CN112057493A

CN112057493A - Sophora flavescens extract and preparation method and application thereof

Info

Publication number: CN112057493A
Application number: CN202011078469.9A
Authority: CN
Inventors: 裴运林; 胡露; 魏瑞敬; 郭朝万; 聂艳峰
Original assignee: Guangdong Marubi Biological Technology Co Ltd
Current assignee: Guangdong Marubi Biological Technology Co Ltd
Priority date: 2020-10-10
Filing date: 2020-10-10
Publication date: 2020-12-11

Abstract

The invention relates to a lightyellow sophora root extract and a preparation method and application thereof. The preparation method of the sophora flavescens extract comprises the following steps: extracting radix Sophorae Flavescentis with ethanol water solution, concentrating the extractive solution, and preparing into first extract; adding ammonia water into the first extract for alkalization, extracting the obtained alkalized solution with chloroform, collecting a chloroform layer, and concentrating to dryness to obtain a second extract; purifying the second extract with macroporous adsorbent resin. The radix Sophorae Flavescentis extract prepared by the preparation method has good antioxidant and antibacterial effects, and has low irritation and good antiallergic effects.

Description

Sophora flavescens extract and preparation method and application thereof

Technical Field

The invention relates to the technical field of plant extraction, and particularly relates to a sophora flavescens extract and a preparation method and application thereof.

Background

Antioxidation is the short term for an antioxidant free radical, which is generated in a human body continuously due to the continuous contact of the human body with the outside, including respiration (oxidation reaction), external pollution, radiation irradiation and other factors. Scientific studies have shown that cancer, aging or other diseases are mostly associated with the production of excess free radicals. Research on antioxidation can effectively overcome the harm caused by the antioxidation, so the antioxidation is listed as one of the main research and development directions by health-care products and cosmetic enterprises, and is also one of the most important functional requirements of the market.

In addition, acne is a chronic inflammatory skin disease of hair follicles and sebaceous glands, and is one of the most common diseases of the cosmetology dermatology. In cosmetics, acne prevention or removal mainly depends on active components with bacteriostatic efficacy, and most of the active components have the problems of high irritation and high sensitivity, so that the market acceptance of corresponding products is low.

Radix Sophorae Flavescentis (Sophora flavescens), also called Japanese pagodatree, Japanese holly twig, Japanese pagodatree seed, and Japanese pagodatree. The radix sophorae flavescentis has wide application, wherein the radix sophorae flavescentis contains matrine (matrine), cytisine (cytisine) and the like, has the effects of clearing heat and promoting diuresis, resisting bacteria and diminishing inflammation, invigorating stomach and expelling parasites when being used as a medicine, and is commonly used for treating skin pruritus, neurasthenia, dyspepsia, constipation and other symptoms; the sophora flavescens seeds can be used as pesticide; the radix Sophorae Flavescentis stem bark fiber can be woven into gunny bag. Currently, matrine is the most studied component of sophora flavescens. The matrine is a natural or new substance with obvious health care function to human health, and the research proves that the matrine has the functions of resisting tumor, raising leucocyte, relieving asthma, eliminating phlegm, easing pain, resisting allergy, suppressing immunity and the like. However, the traditional sophora flavescens extract has difficulty in combining good antioxidant and antibacterial effects.

Disclosure of Invention

Accordingly, there is a need for a method for preparing sophora flavescens extracts. The radix Sophorae Flavescentis extract prepared by the preparation method has good antioxidant and antibacterial effects, and has low irritation and good antiallergic effects.

The specific technical scheme is as follows:

a preparation method of a radix sophorae flavescentis extract comprises the following steps:

taking the roots of the sophora flavescens ait, extracting with 50-70% ethanol water solution by volume concentration, concentrating the obtained extracting solution, and preparing a first extract; the extraction is carried out for 25-35 min by leaching at the temperature of 30-50 ℃;

adding ammonia water into the first extract for alkalization till the pH value is 8-11, extracting the obtained alkalized liquid by using chloroform, collecting a chloroform layer, and concentrating to dryness to prepare a second extract; the extraction is carried out according to the volume ratio of the alkalized liquid to chloroform of 1: (0.5-2.5) adding chloroform for extraction;

and purifying the second extract by using macroporous adsorption resin, wherein the adopted resolving liquid is an ethanol water solution with the volume concentration of 55-65%.

In one embodiment, the extraction is performed according to the volume ratio of the alkalized liquid to chloroform being 1: (0.8-1.2) adding chloroform for extraction.

In one embodiment, the alkalization is alkalization to pH 9-10.

In one embodiment, the volume concentration of ethanol in the ethanol aqueous solution is 58-62%.

In one embodiment, the extraction is carried out at 35-45 ℃ for 25-35 min.

In one embodiment, before the extraction, the kuh-seng root is crushed and sieved, so that the particle size of the obtained kuh-seng root powder is not more than 425 μm.

The invention also provides a sophora flavescens extract which is prepared by adopting the preparation method of the sophora flavescens extract.

In one embodiment, the sophora flavescens extract contains 60-85% of total matrines by weight of active ingredients.

The invention also provides application of the sophora flavescens extract in preparing cosmetics.

In one embodiment, the cosmetic is a cosmetic with acne removing, anti-aging or anti-oxidation effects.

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

the invention accidentally discovers that the radix sophorae flavescentis extract is extracted by an ammonia-chlorine method in the process of researching the radix sophorae flavescentis extract, namely, the extract obtained by extracting an ethanol water solution is alkalized by ammonia water, extracted by chloroform and purified by combining macroporous adsorption resin, so that the obtained radix sophorae flavescentis extract contains total matrine and other active ingredients.

Meanwhile, parameters such as the concentration of the ethanol aqueous solution, the extraction process, the alkalization degree, the chloroform extraction ratio and the like are reasonably regulated and controlled on the basis, so that better antioxidant and antibacterial effects can be obtained. In addition, the preparation method is short in time consumption and low in cost, and can realize industrial large-scale production.

Drawings

FIG. 1 is a 200-800nm absorbance spectrum of the Sophora flavescens Aiton extract prepared in example 1;

FIG. 2 is a matrine standard curve;

FIG. 3 shows the selection of optimal dilution factor for Staphylococcus aureus zone of inhibition experiments;

FIG. 4 shows the selection of optimal dilution factor for Staphylococcus epidermidis zone of inhibition experiments;

FIG. 5 is a measurement of the diameter of the zone of inhibition of Propionibacterium acnes;

FIG. 6 is the determination of the diameter of the zone of inhibition of Staphylococcus aureus;

FIG. 7 is a graph showing the measurement of the inhibition zone diameter of Staphylococcus epidermidis.

Detailed Description

The sophora flavescens extract of the present invention, the preparation method thereof, and the use thereof will be described in further detail below with reference to specific examples. The present invention may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

As used herein, "%" is a volume concentration unless otherwise specified.

"aqueous ammonia" refers to an aqueous solution containing 25% to 28% ammonia, and in particular embodiments, an aqueous solution containing 28% ammonia may be used.

The invention provides a preparation method of a sophora flavescens extract, which comprises the following steps:

adding ammonia water into the first extract for alkalization till the pH value is 8-11, extracting the obtained alkalized liquid by using chloroform, collecting a chloroform layer, and concentrating to dryness to prepare a second extract; the extraction is carried out according to the volume ratio of the alkalized liquid to the chloroform of 1: (0.5-2.5) adding chloroform for extraction;

As will be appreciated, the term "concentration of the resulting extract" refers to concentration to remove most or all of the ethanol.

In one specific example, the extraction is performed according to the volume ratio of the alkalized liquid to the chloroform of 1: (0.5-2.5) adding chloroform for extraction. Further, the volume ratio of the alkalizing liquid to the chloroform includes, but is not limited to, the following ratios: 1:0.5, 1: 0.8, 1: 0.9, 1:1. 1: 1.1, 1: 1.2, 1:1.5, 1:2. 1: 2.5. preferably, the volume ratio of the alkalized liquid to chloroform is 1: (0.8 to 1.2).

In one specific example, the alkalization is alkalization to pH 9-10.

In one specific example, the volume concentration of ethanol in the ethanol aqueous solution is 50-70%. Further, the volume concentration of ethanol in the ethanol aqueous solution includes, but is not limited to, the following volume concentrations: 50%, 55%, 58%, 60%, 62%, 65%, 70%. Preferably, the volume concentration of the ethanol in the ethanol water solution is 58-62%.

In one particular example, the extraction is leaching. The traditional sophora flavescens extraction method aiming at the total matrines usually adopts ultrasonic extraction, but in the research process of the preparation method of the sophora flavescens extract, the result of single-factor investigation of ultrasonic power shows that the stage is from 0W to 380W of ultrasonic power, namely, the effect of ultrasonic extraction on the efficacy of the sophora flavescens extract is not obvious, and the preparation of the sophora flavescens extract can be carried out by adopting an extraction method from the aspects of production cost and energy conservation.

Preferably, the extraction is carried out at 35-45 deg.C for 25-35 min. Further, the temperature of extraction includes, but is not limited to, the following: 35 deg.C, 38 deg.C, 39 deg.C, 40 deg.C, 41 deg.C, 42 deg.C, 45 deg.C. The time of extraction includes, but is not limited to, the following times: 25min, 28min, 30min, 32min and 35 min.

In one specific example, the volume ratio of the methanol aqueous solution to the sophora flavescens root is (10-20): 1. further, the volume ratio of the methanol aqueous solution to the sophora flavescens root includes, but is not limited to, the following ratios: 10: 1. 12: 1. 14: 1. 15: 1. 16: 1. 18: 1. 20: 1.

in one specific example, the purification comprises the steps of:

and (3) re-dissolving the second extract, adsorbing the second extract to macroporous adsorption resin, eluting by taking ethanol water solution with volume concentration of 55-65% as an analytic solution, and collecting eluent.

Further, the volume concentration of the analysis solution (55-65% ethanol aqueous solution) includes, but is not limited to, the following volume concentrations: 55%, 58%, 60%, 62% and 65%.

In one specific example, the macroporous adsorbent resin is HPD-100 resin.

In one specific example, after the second extract is reconstituted, the concentration of the second extract in the reconstituted solution is 0.8-1.2 mg/mL. Further, the solvent used for reconstitution is water.

In one specific example, the flow rate of the desorption solution is 1.5-2 BV/h.

In one specific example, before extraction, the sophora flavescens root is pulverized and sieved so that the particle size of the obtained sophora flavescens root powder is not more than 425 μm, or the sophora flavescens root is pulverized and sieved by a 40-mesh sieve.

In addition, in one specific example, the eluate after the macroporous adsorbent resin is collected, concentrated to remove most or all of ethanol, and lyophilized to prepare lyophilized powder of the active ingredient.

The invention also provides a sophora flavescens extract which is prepared by adopting the preparation method of the sophora flavescens extract. Further, the sophora flavescens extract contains 60-85% of total matrines in percentage by weight of active ingredients.

The invention also provides application of the sophora flavescens extract in preparing cosmetics. Further, the cosmetic has acne removing, anti-aging or antioxidant effects.

Hereinafter, specific examples are described, and the raw materials used in the examples are all commercially available products unless otherwise specified.

Example 1

This example provides a preparation method of a sophora flavescens extract, comprising the following steps:

(1) pulverizing dried radix Sophorae Flavescentis, and sieving with 40 mesh sieve to obtain radix Sophorae Flavescentis powder.

(2) Taking the sophora flavescens powder obtained in the step (1), adding 60% ethanol water solution according to the volume ratio of 1:15, extracting for 30min by an extraction method at the temperature of 40 ℃, centrifuging, filtering, taking filtrate, and concentrating under reduced pressure until no ethanol smell exists, thus obtaining the dealcoholized concentrated solution.

(3) Taking the concentrated solution subjected to alcohol removal in the step (2), adding a proper amount of concentrated ammonia test solution to alkalize until the pH value is 9-10, and adding chloroform into the concentrated solution in a volume ratio of 1:1, extracting for multiple times until the chloroform layer is colorless, combining the chloroform layers, concentrating and recovering the solvent to dryness to obtain dry paste.

(4) Taking the dry paste prepared in the step (3), adding water for redissolution, enabling the concentration of a sample solution to be 1.031mg/mL, washing the HPD-100 resin subjected to pretreatment (soaking the HPD-100 resin for 24 hours by using absolute ethyl alcohol) by using distilled water until the taste of the ethyl alcohol does not exist, filling the resin in a chromatographic column of 1.7cm multiplied by 30cm, enabling the resin to be tightly filled, enabling the sample solution to flow through the resin at a fixed flow rate of 0.9BV/h, and enabling the sample solution to flow through the column of 950 mL.

(5) Washing the resin subjected to sample adsorption in the step (4) with a proper amount of water, discarding the effluent of water washing, then analyzing with 60% ethanol analytic solution by volume concentration, allowing the resin to flow through the fixed analytic solution at a flow rate of 1.8BV/h, allowing the analytic solution to flow through a column of 250mL, and collecting the eluent.

(6) And (5) concentrating the eluent in the step (5) under reduced pressure until no ethanol exists, and then freeze-drying.

Examples 2 to 5

Examples 2 to 5 provide four methods for preparing sophora flavescens extracts, which have the same steps as example 1, and mainly differ from the following steps: in the step (2), the extraction is carried out by chloroform according to different volume ratios, and the volume ratio of the alkalized liquid to the chloroform is 1:0.5, 1:1.5, 1:2 and 1:2.5 in sequence.

The method comprises the following specific steps:

(3) And (3) taking the concentrated solution subjected to alcohol removal in the step (2), adding a proper amount of concentrated ammonia solution to alkalize until the pH value is 9-10, then extracting for multiple times by using chloroform according to a corresponding volume ratio until a chloroform layer is colorless, combining the chloroform layers, concentrating and recovering the solvent to be dry to obtain dry paste.

(4) Taking the dry paste prepared in the step (3), adding water for redissolution, enabling the concentration of the sample solution to be 1.031mg/mL, washing the pretreated HPD-100 resin with distilled water until no ethanol smell exists, filling the resin in a chromatographic column of 1.7cm multiplied by 30cm, enabling the resin to be tightly filled, enabling the sample solution to flow through the resin at a fixed flow rate of 0.9BV/h, and enabling the sample solution to flow through the column of 950 mL.

Comparative example 1

The comparative example provides a preparation method of a sophora flavescens extract, which has the same steps as example 1, and mainly comprises the following steps: in the step (2), the temperature of extraction was 60 ℃.

The method comprises the following specific steps:

(2) Taking the sophora flavescens powder obtained in the step (1), adding 60% ethanol water solution according to the volume ratio of 1:15, extracting for 30min by an extraction method at the temperature of 60 ℃, centrifuging, filtering, taking filtrate, and concentrating under reduced pressure until no ethanol smell exists, thus obtaining the alcohol-removed concentrated solution.

(3) And (3) taking the concentrated solution subjected to alcohol removal in the step (2), adding a proper amount of concentrated ammonia solution to alkalize until the pH value is 9-10, extracting for multiple times by using chloroform at a volume ratio of 1:1 until a chloroform layer is colorless, combining the chloroform layers, concentrating, and recovering the solvent to dryness to obtain dry paste.

Comparative example 2

Comparative example 1 provides a method for preparing a sophora flavescens extract, which has the same steps as example 1, and mainly differs from the following steps: step (3) was not performed.

The method comprises the following specific steps:

(2) Taking the sophora flavescens powder obtained in the step (1), adding 60% ethanol water solution according to the volume ratio of 1:15, extracting for 30min by an extraction method at 40 ℃, centrifuging, filtering, taking the filtrate, and concentrating under reduced pressure until no ethanol smell exists, thus obtaining the alcohol-removed concentrated solution.

(4) Taking the alcohol-removed concentrated solution, adjusting the concentration of the sample solution to be 1.031mg/mL by water, washing the pretreated HPD-100 resin by distilled water until no ethanol smell exists, filling the resin in a chromatographic column of 1.7cm multiplied by 30cm, enabling the resin to be densely filled, enabling the sample solution to flow through the resin at a fixed flow rate of 0.9BV/h, and enabling the sample solution to flow through the column of 950 mL.

The sophora flavescens extracts prepared in examples 1 to 5 and comparative examples 1 to 2 were examined.

1. Method for measuring content of total matrines by acid dye colorimetry

1.1 preparation of reagents

(1) Phthalic acid-hydrochloric acid buffer solution: 10.211g of potassium hydrogen phthalate is precisely weighed, water is added to the solution to a constant volume of 250mL to prepare a 0.2mol/L solution, 50mL of the solution is taken, 1.4584g of hydrochloric acid (36% -38% of concentrated hydrochloric acid) is weighed to prepare a 200mL solution, 20mL of the solution is taken, 130mL of water is added to the solution, the mixture is uniformly mixed, and the pH value is precisely measured and adjusted to 3.

(2) Bromocresol green solution (BCG): precisely weighing 140mg of bromocresol green, adding into 2.5m1)0.1mol/L sodium hydroxide (0.4g sodium hydroxide is dissolved in 100mL water) solution for dissolving, and grinding and adding distilled water to 100 mL.

(3) Preparation of standard solution: accurately weighing 10mg of matrine standard substance, adding 50% ethanol to constant volume to 50mL, and preparing into standard substance solution with concentration of 200ug/mL for use.

1.2 selection of detection wavelength

Putting 0.5mL of the prepared standard substance solution into a test tube with a plug, adding 4mL of phthalic acid-hydrochloric acid buffer solution with the pH value of 3 and 0.5mL of bromocresol green, shaking, adding 7mL of chloroform, shaking vigorously for 1min, standing for 1h, and separating a chloroform layer after two phases are separated completely. A sample of the Sophora flavescens Aiton extract (example 1) was prepared as above, and absorbance of the corresponding wavelength was measured at 1 interval in the wavelength range of 200-800nm, and the maximum absorption wavelength was determined to be 414nm as shown in FIG. 1.

1.3 drawing of matrine Standard Curve

Precisely weighing 0mL, 0.1mL, 0.15mL, 0.2mL, 0.25mL and 0.3mL of 200ug/mL standard substance, dissolving in 20mL of test tube with plug, respectively adding 0.5mL, 0.4mL, 0.35mL, 0.3mL, 0.25mL and 0.2mL of 50% ethanol (respectively adding to 0.5mL), respectively adding 4mL of pH 3.0 phthalic acid buffer solution and 0.5mL of bromocresol green solution, shaking, adding 7mL of chloroform, shaking for 1min, standing for 1h, separating chloroform layer after two phases are completely separated, measuring absorbance at 414nm, and drawing standard curve of matrine content, as shown in figure 2.

1.4 determination of purified lyophilized powder sample

Dissolving and diluting the sample to a certain multiple, accurately measuring the sample solution to be 0.5m1, and carrying out the rest operations in the same way as 3, wherein the correspondingly diluted solvent is used as a blank control. The concentration of the diluted sample solution can be calculated through a standard curve of matrine, and the content, extraction rate and freeze-dried powder total matrine of freeze-dried powder are calculated according to the following formula.

The results are shown in table 1 below:

TABLE 1

	Content of Sophora flavescens Total alkaloid	Extraction rate of total matrines
			Example 1	80.26％	1.086％
Example 2	64.56％	1.059％
			Example 3	79.43％	1.064％
Example 4	72.49％	1.026％
			Example 5	67.15％	0.947％
Comparative example 1	65.12％	1.023％
			Comparative example 2	67.16％	1.045％
Comparative example 3	59.49％	1.029％

2. Qualitative detection of bacteriostatic activity of Oxford cup bacteriostatic ring method on three acne pathogenic bacteria

Staphylococcus aureus, staphylococcus epidermidis and propionibacterium acnes are the main pathogens. Propionibacterium acnes, also known as acne bacilli, Propionibacterium acnes or gram-positive anaerobic bacilli, is an intracellular parasitic bacterium, belongs to the normal flora of the skin, generally parasitizes in the hair follicles and sebaceous glands of the skin, can secrete components such as proteins, enzymes, lipopolysaccharides and the like, is a conditional pathogen, causes infections which are endogenous infections, and is a main bacterium causing whelk. Staphylococcus aureus is the most common pathogenic bacterium in human pyogenic infection, can cause local pyogenic infection, can aggravate infection inflammation on acne focus, and leads to more serious acne. The staphylococcus epidermidis is one of normal flora colonized on the skin and mucous membrane of a human body, pathogenic bacteria of the staphylococcus epidermidis are separated from acne patients, the detection rate of the staphylococcus in the acne diseases is found to be close to that of propionibacterium acnes, and a good effect of treating acne by applying the staphylococcus vaccine is achieved, so that the staphylococcus is closely related to the attack of the acne.

2.1 materials and test strains:

propionibacterium acnes (ATCC 11827), staphylococcus aureus (ATCC 10231), staphylococcus epidermidis (ATCC 49134), BHI (brain heart infusion broth medium), BHIA (brain heart infusion agar medium), TSA (soybean casein agar medium), TSB (soybean casein broth medium), 3.5L anaerobic gas bag (mitsubishi gas chemical co., ltd.), anaerobic indicator (mitsubishi gas chemical co., ltd.), 3.5L anaerobic bag (mitsubishi gas chemical co., ltd.), kanamycin, roxithromycin.

2.2 method steps:

bacterial culture and bacterial suspension preparation: the Propionibacterium acnes is inoculated to a brain heart infusion broth culture medium from an agar plate culture medium by using a sterile inoculating loop, placed in an anaerobic culture bag, added with an anaerobic generation bag 1 bag, cultured in a bacterial incubator at 37 ℃ for 48 hours, inoculated in TSB from a slant agar culture medium by using a sterile inoculating loop, and placed in a bacterial incubator at 37 ℃ for 24 hours. Sequentially diluting the cultured acne propionic acid culture medium by 10^-1、10^-2、10^-3. Under the aseptic operation condition, 100 mu L of diluted propionibacterium acnes liquid is uniformly coated on a culture medium, the propionibacterium acnes liquid is placed in an anaerobic culture bag, 1 bag of anaerobic generation bag is added, the propionibacterium acnes liquid is cultured in a bacterial incubator at 37 ℃ for 48 hours, and the propionibacterium acnes liquid is selected to be covered with compact bacteria in a single layer to be the optimal dilution multiple of the bacteriostatic circle experiment, wherein the dilution multiple is 10^-1The optimal dilution multiple for the propionibacterium acnes bacteriostasis zone experiment. Sequentially diluting cultured culture medium of Staphylococcus aureus and Staphylococcus epidermidis by 10^-3、10^-4、10^-5. Under the aseptic operation condition, 100 mu L of diluted staphylococcus aureus and staphylococcus epidermidis bacterial liquid are respectively taken and evenly coated on a culture medium, and the culture medium is placed in a bacterial incubator at 37 ℃ for 24 hours. Observing results, selecting a monolayer of confluent compact bacteria as the optimal dilution multiple of the inhibition zone experiment as shown in figure 3, 10^-3Selecting 10 for optimal dilution times in Staphylococcus aureus zone experiment as shown in FIG. 4^-4The optimal dilution times are used for the inhibition zone experiment of the staphylococcus epidermidis, and bacterial suspensions are prepared by dilution according to the corresponding dilution times.

2.3 determination of bacteriostatic diameter: is divided into 3 groups of staphylococcus aureus group, propionibacterium acnes group and staphylococcus epidermidis group. Under the aseptic operation condition, 100 mu L of the prepared propionibacterium acnes suspension is uniformly coated on a culture medium, and three oxford cups are placed on the surface of the culture medium, and are numbered 1, 2 and 3. 200 μ L of the Sophora flavescens Aiton extract of example 1 was dissolved in 20% ethanol aqueous solution to prepare a sample solution with a concentration of 50mg/mL, Oxford cups (No. 1) were added, 200 μ L of 20% ethanol blank and physiological saline (No. 2 and No. 3) were added to the other two Oxford cups, respectively, as blank control and negative control, 200 μ L of erythromycin positive control (200ug/mL) was added to each of the three Oxford cups on the other plate, and the results obtained by the anaerobic culture procedure were as shown in FIG. 5. And in the staphylococcus aureus group, 100 mu L of prepared staphylococcus aureus suspension is uniformly coated on a culture medium, four oxford cups are placed on the surface of the culture medium, 200 mu L of sample liquid is taken and added into one of the oxford cups with the number of 1, the numbers of 1, 2, 3 and 4, 200 mu L of 20% ethanol blank, physiological saline and kanamycin (100ug/ml) with the numbers of 2, 3 and 4 are respectively added into the other three oxford cups to serve as blank control, negative control and positive control, and after the mixture is cultured in a 37 ℃ incubator for 24 hours, the result is shown in figure 6. The Staphylococcus epidermidis group was operated as the Staphylococcus aureus group, and the concentrations of the sample solutions were set to 12.5mg/mL and 6.25mg/mL, respectively, and the results are shown in FIG. 7.

Examples 2 to 5 and comparative examples 1 to 2 were examined in the same manner. Meanwhile, the group of 'example 1+ matrine' is detected, which is to mix a proper amount of matrine standard substance into the sophora flavescens extract prepared in example 1, so that the content of matrine in the sophora flavescens extract is 90%. The drug sensitivity test criteria are shown in table 2 below:

TABLE 2

Diameter of antibacterial ring (millimeter)	Sensitivity of the device
		20 toOn the upper part	Extreme sensitivity
15～20	High sensitivity
		10～14	Mianmin
Less than 10	Hyposensitivity
		0	Is not sensitive to

The detection results of the groups are summarized in the following tables 3-5:

TABLE 3

TABLE 4

TABLE 5

As can be seen from tables 3-5, the sophora flavescens extract extracted by the invention has an inhibition effect on three acne pathogenic bacteria, and particularly has a very strong inhibition effect on acne main pathogenic bacteria Propionibacterium acnes and Staphylococcus epidermidis.

3. Method for measuring anti-allergy performance by hyaluronidase in vitro inhibition method

The antiallergic effect of the cosmetic raw materials and the cosmetic products is determined by adopting a hyaluronidase in-vitro inhibition method. Hyaluronidase is a participant of anaphylaxis, decomposes Hyaluronic Acid (HA) in vivo to become an acidic stimulus with low molecular weight, causes histamine release, induces the organism to generate sensitive symptoms, and researches show that hyaluronidase HAs strong correlation with inflammation and allergy, and many antiallergic drugs have strong effect of inhibiting the activity of hyaluronidase.

The following reagents were prepared: 1200U/mL hyaluronidase solution; (ii) acetic acid buffer (pH 5.6); ③ 2.5mmol/ml CaCl₂A solution; fourthly, 0.5mg/mL sodium hyaluronate solution; 0.4mol/LNaOH solution; sixthly, acetylacetone solution; seventhly, P-DAB color developing agent; dissolving in 20% ethanol water solution to prepare a sample solution to be tested with a concentration of 5mg/mL (examples 1-5, comparisons 1-3 and "example 1+ matrine").

The experimental procedure was as follows:

respectively taking the tubes A to D, and performing component preparation and step treatment according to the following table 6:

TABLE 6

3 replicates were made for each sample. The hyaluronidase inhibition rate was calculated according to the following formula:

hyaluronidase inhibition (%) - (C-D) - (A-B) ]/(C-D) × 100%

In the formula:

OD value of A- (hyaluronidase + sample + sodium hyaluronate) sample solution;

the OD value of a blank of the B- (acetic acid buffer solution + sample + acetic acid buffer solution) sample;

OD value of C — (hyaluronidase + deionized water + sodium hyaluronate) control solution; d- (acetate buffer + deionized water + acetate buffer) control blank OD value. The results are shown in table 7 below:

TABLE 7

4. Determination of ABTS +. scavenging Capacity antioxidant

ABTS is oxidized into green ABTS under the action of proper oxidant⁺ABTS in the Presence of an antioxidant⁺The production of (A) is inhibited, and the total antioxidant capacity of the sample can be determined and calculated by measuring the absorbance of ABTS at 734 nm.

The following reagents were prepared separately: 4, 2.45mmol/L potassium persulfate; 7mmol/L ABTS stock solution; (iii) 0.2mol/L PBS (pH 7.4); dissolving with 20% ethanol water solution to prepare 1mg/mL sample solution (examples 1-5, comparative examples 1-3 and "example 1+ matrine").

Taking ABTS stock solution, diluting by 40-50 times with absolute ethyl alcohol or 10mmol/L PBS (pH 7.4) to make the light absorption value of the ABTS stock solution at 734nm be 0.7 +/-0.02 (30 ℃), and obtaining ABTS working solution;

respectively sucking 5ml of LABTS working solution and 50 μ L of sample solution into a test tube with a plug, oscillating for 10s, standing at 30 deg.C for 6min, and measuring absorbance A at 734nm₁(ii) a Respectively sucking 5ml of buffer solution and 50 μ L of sample solution into a test tube with plug, oscillating for 10s, standing at 30 deg.C for 6min, and measuring absorbance A at 734nm₂(ii) a Respectively sucking 5ml of LABTS working solution and 50 μ of LPBS buffer solution into a test tube with plug, oscillating for 10s, standing at 30 deg.C for 6min, and measuring absorbance A at 734nm₃。

3 replicates were made for each sample. ABTS clearance was calculated according to the following formula:

in the formula:

A₁——(ABTSworking solution + sample solution) OD value of the sample solution;

A₂OD value of sample blank — (PBS buffer + sample solution);

A₃OD value of reagent blank (ABTS working solution + PBS buffer).

The results are shown in table 8 below:

TABLE 8

Sample (1mg/mL)	ABTS clearance
		Example 1	65.16％
Example 2	62.06％
		Example 3	54.24％
Example 4	61.42％
		Example 5	63.79％
Comparative example 1	54.71％
		Comparative example 2	63.12％
Comparative example 3	65.37％
		Example 1+ Total matrine	63.15％

5. DPPH-scavenging experiment determination of antioxidation

DPPH, also known as 1, 1-diphenyl-2-trinitrophenylhydrazine, is a very stable free radical with a nitrogen center, and the stability of DPPH is mainly caused by steric hindrance of 3 benzene rings with resonance stabilization, so that unpaired electrons on the nitrogen atom in the middle cannot play the role of electron pairing. Its absolute ethyl alcohol solution is purple, and has maximum absorption at wavelength of 517nm, and its absorbance and concentration are in linear relation. When a radical scavenger is added thereto, DPPH may be combined with or substituted for the radical scavenger, whereby the radical scavenging ability can be evaluated by decreasing the number of radicals, decreasing the absorbance, and decreasing the color of the solution.

Preparing 0.2mmol/L DPPH-methanol (or absolute ethyl alcohol) solution; a sample solution with a concentration of 1mg/mL (examples 1 to 5, comparative examples 1 to 3, and "example 1+ matrine") was prepared by dissolving in a 20% ethanol aqueous solution.

Respectively sucking 2mL of sample solution and 2mL of LDPPH solution into a test tube with a plug, mixing uniformly, reacting for 30min in a dark place, and measuring the light absorption value A at the wavelength of 517nm₁(ii) a Respectively absorbing 2mL of sample solution and 2mL of methanol in a test tube with a plug, uniformly mixing, reacting for 30min in a dark place, and measuring the light absorption value A at the wavelength of 517nm₂(ii) a Respectively sucking 2mL of the PPH solution and 2mL of methanol into a test tube with a plug, uniformly mixing, reacting for 30min in a dark place, and measuring the light absorption value A at the wavelength of 517nm₀。

3 replicates were made for each sample. DPPH-clearance was calculated according to the following formula:

in the formula:

A₁OD value of- (DPPH. solution + sample solution) sample solution;

A₂OD value of sample blank- (sample solution + methanol);

A₃OD of reagent blank- (DPPH. solution + methanol).

The results are shown in table 9 below:

TABLE 9

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. A preparation method of a lightyellow sophora root extract is characterized by comprising the following steps:

2. The method for preparing the Sophora flavescens extract according to claim 1, wherein the extraction is performed according to a volume ratio of the alkalizing solution to chloroform of 1: (0.8-1.2) adding chloroform for extraction.

3. The method for preparing the Sophora flavescens extract according to claim 1, wherein the alkalization is performed to pH 9-10.

4. The method of claim 1, wherein the volume concentration of ethanol in the ethanol aqueous solution is 58-62%.

5. The method of claim 1, wherein the extraction is carried out at 35-45 deg.C for 25-35 min.

6. The method for preparing the Sophora flavescens Aiton extract according to any one of claims 1 to 5, wherein the Sophora flavescens Aiton root is pulverized and sieved before the extraction, so that the particle size of the obtained Sophora flavescens Aiton root powder is not more than 425 μm.

7. A Sophora flavescens extract, which is characterized by being prepared by the preparation method of the Sophora flavescens extract as claimed in any one of claims 1 to 6.

8. The Sophora flavescens extract according to claim 7, which comprises 60 to 85% of total matrines by weight of active ingredients.

9. Use of the sophora flavescens extract as claimed in claim 7 or 8 for preparing cosmetics.

10. Use according to claim 9, characterized in that the cosmetic product is a cosmetic product with acne-removing, anti-ageing or anti-oxidant efficacy.