, and uniformly mixing to prepare a test solution with the concentration of 0.01%.

The animals and breeding environment were as follows:

the test method comprises the following steps:

the detection basis is as follows: disinfection Specification (2002 edition) 2.3.4

Number of experimental animals: 3 pieces of

Preliminary examination and examination of experimental animals: both eyes of the test animals were examined 24 hours before the start of the test, and animals with eye irritation symptoms, corneal defects, and conjunctival lesions were excluded.

The operation procedure is as follows:

firstly, slightly pulling the lower eyelid of one eye of the rabbit, dripping 0.1mL of test solution into the conjunctival sac of the test object, and dripping physiological saline into the eye of the other side to serve as a normal control.

Then, after the test solution was dropped, the upper and lower eyelids were passively closed for 4 seconds, and after 30 seconds, the upper and lower eyelids were rinsed with physiological saline.

Clinical examination and scoring:

the eyes of the animals are examined 1h, 24h, 48h, 72h, 7d, 14d and 21d after the test solution is dripped, and the injury and recovery of the conjunctiva, iris and cornea of the eyes of the rabbits are observed by naked eyes. If no irritation response occurred for 72h, or the eye irritation response was completely restored at 7d or 14d, the test was terminated prematurely.

The result evaluation method comprises the following steps:

rabbits were scored for acute corneal, iris and conjunctival irritation responses according to the ocular damage scoring criteria, and the "mean score" for each animal was calculated for each animal in terms of corneal damage, iris damage, conjunctival hyperemia and conjunctival edema at three different observation times (24h, 48h and 72h), respectively (i.e., the sum of 24h, 48h and 72h scores for each animal divided by the observation number 3).

And (3) test results:

the toxic manifestation of the infected animals: none.

The ocular irritation response scores are shown in table 3.

TABLE 3 Rabbit eye irritation (within 3 days) response score

And (4) conclusion:

according to the judgment standard of eye irritation response, the intensity of the test object to the acute eye irritation response of the rabbit is nonirritant.

The above embodiments are only preferred embodiments of the present invention, and the protection scope of the present invention is not limited thereby, and any insubstantial changes and substitutions made by those skilled in the art based on the present invention are within the protection scope of the present invention.

Claims

1. A preparation method of a refined propolis flavone product is characterized by comprising the following steps:

2. The method for producing a refined propolis flavone product according to claim 1, wherein in step (1), the mass fraction of sodium bicarbonate is 5%, and the ratio of propolis in grams to a 5% sodium bicarbonate aqueous solution in milliliters is 1: (8-20).

3. The method for producing a refined propolis flavone product according to claim 1, wherein in step (1), the mass fraction of sodium hydroxide is 2%, and the mass ratio of propolis to 2% sodium hydroxide in grams is 1: (5-10).

4. The method for preparing a refined propolis flavone product according to claim 1, wherein in the step (1), the propolis is boiled in an aqueous solution of sodium bicarbonate for 25 to 35 minutes, the temperature is maintained at 80 to 90 ℃, and the stirring is maintained for 50 to 70 minutes.

5. The method for preparing a refined propolis flavone product according to claim 1, wherein the hydrochloric acid is used in an amount of 10% by mass and the standing time is 20 to 30 hours.

6. The method for preparing a refined propolis flavone product as claimed in claim 1, wherein in step (3), the mass fraction of ethanol is 40-60%, and the density of the diluted solution is 0.1-0.5 g/mL.

7. The method for preparing refined propolis flavone as claimed in claim 1, wherein the amount of the diluent and the macroporous adsorbent resin is 0.5-1.0 mL/g, and the standing time is 1-2 hours.

8. The method for preparing refined propolis flavone as claimed in claim 1, wherein the mass fraction of absolute ethyl alcohol in step (4) is 75-95%.

9. A refined propolis flavone product characterized by being prepared by the method for preparing a refined propolis flavone product according to any one of claims 1 to 8, wherein the total flavone content in the refined propolis flavone product is 90% or more in terms of rutin, and the 9 flavone monomers having the highest propolis flavone content are sequentially: quercetin, 3, 4-dimethoxy cinnamic acid, pinobanksin, naringenin, pinocembrin, kaempferol, apigenin, chrysin and galangin.

10. Use of the refined propolis flavone as claimed in claim 9 in cosmetics.