Traditional Chinese medicine and trace element composition capable of blacking hair and preventing hair loss as well as preparation method and application of traditional Chinese medicine and trace element composition

Abstract

The invention relates to the technical field of compositions, and provides a hair-blacking and hair-loss-preventing traditional Chinese medicine and trace element composition and a preparation method and application thereof.The preparation method includes the steps that traditional Chinese medicinal materials with the effects of nourishing the liver and kidney, nourishing blood and growing hair are selected, and a multi-step water extraction and alcohol extraction combined technology is adopted to prepare high-purity traditional Chinese medicine extract dry powder; meanwhile, soluble trace elements such as zinc, iron and copper are selected, the optimal proportion of the soluble trace elements, the iron and the copper is determined through a response surface optimization algorithm, and a synergistic activity system is formed. Then, a mixture of traditional Chinese medicines and trace elements is dissolved in a buffer solution, and the mixture is entrapped in lipidosome composed of phospholipid and cholesterol through an ultrasonic nanocrystallization process, so that a stable nanoscale composite carrier is prepared; and finally, uniformly mixing the liposome suspension with a gel matrix to prepare the liposome gel dosage form for scalp external use. The problems of random component proportion, poor permeability and lack of mechanism verification in the prior art are solved.

Description

Technical Field

[0001] The present invention relates to the technical field of compositions, and more specifically, to a traditional Chinese medicine and trace element composition for blackening hair and preventing hair loss, its preparation method and application. Background Art

[0002] The growth state of hair is regulated by a variety of endogenous and exogenous factors, and is particularly closely related to the dynamic regulation of the hair follicle microenvironment, hormone levels, trace nutrients and signaling pathways. Traditional Chinese medicine theory holds that "the kidney stores essence, and its brilliance is manifested in the hair", and hair loss is mostly attributed to deficiency of the liver and kidney and deficiency of qi and blood. It is shown in Document 1 (Jinjin Dou, Zhiming Zhang, Xianrong Xu, Xiwu Zhang, Exploring the effects of Chinese herbal ingredients on the signaling pathway of alopecia and the screening of effective Chinese herbal compounds, Journal of Ethnopharmacology, Volume 294, 2022, 115320, ISSN 0378 - 8741) that certain active ingredients in traditional Chinese medicine can regulate the growth cycle and melanin synthesis of hair follicle cells through signaling pathways such as Wnt / β - catenin, TGF - β, JAK / STAT, thereby playing the roles of blackening hair and preventing hair loss. For example, ingredients such as baicalin, stilbene glucoside (TSG), and ginsenosides have been proven to up - regulate the Wnt signaling pathway, activate the activity of dermal papilla cells, and promote the hair follicles to be in the growth phase.

[0003] On the other hand, it is pointed out in Document 2 (D. Pozebon, G. L. Scheffler, V. L. Dressler, Elemental hair analysis: A review of procedures and applications, Analytica Chimica Acta, Volume 992, 2017, Pages 1 - 23, ISSN 0003 - 2670) that the content of trace elements in hair is an important indicator of the nutritional status of hair follicles and the level of pigment metabolism. Multiple studies have shown that zinc ions can inhibit the activity of 5α - reductase and promote the proliferation of keratinocytes. Iron is an essential factor for oxygen delivery in hair follicles and the stable synthesis of melanin. Copper is a key cofactor of tyrosinase and directly participates in the process of melanin synthesis. Modern detection methods such as inductively coupled plasma mass spectrometry (ICP - MS) have been widely used to evaluate the level of trace elements in hair. However, in the existing technology, there is still a lack of a mature solution for optimizing the ratio and formulating preparations of traditional Chinese medicine and trace elements based on the synergistic action mechanism of signal pathways. Traditional products mainly rely on empirical compatibility, lacking scientific basis and systematic verification.

[0004] Therefore, there is an urgent need for a method to improve the delivery efficiency through modern pharmaceutical means on the basis of the nutritional complementarity between traditional Chinese medicine compounds and trace elements, so as to achieve the synergistic effect and scientific quantification of traditional Chinese medicine and trace elements. Summary of the Invention

[0005] In order to overcome the above - mentioned defects of the existing technology, the present invention provides a composition of traditional Chinese medicine for preventing hair loss and promoting hair growth and trace elements, its preparation method and application. The response surface optimization algorithm is used to determine the synergistic ratio of zinc, iron and copper trace elements, and they are mixed with the traditional Chinese medicine extract according to the specified mass ratio. After ultrasonic nanonization, they are encapsulated in phospholipid - cholesterol nanoliposomes to form a gel preparation. By synergistically regulating the Wnt / β - catenin pathway and the melanin synthesis process, the activity of hair follicles and the efficiency of pigment generation are improved, overcoming the defects of empirical compatibility and low penetration efficiency of traditional Chinese medicine, and achieving a stable effect of preventing hair loss and promoting hair growth.

[0006] To achieve the above - mentioned purpose, the present invention provides the following technical solutions:

[0007] A preparation method of a composition of traditional Chinese medicine for preventing hair loss and promoting hair growth and trace elements, wherein the composition comprises the following components:

[0008] Dry powder of traditional Chinese medicine extract selected from Polygonum multiflorum Thunb., Glossy Privet Fruit, Eclipta prostrata L., and Angelica sinensis (Oliv.) Diels;

[0009] Compounds of zinc element, iron element, and copper element, added in the form of soluble salts;

[0010] Nanoliposomes formed by phospholipids and cholesterol at a mass ratio of 5:1 are used as carriers, where the phospholipids form the main body of the bilayer membrane, and cholesterol is embedded in the phospholipid bilayer structure to improve the physical density and integrity of the membrane structure;

[0011] Among them, the mass ratio range of the traditional Chinese medicine extract to zinc, iron, and copper elements is 10:1 to 20:1; the relative ratio among zinc, iron, and copper is determined by the synergistic ratio interval obtained based on the response surface optimization algorithm;

[0012] The preparation method includes the following steps:

[0013] Step S1, prepare the dry powder of the traditional Chinese medicine extract and the trace element solution respectively;

[0014] Step S2, preset the mass ratio of the traditional Chinese medicine extract to the trace elements to be 10:1 to 20:1, and at the same time use the melanin metabolism response surface optimization algorithm to determine the molar ratio of the three trace elements of zinc, iron, and copper;

[0015] Step S3, combine the traditional Chinese medicine extract and the trace elements according to the determined ratio to form an active ingredient mixture;

[0016] Step S4, use the ultrasonic nanonization technology to encapsulate the active ingredient mixture in the liposome carrier to form a nanoliposome composition with an average particle size of 100 nm and an encapsulation efficiency greater than 70%;

[0017] Step S5, further prepare the nanoliposome composition into a gel according to the application scenario requirements.

[0018] As a further scheme of the present invention, the traditional Chinese medicine extract is selected from at least three of Polygonum multiflorum, Ligustrum lucidum, Eclipta prostrata, and Angelica sinensis. These herbs have the traditional uses of tonifying the liver and kidney and promoting hair growth. Among them, Polygonum multiflorum contains stilbene glycoside components, which have been proven in multiple studies to activate the Wnt / β-catenin signaling pathway and promote hair follicle cells to enter the growth phase; Ligustrum lucidum is rich in oleanolic acid, which can promote the accumulation of β-catenin in hair follicle stem cells and prolong the active cycle of hair follicles; ferulic acid in Angelica sinensis has antioxidant effects, which helps to improve the local microcirculation of the scalp and thus provide a better nutritional environment for hair follicles.

[0019] As a further scheme of the present invention, the traditional Chinese medicine extract and the trace elements act synergistically to produce a synergistic effect. The traditional Chinese medicine compound provides bioactive substances to activate hair follicles, antioxidant and improve microcirculation, while the trace elements directly participate in melanin synthesis and cell proliferation as enzyme cofactors and nutritional substrates, and act together on the hair follicle growth environment.

[0020] As a further scheme of the present invention, the preparation of the traditional Chinese medicine extract includes the following steps:

[0021] Step A1: Crush the traditional Chinese medicine raw materials to a particle size passing through an 80-mesh sieve, add 10 times the volume of pure water, and soak for 1 hour to fully moisten the medicinal materials;

[0022] Step A2: Place the moistened medicinal materials in an extraction tank, heat to boiling, and then reflux and extract with low heat for 2 hours, filter and collect the extract I;

[0023] Step A3: Add 8 times the volume of pure water to the filter residue and continue to reflux and extract for 1 hour, filter and collect the extract II;

[0024] Step A4: Combine extract I and II, add 70% by volume of ethanol to the medicinal residue containing lipophilic active ingredients and reflux and extract for 1 hour to obtain extract III and combine it with the previous two extracts;

[0025] Step A5: Concentrate the combined extract under reduced pressure at 50 °C to a relative density of 1.10 to 1.20;

[0026] Step A6: Place the concentrated solution in a freeze dryer, freeze to -80 °C, and vacuum sublimation dry for 48 hours to obtain a dry powder of the traditional Chinese medicine extract, grind it through an 80-mesh sieve, and store it sealed for later use.

[0027] As a further solution of the present invention, the zinc element compound is zinc sulfate, which is a colorless or slightly white crystalline powder and is easily soluble in water. In hair follicle metabolism, zinc not only participates in DNA synthesis and cell cycle regulation, but also inhibits the activity of 5α-reductase and regulates sebum secretion, thereby maintaining the stability of the hair follicle structure. Zinc ions have also been found to have the ability to promote the migration and proliferation of keratinocytes, which helps the hair follicles to resume their growth state. The iron element compound is ferrous sulfate, and its divalent iron ions can stably exist in a weakly acidic environment. It is not only an important component for oxygen transfer, but also can form a complex structure with phenolic carboxyl groups in the traditional Chinese medicine extract, which helps to improve the stability and availability of pigment precursor substances; the copper element compound is copper sulfate, which is a blue crystal and is easily soluble in water. Copper ions are the active center components of tyrosinase and are an indispensable cofactor in the process of melanin synthesis. The three trace elements are added in the form of soluble inorganic salts in the present invention, and are added synergistically with the traditional Chinese medicine extract according to the molar ratio during the preparation process. The synergistic ratio range is determined through the optimized response surface model to maximize the enzyme activity without antagonism.

[0028] As a further solution of the present invention, the composition exists in the form of a liposome gel dosage form, and its preparation method includes:

[0029] Step B1: Weigh and mix soy lecithin and cholesterol at a mass ratio of 5:1, add an appropriate amount of absolute ethanol to a flask, and stir well in a 37°C water bath until completely dissolved to form a homogeneous organic phase solution. This ratio constitutes the bilayer membrane material of the liposome. Cholesterol can increase the stability and rigidity of the membrane and prevent the leakage of the phospholipid bilayer;

[0030] Step B2: Use the melanin metabolism response surface optimization algorithm to determine the ratio of traditional Chinese medicine extract to trace elements; this algorithm finds the ratio range of each component that is most conducive to melanin synthesis through systematic experiments and mathematical models;

[0031] Step B3: Weigh zinc sulfate, ferrous sulfate, and copper sulfate according to the determined ratio, and dissolve them together with the dry powder of the traditional Chinese medicine extract in a phosphate buffer solution with a pH value of 7.4;

[0032] Step B4: Ultrasonically vibrate the prepared aqueous solution to fully dissolve it, and heat the temperature not exceeding 50°C to avoid the degradation of active ingredients, forming an aqueous solution containing traditional Chinese medicine active ingredients and trace elements;

[0033] Step B5: Slowly drip the aqueous solution into the phospholipid organic phase solution, stir magnetically while dripping, and control the volume ratio of the aqueous phase to the phospholipid organic phase to be about 5:1 to form a primary emulsion;

[0034] Step B6: Place the primary emulsion in an ice bath and ultrasonically treat it at a power of 200W, with an ultrasonic time of 2 seconds and an interval of 3 seconds, for a total of 10 minutes, so that the phospholipid molecules self-assemble in the aqueous phase to form nano-liposomes loaded with traditional Chinese medicine and trace elements;

[0035] Step B7: Place the obtained liposome suspension on a rotary evaporator under reduced pressure and remove the residual ethanol at a low temperature to fully hydrate the liposomes;

[0036] Step B8: Take a high molecular gel matrix (such as Carbomer 940) and slowly stir and swell it in pure water for 12 hours, add triethanolamine dropwise to neutralize it to a pH of about 6.5 - 7.0 to form a transparent matrix gel;

[0037] Step B9: Slowly add the liposome suspension to the gel matrix and stir evenly at a low speed to avoid generating bubbles, and thus obtain the liposome gel preparation.

[0038] As a further solution of the present invention, the melanin metabolism response surface optimization algorithm includes:

[0039] Step B21: Set the molar ratio range of zinc, iron, and copper elements and construct a three-factor orthogonal experimental matrix;

[0040] Step B22: Apply each combined treatment to B16 melanocytes respectively. After culturing for 72 hours, measure the tyrosinase activity and melanin content in the cells. Take the tyrosinase activity as the main evaluation index and the melanin content as the secondary evaluation index. Among them, B16 cells are the standard cell model for studying melanin synthesis, and the tyrosinase activity directly reflects the melanin synthesis ability.

[0041] Step B23: Use the tyrosinase activity value as the dependent variable and the molar ratio combinations of the three elements as the independent variables to establish a quadratic polynomial fitting equation and draw a three-dimensional response surface graph to analyze the interaction between the elements. Among them, the formula of the quadratic polynomial fitting equation is:

[0042] Y = β0 + β1X1 + β2X2 + β3X3 + β 12 X1X2 + β 13 X1X3 + β 23 X2X3 + β 11 X1 2 + β 22 X2 2 + β 33 X3 2 ;

[0043] In the formula, Y is the tyrosinase activity value; X1, X2, and X3 are the molar concentrations or molar ratios of zinc, iron, and copper elements respectively; β0 is the constant term; β1, β2, and β3 are the linear effect coefficients; β 12 、β 13 、β 23 are the interaction effect coefficients; β 11 、β 22 、β 33 are the quadratic effect coefficients, which are used to characterize the influence of each element on the tyrosinase activity and the interaction between the elements.

[0044] Step B24: Obtain the molar ratio combination of the elements that makes the tyrosinase activity the highest under the experimental conditions through gradient search or response surface maximum positioning.

[0045] Step B25: Conduct three repeated experiments on the obtained optimal molar ratio combination to verify that its influence on the tyrosinase activity is repeatable and stable, and the coefficient of variation of error CV < 10%.

[0046] Step B26: Convert the verified optimal molar ratio of the elements into a mass ratio for the formulation design of the composition.

[0047] As a further solution of the present invention, there is provided an application of the preparation method of the traditional Chinese medicine and trace element composition for preventing hair loss and turning hair black in preventing hair loss and turning hair black.

[0048] Compared with the prior art, the beneficial effects of a traditional Chinese medicine and trace element composition for preventing hair loss and blackening hair, its preparation method and application of the present invention are as follows:

[0049] The present invention synergistically integrates a traditional Chinese medicine compound and a trace element supplementation strategy, and determines the synergistic ratio range of three trace elements, namely zinc, iron, and copper, through a response surface optimization algorithm system, establishing a quantifiable and repeatable optimization model, fundamentally improving the scientificity and functional targeting of the formula design. This strategy not only avoids potential antagonistic risks between trace elements but also fully exploits the synergistic effect of traditional Chinese medicine components on follicle activity and melanin metabolism.

[0050] The present invention uses nano-liposomes as carriers to co-encapsulate traditional Chinese medicine extracts and trace elements with optimized ratios to form a nano-composite preparation, which is further prepared into a gel dosage form. This carrier system significantly improves the permeability and retention of active ingredients in the follicle area, solving the problems of low penetration efficiency and poor bioavailability of traditional Chinese medicine aqueous extracts or simple nutritional supplements. The results of animal experiments and cell experiments show that the composition can effectively promote follicle regeneration and enhance melanin synthesis without causing abnormal hyperplasia of follicle cells, demonstrating good functionality and safety. Description of the Drawings

[0051] Figure 1 It is a diagram showing the mechanism of action of the Wnt / β-catenin signaling pathway in the treatment of alopecia with traditional Chinese medicine in Document 1.

[0052] Figure 2 It is a diagram showing the mechanism of androgenetic alopecia mediated by 5α-reductase / DHT and the intervention effect of traditional Chinese medicine in Document 1.

[0053] Figure 3 It is a schematic flow chart of the preparation method of a traditional Chinese medicine and trace element composition for preventing hair loss and blackening hair of the present invention.

[0054] Figure 4 For Cu of the present invention 2+ Fixed (2 μM), Zn 2+ -Fe 2+ Response surface diagram.

[0055] Figure 5 It is a schematic diagram of the coverage area and hair length of the newly generated hair of the present invention.

[0056] In the figure, Wnt: Wnt signaling factor; Frz: Frizzled receptor; Dvl: Dishevelled protein; LRP5 / 6: low density lipoprotein receptor-related protein 5 / 6; CK1: casein kinase 1; GSK-3β: glycogen synthase kinase 3β; Axin: axin protein; APC: adenomatous polyposis coli protein; β-catenin: β-catenin; TCF: T cell factor; c-myc: cellular oncogene c-myc; Cyclin D1: cyclin D1; LEF: lymphoid enhancer factor; DKK-1: Dickkopf-related protein 1; Baicalin: baicalin; OA: oleanolic acid; LA: ursolic acid; SITOS: soy isoflavones; TSG: stilbene glycoside (mainly derived from Polygonum multiflorum Thunb.); Morronside: aucubin; Loureirin A: loureirin A; S8, S9: serial numbers of traditional Chinese medicine extracts (S8, S9); AK10: serial number of active compound AK10; octaphlorethol A: octaphlorethol A; Ginsenoside F2: ginsenoside F2; Rb1: ginsenoside Rb1; Re: ginsenoside Re; Rg1: ginsenoside Rg1; 5α-reductase: 5α-reductase; T: testosterone; DHT: dihydrotestosterone; AR: androgen receptor; TGF-β1, β2: transforming growth factor β1 and β2; Policosanol: policosanol; Ginsenoside Ro: ginsenoside Ro; Ginsenoside Rg3: ginsenoside Rg3; Physcion: physcion; ginsenoside Rd: ginsenoside Rd. Detailed implementation manners

[0057] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0058] A preparation method of a traditional Chinese medicine and trace element composition for preventing hair loss and blackening hair includes the following steps:

[0059] Step S1, prepare dry powder of traditional Chinese medicine extract and trace element solution respectively. The traditional Chinese medicine extract is selected from at least three of Polygonum multiflorum Thunb., Ligustrum lucidum Ait., Eclipta prostrata L., and Angelica sinensis (Oliv.) Diels. The raw medicinal materials are pulverized to pass through an 80-mesh sieve, added with 10 times the volume of purified water, and soaked at room temperature for 1 hour to fully penetrate the cell structure of the medicinal materials. Two-stage reflux extraction is carried out successively: for the first time, reflux with gentle boiling for 2 hours to collect extract I; then add 8 times the water and reflux for 1 hour to collect extract II. To improve the extraction efficiency of fat-soluble components, 70% ethanol is added for reflux extraction for 1 hour to obtain extract III. The above three-stage extracts are combined and concentrated under reduced pressure using a rotary evaporator to a relative density of 1.10 - 1.20, and then placed in a freeze dryer and freeze-dried at -80°C for 48 hours to obtain dry powder of traditional Chinese medicine extract.

[0060] Polygonum multiflorum Thunb. contains stilbene glycoside components, which have been confirmed in multiple studies to activate the Wnt / β-catenin signaling pathway and promote hair follicle cells to enter the growth phase; Ligustrum lucidum Ait. is rich in oleanolic acid, which can promote the accumulation of β-catenin in hair follicle stem cells and prolong the active cycle of hair follicles; ferulic acid in Angelica sinensis (Oliv.) Diels has antioxidant effects, which helps to improve the local microcirculation of the scalp and thus provide a better nutritional environment for hair follicles.

[0061] The zinc, iron, and copper trace elements are added in the form of soluble inorganic salts, preferably zinc sulfate, ferrous sulfate, and copper sulfate. Weigh the required metal salts and dissolve them in phosphate buffer (pH 7.4) respectively to form a colorless and clear trace element solution. The above three elements are added according to the molar ratio and used as an active ingredient mixture together with the dry powder of traditional Chinese medicine extract for subsequent carrier encapsulation.

[0062] Zinc sulfate, a colorless or slightly white crystalline powder, is easily soluble in water and is used to inhibit hair follicles from prematurely entering the regression phase and promote their recovery growth (Zou P, Du Y, Yang C, Cao Y. Trace element zinc and skin disorders. Front Med (Lausanne). 2023 Jan 17; 9: 1093868. doi: 10.3389 / fmed.2022.1093868. PMID: 36733937; PMCID: PMC9887131.).

[0063] Ferrous sulfate, in which ferrous ions can stably exist in a weakly acidic environment, serves not only as an important component for oxygen transfer but also forms a complex structure with phenolic carboxyl groups in traditional Chinese medicine extracts, contributing to enhancing the stability and availability of pigment precursor substances (Podgórska A, Kicman A, Naliwajko S, Wacewicz-Muczyńska M, Niczyporuk M. Zinc, Copper, and Iron in Selected Skin Diseases. Int J Mol Sci. 2024 Mar 29;25(7):3823. doi: 10.3390 / ijms25073823. PMID: 38612631; PMCID: PMC11011755.).

[0064] Copper sulfate, a blue crystal, is easily soluble in water. Copper ions are the active center components of tyrosinase and are essential cofactors in the process of melanin synthesis, used to maintain the activity of the key enzyme tyrosinase for melanin synthesis (Podgórska A, Kicman A, Naliwajko S, Wacewicz-Muczyńska M, Niczyporuk M. Zinc, Copper, and Iron in Selected Skin Diseases. Int J Mol Sci. 2024 Mar 29;25(7):3823. doi: 10.3390 / ijms25073823. PMID: 38612631; PMCID: PMC11011755.).

[0065] The three trace elements are added in the form of soluble inorganic salts in the present invention and are added synergistically with the traditional Chinese medicine extract according to the molar ratio during the preparation process. The synergistic ratio range is determined through the optimized response surface model to maximize the enzyme activity without antagonism.

[0066] In step S2, the preset mass ratio of the traditional Chinese medicine extract to the trace elements is 10:1 to 20:1, and at the same time, the molar ratio of the three trace elements of zinc, iron, and copper is determined by using the melanin metabolism response surface optimization algorithm.

[0067] The melanin metabolism response surface optimization algorithm includes the following steps:

[0068] In step B21, set the molar ratio range of zinc, iron, and copper elements and construct a three-factor orthogonal experimental matrix;

[0069] Step B22: Apply each combined treatment to B16 melanocytes respectively. After culturing for 72 hours, measure the tyrosinase activity and melanin content in the cells. Take the tyrosinase activity as the main evaluation index and the melanin content as the secondary evaluation index. Among them, B16 cells are the standard cell model for studying melanin synthesis, and the tyrosinase activity directly reflects the melanin synthesis ability.

[0070] Step B23: Take the tyrosinase activity value as the dependent variable and the molar ratio combinations of the three elements as the independent variables to establish a quadratic polynomial fitting equation and draw a three-dimensional response surface graph to analyze the interaction between the elements. Among them, the formula of the quadratic polynomial fitting equation is:

[0071] Y = β0 + β1X1 + β2X2 + β3X3 + β 12 X1X2 + β 13 X1X3 + β 23 X2X3 + β 11 X1 2 + β 22 X2 2 + β 33 X3 2 ;

[0072] In the formula, Y is the tyrosinase activity value; X1, X2, and X3 are the molar concentrations or molar ratios of zinc, iron, and copper elements respectively; β0 is the constant term; β1, β2, and β3 are the linear effect coefficients; β 12 , β 13 , β 23 are the interaction effect coefficients; β 11 , β 22 , β 33 are the quadratic effect coefficients, which are used to characterize the influence of each element on the tyrosinase activity and the interaction between the elements.

[0073] Step B24: Obtain the molar ratio combination of the elements that makes the tyrosinase activity the highest under the experimental conditions through gradient search or response surface maximum positioning.

[0074] Step B25: Conduct three repeated experiments on the obtained optimal molar ratio combination to verify that its influence on the tyrosinase activity is repeatable and stable, and the coefficient of variation of error CV < 10%.

[0075] Step B26: Convert the verified optimal molar ratio of the elements into a mass ratio for the formulation design of the composition.

[0076] The following is a Python code example for determining the ratio of zinc, iron, and copper elements using the melanin metabolism response surface optimization algorithm. Please note that this example is only a starting point and may need to be adjusted according to the actual situation and device interface in actual applications;

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[0090] This code is only for example, and appropriate modifications and adjustments need to be made according to specific situations in actual applications.

[0091] Step S3: Combine the traditional Chinese medicine extract and trace elements according to the determined ratio to form an active ingredient mixture. This mixture is prepared in a phosphate buffer solution, and the pH is controlled to be neutral to weakly acidic, which is beneficial to the dissolution of traditional Chinese medicine components and the stable existence of metal ions. Use magnetic stirring (IKA C-MAG HS7) to mix thoroughly for 30 minutes to form a uniform aqueous phase system.

[0092] Step S4: Use ultrasonic nanonization technology to encapsulate the active ingredient mixture in a liposome carrier. The liposome is composed of soy lecithin and cholesterol in a mass ratio of 5:1. First, dissolve it in absolute ethanol and stir in a 37°C water bath to form an organic phase. Then, slowly drip the above aqueous solution into the organic phase while stirring, and control the volume ratio of the aqueous phase to the organic phase to be 5:1 to form a primary emulsion. Place this emulsion in an ultrasonic instrument, under ice bath conditions, with a power of 200W, and ultrasonic for 10 minutes at an interval of 2 seconds / 3 seconds to obtain a nano-liposome dispersion with a particle size of about 100nm. Then use a Buchi rotary evaporator to remove ethanol at a low temperature and add purified water to the original volume.

[0093] Step S5: According to the application scenario requirements, further prepare the nano-liposome composition into a gel.

[0094] The preparation method of the liposome gel dosage form in the embodiment of the present invention includes:

[0095] Step B1: Weigh and mix soybean lecithin and cholesterol at a mass ratio of 5:1, add an appropriate amount of absolute ethanol into a flask, and stir well in a 37°C water bath until completely dissolved to form a uniform organic phase solution. This ratio constitutes the bimolecular membrane material of the liposome, where cholesterol can increase the stability and rigidity of the membrane and prevent the leakage of the phospholipid bilayer;

[0096] Step B2: Use the melanin metabolism response surface optimization algorithm to determine the ratio of the traditional Chinese medicine extract to trace elements; this algorithm finds the ratio range of each component that is most beneficial to melanin synthesis through systematic experiments and mathematical models;

[0097] Step B3: Weigh zinc sulfate, ferrous sulfate, and copper sulfate according to the determined ratio, and dissolve them together with the dry powder of the traditional Chinese medicine extract in a phosphate buffer solution with a pH value of 7.4;

[0098] Step B4: Ultrasonically vibrate the prepared aqueous solution to make it fully dissolved, and heat the temperature not exceeding 50°C to avoid the degradation of active ingredients, forming an aqueous solution containing traditional Chinese medicine active ingredients and trace elements;

[0099] Step B5: Slowly drip the aqueous solution into the phospholipid organic phase solution, stir magnetically while dripping, and control the volume ratio of the aqueous phase to the phospholipid organic phase to be about 5:1 to form a primary emulsion;

[0100] Step B6: Place the primary emulsion under ice bath conditions and perform ultrasonic treatment with a power of 200W, ultrasonic for 2 seconds / interval 3 seconds, for a total of 10 minutes, so that the phospholipid molecules self-assemble in the aqueous phase to form nano-liposomes carrying traditional Chinese medicine and trace elements;

[0101] Step B7: Place the obtained liposome suspension on a rotary evaporator under reduced pressure and remove the residual ethanol at low temperature to fully hydrate the liposomes;

[0102] Step B8: Take a high molecular gel matrix (such as Carbomer 940) and slowly stir and swell it in pure water for 12 hours, add triethanolamine drop by drop and neutralize it to a pH of about 6.5 - 7.0 to form a transparent matrix gel;

[0103] Step B9: Slowly add the liposome suspension into the gel matrix and stir evenly at low speed to avoid generating bubbles, thus obtaining the liposome gel preparation.

[0104] Example 1

[0105] Step S1: Prepare the dry powder of the traditional Chinese medicine extract and the trace element solution respectively;

[0106] In step S2, the preset mass ratio of the traditional Chinese medicine extract to the trace elements is 10:1 to 20:1. At the same time, the molar ratio of three trace elements, zinc, iron, and copper, is determined by using the melanin metabolism response surface optimization algorithm.

[0107] In step S3, the traditional Chinese medicine extract and the trace elements are combined according to the determined ratio to form an active ingredient mixture.

[0108] In step S4, the active ingredient mixture is encapsulated in a liposome carrier by using the ultrasonic nanonization technology to form a nano-liposome composition with an average particle size of 100 nm and an encapsulation efficiency greater than 70%.

[0109] In step S5, according to the application scenario requirements, the nano-liposome composition is further prepared into a gel.

[0110] In the embodiment of the present invention, B16 melanocytes are used as a model to carry out a three-factor and three-level response surface experiment, and a quadratic polynomial regression model between the molar ratio of trace elements and tyrosinase activity is established.

[0111] First, as shown in Table 1, Zn 2+ , Fe 2+ , and Cu 2+ are selected as 3 factors, and each factor is set at 3 levels.

[0112] Table 1

[0113]

[0114] A total of 15 groups of combinations are set in the experiment, and each group is set with 3 duplicate wells. The tyrosinase activity is expressed by the OD 475 absorbance value. The results are shown in Table 2:

[0115] Table 2

[0116]

[0117] Taking the tyrosinase activity (OD 475 ) as the response value, Zn 2+ (X1), Fe 2+ (X2), and Cu 2+ (X3) as the independent variables, the fitted quadratic polynomial regression equation is:

[0118]

[0119] In the formula, Y represents the tyrosinase activity, and the unit is OD 475 absorbance value. The goodness of fit R 2= 0.961. The analysis of variance result shows F = 19.78 and P < 0.001, indicating that the model fits well and can effectively predict the tyrosinase reaction intensity under various element ratio combinations.

[0120] According to the response surface plot and regression model prediction, the optimal conditions are: Zn 2+ : 5.4 μM; Fe 2+ : 5.7 μM; Cu 2+ : 2.4 μM; Under these conditions, the predicted tyrosinase activity value is 0.392 OD 475 .

[0121] At the optimal molar ratio (Zn 2+ :Fe 2+ :Cu 2+ = 5.4:5.7:2.4), three independent experiments were repeated. The obtained tyrosinase activity values were 0.394, 0.389, and 0.391; their average value was 0.391 OD 475 , CV = 0.65%, and the verification result was stable with good repeatability.

[0122] Convert the above optimal molar ratio to a mass ratio (using zinc sulfate, ferrous sulfate, and copper sulfate as the sources of Zn 2+ , Fe 2+ , Cu 2+ respectively):

[0123] Zn 2+ : 5.4 μM × 161.5 g / mol (zinc sulfate) ≈ 0.872 mg;

[0124] Fe 2+ : 5.7 μM × 278.0 g / mol (ferrous sulfate) ≈ 1.585 mg;

[0125] Cu 2+ : 2.4 μM × 249.7 g / mol (copper sulfate) ≈ 0.599 mg.

[0126] Example 2

[0127] Step S1, prepare dry powder of traditional Chinese medicine extract and trace element solution respectively;

[0128] Step S2, preset the mass ratio of traditional Chinese medicine extract to trace elements as 10:1 to 20:1, and at the same time use the melanin metabolism response surface optimization algorithm to determine the molar ratio of three trace elements of zinc, iron, and copper;

[0129] Step S3, combine the traditional Chinese medicine extract and trace elements according to the determined ratio to form an active ingredient mixture;

[0130] Step S4: Using ultrasonic nanonization technology, the active ingredient mixture is encapsulated in a liposome carrier to form a nano-liposome composition with an average particle size of 100 nm and an encapsulation efficiency greater than 70%.

[0131] Step S5: According to the application scenario requirements, the nano-liposome composition is further prepared into a gel.

[0132] In this embodiment, Polygonum multiflorum Thunb., Glossy Privet Fruit, Eclipta prostrata L., and Angelica sinensis (Oliv.) Diels are selected as traditional Chinese medicine raw materials. 30 grams of each medicinal material are taken, washed, air-dried, crushed, and sieved through 80 meshes to obtain uniformly sized traditional Chinese medicine powder. A total of 120 grams of traditional Chinese medicine powder is placed in an extraction tank. First, pure water (10 times the volume) is added and soaked for 1 hour to fully moisten the medicinal materials; then it is heated to boiling and refluxed with a small fire for 2 hours, and the extract I is obtained after filtration; then 8 times the volume of pure water is added to the filter residue and refluxed for 1 hour, and the extract II is obtained by filtration; after combining the extract I and II, 70% ethanol is added to the residue containing fat-soluble active ingredients and refluxed for 1 hour, the extract III is collected, and the three extracts are mixed. The mixed solution is concentrated by reduced-pressure rotary evaporation at 50 °C to a relative density of about 1.15, then frozen at -80 °C and vacuum sublimation dried for 48 hours, and sieved through 80 meshes to obtain the dry powder of the traditional Chinese medicine extract.

[0133] Meanwhile, zinc sulfate, ferrous sulfate, and copper sulfate are pre-dissolved in ultrapure water. According to the optimized results, the usage concentrations of the three are adjusted to Zn 2+ 5.4 μM, Fe 2+ 5.7 μM, Cu 2+ 2.4 μM, and based on this, the addition mass ratio of trace elements to the dry powder of the traditional Chinese medicine extract is determined to be 15:1. The dry powder of the traditional Chinese medicine extract is fully mixed with the above trace element solution to obtain an active ingredient mixture with the effects of synergistically activating hair follicles, antioxidant, and promoting melanin synthesis.

[0134] In terms of carrier preparation, soybean lecithin and cholesterol are used in a mass ratio of 5:1, dissolved in absolute ethanol, and stirred in a 37 °C water bath to form a homogeneous organic phase. The active ingredient mixture obtained in the previous step is dissolved in a phosphate buffer solution with a pH of 7.4, and after ultrasonic mixing, it is slowly mixed with the organic phase at a volume ratio of the aqueous phase to the organic phase of 5:1 while maintaining magnetic stirring to form a primary emulsion. The primary emulsion is placed under ice bath conditions and treated with an ultrasonic emulsification device (set power 200 W, ultrasonic for 2 seconds, interval 3 seconds, cumulative 10 minutes) to cause the phospholipids to self-assemble to form nano-liposomes. Subsequently, the residual ethanol is removed by reduced-pressure rotary evaporation, and an appropriate amount of distilled water is added to adjust to the original volume.

[0135] To prepare a dosage form suitable for topical application, the above liposome suspension was mixed with a gel matrix that had been fully swollen with Carbomer 940 in pure water and adjusted to a pH of 6.8 - 7.0 with triethanolamine. After stirring evenly at low speed, a liposome gel was obtained.

[0136] Male C57BL / 6 mice (6 - 7 weeks old, weighing 20 ± 2 g) were selected for animal experiments. The mice were randomly divided into four groups (6 mice in each group): a blank control group, a group treated with the traditional Chinese medicine extract alone, a group treated with trace elements alone, and a group treated with a combination of traditional Chinese medicine and trace elements. Before the experiment, the hair in an area of about 3 cm × 3 cm on the back of the mice was shaved off with an electric hair clipper, and the remaining hair was completely removed with depilatory cream to make the hair follicles enter the resting phase. From the day after hair removal, 0.2 mL of each respective preparation was evenly applied to the treatment area of each group at 10 am every day, and gently massaged for 1 minute to promote absorption. After continuous treatment for 21 days, the coverage area and hair length of the newly grown hair were recorded by taking pictures and on-site measurement on the 7th, 14th, and 21st days respectively. As Figure 5 shown, in the combination group, the coverage area of the newly grown hair was about 35% on the 7th day, reached 62% on the 14th day, and was about 85% on the 21st day. The average hair lengths were 2.5 mm, 4.8 mm, and 7.2 mm respectively; while in the blank control group, the coverage area of the newly grown hair was only 15% - 20%, and the average hair length was 2.1 mm. After observing the skin tissue by H&E and Masson - Fontana staining, it was found that the number of hair follicles in the treatment area of the combination was significantly increased, most of the hair follicles were in the growth phase, and the melanin deposition was significantly better than that of other groups. At the same time, the content of trace elements in the newly grown hair was detected by ICP - MS. In the combination group, the measured Zn was 210 ± 15 μg / g, Fe was 180 ± 12 μg / g, and Cu was 35 ± 3 μg / g, while in the control group, they were 150 ± 10 μg / g, 130 ± 9 μg / g, and 22 ± 2 μg / g respectively, indicating that the composition of the present invention significantly improved the hair nutritional status.

[0137] Example 3

[0138] Step S1, prepare the dry powder of the traditional Chinese medicine extract and the trace element solution respectively;

[0139] Step S2, preset the mass ratio of the traditional Chinese medicine extract to the trace elements to be 10:1 to 20:1, and at the same time use the melanin metabolism response surface optimization algorithm to determine the molar ratio of the three trace elements of zinc, iron, and copper;

[0140] Step S3, combine the traditional Chinese medicine extract and the trace elements according to the determined ratio to form an active ingredient mixture;

[0141] Step S4, use the ultrasonic nanonization technology to encapsulate the active ingredient mixture in a liposome carrier to form a nano - liposome composition with an average particle size of 100 nm and an encapsulation efficiency greater than 70%;

[0142] Step S5: According to the requirements of the application scenario, the nano-liposome composition is further prepared into a gel.

[0143] In this embodiment, Polygonum multiflorum Thunb., Ligustrum lucidum Ait., Eclipta prostrata L., and Angelica sinensis (Oliv.) Diels are selected as traditional Chinese medicine raw materials. 30 grams of each medicinal material are taken, washed, air-dried, crushed, and sieved through 80 meshes to obtain traditional Chinese medicine powders with a uniform particle size. A total of 120 grams of traditional Chinese medicine powders are placed in an extraction tank. First, pure water (10 times the volume) is added and soaked for 1 hour to fully moisten the medicinal materials; then it is heated to boiling and reflux-extracted with a small fire for 2 hours, and the extract I is obtained after filtration; then 8 times the volume of pure water is added to the filter residue and reflux-extracted for 1 hour, and the extract II is obtained by filtration; after combining extract I and II, 70% ethanol is added to the residue containing liposoluble active ingredients and reflux-extracted for 1 hour, the extract III is collected, and the three extracts are mixed. The mixed solution is concentrated by reduced-pressure rotary evaporation at 50 °C to a relative density of about 1.15, then frozen at -80 °C and vacuum sublimation-dried for 48 hours, and after sieving through 80 meshes, the dry powder of the traditional Chinese medicine extract is prepared.

[0144] Meanwhile, zinc sulfate, ferrous sulfate, and copper sulfate are pre-dissolved in ultrapure water. According to the optimized results, the usage concentrations of the three are adjusted to Zn 2+ 5.4 μM, Fe 2+ 5.7 μM, Cu 2+ 2.4 μM, and based on this, the mass ratio of the trace elements to the dry powder of the traditional Chinese medicine extract is determined to be 15:1. The dry powder of the traditional Chinese medicine extract is fully mixed with the above trace element solution to obtain an active ingredient mixture with the functions of synergistically activating hair follicles, antioxidation, and promoting melanin synthesis.

[0145] In terms of carrier preparation, soybean lecithin and cholesterol are used in a mass ratio of 5:1, dissolved in absolute ethanol, and stirred in a 37 °C water bath to form a homogeneous organic phase. The active ingredient mixture obtained in the step is dissolved in a phosphate buffer solution with a pH of 7.4, and after being ultrasonically mixed thoroughly, it is slowly mixed with the organic phase at a volume ratio of the aqueous phase to the organic phase of 5:1 while maintaining magnetic stirring to form a primary emulsion. The primary emulsion is placed under ice bath conditions and treated with an ultrasonic emulsification device (set power 200 W, ultrasonic for 2 seconds, interval 3 seconds, cumulative 10 minutes) to enable the self-assembly of phospholipids to form nano-liposomes. Subsequently, the residual ethanol is removed by reduced-pressure rotary evaporation, and an appropriate amount of distilled water is added to adjust to the original volume.

[0146] To prepare a dosage form suitable for topical application, the above liposome suspension is mixed with a gel matrix obtained by fully swelling carbomer 940 in pure water and adjusted to a pH of 6.8 - 7.0 with triethanolamine, and stirred evenly at a low speed to obtain a liposome gel.

[0147] In in vitro cell experiments, B16 melanoma cells were used as a model. The B16 cells were treated in a control group, a group with a single traditional Chinese medicine extract (final concentration 100 μg / mL), a group with a single trace element group (using optimized concentrations of Zn 2+ 5.4 μM, Fe 2+ 5.7 μM, Cu 2+ 2.4 μM), and a composition group. After 72 hours of culture, the melanin content in the cells was determined by the NaOH method. The melanin content per million cells in the composition group was 8.1 ± 0.6 μg, while that in the control group was only 5.0 ± 0.4 μg. At the same time, the tyrosinase activity was detected by the L-DOPA method. The OD 475 value of the composition group was significantly higher than that of each control group (P < 0.05). In addition, MTT assays were performed on human hair follicle dermal papilla cells. The results showed that the cell viability of the composition treatment group remained at 105% ± 4%, showing a certain protective effect compared with the control group (100%). This indicates that the composition can effectively prevent the decline of hair follicle function without stimulating excessive hyperplasia.

[0148] As described above, this is only the specific implementation manner of the present application, but the protection scope of the present application is not limited thereto. Any person skilled in the art within the technical scope disclosed in the present application can easily think of changes or substitutions, which should all be covered by the protection scope of the present application. Therefore, the protection scope of the present application shall be subject to the protection scope of the claimed rights.

[0149] Finally: The above are only the preferred embodiments of the present invention and are not used to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection scope of the present invention.

Claims

1. A preparation method of a traditional Chinese medicine and trace element composition for blackening hair and preventing hair loss, characterized in that, The composition comprises the following components: Dry powder of traditional Chinese medicine extract, selected from Polygonum multiflorum Thunb., Glossy Privet Fruit, Eclipta prostrata L. and Angelica sinensis (Oliv.) Diels; Zinc element compound, iron element compound, copper element compound, added in the form of soluble salts; Nanoliposomes formed by phospholipids and cholesterol in a mass ratio of 5:1 as a carrier; Among them, the added mass ratio range of the traditional Chinese medicine extract to zinc, iron and copper elements is 10:1 to 20:1; the relative ratio among zinc, iron and copper is determined by the synergistic ratio interval obtained based on the response surface optimization algorithm; The preparation method comprises the following steps: Step S1, separately prepare the dry powder of traditional Chinese medicine extract and the trace element solution; Step S2, preset the mass ratio of the traditional Chinese medicine extract to the trace elements to be 10:1 to 20:1, and simultaneously determine the molar ratio of the three trace elements of zinc, iron and copper by using the melanin metabolism response surface optimization algorithm; Step S3, combine the traditional Chinese medicine extract and the trace elements according to the determined ratio to form an active ingredient mixture; Step S4, adopt the ultrasonic nanonization technology to encapsulate the active ingredient mixture in the liposome carrier to form a nanoliposome composition with an average particle size of 100 nm and an encapsulation efficiency greater than 70%; Step S5, further prepare the nanoliposome composition into a gel according to the application scenario requirements.

2. The preparation method of a traditional Chinese medicine and trace element composition for blackening hair and preventing hair loss according to claim 1, characterized in that, The preparation of the traditional Chinese medicine extract comprises the following steps: Step A1, crush the traditional Chinese medicine raw materials to pass through an 80-mesh sieve, add 10 times the volume of pure water and soak for 1 hour to fully moisten the medicinal materials; Step A2, place the moistened medicinal materials in an extraction tank, heat to boiling and then reflux extract with low heat for 2 hours, filter and collect the extract I; Step A3, add 8 times the volume of pure water to the filter residue and continue to reflux extract for 1 hour, filter and collect the extract II; Step A4, combine the extract I and II, add 70% volume fraction ethanol to the medicinal material residue containing liposoluble active ingredients and reflux extract for 1 hour to obtain the extract III and combine it with the previous two extracts; Step A5, concentrate the combined extract under reduced pressure at 50 °C to a relative density of 1.10 to 1.20; Step A6, place the concentrated solution in a freeze dryer, freeze it to -80 °C and vacuum sublimation dry it for 48 hours to obtain the dry powder of traditional Chinese medicine extract, grind it through an 80-mesh sieve, and seal it for storage for later use.

3. The preparation method of a traditional Chinese medicine and trace element composition for blackening hair and preventing hair loss according to claim 1, characterized in that, The zinc element compound is zinc sulfate, which is added together with the iron element compound and the copper element compound in a molar ratio during the preparation process to form a trace metal element synergistic system; The iron element compound is ferrous sulfate; the copper element compound is copper sulfate, which is used to maintain the activity of tyrosinase.

4. The preparation method of a traditional Chinese medicine and trace element composition for blackening hair and preventing hair loss according to claim 1, characterized in that, The composition exists in the form of a liposome gel dosage form, and its preparation method comprises: Step B1, weigh and mix soybean lecithin and cholesterol in a mass ratio of 5:1, add an appropriate amount of absolute ethanol to a flask, and fully stir in a 37 °C water bath until completely dissolved to form a uniform organic phase solution; Step B2, determine the ratio of the traditional Chinese medicine extract to the trace elements by using the melanin metabolism response surface optimization algorithm; Step B3, weigh zinc sulfate, ferrous sulfate and copper sulfate according to the determined ratio, and dissolve them together with the dry powder of traditional Chinese medicine extract in a phosphate buffer solution with a pH value of 7.4; Step B4: Ultrasonically oscillate the prepared aqueous solution to fully dissolve it, and heat it at a temperature not exceeding 50 °C to form an aqueous solution containing traditional Chinese medicine active ingredients and trace elements; Step B5: Slowly drip the aqueous solution into the phospholipid organic phase solution, and stir magnetically while dripping. Control the volume ratio of the aqueous phase to the phospholipid organic phase to be about 5:1 to form a primary emulsion; Step B6: Place the primary emulsion under ice bath conditions and perform ultrasonic treatment at a power of 200 W, with ultrasonic waves for 2 seconds / interval of 3 seconds, for a total of 10 minutes, so that phospholipid molecules self-assemble in the aqueous phase to form nano-liposomes loaded with traditional Chinese medicine and trace elements; Step B7: Place the obtained liposome suspension on a rotary evaporator under reduced pressure and remove the residual ethanol at low temperature to fully hydrate the liposomes; Step B8: Take the high molecular gel matrix and slowly stir and swell it in pure water for 12 hours, and add triethanolamine drop by drop to neutralize it to a pH of about 6.5 - 7.0 to form a transparent matrix gel; Step B9: Slowly add the liposome suspension to the gel matrix and stir evenly at low speed to avoid generating bubbles, thus obtaining the liposome gel preparation.

5. The preparation method of a traditional Chinese medicine and trace element composition for blackening hair and preventing hair loss according to claim 1 or 3, characterized in that, The melanin metabolism response surface optimization algorithm includes: Step B21: Set the molar ratio range of zinc, iron, and copper elements and construct a three-factor orthogonal experimental matrix; Step B22: Apply each combination treatment to B16 melanoma cells respectively. After culturing for 72 hours, measure the tyrosinase activity and melanin content in the cells. Take the tyrosinase activity as the main evaluation index and the melanin content as the secondary evaluation index; Step B23: Take the tyrosinase activity value as the dependent variable and the molar ratio combination of the three elements as the independent variable, establish a quadratic polynomial fitting equation, and draw a three-dimensional response surface diagram to analyze the interaction between the elements; Step B24: Through gradient search or response surface maximum positioning, obtain the molar ratio combination of elements that makes the tyrosinase activity the highest under the experimental conditions; Step B25: Perform three repeated experiments on the obtained molar ratio combination that makes the tyrosinase activity the highest to verify that its influence on the tyrosinase activity is repeatable and stable, with an error coefficient of variation CV < 10%; Step B26: Convert the verified molar ratio of elements to a mass ratio for the formulation design of the composition.

6. Use of the preparation method of a traditional Chinese medicine and trace element composition for preventing hair loss and turning hair black according to any one of claims 1 - 5 in preventing hair loss and turning hair black.

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