Separation and purification process of active ingredients of polygonum multiflorum

Abstract

The invention provides a separation and purification process of active ingredients of polygonum multiflorum, and belongs to the technical field of plant extraction. The separation and purification process comprises the steps of ethanol ultrasonic extraction of polygonum multiflorum, activated carbon adsorption, HPD-100 macroporous resin purification, membrane filtration, ultrafiltration, vacuum concentration and crystallization, and the polygonum multiflorum extract with high 5alpha-reductase inhibitory activity can be efficiently extracted. The anti-hair loss effect can be obviously enhanced after the compound is compounded with the eucommia ulmoides vine extract obtained through specific microbial fermentation. In-vitro experiment results show that both the polygonum multiflorum extract and the eucommia ulmoides vine extract have remarkable 5alpha-reductase inhibitory activity. The anti-hair loss composition can effectively promote hair growth and improve the hair loss condition, and the effect of the anti-hair loss composition is close to that of a positive control drug. The process is stable, the purity is high, the activity is strong, the method is suitable for industrial production, and the market application prospect is good.

Description

Technical Field

[0001] This invention relates to the field of plant extraction technology, specifically to a process for separating and purifying the active ingredients of Polygonum multiflorum. Background Technology

[0002] Polygonum multiflorum (He Shou Wu) is a renowned traditional Chinese medicine, hailed since ancient times as a superior remedy for "nourishing blood, darkening hair, tonifying the kidneys, and strengthening essence." Its effects on "darkening hair, strengthening muscles and bones, and consolidating essence" are recorded in classic texts such as the *Compendium of Materia Medica*. Therefore, consumers have a high level of awareness and trust in its hair-darkening and hair-preventing efficacy. With increasing life pressures and environmental changes, hair loss and graying are becoming increasingly common among younger people, creating a huge market demand. Consumers are increasingly inclined to choose "natural," "plant-extracted," and "gentle yet effective" hair care products, making Polygonum multiflorum, as a traditional Chinese medicine ingredient, a market hotspot.

[0003] However, current separation and purification processes for Polygonum multiflorum still primarily rely on traditional methods. These methods easily damage heat-sensitive active ingredients, resulting in complex crude extracts, low levels of effective components, and incomplete impurity removal, thus affecting product safety and efficacy. Furthermore, existing processes lack efficient, stable, and industrially scalable precise separation and purification solutions, leading to significant quality variations and unstable active ingredient content between different batches of products.

[0004] Furthermore, other ingredients in shampoo products, such as peptides and amino acid-based conditioning aids, suffer from poor hair penetration, instability, easy loss, and low conditioning effects, and can irritate the skin and mucous membranes. Therefore, a highly efficient process for separating and purifying the active ingredients of Polygonum multiflorum is needed to obtain high-purity, high-activity Polygonum multiflorum active ingredients for application in shampoo products. This would simultaneously address the problems associated with other ingredients in existing shampoos, thereby improving the quality and effectiveness of shampoo products. Summary of the Invention

[0005] The purpose of this invention is to provide a process for separating and purifying the active ingredients of Polygonum multiflorum. The Polygonum multiflorum extract prepared by the purification process of this invention can inhibit 5α-reductase activity and has a positive effect on preventing hair loss.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a process for separating and purifying the active ingredients of Polygonum multiflorum, comprising: pulverizing Polygonum multiflorum, adding ethanol solution for ultrasonic extraction, centrifuging, and sequentially passing the supernatant through activated carbon adsorption, macroporous adsorption resin purification, membrane filtration, ultrafiltration, vacuum concentration, and crystallization to obtain Polygonum multiflorum extract.

[0007] Preferably, the volume fraction of the ethanol solution is 55%-65%.

[0008] Preferably, the ultrasonic extraction has a material-to-liquid ratio of 1:(10-15)g / mL, a temperature of 50-60℃, a power of 300-400W, and is performed 1-3 times, each time for 20-60 minutes.

[0009] Preferably, the amount of activated carbon added in the activated carbon adsorption is 0.8%-1.5% of the supernatant mass, the rotation speed is 80-120 rpm, and the time is 20-40 min.

[0010] Preferably, the macroporous adsorption resin purification includes: adsorbing the solution after activated carbon adsorption onto HPD-100 macroporous adsorption resin, then eluting it sequentially with water, 20%-30% ethanol solution (v / v), and 60%-70% ethanol solution (v / v), and collecting the eluent of 60%-70% ethanol solution (v / v).

[0011] Preferably, 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.

[0012] Preferably, the ultrafiltration includes: passing the filtrate after membrane filtration through a 3000-5000 Da ultrafiltration membrane at a pressure of 0.2-0.5 MPa to obtain a permeate.

[0013] The present invention also provides a Polygonum multiflorum extract obtained according to the above separation and purification process.

[0014] The present invention also provides an application of the above-mentioned Polygonum multiflorum extract in anti-hair loss and shampoo products.

[0015] Preferably, the shampoo product further includes Eucommia ulmoides extract, which is obtained by fermenting Eucommia ulmoides through compound microorganisms.

[0016] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a process for separating and purifying the active ingredients of Polygonum multiflorum, comprising: pulverizing Polygonum multiflorum, adding ethanol solution for ultrasonic extraction, centrifuging, and sequentially passing the supernatant through activated carbon adsorption, macroporous adsorption resin purification, membrane filtration, ultrafiltration, vacuum concentration, and crystallization to obtain Polygonum multiflorum extract. The Polygonum multiflorum extract and Eucommia ulmoides extract obtained using this separation and purification process can be used to prepare anti-hair loss shampoo. Specifically, the Polygonum multiflorum extract can directly promote the proliferation and differentiation of dermal papilla cells, prolong the hair follicle growth phase, induce the hair follicle to transition from the resting phase to the growth phase, and inhibit 5α-reductase activity, thus preventing hair loss from its root cause. The Eucommia ulmoides extract improves hair follicle microcirculation, increases local blood flow, and improves the permeability of the scalp stratum corneum, creating conditions for the effective components and nutrients of Polygonum multiflorum and Eucommia ulmoides to reach their target sites, thereby improving the bioavailability of the active ingredients. Detailed Implementation

[0017] This invention provides a process for separating and purifying the active ingredients of Polygonum multiflorum, comprising: pulverizing Polygonum multiflorum, adding ethanol solution for ultrasonic extraction, centrifuging, and sequentially passing the supernatant through activated carbon adsorption, macroporous adsorption resin purification, membrane filtration, ultrafiltration, vacuum concentration, and crystallization to obtain Polygonum multiflorum extract.

[0018] The volume fraction of the ethanol solution described in this invention is preferably 55%-65%, more preferably 60%; the material-to-liquid ratio for ultrasonic extraction is preferably 1:(10-15) g / mL, more preferably 1:12 g / mL; the temperature is preferably 50-60℃, more preferably 55℃; the power is preferably 300-400W, more preferably 350W; the extraction is preferably performed 1-3 times, more preferably 2 times; each extraction is preferably performed for 20-60 minutes, more preferably 30 minutes. Ethanol ultrasonic extraction can improve the solubility of stilbene glycoside, an active ingredient in Polygonum multiflorum, accelerate the dissolution and diffusion of the effective components from plant cells, significantly shorten the extraction time, and improve the extraction efficiency, laying the foundation for subsequent fine separation.

[0019] In the activated carbon adsorption process described in this invention, the preferred amount of activated carbon added is 0.8%-1.5% of the supernatant mass, more preferably 1%, the preferred rotation speed is 80-120 rpm, more preferably 100 rpm, and the preferred time is 20-40 min, more preferably 30 min. The extract of Polygonum multiflorum has a dark color. Activated carbon can effectively adsorb macromolecular pigments such as chlorophyll, tannins, and some polyphenols, making the liquid color lighter. It can also adsorb high-molecular-weight colloids, resin impurities, and a small amount of fat-soluble impurities, reducing the burden on the subsequent macroporous resin adsorption and improving the resin's adsorption capacity and purification effect.

[0020] The preferred purification method using macroporous adsorption resin of this invention includes: adsorbing the solution after activated carbon adsorption onto HPD-100 macroporous adsorption resin, followed by sequential elution with water, 20%-30% (v / v) ethanol solution, and 60%-70% (v / v) ethanol solution, collecting the eluent of the 60%-70% (v / v) ethanol solution. The macroporous resin separates substances through surface adsorption and molecular sieving, achieving efficient separation of active ingredients such as stilbene glycosides. HPD-100 resin is a non-polar resin with good adsorption capacity for compounds with benzene ring structures (such as stilbene glycosides and anthraquinones). Water washing removes unadsorbed strongly polar impurities such as sugars, amino acids, and inorganic salts. 20%-30% ethanol elution selectively elutes relatively polar anthraquinone components (such as emodin and rhein), while 60%-70% ethanol elution effectively elutes and collects active ingredients such as stilbene glycosides.

[0021] The membrane filtration method of the present invention preferably includes: filtering the eluent purified by macroporous adsorption resin through a 0.22 μm or 0.45 μm filter membrane at a pressure of 0.1-0.3 MPa; more preferably, it includes: filtering the eluent purified by macroporous adsorption resin through a 0.22 μm filter membrane at a pressure of 0.2 MPa. This step can precisely remove bacteria and tiny particles, serving as a pretreatment before ultrafiltration, protecting the subsequent ultrafiltration membrane, preventing it from becoming clogged, and further improving the clarity of the liquid.

[0022] The ultrafiltration method of this invention preferably includes: passing the filtrate after membrane filtration through a 3000-5000 Da ultrafiltration membrane at a pressure of 0.2-0.5 MPa to obtain a permeate; more preferably, it includes: passing the filtrate after membrane filtration through a 4000 Da ultrafiltration membrane at a pressure of 0.3 MPa to obtain a permeate. This step further purifies the active ingredients such as stilbene glycosides and removes biomolecules that may cause allergies or instability in the formulation, making the final Polygonum multiflorum extract purer and safer.

[0023] The present invention also provides a Polygonum multiflorum extract obtained according to the above separation and purification process.

[0024] The present invention also provides an application of the above-mentioned Polygonum multiflorum extract in anti-hair loss and shampoo products, wherein the shampoo product is preferably an anti-hair loss shampoo product.

[0025] The shampoo product of this invention also includes Eucommia ulmoides vine extract, which is preferably obtained by fermenting Eucommia ulmoides vine with compound microorganisms. A more preferred method for preparing the Eucommia ulmoides vine extract includes: pulverizing Eucommia ulmoides vine stem bark through a 50-100 mesh sieve, adding it to a liquid fermentation culture medium at a ratio of 1:15-20 g / mL, sterilizing, inoculating with 3%-8% of the weight of the Eucommia ulmoides vine stem bark into a compound microorganism, anaerobic fermenting at 35-39℃ for 54-72 h, filtering, sterilizing, concentrating the filtrate, and then freeze-drying under vacuum to a water content of 2wt%-5wt% to obtain the Eucommia ulmoides vine extract; the compound microorganisms are preferably composed of Bifidobacterium adolescentis CICC 24573 and Lactobacillus johnsonii CICC 10862, and the preferred bacterial count of Bifidobacterium adolescentis CICC 24573 in the compound microorganisms is (3-7)×10⁻⁶. 8 CFU / g, more preferably 5×10 8 The optimal CFU / g count for *Lactobacillus johnsonii* CICC 10862 is (2-4) × 10⁻⁶. 8 CFU / g, more preferably 3×10 8 CFU / g.

[0026] The various enzyme systems secreted by microorganisms can break down the tough cell wall structure of Eucommia ulmoides, making the inherent active ingredients within the cells more easily dissolved. The fermentation process converts these active ingredients, existing in glycoside form, into aglycones, which typically have smaller molecular weights, higher lipid solubility, stronger biological activity, and are more easily absorbed by the skin. Furthermore, microorganisms produce abundant secondary metabolites such as organic acids, amino acids, vitamins, polysaccharides, and bacteriocins, which have moisturizing, scalp-nourishing, and hair-growth-promoting functions. The Eucommia ulmoides extract of this invention stimulates capillary dilation and improves local blood circulation in the scalp, delivering sufficient oxygen and nutrients (amino acids, vitamins, etc.) to hair papilla cells. It has good antioxidant activity, scavenging free radicals and reducing oxidative stress, and possesses certain anti-inflammatory effects, soothing the scalp. It can directly penetrate the hair shaft, replenishing nutrients, improving hair quality, increasing hair shine and resilience, and reducing breakage.

[0027] The Polygonum multiflorum extract of this invention can directly promote the proliferation and differentiation of hair papilla cells, prolong the hair follicle growth phase, and induce the hair follicle to transition from the resting phase to the growth phase, while inhibiting 5α-reductase activity, thus preventing hair loss from the root cause. The Eucommia ulmoides extract improves the microcirculation of hair follicles, increases local blood flow, and improves the permeability of the scalp stratum corneum, creating conditions for the effective components and nutrients of Polygonum multiflorum and Eucommia ulmoides to reach the target site, thereby improving the bioavailability of the active ingredients.

[0028] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0029] Unless otherwise specified, the following embodiments are all conventional methods.

[0030] Unless otherwise specified, all materials and reagents used in the following examples are commercially available. *Bifidobacterium adolescentis* CICC 24573 is *Bifidobacterium adolescentis*. Bifidobacterium adolescentis CICC ® 24573, Lactobacillus johnsonii CICC 10862 is Lactobacillus johnsonii. Lactobacillus johnsonii CICC ® 10862, Bifidobacterium breve CICC6182 is Bifidobacterium breve. Bifidobacterium breve CICC ® 6182, Lactobacillus helveticus CICC 24943 is Lactobacillus helveticus Lactobacillus helveticus CICC ® All 24943 samples were purchased from the China Industrial Microbial Culture Collection Center.

[0031] Example 1 Separation and purification process of active ingredients of Polygonum multiflorum (1) Ethanol ultrasonic extraction: Polygonum multiflorum was pulverized and passed through a 150-mesh sieve. 60% ethanol solution was added at a ratio of 1:12 g / mL. The mixture was ultrasonically extracted twice at a temperature of 55℃ and a power of 350W for 30 min each time. The mixture was then centrifuged at 5000 rpm for 10 min. The supernatants were combined. (2) Activated carbon adsorption: Add 1% by weight of activated carbon to the supernatant, adsorb at 100 rpm for 30 min, filter, and obtain filtrate; (3) Purification with macroporous adsorption resin: After the filtrate is adsorbed by HPD-100 macroporous adsorption resin, it is eluted sequentially with water, 25% ethanol solution and 65% ethanol solution, and the eluent of 65% ethanol solution is collected. (4) Membrane filtration: The eluent is filtered through a 0.22 μm filter membrane at a pressure of 0.2 MPa to obtain the filtrate; (5) Ultrafiltration: The filtrate after membrane filtration is passed through a 4000 Da ultrafiltration membrane at a pressure of 0.3 MPa to obtain the permeate; (6) Concentration and crystallization: The permeate was concentrated under reduced pressure to 1 / 4 of the permeate volume to obtain a concentrate. Anhydrous ethanol with a volume of 10 times that of the concentrate was added. The mixture was stirred at 58°C and 120 rpm for 20 min. After naturally cooling to 23°C, it was allowed to stand at 2°C for 24 h. The mixture was then filtered and washed three times with anhydrous ethanol. After each washing, the mixture was filtered again and the crystals were collected. The mixture was then freeze-dried under vacuum to a water content of 3 wt% to obtain the Polygonum multiflorum extract.

[0032] Example 2

[0033] Separation and purification process of active ingredients of Polygonum multiflorum (1) Ethanol ultrasonic extraction: Polygonum multiflorum was pulverized and passed through a 120-mesh sieve. 55% ethanol solution was added at a ratio of 1:10 g / mL. The mixture was ultrasonically extracted 3 times at a temperature of 50℃ and a power of 300W for 20 min each time. The mixture was then centrifuged at 2000 rpm for 12 min and the supernatants were combined. (2) Activated carbon adsorption: Add 0.8% by weight of activated carbon to the supernatant, adsorb at 80 rpm for 40 min, filter, and obtain filtrate; (3) Purification with macroporous adsorption resin: After the filtrate is adsorbed by HPD-100 macroporous adsorption resin, it is eluted sequentially with water, 20% ethanol solution and 60% ethanol solution, and the eluent of 60% ethanol solution is collected. (4) Membrane filtration: The eluent is filtered through a 0.22 μm filter membrane at a pressure of 0.1 MPa to obtain the filtrate; (5) Ultrafiltration: The filtrate after membrane filtration is passed through a 3000 Da ultrafiltration membrane at a pressure of 0.2 MPa to obtain the permeate; (6) Concentration and crystallization: The permeate is concentrated under reduced pressure to 1 / 3 of the permeate volume to obtain a concentrate. Anhydrous ethanol with a volume of 12 times that of the concentrate is added. The mixture is stirred at 55°C and 100 rpm for 30 min. After naturally cooling to 20°C, it is allowed to stand at 1°C for 22 h. The mixture is then filtered and washed twice with anhydrous ethanol. After each washing, the mixture is filtered again to collect the crystals. The crystals are then freeze-dried under vacuum to a water content of 4 wt% to obtain the Polygonum multiflorum extract.

[0034] Example 3 Separation and purification process of active ingredients of Polygonum multiflorum (1) Ethanol ultrasonic extraction: Polygonum multiflorum was pulverized and passed through a 180-mesh sieve. 65% ethanol solution was added at a ratio of 1:15 g / mL. The mixture was ultrasonically extracted at 60℃ and 400W for 60 min. The mixture was then centrifuged at 5500 rpm for 8 min. The supernatants were combined. (2) Activated carbon adsorption: Add 1.5% by weight of activated carbon to the supernatant, adsorb at 120 rpm for 20 min, filter, and obtain filtrate; (3) Purification with macroporous adsorption resin: After the filtrate is adsorbed by HPD-100 macroporous adsorption resin, it is eluted sequentially with water, 30% ethanol solution and 70% ethanol solution, and the eluent of 70% ethanol solution is collected. (4) Membrane filtration: The eluent is filtered through a 0.45 μm filter membrane at a pressure of 0.3 MPa to obtain the filtrate; (5) Ultrafiltration: The filtrate after membrane filtration is passed through a 5000 Da ultrafiltration membrane at a pressure of 0.5 MPa to obtain the permeate; (6) Concentration and crystallization: The permeate was concentrated under reduced pressure to 1 / 5 of the permeate volume to obtain a concentrate. Eight times the volume of anhydrous ethanol was added to the concentrate. The mixture was stirred at 60°C and 150 rpm for 18 min. After naturally cooling to 25°C, it was allowed to stand at 4°C for 26 h. The mixture was then filtered and washed four times with anhydrous ethanol. After each washing, the mixture was filtered again to collect the crystals. The crystals were then freeze-dried under vacuum to a water content of 2 wt% to obtain the Polygonum multiflorum extract.

[0035] Example 4 Anti-hair loss composition (1) Preparation of Eucommia ulmoides extract Glucose and beef extract powder were added to deionized water to obtain a liquid fermentation medium with a glucose concentration of 5 g / L and a beef extract powder concentration of 10 g / L. The bark of Eucommia ulmoides stem was crushed and passed through an 80-mesh sieve. It was added to a liquid fermentation medium at a ratio of 1:18 g / mL. After sterilization, 5% of the weight of the bark of Eucommia ulmoides stem was inoculated with compound microorganisms. The mixture was anaerobic fermented at 37°C for 64 hours. After filtration and sterilization, the filtrate was concentrated and then freeze-dried under vacuum to a water content of 3 wt% to obtain Eucommia ulmoides extract. The compound microbial system consists of *Bifidobacterium adolescentis* CICC 24573 and *Lactobacillus johnsonii* CICC 10862, with the *Bifidobacterium adolescentis* CICC 24573 comprising 5 × 10⁻⁶ bacteria. 8 CFU / g, Lactobacillus johnsonii CICC 10862 bacterial count was 3×10⁻¹⁰. 8 CFU / g.

[0036] (2) Composition The extracts of Polygonum multiflorum and Eucommia ulmoides in Example 1 were mixed at a weight ratio of 1:1 to obtain an anti-hair loss composition.

[0037] Example 5 Anti-hair loss composition (1) Preparation of Eucommia ulmoides extract Glucose and beef extract powder were added to deionized water to obtain a liquid fermentation medium with a glucose concentration of 3 g / L and a beef extract powder concentration of 8 g / L. The bark of Eucommia ulmoides stem was crushed and passed through a 50-mesh sieve. It was added to a liquid fermentation medium at a ratio of 1:15 g / mL. After sterilization, 3% of the weight of the bark of Eucommia ulmoides stem was inoculated with compound microorganisms. The mixture was anaerobic fermented at 35°C for 72 hours. After filtration and sterilization, the filtrate was concentrated and then freeze-dried under vacuum to a water content of 2 wt% to obtain Eucommia ulmoides extract. The compound microorganisms are preferably composed of Bifidobacterium adolescentis CICC 24573 and Lactobacillus johnsonii CICC 10862, with the number of Bifidobacterium adolescentis CICC 24573 in the compound microorganisms being 7 × 10⁻⁶. 8 CFU / g, Lactobacillus johnsonii CICC 10862 bacterial count was 4 × 10⁻⁶. 8 CFU / g.

[0038] (2) Composition In Example 2, Polygonum multiflorum extract and Eucommia ulmoides extract were mixed at a weight ratio of 1:0.8 to obtain an anti-hair loss composition.

[0039] Example 6 Anti-hair loss composition (1) Preparation of Eucommia ulmoides extract Glucose and beef extract powder were added to deionized water to obtain a liquid fermentation medium with a glucose concentration of 8 g / L and a beef extract powder concentration of 12 g / L. The bark of Eucommia ulmoides stem was crushed and passed through a 100-mesh sieve. It was added to a liquid fermentation medium at a ratio of 1:20 g / mL. After sterilization, it was inoculated with 8% of the weight of the Eucommia ulmoides stem bark compound microorganisms. It was anaerobic fermented at 39°C for 54 hours. After filtration and sterilization, the filtrate was concentrated and then freeze-dried under vacuum to a water content of 5 wt% to obtain Eucommia ulmoides extract. The composite microorganisms preferably consist of *Bifidobacterium adolescentis* CICC 24573 and *Lactobacillus johnsonii* CICC 10862, with the *Bifidobacterium adolescentis* CICC 24573 comprising 3 × 10⁻⁶ bacteria. 8 CFU / g, Lactobacillus johnsonii CICC10862 bacterial count was 2×10⁻⁶. 8 CFU / g.

[0040] (2) Composition In Example 3, Polygonum multiflorum extract and Eucommia ulmoides extract were mixed at a weight ratio of 1:1.2 to obtain an anti-hair loss composition.

[0041] Comparative Example 1 The specific implementation method is the same as in Example 1, except that the 60% ethanol solution in step (1) is replaced with deionized water.

[0042] Comparative Example 2 The specific implementation method is the same as that in Example 1, except that the HPD-100 macroporous adsorption resin in step (3) is replaced with D101 macroporous adsorption resin.

[0043] Comparative Example 3 The specific implementation method is the same as that in Example 1, except that steps (4) and (5) are omitted, and the permeate in step (6) is replaced with the elution solution.

[0044] Comparative Example 4 The specific implementation method is the same as in Example 1, except that step (6) is omitted. After the permeate is concentrated under reduced pressure to 1 / 4 of the permeate volume, it is freeze-dried under vacuum to obtain Polygonum multiflorum extract.

[0045] Comparative Example 5 The specific implementation method is the same as in Example 4, except that the anti-hair loss composition omits the Polygonum multiflorum extract and only contains Eucommia ulmoides extract.

[0046] Comparative Example 6 The specific implementation method is the same as in Example 4, except that in step (1), the composite microorganism consists of Bifidobacterium breve CICC6182 and Lactobacillus helveticus CICC 24943, and the number of Bifidobacterium breve CICC 6182 in the composite microorganism is 5 × 10⁻⁶. 8 CFU / g, Lactobacillus helveticus CICC 24943 count was 3×10⁻⁶. 8CFU / g, other fermentation conditions remain unchanged.

[0047] Comparative Example 7 The specific implementation method is the same as that in Example 4, except that the Polygonum multiflorum extract in Example 1 is replaced with the Polygonum multiflorum extract in Comparative Example 1.

[0048] Comparative Example 8 The specific implementation method is the same as that in Example 4, except that the Polygonum multiflorum extract in Example 1 is replaced with the Polygonum multiflorum extract in Comparative Example 2.

[0049] Comparative Example 9 The specific implementation method is the same as that in Example 4, except that the Polygonum multiflorum extract in Example 1 is replaced with the Polygonum multiflorum extract in Comparative Example 3.

[0050] Comparative Example 10 The specific implementation method is the same as that in Example 4, except that the Polygonum multiflorum extract in Example 1 is replaced with the Polygonum multiflorum extract in Comparative Example 4.

[0051] Experimental Example 1 Detection of 5α-reductase inhibition rate 5α-reductase is a key enzyme that causes hair loss. Excessive 5α-reductase activity can promote the conversion of testosterone into dihydrotestosterone, thereby inhibiting hair follicle growth. Therefore, the inhibition rate of 5α-reductase is an important indicator for evaluating the effectiveness of hair loss prevention.

[0052] The 5α-reductase inhibition rate of the test samples (Polygonum multiflorum extract of Example 1 and Comparative Examples 1-4, and anti-hair loss composition of Example 4 and Comparative Examples 5-10) was detected respectively, and the specific results are shown in Table 2.

[0053] The method for detecting 5α-reductase inhibition rate is as follows: The experimental groupings are shown in Table 1.

[0054] Table 1 Experimental grouping and system composition Grouping System composition (total volume 200 μL, made up with enzyme buffer). Blank group Enzyme buffer + 500 nM testosterone + 2 mM NADPH Enzyme inactivation group 1 U / mL 5α-reductase (inactivated by boiling for 10 min) + 500 nM testosterone + 2 mM NADPH Test group 1 U / mL 5α-reductase + 500 nM testosterone + 2 mM NADPH + 1% of the sample to be tested

[0055] The concentrations mentioned in Table 1 are all 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 test sample means that the mass percentage of the test sample in the system is 1%.

[0056] Prepare the system according to Table 1, incubate at 37℃ for 30 min, add 50 μL 1M HCl to terminate enzyme activity, add 2 mL ethyl acetate (to extract dihydrotestosterone), vortex for 1 min; centrifuge at 12000 rpm for 5 min, take the upper ethyl acetate phase (containing dihydrotestosterone), and dry it with nitrogen gas; add 100 μL anhydrous ethanol to dissolve the residue, filter through a 0.22 μm organic filter membrane (to remove impurities and protect the HPLC column), and then proceed with the analysis.

[0057] HPLC detection: Column: Agilent ZORBAX SB-C18, 4.6 × 250 mm, 5 μm; Mobile phase: methanol-water (70:30, v / v), flow rate 1.0 mL / min; Detection wavelength: 245 nm; Column temperature: 30℃; Injection volume: 20 μL; Standard curve plotting: Inject 0-100 nM dihydrotestosterone standard solution, record the peak area, and plot the dihydrotestosterone concentration on the x-axis and the peak area on the y-axis to fit a linear regression equation (R²). 2 ≥0.999).

[0058] The 5α-reductase inhibition rate of the sample to be tested is calculated using the following formula.

[0059] 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%; Among them, C 待测样品 C represents the dihydrotestosterone concentration in the sample group to be tested. 酶失活组 The concentration of dihydrotestosterone in the enzyme-inactivated group, C 空白组 The concentration of dihydrotestosterone in the control group is shown.

[0060] Table 2. 5α-reductase inhibition rate (%) of the test samples Sample to be tested 5α-Reductase Inhibition Rate (%) Sample to be tested 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 Table 2 shows that, overall, the 5α-reductase inhibition rate of the anti-hair loss composition is higher than that of the Polygonum multiflorum extract. A comparison of the data from Example 1, Comparative Example 5, and Example 4 indicates that the Polygonum multiflorum extract and Eucommia ulmoides extract have a synergistic effect in inhibiting 5α-reductase activity. A comparison of the data from Example 1 with Comparative Examples 1-4, and Example 4 with Comparative Examples 7-10, shows that in the preparation of the Polygonum multiflorum extract, the type of solvent, the type of macroporous adsorption resin, the omission of membrane filtration / ultrafiltration, and the omission of the crystallization step all affect the 5α-reductase inhibition rate. A comparison of the data from Example 4 and Comparative Example 6 shows that changing the type of compound microorganisms also affects the 5α-reductase inhibition rate of the anti-hair loss composition. Therefore, the Polygonum multiflorum extract and Eucommia ulmoides extract obtained using the extraction method of the present invention can be used together in anti-hair loss products.

[0061] Experimental Example 2 Animal experiments Eight-week-old male C57BL / 6 mice of SPF grade were selected and fed standard hormone-free diet under an SPF-grade barrier environment (temperature 22-25℃, humidity 50%-60%, 12h light-dark cycle), with free access to food and water. The experiment was started after one week of acclimatization to reduce the impact of environmental stress on the results.

[0062] On day 0 of the experiment, the hair in the interscapular region of the back of all mice was shaved using a small animal shaver (area 2cm×2cm, hair removal area) to avoid scratching the skin (skin damage will initiate wound healing and interfere with the hair follicle cycle). After shaving, the mice were fed for another 3 days to allow the hair follicles to uniformly enter the growth phase and ensure that the hair follicles responded to androgens consistently during modeling.

[0063] After shaving, the mice were 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. There were no significant differences in weight, health status, etc. among the groups, and subsequent experimental steps could be carried out.

[0064] Model establishment: Every morning at 9:00 AM, mice in each group were subcutaneously injected with testosterone propionate (5 mg / mL oil solution prepared by dissolving testosterone propionate in sesame oil) in their backs for 28 consecutive days. Testosterone propionate induces hair follicles to enter the resting phase prematurely by increasing dihydrotestosterone levels in the body, leading to thinning and hair loss.

[0065] One hour after the injection of testosterone propionate, the shaved area on the back of each group of mice was smeared (using a pipette to draw the corresponding dose of product (0.1 mL / mouse)). The model group was smeared with physiological saline, the positive group was smeared with 2% minoxidil tincture, and the other groups were smeared with the corresponding test sample solution.

[0066] The method for preparing the test sample solution is as follows: add 10 mL of purified water to 1 g of Polygonum multiflorum extract / anti-hair loss composition to obtain the test sample solution.

[0067] The degree of hair thinning was observed using a stereomicroscope (10× objective lens), and the hair appearance was scored. The scoring criteria are as follows: 0 points: Thick hair, no exposed skin; 1 point: Sparse hair with a small amount of hair, and the exposed area is less than 10%; 2 points: Moderately sparse, with 10%-30% of the area exposed; 3 points: Severely sparse, with 30%-50% of the area exposed; 4 points: Almost hairless, with more than 50% of the body exposed.

[0068] After scoring the appearance of the hair, the mice were sacrificed, the hair in the bald areas was shaved off, and the weight of the hair in each group of mice was measured.

[0069] The hair appearance scores and hair weights of the mice in each group are shown in Table 3.

[0070] Table 3. Hair appearance scores and hair weight of mice in each group. Group Hair appearance rating (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 Table 3 shows that the hair appearance score and hair weight of the positive group, the example group, and the comparative group were significantly better than those of the model group. The results of Example 1 were significantly better than those of the model group, indicating that the Polygonum multiflorum extract can effectively improve hair loss induced by testosterone propionate and promote hair growth. The effects of Comparative Examples 1-4 were all worse than those of Example 1, consistent with the 5α-reductase inhibition rate results in Table 2, further proving that the extraction process of Example 1 is superior and can prepare more effective anti-hair loss active ingredients. The hair appearance score and hair weight of Example 4 were close to those of the positive group, indicating that the anti-hair loss composition of the present invention is significantly effective in preventing hair loss and promoting hair growth. The effect of Comparative Example 5 was 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 enhance the anti-hair loss effect, while the effect of Eucommia ulmoides extract alone is limited. The effects of Comparative Examples 6 and 7-10 were all worse than those of Example 4, further demonstrating that the Polygonum multiflorum extraction process of Example 1 and the Eucommia ulmoides extract fermented by specific compound microorganisms are the key to ensuring the excellent effect of the anti-hair loss composition.

[0071] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A process for separating and purifying the active ingredients of Polygonum multiflorum, characterized in that, include: Polygonum multiflorum was pulverized, added to ethanol solution for ultrasonic extraction, centrifuged, and the supernatant was successively purified by activated carbon adsorption, macroporous adsorption resin, membrane filtration, ultrafiltration, vacuum concentration, and crystallization to obtain Polygonum multiflorum extract.

2. The separation and purification process according to claim 1, characterized in that, The volume fraction of the ethanol solution is 55%-65%.

3. The separation and purification process 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 separation and purification process 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 separation and purification process according to claim 1, characterized in that, The purification process using the macroporous adsorption resin includes: adsorbing the solution after adsorption by activated carbon by HPD-100 macroporous adsorption resin, followed by elution with water, 20%-30% ethanol solution (v / v), and 60%-70% ethanol solution (v / v), and collecting the eluent of 60%-70% ethanol solution (v / v).

6. The separation and purification process 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.

7. The separation and purification process according to claim 1, characterized in that, The ultrafiltration process includes passing the filtrate after membrane filtration through a 3000-5000 Da ultrafiltration membrane at a pressure of 0.2-0.5 MPa to obtain a permeate.

8. A Polygonum multiflorum extract obtained by the separation and purification process according to any one of claims 1-7.

9. The application of the Polygonum multiflorum extract according to claim 8 in anti-hair loss and shampoo products.

10. The application according to claim 9, characterized in that, The shampoo product also includes Eucommia ulmoides extract, which is obtained by fermenting Eucommia ulmoides through compound microorganisms.