A process for separating and purifying active components of radix foetid bupleuri

Abstract

The present application provides a kind of Radix Polygoni Multiflori active ingredient separation and purification process, belong to plant extraction technical field.The present application separation and purification process includes: Radix Polygoni Multiflori ethanol ultrasonic extraction, activated carbon adsorption, HPD-100 macroporous resin purification, membrane filtration, ultrafiltration, reduced pressure concentration and crystallization step, can efficiently extract Radix Polygoni Multiflori extract with high 5 alpha-reductase inhibitory activity.After compounding with the extract of Euonymus alatus obtained by specific microbial fermentation, the anti-hair loss effect can be significantly enhanced.The in vitro experimental results show that the Radix Polygoni Multiflori extract and the extract of Euonymus alatus both have significant 5 alpha-reductase inhibitory activity.The present application can effectively promote hair growth and improve hair loss, and the effect is close to the positive control drug.The present application process is stable, high in purity, strong in activity, suitable for industrial production, and has good market application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of plant extraction, in particular to a separation and purification process of active ingredients of Radix Polygoni Multiflori. BACKGROUND

[0002] Radix Polygoni Multiflori is a famous traditional Chinese medicinal material, which has been praised as a good medicine for nourishing blood and hair, tonifying kidney and astringing essence since ancient times. It is recorded in the classics such as Bencao Gangmu that it can blacken the hair and bones, and astringe essence. Therefore, consumers have a high degree of recognition and trust in its hair blackening and hair loss prevention effects. With the increase of life pressure and environmental changes, the problem of hair loss and white hair is becoming younger, and the market demand is huge. Consumers are increasingly inclined to choose "natural", "plant extraction", "mild and effective" hair care products. Radix Polygoni Multiflori as a traditional Chinese medicine ingredient has become a market hotspot.

[0003] However, the current separation and purification process of Radix Polygoni Multiflori is mainly based on traditional methods. Such methods are easy to damage heat-sensitive active ingredients, resulting in complex components of crude extracts, low content of effective components, incomplete removal of impurities, and thus affecting the safety and efficacy of the product. At the same time, the existing process lacks efficient, stable and industrialized separation and purification schemes, resulting in large differences in product quality between different batches and unstable content of active ingredients.

[0004] In addition, other ingredients such as polypeptides and amino acid care agents in hair care products have the problems of poor penetration into hair, unstable properties, easy loss, low care effect, and irritation to the skin mucosa. Therefore, an efficient separation and purification process of active ingredients of Radix Polygoni Multiflori is needed to obtain high-purity and high-activity active ingredients of Radix Polygoni Multiflori and apply them to hair care products, while solving the problems of other ingredients in existing hair care products, thereby improving the quality and effect of hair care products. SUMMARY

[0005] The purpose of the present application is to provide a separation and purification process of active ingredients of Radix Polygoni Multiflori. The Radix Polygoni Multiflori extract prepared by the purification process of the present application can inhibit the activity of 5α-reductase and has a positive effect on hair loss prevention.

[0006] In order to achieve the above-mentioned purpose of the application, the present application provides the following technical solutions:

[0007] The present application provides a separation and purification process of active ingredients of Radix Polygoni Multiflori, comprising: crushing Radix Polygoni Multiflori, adding an ethanol solution for ultrasonic extraction, centrifuging, and sequentially subjecting the supernatant to activated carbon adsorption, macroporous adsorption resin purification, membrane filtration, ultrafiltration, reduced pressure concentration, and crystallization to obtain a Radix Polygoni Multiflori extract.

[0008] Preferably, the volume fraction of the ethanol solution is 55%-65%.

[0009] Preferably, the ultrasonic extraction is carried out at a material-liquid ratio of 1:(10-15) g / mL, a temperature of 50-60 DEG C, and a power of 300-400 W, and is repeated 1-3 times, each time for 20-60 min.

[0010] Preferably, the activated carbon adsorption is carried out at an activated carbon addition amount of 0.8%-1.5% of the supernatant, a rotation speed of 80-120 rpm, and for 20-40 min.

[0011] Preferably, the macroporous adsorption resin purification comprises: adsorbing the solution after activated carbon adsorption by HPD-100 type macroporous adsorption resin, and then eluting with water, 20%-30% (by volume) ethanol solution and 60%-70% (by volume) ethanol solution in sequence, and collecting the eluate of the 60%-70% (by volume) ethanol solution.

[0012] Preferably, the membrane filtration comprises: filtering the eluate after macroporous adsorption resin purification by a filter membrane with a pore size of 0.22 mu m or 0.45 mu m under a pressure of 0.1-0.3 MPa.

[0013] Preferably, the ultrafiltration comprises: filtering the filtrate after membrane filtration by a 3000-5000 Da ultrafiltration membrane under a pressure of 0.2-0.5 MPa to obtain a permeate.

[0014] The present application also provides a polygonum multiflorum Thunb extract obtained by the above separation and purification process.

[0015] The present application also provides an application of the polygonum multiflorum Thunb extract in anti-hair loss and shampoo products.

[0016] Preferably, the shampoo product further comprises a eucommia ulmoides oliver extract obtained by complex microbial fermentation of eucommia ulmoides oliver.

[0017] Compared with the prior art, the present application has the following beneficial effects:

[0018] The present application provides a separation and purification process of polygonum multiflorum Thunb active ingredients, which comprises: crushing polygonum multiflorum Thunb, adding an ethanol solution for ultrasonic extraction, centrifuging, and sequentially subjecting the supernatant to activated carbon adsorption, macroporous adsorption resin purification, membrane filtration, ultrafiltration, reduced-pressure concentration and crystallization to obtain a polygonum multiflorum Thunb extract. The polygonum multiflorum Thunb extract and the eucommia ulmoides oliver extract obtained by the separation and purification process can be used for preparing anti-hair loss shampoo. The polygonum multiflorum Thunb extract can directly promote the proliferation and differentiation of hair papilla cells, prolong the anagen phase of hair follicles, induce the conversion of hair follicles from the telogen phase to the anagen phase, inhibit the activity of 5 alpha-reductase, and prevent hair loss from the root; the eucommia ulmoides oliver extract improves the microcirculation of hair follicles, increases local blood flow, and improves the permeability of the stratum corneum of the scalp, thereby creating conditions for the effective components of polygonum multiflorum Thunb and eucommia ulmoides oliver and their own nutrients to reach the target, and improving the bioavailability of the active ingredients. DETAILED DESCRIPTION

[0019] The application provides a separation and purification process of active components of Radix Polygoni Multiflori, which comprises: crushing Radix Polygoni Multiflori, adding an ethanol solution for ultrasonic extraction, centrifuging, and sequentially subjecting supernatant to active carbon adsorption, macroporous adsorption resin purification, membrane filtration, ultrafiltration, reduced pressure concentration and crystallization to obtain Radix Polygoni Multiflori extract.

[0020] The volume fraction of the ethanol solution is preferably 55%-65%, and more preferably 60%; the solid-liquid ratio of the ultrasonic extraction is preferably 1:(10-15) g / mL, and more preferably 1:12 g / mL; the temperature is preferably 50-60 DEG C, and more preferably 55 DEG C; the power is preferably 300-400 W, and more preferably 350 W; the extraction is preferably performed 1-3 times, and more preferably 2 times; and each time is preferably 20-60 min, and more preferably 30 min. The use of ethanol ultrasonic extraction can improve the solubility of the active component stilbene glycoside in Radix Polygoni Multiflori, accelerate the dissolution and diffusion of the active component from plant cells, significantly shorten the extraction time, improve the extraction efficiency, and lay a foundation for subsequent fine separation.

[0021] The addition amount of the active carbon in the active carbon adsorption is preferably 0.8%-1.5% of the mass of the supernatant, and more preferably 1%; the rotation speed is preferably 80-120 rpm, and more preferably 100 rpm; and the time is preferably 20-40 min, and more preferably 30 min. The Radix Polygoni Multiflori extract has a deep color, and the active carbon can effectively adsorb macromolecular pigments such as chlorophyll, tannins and part of polyphenols, so that the color of the liquid is lightened, and high molecular colloids, resin impurities and a small amount of fat-soluble impurities can also be adsorbed, thereby reducing the burden of the next step of macroporous resin adsorption, improving the adsorption capacity and purification effect of the resin.

[0022] The macroporous adsorption resin purification preferably comprises: sequentially subjecting the solution after the active carbon adsorption to HPD-100 type macroporous adsorption resin adsorption, water elution, 20%-30% ethanol solution elution and 60%-70% ethanol solution elution, and collecting the eluate of the 60%-70% ethanol solution. The macroporous resin separates substances through surface adsorption and molecular sieve effect, realizes high-efficiency separation of active components such as stilbene glycoside, the HPD-100 type resin is a non-polar resin, has good adsorption capacity for compounds with benzene ring structure (such as stilbene glycoside and anthraquinone), water washing can wash away strong polar impurities such as sugars, amino acids and inorganic salts, 20%-30% ethanol elution can selectively elute anthraquinone components (such as emodin and rhein acid) with relatively large polarity, and 60%-70% ethanol elution can effectively elute active components such as stilbene glycoside and collect them.

[0023] The membrane filtration preferably comprises filtering the eluate purified by the macroporous adsorption resin through a filter membrane with a pore size of 0.22 μm or 0.45 μm under a pressure of 0.1-0.3 MPa, and more preferably comprises filtering the eluate purified by the macroporous adsorption resin through a filter membrane with a pore size of 0.22 μm under a pressure of 0.2 MPa. This step can precisely remove bacteria and small particles, and serves as a pretreatment before ultrafiltration, protects the subsequent ultrafiltration membrane from being blocked, and further improves the clarity of the liquid.

[0024] The ultrafiltration preferably comprises passing the filtrate filtered by the membrane through an ultrafiltration membrane with a molecular weight cut-off of 3000-5000 Da under a pressure of 0.2-0.5 MPa to obtain a permeate, and more preferably comprises passing the filtrate filtered by the membrane through an ultrafiltration membrane with a molecular weight cut-off of 4000 Da under a pressure of 0.3 MPa to obtain a permeate. This step further purifies the active ingredients such as stilbene glucoside, removes biological macromolecules that may cause allergy or instability of the preparation, and makes the final extract of Polygoni multiflori Radix purer and safer.

[0025] The application further provides an extract of Polygoni multiflori Radix obtained by the above separation and purification process.

[0026] The application further provides an application of the extract of Polygoni multiflori Radix in a hair loss prevention product, which is preferably a hair loss prevention shampoo.

[0027] The shampoo further comprises an extract of Euonymus alatus, which is preferably obtained by fermenting Euonymus alatus with complex microorganisms. The preparation method of the extract of Euonymus alatus more preferably comprises the following steps: crushing Euonymus alatus bark to pass through a 50-100 mesh sieve, adding a liquid fermentation medium at a ratio of 1:15-20 g / mL, sterilizing, inoculating complex microorganisms at a ratio of 3%-8% of the weight of Euonymus alatus bark, anaerobic fermentation at 35-39℃ for 54-72 h, filtering, sterilizing, concentrating the filtrate, and vacuum freeze-drying to a water content of 2wt%-5wt% to obtain the extract of Euonymus alatus; the complex microorganisms preferably consist of Bifidobacterium adolescentis CICC 24573 and Lactobacillus johnsonii CICC 10862, the number of Bifidobacterium adolescentis CICC 24573 in the complex microorganisms is preferably (3-7)×10 8 CFU / g, and more preferably 5×10 8 CFU / g, and the number of Lactobacillus johnsonii CICC 10862 is preferably (2-4)×10 8 CFU / g, and more preferably 3×10 8 CFU / g.

[0028] The various enzyme systems secreted by microorganisms can destroy the tough cell wall structure of Eucommia ulmoides Oliv. vine, so that the inherent active ingredients in the cells are more easily dissolved. The fermentation process can convert the active ingredients in the form of glycosides into aglycones, which usually have smaller molecular weights, higher lipid solubility, stronger biological activity, and are more easily absorbed by the skin. Microorganisms can also produce a large number of secondary metabolites such as organic acids, amino acids, vitamins, polysaccharides, bacteriocins, etc., which have the functions of moisturizing, nourishing the scalp, and promoting hair growth. The Eucommia ulmoides Oliv. vine extract has the effects of stimulating capillary expansion and improving local blood circulation of the scalp, can transport sufficient oxygen and nutrients (amino acids, vitamins, etc.) to the hair papilla cells, has good antioxidant activity, can scavenge free radicals, reduce oxidative stress, has a certain anti-inflammatory effect, can soothe the scalp, can directly penetrate the hair shaft, supplement nutrients, improve hair quality, increase the luster and toughness of the hair, and reduce hair breakage.

[0029] The Radix Polygoni Multiflori extract can directly promote the proliferation and differentiation of hair papilla cells, prolong the anagen phase of hair follicles, induce the conversion of hair follicles from the telogen phase to the anagen phase, and inhibit the activity of 5α-reductase, thereby preventing hair loss from the root. The Eucommia ulmoides Oliv. vine extract improves the microcirculation of hair follicles, increases local blood flow, and improves the permeability of the stratum corneum of the scalp, thereby creating conditions for the active ingredients of Radix Polygoni Multiflori and Eucommia ulmoides Oliv. vine and their own nutrients to reach the target, and improving the bioavailability of the active ingredients.

[0030] The technical solutions in the present application will be described clearly and completely below in combination with the embodiments in the present application. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by a person of ordinary skill in the art without creative work fall within the protection scope of the present application.

[0031] In the following examples, if not specifically stated, all are conventional methods.

[0032] In the following examples, if not specifically stated, all the materials and reagents used can be obtained from commercial channels. Bifidobacterium adolescentis CICC 24573 is Bifidobacterium adolescentis Bifidobacterium adolescentis CICC ® 24573, Lactobacillus johnsonii CICC 10862 is Lactobacillus johnsonii Lactobacillus johnsonii CICC ® 10862, Bifidobacterium breve CICC 6182 is Bifidobacterium breve Bifidobacterium breve CICC ® 6182, Lactobacillus helveticus CICC 24943 is Lactobacillus helveticus Lactobacillus helveticus CICC ® 24943, all purchased from China Industrial Microbial Culture Collection Center.

[0033] Example 1

[0034] Separation and purification process of active ingredient of radix polygoni multiflori

[0035] (1) Ethanol ultrasonic extraction: the radix polygoni multiflori is crushed to pass through a 150-mesh sieve, and 60% ethanol solution by volume is added at a ratio of 1:12 g / mL, ultrasonic extraction is carried out at a temperature of 55℃ and a power of 350W for 2 times, each for 30 min, centrifugation is carried out at 5000 rpm for 10 min, and the supernatant is combined;

[0036] (2) Activated carbon adsorption: 1% activated carbon by weight is added to the supernatant, adsorption is carried out at 100 rpm for 30 min, filtration is carried out, and a filtrate is obtained;

[0037] (3) Macroporous adsorption resin purification: the filtrate is adsorbed by HPD-100 type macroporous adsorption resin, and then water, 25% ethanol solution by volume and 65% ethanol solution by volume are used for elution in sequence, and the eluate of the 65% ethanol solution is collected;

[0038] (4) Membrane filtration: the eluate is filtered through a 0.22μm filter membrane under a pressure of 0.2MPa to obtain a filtrate;

[0039] (5) Ultrafiltration: the filtrate after membrane filtration is filtered through a 4000Da ultrafiltration membrane under a pressure of 0.3MPa to obtain a permeate;

[0040] (6) Concentration and crystallization: the permeate is concentrated under reduced pressure to 1 / 4 of the volume of the permeate to obtain a concentrated solution, 10 times the volume of anhydrous ethanol is added to the concentrated solution, stirring is carried out at 58℃ and 120 rpm for 20 min, natural cooling is carried out to 23℃, and then standing is carried out at 2℃ for 24 h, suction filtration is carried out, the crystals are collected, and vacuum freeze-drying is carried out until the water content is 3wt% to obtain the radix polygoni multiflori extract.

[0041] Example 2

[0042] Separation and purification process of active ingredient of radix polygoni multiflori

[0043] (1) Ethanol ultrasonic extraction: the radix polygoni multiflori is crushed to pass through a 120-mesh sieve, and 55% ethanol solution by volume is added at a ratio of 1:10 g / mL, ultrasonic extraction is carried out at a temperature of 50℃ and a power of 300W for 3 times, each for 20 min, centrifugation is carried out at 2000 rpm for 12 min, and the supernatant is combined;

[0044] (2) Activated carbon adsorption: 0.8% activated carbon by weight is added to the supernatant, adsorption is carried out at 80 rpm for 40 min, filtration is carried out, and a filtrate is obtained;

[0045] (3) macroporous adsorption resin purification: the filtrate is adsorbed by HPD-100 type macroporous adsorption resin, and then eluted by water, 20% ethanol solution by volume and 60% ethanol solution by volume in sequence, and the eluate of the 60% ethanol solution by volume is collected;

[0046] (4) membrane filtration: the eluate is filtered by a 0.22 μm filter membrane under a pressure of 0.1 MPa to obtain a filtrate;

[0047] (5) ultrafiltration: the filtrate after membrane filtration is filtered by a 3000 Da ultrafiltration membrane under a pressure of 0.2 MPa to obtain a permeate;

[0048] (6) concentration and crystallization: the permeate is concentrated under reduced pressure to 1 / 3 of the volume of the permeate to obtain a concentrated solution, 12 times the volume of anhydrous ethanol is added to the concentrated solution, stirring is carried out at 55°C and 100 rpm for 30 min, natural cooling is carried out to 20°C, and standing is carried out at 1°C for 22 h, suction filtration is carried out twice after each washing, the crystals are collected, and vacuum freeze-drying is carried out until the water content is 4 wt% to obtain the extract of Radix Polygoni Multiflori.

[0049] Example 3

[0050] Separation and purification process of active ingredients of Radix Polygoni Multiflori

[0051] (1) ethanol ultrasonic extraction: Radix Polygoni Multiflori is crushed to pass through a 180-mesh sieve, 65% ethanol solution by volume is added at 1:15 g / mL, ultrasonic extraction is carried out at 60°C and 400 W for 60 min, and centrifugation is carried out at 5500 rpm for 8 min, and the supernatant is combined;

[0052] (2) activated carbon adsorption: 1.5% activated carbon by weight is added to the supernatant, adsorption is carried out at 120 rpm for 20 min, and filtration is carried out to obtain a filtrate;

[0053] (3) macroporous adsorption resin purification: the filtrate is adsorbed by HPD-100 type macroporous adsorption resin, and then eluted by water, 30% ethanol solution by volume and 70% ethanol solution by volume in sequence, and the eluate of the 70% ethanol solution by volume is collected;

[0054] (4) membrane filtration: the eluate is filtered by a 0.45 μm filter membrane under a pressure of 0.3 MPa to obtain a filtrate;

[0055] (5) ultrafiltration: the filtrate after membrane filtration is filtered by a 5000 Da ultrafiltration membrane under a pressure of 0.5 MPa to obtain a permeate;

[0056] (6) Concentration and crystallization: the permeate was concentrated under reduced pressure to 1 / 5 of the volume of the permeate to obtain a concentrated solution, 8 times the volume of anhydrous ethanol was added to the concentrated solution, and the solution was stirred at 60°C and 150 rpm for 18 min. After natural cooling to 25°C, it was placed at 4°C for 26 h, and then filtered under suction. The crystals were collected and freeze-dried under vacuum to obtain the extract of Radix Polygoni Multiflori with a water content of 2wt%.

[0057] Example 4

[0058] Anti-shedding composition

[0059] (1) Preparation of Euonymus alatus extract

[0060] Glucose and beef extract were added to deionized water to obtain a liquid fermentation medium, and the concentration of glucose in the liquid fermentation medium was 5 g / L, and the concentration of beef extract was 10 g / L.

[0061] The Euonymus alatus stem bark was crushed to pass through an 80-mesh sieve, and was added to the liquid fermentation medium at a ratio of 1:18 g / mL. After sterilization, the compound microorganism was inoculated at a weight of 5% of the Euonymus alatus stem bark, and was anaerobically fermented at 37°C for 64 h. After filtration and sterilization, the filtrate was concentrated and freeze-dried under vacuum to obtain the Euonymus alatus extract with a water content of 3wt%.

[0062] The compound microorganism was composed of Bifidobacterium adolescentis CICC 24573 and Lactobacillus johnsonii CICC 10862, and the number of Bifidobacterium adolescentis CICC 24573 in the compound microorganism was 5×10 8 CFU / g, and the number of Lactobacillus johnsonii CICC 10862 was 3×10 8 CFU / g.

[0063] (2) Composition

[0064] The extract of Radix Polygoni Multiflori of Example 1 and the Euonymus alatus extract were mixed at a weight ratio of 1:1 to obtain an anti-shedding composition.

[0065] Example 5

[0066] Anti-shedding composition

[0067] (1) Preparation of Euonymus alatus extract

[0068] Glucose and beef extract were added to deionized water to obtain a liquid fermentation medium, and the concentration of glucose in the liquid fermentation medium was 5 g / L, and the concentration of beef extract was 10 g / L.

[0069] The Eucommia ulmoides Oliv. stem bark is crushed to pass through a 50-mesh sieve, and is added to a liquid fermentation medium at a ratio of 1:15 g / mL, and after sterilization, is inoculated with 3% of the compound microorganism by weight, and is anaerobically fermented at 35°C for 72 h, filtered, sterilized, and concentrated, and then vacuum freeze-dried to a water content of 2 wt% to obtain the Eucommia ulmoides Oliv. extract;

[0070] Preferably, the compound microorganism is composed of Bifidobacterium adolescentis CICC 24573 and Lactobacillus johnsonii CICC 10862, and the number of Bifidobacterium adolescentis CICC 24573 in the compound microorganism is 7×10 8 CFU / g, and the number of Lactobacillus johnsonii CICC 10862 is 4×10 8 CFU / g.

[0071] (2) Composition

[0072] The Eucommia ulmoides Oliv. extract of Example 2 and the extract of Eucommia ulmoides Oliv. are mixed at a weight ratio of 1:0.8 to obtain the anti-hair loss composition.

[0073] Example 6

[0074] Anti-hair loss composition

[0075] (1) Preparation of Eucommia ulmoides Oliv. extract

[0076] Glucose and beef extract powder are added to deionized water to obtain a liquid fermentation medium, and the concentration of glucose in the liquid fermentation medium is 8 g / L, and the concentration of beef extract powder is 12 g / L;

[0077] The Eucommia ulmoides Oliv. stem bark is crushed to pass through a 100-mesh sieve, and is added to a liquid fermentation medium at a ratio of 1:20 g / mL, and after sterilization, is inoculated with 8% of the compound microorganism by weight, and is anaerobically fermented at 39°C for 54 h, filtered, sterilized, and concentrated, and then vacuum freeze-dried to a water content of 5 wt% to obtain the Eucommia ulmoides Oliv. extract;

[0078] Preferably, the compound microorganism is composed of Bifidobacterium adolescentis CICC 24573 and Lactobacillus johnsonii CICC 10862, and the number of Bifidobacterium adolescentis CICC 24573 in the compound microorganism is 3×10 8 CFU / g, and the number of Lactobacillus johnsonii CICC 10862 is 2×10 8 CFU / g.

[0079] (2) Composition

[0080] The Eucommia ulmoides Oliv. extract of Example 3 and the extract of Eucommia ulmoides Oliv. are mixed at a weight ratio of 1:1.2 to obtain the anti-hair loss composition.

[0081] Comparative Example 1

[0082] The specific embodiment is the same as that of Example 1, except that the 60% ethanol solution in step (1) is replaced by deionized water.

[0083] Comparative Example 2

[0084] The specific embodiment is the same as that of Example 1, except that the HPD-100 macroporous adsorption resin in step (3) is replaced by D101 macroporous adsorption resin.

[0085] Comparative Example 3

[0086] The specific embodiment is the same as that of Example 1, except that steps (4) and (5) are omitted, and the permeate in step (6) is replaced by the eluent.

[0087] Comparative Example 4

[0088] The specific embodiment is the same as that of Example 1, except that step (6) is omitted, and the permeate is concentrated under reduced pressure to 1 / 4 of the volume of the permeate, and then vacuum freeze-dried to obtain the extract of Radix Polygoni Multiflori.

[0089] Comparative Example 5

[0090] The specific embodiment is the same as that of Example 4, except that the anti-shedding composition omits the extract of Radix Polygoni Multiflori and only has the extract of Eucommia ulmoides Oliv.

[0091] Comparative Example 6

[0092] The specific embodiment is the same as that of Example 4, except that the composite microorganism in step (1) is composed of Bifidobacterium breve CICC 6182 and Lactobacillus helveticus CICC 24943, the number of Bifidobacterium breve CICC 6182 in the composite microorganism is 5×10 8 CFU / g, and the number of Lactobacillus helveticus CICC 24943 is 3×10 8 CFU / g, and the other fermentation conditions remain unchanged.

[0093] Comparative Example 7

[0094] The specific embodiment is the same as that of Example 4, except that the extract of Radix Polygoni Multiflori in Example 1 is replaced by the extract of Radix Polygoni Multiflori in Comparative Example 1.

[0095] Comparative Example 8

[0096] The specific embodiment is the same as that of Example 4, except that the extract of Radix Polygoni Multiflori in Example 1 is replaced by the extract of Radix Polygoni Multiflori in Comparative Example 2.

[0097] Comparative Example 9

[0098] The specific embodiment is the same as that of Example 4, except that the extract of Radix Polygoni Multiflori in Example 1 is replaced by the extract of Radix Polygoni Multiflori in Comparative Example 3.

[0099] Comparative Example 10

[0100] The specific implementation is the same as that of Example 4, except that the Polygonum multiflorum extract of Example 1 is replaced by the Polygonum multiflorum extract of Comparative Example 4.

[0101] Test Example 1

[0102] Detection of 5α-reductase inhibition rate

[0103] 5α-reductase is a key enzyme leading to hair loss, and its excessive activity can promote the conversion of testosterone into dihydrotestosterone, thereby inhibiting hair follicle growth. Therefore, the 5α-reductase inhibition rate is an important indicator for evaluating the hair loss prevention effect.

[0104] The 5α-reductase inhibition rates of the samples to be tested (Polygonum multiflorum extracts of Example 1 and Comparative Examples 1-4, hair loss prevention compositions of Example 4 and Comparative Examples 5-10) were detected, and the specific results are shown in Table 2.

[0105] The detection method of 5α-reductase inhibition rate is as follows:

[0106] The experimental grouping is shown in Table 1.

[0107] Table 1 Experimental grouping and system composition

[0108] grouping System composition (total volume 200 μL, supplemented with enzyme buffer) blank group enzyme buffer + 500 nM testosterone + 2 mM NADPH enzyme inactivation group 1 U / mL 5α-reductase (boiled for 10 min, inactivated) + 500 nM testosterone + 2 mM NADPH test group 1 U / mL 5α-reductase + 500 nM testosterone + 2 mM NADPH + 1% test sample

[0109] The concentrations involved in Table 1 are the final concentrations in the system; the enzyme buffer is 50 mM Tris-HCl (pH 7.4) + 1 mM EDTA + 10% glycerol (volume ratio); 1% of the sample to be tested means that the mass percentage of the sample to be tested in the system is 1%.

[0110] The system was configured according to Table 1, incubated at 37°C for 30 min, 50 μL of 1M HCl was added to terminate the enzyme activity, 2 mL of ethyl acetate was added (to extract dihydrotestosterone), and vortexed for 1 min; centrifuged at 12000 rpm for 5 min, and the upper ethyl acetate phase (containing dihydrotestosterone) was taken, and dried with nitrogen; 100 μL of anhydrous ethanol was added to dissolve the residue, and passed through a 0.22 μm organic filter membrane (to remove impurities and protect the HPLC chromatographic column) for detection.

[0111] HPLC detection:

[0112] Chromatographic column: Agilent ZORBAX SB-C18, 4.6x250mm, 5μm;

[0113] Mobile phase: methanol-water (70:30, volume ratio), flow rate 1.0 mL / min;

[0114] Detection wavelength: 245 nm;

[0115] Column temperature: 30℃;

[0116] Injection volume: 20 μL;

[0117] Standard curve drawing: injection of 0-100 nM dihydrotestosterone standard solution, record the peak area, take dihydrotestosterone concentration as the abscissa and the peak area as the ordinate, and fit the linear regression equation (R 2 ≥0.999).

[0118] The 5α-reductase inhibition rate of the sample to be tested was calculated according to the following formula.

[0119] 5α-reductase inhibition rate = [1- (C 待测样品 -C 酶失活组 ) / (C 空白组 -C 酶失活组 )] × 100%;

[0120] Wherein, C 待测样品 is the dihydrotestosterone concentration of the sample group to be tested, C 酶失活组 is the dihydrotestosterone concentration of the enzyme inactivation group, and C 空白组 is the dihydrotestosterone concentration of the blank group.

[0121] Table 2 5α-reductase inhibition rate (%) of the sample to be tested

[0122] test sample 5α-reductase inhibition rate (%) test sample 5α-reductase inhibition rate (%) Example 1 33.27 Comparative Example 5 20.43 Comparative Example 1 25.80 Comparative Example 6 42.96 Comparative Example 2 27.32 Comparative Example 7 35.35 Comparative Example 3 28.06 Comparative Example 8 37.70 Comparative Example 4 30.81 Comparative Example 9 38.14 Example 4 49.69 Comparative Example 10 40.58

[0123] The results in Table 2 show that, overall, the 5α-reductase inhibition rate of the anti-hair loss composition is higher than that of the polygonum multiflorum extract. As can be seen from the comparison of the data of Example 1, Comparative Example 5 and Example 4, the polygonum multiflorum extract and the eucommia ulmoides extract have a synergistic effect on inhibiting the activity of 5α-reductase. As can be seen from the comparison of the data of Example 1 and Comparative Examples 1-4, Example 4 and Comparative Examples 7-10, in the preparation of the polygonum multiflorum extract, the type of solvent, the type of macroporous adsorption resin, the omission of membrane filtration and ultrafiltration, and the omission of the crystallization step all affect the 5α-reductase inhibition rate. As can be seen from the comparison of the data of Example 4 and Comparative Example 6, changing the type of complex microorganisms also affects the 5α-reductase inhibition rate of the anti-hair loss composition. Therefore, the polygonum multiflorum extract and the eucommia ulmoides extract obtained by the extraction method of the present application can be used together in an anti-hair loss product.

[0124] Test Example 2

[0125] Animal Experiment

[0126] SPF level 8-week-old C57BL / 6 male mice were selected, and the mice were fed with standard hormone-free feed in a SPF level barrier environment (temperature 22-25℃, humidity 50%-60%, 12h light-dark cycle), free to eat and drink, and adaptively fed for 1 week before starting the experiment to reduce the influence of environmental stress on the results.

[0127] On the 0th day of the experiment, the hair on the back of all mice between the shoulder blades was shaved off (area 2cm x 2cm, depilation area) using a small animal hair clipper to avoid scratching the skin (skin damage can start wound healing and interfere with hair follicle cycles). After shaving, the mice were continued to be fed for 3 days to make the hair follicles enter the growth phase uniformly and ensure that the hair follicles respond to androgens consistently when modeling.

[0128] The shaved mice were evenly divided into 14 groups (model group, positive group (2% minoxidil tincture), Example 1 group, Comparative Example 1 group, Comparative Example 2 group, Comparative Example 3 group, Comparative Example 4 group, Example 4 group, Comparative Example 5 group, Comparative Example 6 group, Comparative Example 7 group, Comparative Example 8 group, Comparative Example 9 group, and Comparative Example 10 group), with 10 mice in each group, and there were no significant differences in the body weight and health status of the mice in each group, so that the subsequent experimental steps could be performed.

[0129] Modeling: At 9am every day, the mice in each group were subcutaneously injected with testosterone propionate (5mg / kg) on the back according to the dose (testosterone propionate was dissolved in sesame oil to prepare a 5mg / mL oil agent), for 28 consecutive days. Testosterone propionate induces hair follicles to enter the resting phase prematurely by increasing the level of dihydrotestosterone in the body, resulting in sparse hair loss.

[0130] 1h after the injection of testosterone propionate, the back of each group of mice was shaved and applied (0.1mL per mouse was taken with a pipette), the model group was applied with normal saline, the positive group was applied with 2% minoxidil tincture, and the other groups were applied with the corresponding sample solution to be tested.

[0131] The preparation method of the sample solution to be tested is as follows: 10mL of pure water is added to 1g of radix polygoni multiflori extract / anti-hair loss composition to obtain the sample solution to be tested.

[0132] The sparse degree of hair was observed with a stereomicroscope (10x objective lens), and the hair appearance score was determined,

[0133] The scoring criteria are as follows:

[0134] 0 points: dense hair, no exposed skin;

[0135] 1 point: a small amount of sparse hair, exposed area <10%;

[0136] 2 points: moderate sparse, exposed area 10%-30%;

[0137] 3 points: severe sparse, exposed area 30%-50%;

[0138] 4 points: almost no hair, bare area > 50%.

[0139] After the hair appearance score was performed, the mice were sacrificed, the hair in the depilation area was scraped off, and the hair weight of each group of mice was weighed.

[0140] The hair appearance score and hair weight of each group of mice are shown in Table 3.

[0141] Table 3 Hair appearance score and hair weight of each group of mice

[0142] group hair appearance score (points) hair weight (g) model group 3.8 0.0972 positive group 1.3 0.1627 Example 1 2.2 0.1251 Comparative Example 1 2.7 0.1039 Comparative Example 2 2.6 0.1080 Comparative Example 3 2.4 0.1126 Comparative Example 4 2.3 0.1194 Example 4 1.4 0.1575 Comparative Example 5 3.1 0.1167 Comparative Example 6 1.7 0.1461 Comparative Example 7 1.9 0.1340 Comparative Example 8 2.0 0.1218 Comparative Example 9 1.9 0.1273 Comparative Example 10 1.8 0.1305

[0143] The results in Table 3 show that the hair appearance score and hair weight of the positive group, the example group and the comparative example group are significantly better than those of the model group. The results of Example 1 group are obviously better than those of the model group, indicating that the polygonum multiflorum extract can effectively improve the hair loss induced by testosterone propionate and promote hair growth. The effects of Comparative Example 1-4 groups are all worse than that of Example 1, which is consistent with the 5α-reductase inhibition rate results in Table 2, further proving that the extraction process of Example 1 is better, and can prepare more effective anti-hair loss active ingredients. The hair appearance score and hair weight of Example 4 group are close to those of the positive group, indicating that the anti-hair loss composition of the present application has a significant effect on preventing hair loss and promoting hair growth. The effect of Comparative Example 5 is much worse than that of Example 4, indicating that there is a synergistic effect between the polygonum multiflorum extract and the eucommia ulmoides extract, which can greatly improve the anti-hair loss effect, and the single eucommia ulmoides extract has limited effect. The effects of Comparative Example 6, Comparative Example 7-10 are all worse than that of Example 4, further indicating that the polygonum multiflorum extraction process of Example 1 and the eucommia ulmoides extract fermented by specific composite microorganisms are the key to ensure the excellent effect of the anti-hair loss composition.

[0144] The above only describes the preferred embodiments of the present application, and it should be noted that for those skilled in the art, without departing from the principles of the present application, several improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. The application of Polygonum multiflorum extract in the preparation of anti-hair loss shampoo products, characterized in that, The preparation process of Polygonum multiflorum extract includes: pulverizing Polygonum multiflorum, adding 55%-65% ethanol solution by volume for ultrasonic extraction, centrifuging, and then passing the supernatant through activated carbon adsorption, macroporous adsorption resin purification, membrane filtration, ultrafiltration, vacuum concentration, and crystallization to obtain Polygonum multiflorum extract. Purification with macroporous adsorption resin includes: after the solution adsorbed by activated carbon is adsorbed by HPD-100 macroporous adsorption resin, it is eluted sequentially with water, 20%-30% ethanol solution (v / v), and 60%-70% ethanol solution (v / v), and the eluent of 60%-70% ethanol solution (v / v) is collected. The product also includes Eucommia ulmoides extract, which is obtained by fermenting Eucommia ulmoides through compound microorganisms; the compound microorganisms consist of Bifidobacterium adolescentis CICC 24573 and Lactobacillus johnsonii CICC 10862.

2. The application according to claim 1, characterized in that, Ultrafiltration involves passing the filtrate after membrane filtration through a 3000-5000 Da ultrafiltration membrane at a pressure of 0.2-0.5 MPa to obtain the permeate.

3. The application according to claim 1, characterized in that, The ultrasonic extraction process involves a material-to-liquid ratio of 1:(10-15)g / mL, a temperature of 50-60℃, a power of 300-400W, and 1-3 extractions, each lasting 20-60 minutes.

4. The application according to claim 1, characterized in that, The activated carbon added in the activated carbon adsorption process is 0.8%-1.5% of the supernatant mass, the rotation speed is 80-120 rpm, and the time is 20-40 min.

5. The application according to claim 1, characterized in that, The membrane filtration includes: filtering the eluent purified by macroporous adsorption resin through a 0.22μm or 0.45μm filter membrane at a pressure of 0.1-0.3MPa.