CN112516006A

CN112516006A - Nanometer composition with hair loss prevention, hair growth promotion, hair fixing and hair blackening functions and preparation method and application thereof

Info

Publication number: CN112516006A
Application number: CN202011454283.9A
Authority: CN
Inventors: 刘卫; 沈慧慧; 陈家玲; 王曦; 罗丹; 陈丹; 洪延涵
Original assignee: Wuhan Best Carrier Biotechnology Co ltd
Current assignee: Wuhan Best Carrier Biotechnology Co ltd
Priority date: 2020-12-10
Filing date: 2020-12-10
Publication date: 2021-03-19

Abstract

The invention provides a nano composition with functions of preventing hair loss, restoring hair, fixing hair and blackening hair, and a preparation method and application thereof, and relates to the technical field of cosmetics with special functions. The nano composition has good transdermal performance, increases the adsorption capacity of the preparation on scalp, deeply permeates into hair follicles, can be retained in the hair follicles for a long time, can be maintained at effective concentration for a long time, improves bioavailability, reduces the using amount of active ingredients, and ensures that low-content anti-hair loss active substances can also play an excellent anti-hair loss effect.

Description

Nanometer composition with hair loss prevention, hair growth promotion, hair fixing and hair blackening functions and preparation method and application thereof

Technical Field

The invention relates to the technical field of cosmetics with special functions, in particular to a nano composition with functions of preventing hair loss, growing hair, fixing hair and blackening hair, and a preparation method and application thereof.

Background

With the increasingly fierce social competition, the working and living pressure of people is increased, the proportion of people with alopecia and poliosis in modern society is increased due to mental stress, night stay and the like, and the trend of the youthfulness is more and more obvious.

Among them, androgenetic alopecia (AGA) is the most clinically common alopecia disease, accounting for about 90% of alopecia. At present, common chemical pilatory for treating androgenetic alopecia comprises minoxidil, finasteride and the like, but side effects are obvious, the minoxidil can cause scalp dryness, scurf, scalp erythema, inflammation, irritation and the like and can affect blood pressure, and the finasteride has a male hormone activity inhibition effect.

The diaminopyrimidine oxide and the pyrrolidinyl diaminopyrimidine oxide are novel hair growth promoters, are structurally similar to minoxidil, have different action principles, do not belong to the class of medicines, are raw materials for cosmetic approval, and are safe and free of toxic and side effects. However, the diaminopyrimidine oxide and the pyrrolidinyl diaminopyrimidine oxide are crystalline powders and have poor water solubility, so that the hair growth solution contains a large amount of alcohol substances, and uncomfortable experiences such as dry scalp and itching are easily caused after the hair growth solution is used; secondly, due to the skin's own barrier, the active substance is difficult to penetrate into the hair follicle, and cannot directly act on the target site, so that the bioavailability is low.

With the development of biotechnology, active polypeptides for treating alopecia, promoting hair growth and white hair have received increasing attention from experts. The active polypeptide has high biological activity, short treatment period and no toxic or side effect, but has poor stability, easy degradation and inactivation, poor transdermal absorption and difficult penetration into hair follicles, greatly reduces the efficiency of preventing hair loss and blackening hair, has limited action of single active peptide, and can play an ideal role by the mutual synergistic compatibility of a plurality of active peptides.

The problems in the above applications can be effectively solved by adopting the nano-carrier technology. The chinese invention patent CN201910540301.6 discloses a nano composition, its preparation method and application, but still has the following defects and shortcomings: because the nano vesicles are common nano vesicles and do not have positive charges specially in the manufacturing process, the nano vesicles are difficult to adsorb on human hair and scalp with negative charges, so that the adsorption probability of the preparation is reduced, and the probability of the action target point penetrating through the scalp and penetrating into hair follicles is also reduced; secondly, the hair-blackening and hair-restoring shampoo only relates to hair-restoring and hair-fixing effects, does not relate to hair-blackening effects, and has incomplete effects; thirdly, two biological peptides of hair root fixing peptide and hair growth promoting peptide are mainly coated, but no cell biological experiment of hair fixing and hair growth is involved; finally, diaminopyrimidine oxides are incorporated in the patent, but are less effective in use.

Disclosure of Invention

In view of the above, the invention aims to provide a nano composition with functions of preventing hair loss, promoting hair growth, fixing hair and blackening hair, and a preparation method and application thereof.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides a nano composition with functions of preventing hair loss, growing hair, fixing hair and blackening hair, which comprises the following components in percentage by mass:

0.1-10% of diaminopyrimidine oxide, 0.1-10% of pyrrolidinyl diaminopyrimidine oxide, 0.01-1% of trichogen peptide, 0.01-1% of hair-fixing peptide, 0.01-1% of hair-blackening peptide, 0.1-10% of cell penetrant, 0.1-5% of vitamin E polyethylene glycol succinate, 1-20% of phospholipid, 0.1-5% of cholesterol, 10-40% of polyhydric alcohol and the balance of water.

Preferably, the hair growing peptide comprises one or more of oligopeptide-54, decapeptide-18, octapeptide-2, myristoyl pentapeptide-17, myristoyl pentapeptide-16, myristoyl pentapeptide-7, myristoyl pentapeptide-4, myristoyl hexapeptide-16, myristoyl tetrapeptide-12, palmitoyl hexapeptide-25 and tripeptide-2.

Preferably, the hair-fixing peptide comprises one or more of acetyl tetrapeptide-3, biotin tripeptide-1, palmitoyl tripeptide-1 and decapeptide-10.

Preferably, the hair blackening peptide comprises one or more of copper peptide, acetyl hexapeptide-1 and palmitoyl tetrapeptide-10.

Preferably, the cell penetrating agent comprises one or more of hydroxypropyl trimethyl ammonium chloride hydrolyzed soybean protein, lauryl dimethyl ammonium hydroxypropyl hydrolyzed soybean protein, soybean oil-based trimethyl ammonium chloride, polyquaternary ammonium salt-51, polyquaternary ammonium salt-37, polyquaternary ammonium salt-11, polyquaternary ammonium salt-22, polyquaternary ammonium salt-39, behenyl trimethyl ammonium chloride, dicetyl dimethyl ammonium chloride and hydroxypropyl guar hydroxypropyl trimethyl ammonium chloride.

Preferably, the phospholipid comprises one or more of soybean lecithin, egg yolk lecithin, cephalin, hydrogenated soybean lecithin, hydrogenated egg yolk lecithin and dilauroyl phosphatidylcholine.

Preferably, the polyhydric alcohol comprises one or more of glycerol, butanediol, propylene glycol, dipropylene glycol, 1, 2-pentanediol and 1, 2-hexanediol.

Preferably, the particle size of the nano composition is 10-300 nm, and the Zeta potential is 0-60 mV.

The invention also provides a preparation method of the nano composition in the technical scheme, which comprises the following steps:

(1) mixing the phospholipid, cholesterol, a cell penetrating agent, polyol, diaminopyrimidine oxide, pyrrolidinyl diaminopyrimidine oxide and fat-soluble components in the hair growing peptide, the hair fixing peptide and the hair blackening peptide, and dissolving at 20-60 ℃ to obtain a first mixture;

(2) mixing water-soluble components in the hair growing peptide, the hair fixing peptide and the hair blackening peptide, polyalcohol and vitamin E polyethylene glycol succinate with water, and dissolving at 20-60 ℃ to obtain a second mixture;

(3) dropwise adding the second mixture obtained in the step (2) into the first mixture obtained in the step (1), and then carrying out shearing emulsification treatment to obtain a micron-sized dispersion;

(4) and (4) carrying out high-pressure homogenization or high-pressure microjet treatment on the micron-sized dispersion obtained in the step (3) to obtain the nano composition.

There is no chronological definition between the step (1) and the step (2).

The invention also provides application of the nano composition in the technical scheme in preparing cosmetics for preventing and growing hair, fixing hair and blackening hair and medicines for treating androgenetic alopecia, alopecia areata, telogen alopecia and anagen-phase osteoporosis syndromes.

The components and the proportion of the nano composition provided by the invention are closely related to the promotion of the active ingredients to penetrate through the stratum corneum of the skin, penetrate into hair follicles, improve the retention performance in the hair follicles and improve the stability of the nano composition. The concrete expression is as follows:

(1) the lipid bilayer structure composed of phospholipid and cholesterol in the nanometer composition can enter the inside of skin stratum corneum cells, and interact with keratin, so that the compactness of stratum corneum cells is reduced, lipid channels are formed, and active substances are promoted to penetrate through the stratum corneum;

(2) the addition of a proper amount of cholesterol can stabilize the structure of phospholipid bilayer, increase the membrane strength and facilitate the wrapping of substances, and play the roles of stabilizing lipid membrane and reducing leakage;

(3) the addition of the two flexibilizers, namely the cell penetrating agent and the vitamin E succinic acid polyethylene glycol ester, ensures that the lipid membrane has high deformability, can efficiently penetrate through a pore canal which is a plurality of times smaller than the self of the lipid membrane by taking the skin hydration pressure as power to deeply penetrate into hair follicles, and has good safety and no stimulation to skin due to natural sources of the two flexibilizers;

(4) the cell penetrating agent is a positively charged component, so that the positively charged liposome is more easily adsorbed on negatively charged human hair and scalp, has stronger adhesion with a cell biological membrane, is more easily and deeply penetrated into hair follicles, can fully exert the anti-hair loss and hair blackening effects of active ingredients, greatly improve the bioavailability, reduce the using amount of the active ingredients and ensure that the low-content anti-hair loss active substance can also exert an excellent anti-hair loss effect;

(5) the vitamin E succinic acid polyethylene glycol ester consists of a hydrophilic polar polyethylene glycol chain segment and an lipophilic nonpolar vitamin E succinic acid ester chain segment, can inhibit P-glycoprotein in cells from discharging the anti-hair loss active ingredients into blood, and slows down the removal speed of the anti-hair loss active ingredients in the cells, thereby prolonging the retention time of the anti-hair loss active ingredients in hair follicles.

In conclusion, the nano composition has good transdermal performance, increases the adsorption capacity of the preparation on the scalp, deeply permeates into hair follicles, can be retained in the hair follicles for a long time, can be maintained at an effective concentration for a long time, improves the bioavailability, reduces the using amount of active ingredients, and enables low-content anti-hair loss active substances to play an excellent anti-hair loss effect.

The invention has the beneficial effects that:

(1) most anti-hair loss product preparations sold in the market at present are common, the anti-hair loss mechanism is single, and the anti-hair loss effect is poor. The invention uses the nano-drug targeting carrier preparation technology to prepare the nano-composition, and carries the novel hair growth promoter diaminopyrimidine oxide, the pyrrolidinyl diaminopyrimidine oxide, the hair growing peptide, the hair fixing peptide and the hair blackening peptide together according to different targets of androgenetic alopecia, so that the synergistic effect is realized, and the triple effects of hair loss prevention, hair growing, hair fixing and hair blackening are achieved;

(2) because the skin has a barrier, the anti-hair loss active substance and the active polypeptide are difficult to permeate into hair follicles and cannot directly act on a target site, the lipid bilayer structure of the nano composition can enter the inside of skin stratum corneum cells to promote the active substance to permeate the stratum corneum; but also has high deformability and can deeply penetrate into hair follicles; the liposome with positive charges can increase the adsorption of the preparation on the scalp, greatly improve the bioavailability, reduce the using amount of active ingredients and ensure that low-content anti-dropping active matters can also play an excellent anti-dropping effect;

(3) the vitamin E polyethylene glycol succinate can inhibit P-glycoprotein in cells from discharging the anti-hair loss active ingredients into blood, and slow down the removal speed of the anti-hair loss active ingredients in the cells, so that the retention time of the anti-hair loss active ingredients in hair follicles is prolonged;

(4) the stability of the active polypeptide can be improved, and the influence of the environment on the active polypeptide is reduced;

(5) the accelerated storage test result shows that after the nano composition provided by the invention is placed at normal temperature for 12 months, the properties, the particle size distribution, the Zeta potential and the content of the composite polypeptide are not significantly changed, which indicates that the nano composition has good stability;

(6) the skin irritation test result shows that the nano composition provided by the invention has no irritation to scalp and high safety;

(7) the nanometer composition provided by the invention is easy to dissolve in water, can increase the solubility of the anti-falling active substance in water, is easy to add into anti-falling products of different types, is convenient to use, does not need to add an alcohol solvent in an additional amount, and has good product experience.

Drawings

FIG. 1 shows the in vitro cumulative permeation of the nanocompositions;

FIG. 2 is the results of the in vitro skin retention of the nano-composition;

FIG. 3 shows the proliferation of hair papilla cells in samples No. 1 to No. 5, compared with sample No. 1<0.05; in comparison with the sample No. 2,^#P<0.05；

figure 4 is a graph of the effect of nanocompositions on the rate of increase of VEGF content, P <0.05 compared to sample No. 9;

figure 5 is a graph showing the effect of the nanocompositions on the rate of increase of collagen iii content, P <0.05 compared to sample No. 9;

figure 6 is a graph of the effect of the nanocompositions on melanin content, P <0.05 compared to sample No. 9;

fig. 7 is a nano-composition mouse in vitro hair growth experiment.

Detailed Description

The invention provides a nano composition with functions of preventing hair loss, growing hair, fixing hair and blackening hair, which comprises the following components in percentage by mass: 0.1-10% of diaminopyrimidine oxide, 0.1-10% of pyrrolidinyl diaminopyrimidine oxide, 0.01-1% of trichogen peptide, 0.01-1% of hair-fixing peptide, 0.01-1% of hair-blackening peptide, 0.1-10% of cell penetrant, 0.1-5% of vitamin E polyethylene glycol succinate, 1-20% of phospholipid, 0.1-5% of cholesterol, 10-40% of polyhydric alcohol and the balance of water; preferably 0.5-8% of diaminopyrimidine oxide, 0.5-8% of pyrrolidinyl diaminopyrimidine oxide, 0.05-0.8% of trichogen peptide, 0.05-0.8% of hair-fixing peptide, 0.05-0.8% of hair-blackening peptide, 1-8% of cell penetrant, 0.5-4% of vitamin E succinic acid polyethylene glycol ester, 5-15% of phospholipid, 0.5-4% of cholesterol, 15-35% of polyhydric alcohol and the balance of water; more preferably 2-6% of diaminopyrimidine oxide, 2-5% of pyrrolidinyl diaminopyrimidine oxide, 0.1-0.5% of germinal peptide, 0.1-0.5% of hair-fixing peptide, 0.1-0.5% of hair-blackening peptide, 2-6% of cell penetrant, 1-3% of vitamin E polyethylene glycol succinate, 6-10% of phospholipid, 1-3% of cholesterol, 20-30% of polyhydric alcohol and the balance of water.

In the present invention, the sources of the diaminopyrimidine oxide and the pyrrolidinyl diaminopyrimidine oxide are not particularly limited, and commercially available products may be used. In the present invention, the diaminopyrimidine oxide and pyrrolidinyl diaminopyrimidine oxide are hair growth promoters, which inhibit the activity of 5 α -reductase, thereby reducing the conversion of testosterone to dihydrotestosterone; by influencing potassium ion channels through antagonism of potassium ion channel switch inhibitors, proliferation of epidermal hair papilla cells is promoted; dilating blood vessels around hair follicles, improving microcirculation of scalp, and providing nutrients necessary for hair growth; optimizing the hair cycle, promoting the transformation of the hair from the resting period to the growing period, maintaining the longer growing period, and increasing the growing period/resting period ratio; promote the growth of new hair, act on the deep structure of hair root, and induce new hair.

In the present invention, the hair-growing peptide preferably includes one or more of oligopeptide-54, decapeptide-18, octapeptide-2, myristoyl pentapeptide-17, myristoyl hexapeptide-16, and palmitoyl hexapeptide-25. The source of the germinal peptide is not particularly limited, and the germinal peptide can be prepared by adopting a conventional commercial product. In the invention, the germinal peptide can obviously activate keratin genes, promote the expression of the keratin genes, improve the content of an autocrine growth factor VEGF of hair papillary cells, regulate the proliferation and differentiation of the keratinocyte, increase the growth of eyelashes and hair, and make the hair thicker and tougher.

In the invention, the hair-fixing peptide preferably comprises one or more of acetyl tetrapeptide-3, biotin tripeptide-1 and decapeptide-10. The source of the hair-fixing peptide is not particularly limited, and the hair-fixing peptide can be obtained by adopting a conventional commercial product. In the invention, the hair-fixing peptide accelerates the synthesis of extracellular matrix proteins, such as laminin, collagen III and VII, through fibroblasts, directly acts on tissues around hair follicles, increases the volume and the length of the hair follicles, repairs epidermal-dermal junction (DEJ) and promotes the fixation of hairs in the hair follicles.

In the invention, the hair growing peptide and the hair fixing peptide have high biological activity, can effectively activate keratin genes, make hairs thicker and tougher, accelerate the synthesis of extracellular matrix proteins, repair epidermis-dermis connecting tissues (DEJ), promote the hairs to be fixed in the hair follicles, and can greatly improve the effects of hair loss prevention, hair growth and hair fixing by matching the hair growing peptide and the hair fixing peptide with diaminopyrimidine oxide and pyrrolidinyl diaminopyrimidine oxide, so that the low-content hair growth promoter can also play an excellent hair loss prevention effect.

In the present invention, the hair blackening peptide preferably includes one or more of copper peptide, acetyl hexapeptide-1 and palmitoyl tetrapeptide-10. The source of the hair blackening peptide is not particularly limited, and the hair blackening peptide can be obtained by adopting a conventional commercial product. In the present invention, the melanotide is an agonist of α -MSH, has a chemical structure with good affinity to MC1-R, binds to MC1-R receptor on melanocyte, simultaneously promotes the expression of MC1-R and MITF (melanogenesis associated transfection factor), induces the activity of tyrosinase to increase by activating cyclic adenosine monophosphate (cAMP) channel, promotes melanogenesis, optimizes melanosome maturation and transfer to keratinocytes. Meanwhile, the copper peptide can also promote collagen proliferation, strengthen hair follicles and increase the growth period of hair; inhibiting 5-alpha reductase activity, reducing testosterone conversion to dihydrotestosterone, reducing androgen receptor expression in hair papilla, and inhibiting alopecia.

In the present invention, the cell penetrating agent preferably includes one or more of hydroxypropyl trimethyl ammonium chloride hydrolyzed soybean protein, lauryl dimethyl ammonium hydroxypropyl hydrolyzed soybean protein, soybean oil-based trimethyl ammonium chloride, polyquaternary ammonium salt-51, polyquaternary ammonium salt-37, polyquaternary ammonium salt-11, behenyl trimethyl ammonium chloride and hydroxypropyl guar hydroxypropyl trimethyl ammonium chloride. In the invention, the cell penetrating agent is a positively charged component, so that the positively charged liposome is easier to adsorb on human hair and scalp with negative charges, has stronger adhesion with a cell biological membrane, is easier to deeply penetrate into hair follicles, can fully exert the anti-hair loss and hair blackening effects of active components, and greatly improves the bioavailability.

In the invention, the vitamin E succinic acid polyethylene glycol ester consists of a hydrophilic polar polyethylene glycol chain segment and an lipophilic nonpolar vitamin E succinic acid ester chain segment, can inhibit P-glycoprotein in cells from discharging the anti-hair loss active ingredients into blood, and slows down the removal speed of the anti-hair loss active ingredients in the cells, thereby prolonging the retention time of the anti-hair loss active ingredients in hair follicles.

In the invention, the addition of the two flexibilizers, namely the cell penetrating agent and the vitamin E succinic acid polyethylene glycol ester, ensures that the lipid membrane has high deformability, can efficiently penetrate through a pore canal which is a plurality of times smaller than the self of the lipid membrane by taking the skin hydration pressure as power, and deeply penetrates into hair follicles, and the two flexibilizers are natural in source, good in safety and free from stimulation to skin.

In the present invention, the phospholipid preferably includes one or more of soybean lecithin, egg yolk lecithin, and hydrogenated soybean lecithin. In the invention, the lipid bilayer structure composed of the phospholipid and the cholesterol can enter the inside of skin stratum corneum cells, interact with keratin, reduce the compactness of the stratum corneum cells, form lipid channels and promote active substances to penetrate through the stratum corneum; the addition of a proper amount of cholesterol can stabilize the structure of phospholipid bilayer, increase membrane strength and facilitate the wrapping of substances, and play the roles of stabilizing lipid membrane and reducing leakage.

In the present invention, the polyhydric alcohol preferably includes one or more of glycerin, propylene glycol, 1, 2-pentanediol, and 1, 2-hexanediol. In the present invention, the polyol can impart deformability to the lipid bilayer. It can reduce the interfacial tension of liposome, so that its flowability is raised and its deformability is raised. Meanwhile, the polyhydric alcohol can replace moisture near the head group of the lipid bilayer, so that the flexibility and the fluidity of the lipid bilayer are improved.

In the invention, the particle size of the nano composition is 10-300 nm, preferably 50-250 nm, and more preferably 100-200 nm; the Zeta potential of the nano-composition is 0 to +60mV, preferably +10 to +50mV, and more preferably +20 to +40 mV.

There is no chronological definition between the step (1) and the step (2).

The phospholipid, cholesterol, a cell penetrating agent, polyhydric alcohol, diaminopyrimidine oxide, pyrrolidinyl diaminopyrimidine oxide and fat-soluble components in hair growing peptide, hair fixing peptide and hair blackening peptide are mixed and dissolved at the temperature of 20-60 ℃ to obtain a first mixture. In the invention, the dissolving is preferably carried out in a water bath, and the temperature of the water bath is more preferably 30-50 ℃, and most preferably 35-45 ℃.

The water-soluble components in the hair growing peptide, the hair fixing peptide and the hair blackening peptide, the polyhydric alcohol, the vitamin E succinic acid polyethylene glycol ester and water are mixed and dissolved at the temperature of 20-60 ℃ to obtain a second mixture. In the invention, the dissolving is preferably carried out in a water bath, and the temperature of the water bath is more preferably 30-50 ℃, and most preferably 35-45 ℃.

The obtained second mixture is dripped into the obtained first mixture, and then shearing and emulsifying treatment are carried out to obtain the micron-sized dispersoid. The invention preferably adds the second mixture dropwise to the first mixture with stirring. In the invention, the shearing rotating speed of the shearing emulsification treatment is preferably 3000-10000 rpm, more preferably 4000-8000 rpm, and most preferably 5000-7000 rpm; the treatment time is preferably 1 to 10min, more preferably 3 to 8min, and most preferably 4 to 6 min. In the present invention, the mixing temperature of the first mixture and the second mixture is preferably 20 to 60 ℃, more preferably 30 to 50 ℃, and most preferably 35 to 45 ℃.

The invention carries out high-pressure homogenization or high-pressure microjet treatment on the obtained micron-sized dispersoid to obtain the nano composition. In the invention, the pressure of the high-pressure homogenizing treatment is preferably 200-1600 bar, more preferably 500-1200 bar, and most preferably 700-1000 bar; the number of cycles is preferably 1 to 10, more preferably 2 to 8, and most preferably 3 to 5. In the invention, the pressure of the high-speed micro-jet treatment is preferably 5000-16000 psi, more preferably 6000-14000 psi and most preferably 8000-10000 psi; the number of cycles is preferably 1 to 10, more preferably 2 to 8, and most preferably 3 to 5.

In the invention, when the nano composition is applied to a hair loss prevention and hair growth and hair blacking cosmetic, the mass percentage of the nano composition added to the hair loss prevention and hair growth and hair blacking cosmetic is preferably 1-30%, and more preferably 5-20%.

In the invention, when the nano composition has an auxiliary treatment effect on androgenetic alopecia, alopecia areata, alopecia in resting stage and anagen-phase osteoporosis syndrome, the nano composition can be applied to an external medicinal preparation to improve the medicinal effect. In the invention, the mass percentage of the nano composition in the external pharmaceutical preparation for treating alopecia is preferably 1-40%, and more preferably 5-30%.

The nano composition provided by the invention can be directly added into matrixes such as hair tonic, essence, cream, emulsion, shampoo, hair conditioner and the like and external medicinal preparations, and the preparation can be uniformly cut at 40-50 ℃ for a proper time, so that the nano composition is convenient to use.

In order to further illustrate the present invention, the following detailed description of the invention is given in conjunction with examples, which should not be construed to limit the scope of the invention.

Example 1

Mixing 5% of soybean lecithin, 1% of hydrogenated soybean lecithin, 0.5% of cholesterol, 2% of hydroxypropyl trimethyl ammonium chloride hydrolyzed soybean protein, 1% of soybean oleyl trimethyl ammonium chloride, 20% of propylene glycol, 5% of 1, 2-pentanediol, 4% of diaminopyrimidine oxide, 2% of pyrrolidinyl diaminopyrimidine oxide and 0.1% of palmitoyl hexapeptide-25, and heating and dissolving in a water bath at 35 ℃ to obtain a first mixture;

adding 0.1% octapeptide-2, 0.1% acetyl tetrapeptide-3, 0.1% biotin tripeptide-1, 0.2% acetyl hexapeptide-1, 5% glycerol and 3% vitamin E polyethylene glycol succinate into 50.9% purified water, and heating and dissolving in water bath at 35 deg.C to obtain a second mixture;

dropwise adding the second mixture into the first mixture, continuously stirring, and after mixing, performing high-speed shearing emulsification for 8min under the condition that the rotating speed is 5000rpm to obtain a micron-sized dispersion;

and (3) carrying out high-pressure homogenization treatment on the micron-sized dispersion under the condition that the pressure is 500bar, circulating for 5 times, and cooling to room temperature to obtain the nano composition.

The particle size of the nano composition and the Zeta potential are detected, and the particle size of the nano composition is 141.2nm, and the Zeta potential is +18.3 mV.

Example 2

Mixing 7% of egg yolk lecithin, 1% of cholesterol, 4% of hydroxypropyl trimethyl ammonium chloride hydrolyzed soybean protein, 2% of polyquaternium-51, 15% of propylene glycol, 15% of 1, 2-hexanediol, 5% of diaminopyrimidine oxide, 2.5% of pyrrolidinyl diaminopyrimidine oxide and 0.05% of palmitoyl tripeptide-1, and heating and dissolving in a water bath at 40 ℃ to obtain a first mixture;

adding 0.05% of myristoyl pentapeptide-17, 0.05% of myristoyl pentapeptide-16, 0.05% of decapeptide-10, 0.1% of copper peptide, 5% of butanediol and 4% of vitamin E succinic acid polyethylene glycol ester into 39.2% of purified water, and heating and dissolving in a water bath at 40 ℃ to obtain a second mixture;

dropwise adding the second mixture into the first mixture, continuously stirring, and after mixing, carrying out high-speed shearing emulsification for 6min under the condition that the rotating speed is 7000rpm to obtain a micron-sized dispersion;

and (3) carrying out high-pressure homogenization treatment on the micron-sized dispersion under the condition that the pressure is 800bar, circulating for 3 times, and cooling to room temperature to obtain the nano composition.

The particle size of the nano composition and the Zeta potential are detected, and the particle size of the nano composition is 216.4nm, and the Zeta potential is +41.5 mV.

Example 3

Mixing 10% egg yolk lecithin, 5% hydrogenated egg yolk lecithin, 2% cholesterol, 5% hydroxypropyl trimethyl ammonium chloride hydrolyzed soybean protein, 5% lauryl dimethyl ammonium hydroxypropyl hydrolyzed soybean protein, 20% propylene glycol, 15% dipropylene glycol, 6% diaminopyrimidine oxide, 3% pyrrolidinyl diaminopyrimidine oxide and 0.2% palmitoyl tetrapeptide-10, and heating in a water bath at 50 ℃ for dissolution to obtain a first mixture;

adding 0.1% oligopeptide-54, 0.1% decapeptide-18, 0.1% biotin tripeptide-1, 0.1% decapeptide-10, 5% glycerol and 5% vitamin E polyethylene glycol succinate into 18.4% purified water, and heating and dissolving in a water bath at 50 ℃ to obtain a second mixture;

dropwise adding the second mixture into the first mixture, continuously stirring, and after mixing, performing high-speed shearing emulsification for 7min under the condition that the rotating speed is 10000rpm to obtain a micron-sized dispersion;

and (3) carrying out high-pressure homogenization treatment on the micron-sized dispersion under the condition that the pressure is 1000bar, circulating for 4 times, and cooling to room temperature to obtain the nano composition.

The particle size of the nano composition and the Zeta potential are detected, and the particle size of the nano composition is 290.4nm, and the Zeta potential is +55.3 mV.

Example 4

Mixing 8% of cephalin, 3% of cholesterol, 5% of lauryl dimethyl ammonium hydroxypropyl hydrolyzed soybean protein, 1.5% of polyquaternium-37, 10% of dipropylene glycol, 5% of 1, 2-pentanediol, 3% of diaminopyrimidine oxide and 1.5% of pyrrolidinyl diaminopyrimidine oxide, and heating and dissolving in a water bath at 45 ℃ to obtain a first mixture;

adding 0.3% myristoyl pentapeptide-7, 0.3% myristoyl hexapeptide-16, 0.3% acetyl tetrapeptide-3, 0.3% decapeptide-10, 0.3% copper peptide, 0.3% acetyl hexapeptide-1, 5% propylene glycol and 3% vitamin E succinic acid polyethylene glycol ester into 53.2% purified water, and heating and dissolving in a water bath at 45 ℃ to obtain a second mixture;

dropwise adding the second mixture into the first mixture, continuously stirring, and after mixing, performing high-speed shearing emulsification for 10min at the rotating speed of 6000rpm to obtain a micron-sized dispersion;

and (3) carrying out high-pressure homogenization treatment on the micron-sized dispersion under the condition that the pressure is 700bar, circulating for 8 times, and cooling to room temperature to obtain the nano composition.

The particle size of the nano composition and the Zeta potential are detected, and the particle size of the nano composition is 25.6nm, and the Zeta potential is +36.5 mV.

Example 5

Mixing 5% dilauroyl phosphatidylcholine, 1.5% cholesterol, 3% lauryl dimethyl ammonium hydroxypropyl hydrolyzed soy protein, 0.5% polyquaternium-11, 8% propylene glycol, 2% 1, 2-hexanediol, 2% diaminopyrimidine oxide, 1% pyrrolidinyl diaminopyrimidine oxide, 0.4% palmitoyl hexapeptide-25, and 0.4% palmitoyl tripeptide-1, and heating in water bath at 30 ℃ to dissolve to obtain a first mixture;

adding 0.4% myristoyl pentapeptide-4, 0.4% acetyl tetrapeptide-3, 0.8% acetyl hexapeptide-1, 5% glycerol and 2% vitamin E polyethylene glycol succinate into 67.6% purified water, and heating in water bath at 30 deg.C for dissolving to obtain a second mixture;

dropwise adding the second mixture into the first mixture, continuously stirring, and after mixing, performing high-speed shearing emulsification for 6min under the condition that the rotating speed is 5000rpm to obtain a micron-sized dispersion;

and (3) carrying out high-pressure homogenization treatment on the micron-sized dispersion under the condition that the pressure is 600bar, circulating for 5 times, and cooling to room temperature to obtain the nano composition.

The particle size of the nano composition and the Zeta potential are detected, and the particle size of the nano composition is 108.6nm, and the Zeta potential is +22.1 mV.

Example 6

Mixing 4% of soybean lecithin, 1% of cholesterol, 0.1% of hydroxypropyl trimethyl ammonium chloride hydrolyzed soybean protein, 0.2% of polyquaternium-22, 0.2% of polyquaternium-39, 6% of butanediol, 1% of propylene glycol, 0.1% of diaminopyrimidine oxide and 0.1% of pyrrolidinyl diaminopyrimidine oxide, and heating and dissolving in a water bath at 35 ℃ to obtain a first mixture;

adding 0.5% myristoyl tetrapeptide-12, 0.5% tripeptide-2, 0.5% acetyl tetrapeptide-3, 0.5% biotin tripeptide-1, 0.5% copper peptide, 0.5% acetyl hexapeptide-1, 3% glycerol and 1% vitamin E polyethylene glycol succinate into 80.3% purified water, and heating and dissolving in water bath at 35 deg.C to obtain a second mixture;

dropwise adding the second mixture into the first mixture, continuously stirring, and after mixing, carrying out high-speed shearing emulsification for 5min under the condition that the rotating speed is 4000rpm to obtain a micron-sized dispersion;

and (3) carrying out high-pressure homogenization treatment on the micron-sized dispersion under the condition that the pressure is 700bar, circulating for 6 times, and cooling to room temperature to obtain the nano composition.

The particle size of the nano composition and the Zeta potential are detected, and the particle size of the nano composition is 54.1nm, and the Zeta potential is +33.5 mV.

Example 7

Mixing 8% of soybean lecithin, 2% of egg yolk lecithin, 5% of cholesterol, 6% of hydrolyzed soybean protein of hydroxypropyl trimethyl ammonium chloride, 3% of behenyl trimethyl ammonium chloride, 20% of propylene glycol, 10% of butanediol, 10% of diaminopyrimidine oxide, 5% of pyrrolidinyl diaminopyrimidine oxide and 0.05% of palmitoyl tetrapeptide-10, and heating and dissolving in a water bath at 45 ℃ to obtain a first mixture;

adding 0.05% decapeptide-18, 0.05% acetyl tetrapeptide-3, 5% 1, 2-hexanediol and 5% vitamin E polyethylene glycol succinate into 20.85% purified water, and heating in a water bath at 45 ℃ to dissolve to obtain a second mixture;

dropwise adding the second mixture into the first mixture, continuously stirring, and after mixing, performing high-speed shearing emulsification for 6min at a rotation speed of 8000rpm to obtain a micron-sized dispersion;

and (3) carrying out high-pressure homogenization treatment on the micron-sized dispersion under the condition that the pressure is 1200bar, circulating for 3 times, and cooling to room temperature to obtain the nano composition.

The particle size of the nano composition and the Zeta potential are detected, and the particle size of the nano composition is 198.5nm, and the Zeta potential is +11.3 mV.

Example 8

Mixing 10% of soybean lecithin, 2% of cephalin, 3% of cholesterol, 4% of hydroxypropyl trimethyl ammonium chloride hydrolyzed soybean protein, 4% of lauryl dimethyl ammonium hydroxypropyl hydrolyzed soybean protein, 1% of dicetyl dimethyl ammonium chloride, 20% of 1, 2-pentanediol, 10% of dipropylene glycol, 6% of diaminopyrimidine oxide, 10% of pyrrolidinyl diaminopyrimidine oxide and 0.01% of palmitoyl hexapeptide-25, and heating and dissolving in a water bath at 55 ℃ to obtain a first mixture;

adding 0.01% acetyl tetrapeptide-3, 0.01% copper peptide, 5% 1, 2-hexanediol and 5% vitamin E succinic acid polyethylene glycol ester into 19.97% purified water, and heating and dissolving in a water bath at 55 ℃ to obtain a second mixture;

dropwise adding the second mixture into the first mixture, continuously stirring, and after mixing, carrying out high-speed shearing emulsification for 5min under the condition that the rotating speed is 9000rpm to obtain a micron-sized dispersion;

and (3) carrying out high-pressure homogenization treatment on the micron-sized dispersion under the condition that the pressure is 800bar, circulating for 5 times, and cooling to room temperature to obtain the nano composition.

The particle size of the nano composition and the Zeta potential are detected, and the particle size of the nano composition is 268.3nm, and the Zeta potential is +59.1 mV.

Example 9

Mixing 3% egg yolk lecithin, 3% hydrogenated egg yolk lecithin, 0.5% cholesterol, 4% lauryl dimethyl ammonium hydroxypropyl hydrolyzed soybean protein, 2% hydroxypropyl guar hydroxypropyl trimethyl ammonium chloride, 15% 1, 2-hexanediol, 5% butanediol, 0.5% diaminopyrimidine oxide, 0.5% pyrrolidinyl diaminopyrimidine oxide, and 0.25% palmitoyl tetrapeptide-10, and heating and dissolving in a water bath at 50 ℃ to obtain a first mixture;

adding 0.25% myristoyl pentapeptide-17, 0.25% myristoyl hexapeptide-16, 0.5% decapeptide-10, 0.25% acetyl hexapeptide-1, 5% dipropylene glycol and 3% vitamin E polyethylene glycol succinate into 57% purified water, and heating and dissolving in water bath at 50 ℃ to obtain a second mixture;

dropwise adding the second mixture into the first mixture, continuously stirring, and after mixing, performing high-speed shearing emulsification for 7min at the rotation speed of 8000rpm to obtain a micron-sized dispersion;

and (3) carrying out high-pressure homogenization treatment on the micron-sized dispersion under the condition that the pressure is 900bar, circulating for 4 times, and cooling to room temperature to obtain the nano composition.

The particle size of the nano composition and the Zeta potential are detected, and the particle size of the nano composition is 81.2nm, and the Zeta potential is +30.7 mV.

Example 10

Mixing 6% of soybean lecithin, 2% of hydrogenated soybean lecithin, 1% of cholesterol, 2% of soybean oleyl trimethyl ammonium chloride, 3% of polyquaternium-51, 10% of propylene glycol, 5% of 1, 2-pentanediol, 5% of butanediol, 5% of diaminopyrimidine oxide and 2.5% of pyrrolidinyl diaminopyrimidine oxide, and heating and dissolving in a water bath at 45 ℃ to obtain a first mixture;

adding 0.05% decapeptide-18, 0.05% octapeptide-2, 0.05% acetyl tetrapeptide-3, 0.05% biotin tripeptide-1, 0.05% copper peptide, 0.05% acetyl hexapeptide-1, 5% glycerol and 3% vitamin E polyethylene glycol succinate into 50.2% purified water, and heating and dissolving in a water bath at 45 ℃ to obtain a second mixture;

and (3) carrying out high-pressure homogenization treatment on the micron-sized dispersion under the condition that the pressure is 800bar, circulating for 6 times, and cooling to room temperature to obtain the nano composition.

The particle size of the nano composition and the Zeta potential are detected, and the particle size of the nano composition is 249.8nm, and the Zeta potential is +49.7 mV.

Example 11

Mixing 4% dilauroyl phosphatidylcholine, 5% hydrogenated soybean lecithin, 2% cholesterol, 4% behenyl trimethyl ammonium chloride, 1% hydroxypropyl guar hydroxypropyl trimethyl ammonium chloride, 10% propylene glycol, 10% 1, 2-hexanediol, 2.5% diaminopyrimidine oxide, 1.5% pyrrolidinyl diaminopyrimidine oxide, and 0.5% palmitoyl tripeptide-1, and heating in a water bath at 30 ℃ to dissolve to obtain a first mixture;

adding 0.25% oligopeptide-54, 0.25% decapeptide-18, 0.5% copper peptide, 5% butanediol and 3% vitamin E polyethylene glycol succinate into 50.5% purified water, and heating and dissolving in water bath at 30 ℃ to obtain a second mixture;

dropwise adding the second mixture into the first mixture, continuously stirring, and after mixing, carrying out high-speed shearing emulsification for 7min under the condition that the rotating speed is 4000rpm to obtain a micron-sized dispersion;

and (3) carrying out high-pressure homogenization treatment on the micron-sized dispersion under the condition that the pressure is 700bar, circulating for 3 times, and cooling to room temperature to obtain the nano composition.

The particle size of the nano composition and the Zeta potential are detected, and the particle size of the nano composition is 164.3nm, and the Zeta potential is +15.7 mV.

Example 12

Mixing 7% of egg yolk lecithin, 8% of soybean lecithin, 4.5% of cholesterol, 1.5% of polyquaternium-51, 1.5% of polyquaternium-37, 1% of soybean oil-based trimethyl ammonium chloride, 20% of propylene glycol, 5% of dipropylene glycol, 5% of butanediol, 8% of diaminopyrimidine oxide and 8% of pyrrolidinyl diaminopyrimidine oxide, and heating and dissolving in a water bath at 35 ℃ to obtain a first mixture;

adding 0.15% octapeptide-2, 0.15% myristoyl pentapeptide-17, 0.3% decapeptide-10, 0.3% acetyl hexapeptide-1, 5% glycerol and 4% vitamin E polyethylene glycol succinate into 20.6% purified water, and heating and dissolving in a water bath at 35 ℃ to obtain a second mixture;

dropwise adding the second mixture into the first mixture, continuously stirring, and after mixing, performing high-speed shearing emulsification for 3min at a rotation speed of 8000rpm to obtain a micron-sized dispersion;

and (3) carrying out high-pressure homogenization treatment on the micron-sized dispersion under the condition that the pressure is 900bar, circulating for 2 times, and cooling to room temperature to obtain the nano composition.

The particle size of the nano composition and the Zeta potential are detected, and the particle size of the nano composition is 124.3nm, and the Zeta potential is +28.6 mV.

Example 13

Stability test

After the nano composition with hair loss prevention, hair growth promotion, hair fixing and hair blackening functions prepared in examples 1 to 12 is placed in a closed container at room temperature for 3, 6, 9 and 12 months, the particle size and Zeta potential of a sample are detected, and the properties of the sample are observed. Specific detection results are shown in table 1.

TABLE 1 stability test results of nano-composition with hair loss preventing, hair growth promoting, hair fixing and hair blackening functions

As can be seen from table 1: the particle size of the nano composition with the functions of preventing hair loss, growing hair, fixing hair and blackening hair is 10-300 nm, and the Zeta potential is 0-60 mV, so that the requirement of practical application is met. The sample does not have the phenomena of agglomeration, discoloration and delamination after being placed for 12 months, the particle size and the Zeta potential of the sample do not have significant changes, the practical application requirements are still met, particularly, the sample is still stable under the condition of high concentration of active ingredients, and the phenomenon of crystallization is not found, so that the nano composition with the functions of preventing hair loss, growing hair, fixing hair and blackening hair has good stability.

Comparative example 1

Preparing blank hair restorer: heating and dissolving 5% of propylene glycol, 2% of 1, 2-hexanediol, 1% of panthenol, 2% of nicotinamide, 1% of betaine and the balance of water, and uniformly stirring to obtain the blank hair tonic water.

Comparative example 2

Preparing nano composite hair growth lotion: and (2) adding 10% of the nano composition prepared in the example 1 into the blank hair growth lotion prepared in the comparative example 1, and uniformly stirring to obtain the nano composite hair growth lotion.

The nano composite hair growth promoting water comprises the following functional components in percentage by weight: 0.4% diaminopyrimidine oxide, 0.2% pyrrolidinyl diaminopyrimidine oxide, 0.01% palmitoyl hexapeptide-25, 0.01% octapeptide-2, 0.01% acetyl tetrapeptide-3, 0.01% biotin tripeptide-1, 0.02% acetyl hexapeptide-1.

Comparative example 3

Ordinary hair tonic: heating and dissolving 5% of propylene glycol, 2% of 1, 2-hexanediol, 1% of panthenol, 2% of nicotinamide, 1% of betaine, 0.4% of diaminopyrimidine oxide, 0.2% of pyrrolidinyl diaminopyrimidine oxide, 0.01% of palmitoyl hexapeptide-25, 0.01% of octapeptide-2, 0.01% of acetyl tetrapeptide-3, 0.01% of biotin tripeptide-1, 0.02% of acetyl hexapeptide-1 and the balance of water, and stirring uniformly to obtain the common hair-growing water.

Example 14

Stability test

After the nano-composition with the functions of preventing hair loss, promoting hair growth, fixing hair and blackening hair prepared in examples 1, 5 and 6, the nano-composite hair restorer prepared in comparative example 2 and the common hair restorer prepared in comparative example 3 are placed in a closed container at room temperature for 6 to 12 months, the contents of acetyl tetrapeptide-3 and acetyl hexapeptide-1 in each sample are detected by High Performance Liquid Chromatography (HPLC), the residual content percentages of the acetyl tetrapeptide-3 and the acetyl hexapeptide-1 are calculated, and the stability of the active polypeptide after nano-entrapment is evaluated. Specific detection results are shown in table 2.

TABLE 2 stability test results for active Polypeptides in Nanocompositions

As can be seen from table 2: after the nano-compositions prepared in examples 1, 5 and 6 are placed at room temperature for 6 to 12 months, the contents of acetyl tetrapeptide-3 and acetyl hexapeptide-1 in the nano-compositions do not change significantly, and oil-water separation and layering phenomena do not occur, which indicates that the nano-compositions with functions of preventing hair loss, promoting hair growth, fixing hair and blackening hair prepared by the method have good stability; the contents of acetyl tetrapeptide-3 and acetyl hexapeptide-1 in the nano composite hair restorer prepared in the comparative example 2 are not obviously changed and have no layering and precipitation phenomena after being placed for 6 and 12 months at room temperature, the content of the acetyl tetrapeptide-3 and the content of the acetyl hexapeptide-1 in the comparative example 3 are the same as those of the acetyl tetrapeptide-1 in the comparative example 2, but the contents of the acetyl tetrapeptide-3 and the acetyl hexapeptide-1 in the nano composite hair restorer prepared in the comparative example 3 are obviously reduced after being placed for 6 and 12 months at room temperature, which shows that the stability of active polypeptide is greatly improved after nano entrapment, and simultaneously shows that the nano composition prepared in the application can be stably compounded in cosmetic matrixes such as hair restorer.

Example 15

Irritation test

The nano-composition samples prepared in examples 1 to 6 were compounded with the blank hair restorer in comparative example 1 at a mass ratio of 3:7, respectively, and subjected to a skin irritation test.

Taking 42 healthy rabbits with the weight of 2.0 +/-0.2 kg, randomly dividing the rabbits into 7 groups, removing hairs on two sides of the skin on the back of the rabbits 24 hours before the experiment, checking whether the removed hairs are injured 24 hours after the hairs are removed, and not suitable for skin irritation test of the injured skin. The composite hair growth lotion prepared using the nano-composition prepared in examples 1 to 6 was applied 3 times a day for 7 consecutive days while applying blank hair growth lotion (without any drug) for control, and the test results were observed and listed in table 3.

Table 3 observations of skin irritation in the composite hair restorer and blank groups prepared from the samples of examples 1-6

"+" rabbit skin congestion, red swelling; "+ +" indicates that the congestion and red swelling still exist, but there is an increasing trend; "-" indicates no hyperemia or redness and swelling.

According to the test results in table 3, the nanocomposite hair restorer and the blank hair restorer prepared by using the nano compositions in the examples 1-6 have no hyperemia and redness and swelling after being smeared on the skin of rabbits, which indicates that the nano composition provided by the invention has no irritation to the scalp.

Example 16

In vitro transdermal test

The vertical Franz diffusion cell method is adopted to carry out the transdermal experiment of the in vitro rat skin. SD male rat abdominal skin is fixed between a receiving chamber and a supplying chamber, 1g of each of the nano composite hair growth lotion prepared in comparative example 2 and the common hair growth lotion prepared in comparative example 3 is taken in the supplying chamber, propylene glycol with the mass percentage of 20% and normal saline with the mass percentage of 80% are taken as receiving liquid, and stirring and diffusion are carried out at 37 ℃. 0.5mL of receiving solution was taken at 1,2, 4, 6, 8, 10, 12h and an equal amount of fresh receiving solution was immediately replenished. And (4) performing HPLC analysis, and calculating the cumulative permeation amount of the specific medicament per unit area at different times. After 12h, the skin is taken down, cleaned, cut into pieces, ground into homogenate, added with a proper amount of receiving liquid for centrifugation, and the supernatant is taken for HPLC analysis to calculate the skin retention amount of a specific medicament per unit area. The drugs tested in this experiment were diaminopyrimidine oxide and pyrrolidinyl diaminopyrimidine oxide. The experimental data are shown in fig. 1 and fig. 2.

FIG. 1 is a graph showing the cumulative permeation amount of the skin of the body after 12 hours for the nanocomposite hair-growing water prepared in comparative example 2 and the common hair-growing water prepared in comparative example 3; fig. 2 is an in vitro skin retention of the nanocomposite hair growth water prepared in comparative example 2 and the general hair growth water prepared in comparative example 3.

As can be seen from FIG. 1, the cumulative permeation amounts of diaminopyrimidine oxide and pyrrolidinyl-diaminopyrimidine oxide in ordinary hair growth lotion were 82.3. mu.g/cm, respectively²、47.7μg/cm²The cumulative penetration of the diaminopyrimidine oxide and the pyrrolidinyl diaminopyrimidine oxide in the nano composite hair growth promoting water is 173.8 mu g/cm²、125.6μg/cm²It shows that the cumulative permeation of the anti-dropping active substance in the skin is obviously improved after being coated by the nano composition. The solubility of the diaminopyrimidine oxide and the pyrrolidinyl diaminopyrimidine oxide is extremely poor, the diaminopyrimidine oxide is difficult to penetrate through the stratum corneum of the skin and cannot act on deep structures of hair follicles, so that the bioavailability is low, the prepared nano composition can increase the solubility of the diaminopyrimidine oxide and the pyrrolidyl diaminopyrimidine oxide, and lipid bilayer structures of the diaminopyrimidine oxide can enter the cells of the stratum corneum of the skin and interact with keratin to reduce the compactness of the cells of the stratum corneum, and the pyrrolidyl diaminopyrimidine oxide andforming lipid channels to promote the penetration of actives through the stratum corneum; the nano composition is a flexible liposome, has high deformability and high permeability, enables active ingredients to reach a deeper functional structure of hair follicles, and fully exerts the effects of preventing hair loss and growing hair.

As can be seen from FIG. 2, the skin retentions of diaminopyrimidine oxide and pyrrolidinyl-diaminopyrimidine oxide in ordinary hair growth lotion were 6.8. mu.g/cm²、5.2μg/cm²The skin retention of the diaminopyrimidine oxide and the pyrrolidinyl diaminopyrimidine oxide in the nano composite hair growth promoting water is 18.6 mu g/cm²、15.5μg/cm²The results show that the retention of the anti-dropping active substance in the skin is obviously improved after the anti-dropping active substance is coated by the nano composition. The nano composition prepared by the application is positively charged, is easier to adsorb on hair and scalp with negative charges, has stronger adhesion with a cell biological membrane, can inhibit P-glycoprotein in cells from discharging anti-hair loss active ingredients into blood, and slows down the clearing speed of the anti-hair loss active ingredients in the cells, so that the detention time of the anti-hair loss active ingredients in hair follicles is prolonged, the sustained release and controlled release are realized, the bioavailability is remarkably improved, and the anti-hair loss and hair growth effects are enhanced.

Example 17

In vitro hair follicle growth assay

Isolating and extracting mouse hair follicle for in vitro culture, beginning the next day of culture, diluting the nano composition prepared in example 1-3 5000 times with culture solution, and setting blank and positive control minoxidil with a concentration of 10uM and 5% CO at 37 deg.C₂Incubate in incubator for 14 days. Whisker hair follicle length was photographed every 7 days and measured. Data are presented as mean ± standard deviation. The relative growth rate of hair follicles was calculated by the following formula, and the results are shown in Table 4.

Relative growth rate (%) of hair follicle (average length of hair follicle in each group)_D14Average length of each group of hair follicles_D0) /(blank control group Hair follicle Length_D14Hair follicle length in blank control group_D0)×100％

TABLE 4 results of the in vitro hair follicle growth promotion test

Group of	Hair follicle growth value (mum) for 14 days	Relative growth rate (%)
			Blank control group	53.23±4.67	100.00±2.31
Minoxidil group	72.59±4.21	136.37±2.87
			Example 1	84.56±5.02	158.86±2.45
Example 2	87.14±5.15	163.70±2.56
			Example 3	92.16±5.25	173.14±3.01

According to the results in Table 4, the hair follicle growth value of the minoxidil group in 14 days is obviously greater than that of the blank control group, which indicates that the in vitro culture test of the hair follicle of the mouse is successful.

As can be seen from the data in Table 4, the nano-composition prepared by the method can significantly promote the growth of hair follicles, and the effect is equivalent to or even stronger than that of minoxidil. The new hair growth promoter diaminopyrimidine oxide and pyrrolidinyl diaminopyrimidine oxide are expected to replace minoxidil for treating alopecia and promoting hair growth.

Example 18

Experiments on combinations of anti-alopecia actives

Test samples: the sample No. 1 was prepared at a concentration of 30. mu.g/mL diaminopyrimidine oxide, the sample No. 2 was prepared at a concentration of 30. mu.g/mL pyrrolidinyl diaminopyrimidine oxide, the sample No. 3 was prepared at a concentration of 15. mu.g/mL diaminopyrimidine oxide and 15. mu.g/mL pyrrolidinyl diaminopyrimidine oxide, the sample No. 4 was prepared at a concentration of 10. mu.g/mL diaminopyrimidine oxide and 20. mu.g/mL pyrrolidinyl diaminopyrimidine oxide, and the sample No. 5 was prepared at a concentration of 20. mu.g/mL diaminopyrimidine oxide and 10. mu.g/mL pyrrolidinyl diaminopyrimidine oxide.

Detecting influence of diaminopyrimidine oxide and pyrrolidinyl diaminopyrimidine oxide with different concentrations on growth of hair papilla cells by CCK-8 method, and adjusting hair papilla cell density to 8 × 10⁴Perml, 100. mu.L/well of 96-well cell culture plates, incubated at 37 ℃ with 5% CO₂Culturing in a cell culture box for 24 h. After the culture is finished, the culture medium in the cell plate is sucked out, 100 mu L of test samples are respectively added into the treatment groups, the blank control group is added with the serum-free culture medium with the same volume, the culture is continued for 72h, and each group has 3 duplicate wells. After 72h, 10 μ L of CCK-8 solution is added into each group, after further incubation for 4h, the 96-well plate is taken out, the absorbance of the cells is measured, and the survival rate of the cells is calculated, and the experimental result is shown in FIG. 3.

As can be seen from fig. 3, samples No. 1 to 5 can effectively promote the proliferation of hair papilla cells, and the proliferation rates of the hair papilla cells are 118.5%, 112.8%, 129.9%, 126.5% and 138.4%, respectively, which indicates that the diaminopyrimidine oxide and the pyrrolidinyl diaminopyrimidine oxide can effectively resist alopecia, stimulate hair follicle regeneration, optimize the hair cycle, promote the growth period, shorten the resting period, and accelerate the growth of new hairs. From the results of FIG. 3, it can be seen that the combination of diaminopyrimidine oxide and pyrrolidinyl diaminopyrimidine oxide gives superior hair growth effect than that of itself, and that the hair growth effect is most excellent when the ratio of the contents of diaminopyrimidine oxide and pyrrolidinyl diaminopyrimidine oxide is 2: 1. The application aims at different target points of androgen-induced alopecia, and reasonably prepares different mechanisms of anti-alopecia active substances on the premise of optimal proportion, so that the anti-alopecia and hair-growing effects are optimal.

Example 19

Active polypeptide hair cell assay

Test samples: the nanocomposites obtained in examples 4, 5 and 6 were diluted 500 times with the culture medium to obtain the cultured samples corresponding to Nos. 6, 7 and 8, and the same culture medium was used to prepare the cultured sample of the free polypeptide material having the same type and content of the active polypeptide as those in sample No. 8, and No. 9 was used as a control.

VEGF content was measured by ELISA assay kit and cell culture was performed as in example 18. And respectively adding 100 mu L of test samples into the treatment groups, adding an equal volume of serum-free culture medium into the blank control group, continuously culturing for 72h, collecting cell supernatant after 72h, carrying out VEGF detection according to the specification requirements of an ELISA kit, and calculating the content change rate of VEGF in the cell supernatant, wherein the experimental result is shown in figure 4.

VEGF is an autocrine growth factor of hair papilla cells, and has the effects of regulating the proliferation and differentiation of keratinocytes, promoting the proliferation of hair follicle epithelial cells, inducing the proliferation and migration of hair papilla cells or inducing the formation of blood vessels around hair follicles to regulate the periodic circulation of hair follicles and promote hair growth.

As can be seen from fig. 4, the nano-compositions prepared in examples 4, 5 and 6 all can significantly increase the VEGF content, and the VEGF content increase rates are 78.2%, 82.8% and 92.3%, respectively, which indicates that the nano-compositions prepared in the present application can effectively regulate the proliferation and differentiation of keratinocytes, promote the proliferation of hair follicle epithelial cells, induce the proliferation of hair papilla cells, and have excellent hair growth effects; compared with the VEGF content increase rate of 43.6% of the free polypeptide (sample No. 9), the nano composition (sample No. 8) prepared in example 6 has significant difference on VEFG content increase rate secreted by hair papilla cells, and shows that the trichogenous peptide can promote VEGF secretion after being coated by the nano composition, induce the hair papilla cells to proliferate and has stronger trichogenous effect.

Example 20

Active polypeptide hair-fixing cell experiment

The content of collagen III, the cell culture method and the test samples were measured by ELISA assay kit as in example 19. And respectively adding 100 mu L of test samples into the treatment groups, adding an equal volume of serum-free culture medium into the blank control group, continuously culturing for 72h, collecting cell supernatant after 72h, carrying out collagen III detection according to the specification requirements of the ELISA kit, and calculating the content change rate of the collagen III in the cell supernatant, wherein the experimental result is shown in figure 5.

As can be seen from fig. 5, the nano-compositions prepared in examples 4, 5 and 6 can significantly improve the content of collagen iii, and the increase rates of the content of collagen iii are 134.1%, 139.3% and 167.5%, respectively, which indicates that the nano-composition prepared in the present application has an excellent hair-fixing effect, can directly act on the tissues around hair follicles, accelerates the synthesis of extracellular matrix proteins, and promotes the hair to be fixed in the hair follicles; compared with the collagen III content increase rate of 85.7% of free polypeptide (sample No. 9), the nano composition (sample No. 8) prepared in example 6 has a significant difference in the collagen III content increase rate secreted by hair papilla cells, which indicates that the hair-strengthening peptide can promote the secretion of collagen III after being encapsulated by the nano composition, and has a stronger hair-strengthening effect.

Example 21

Active polypeptide Hair blackening cell assay

The content of melanin in cells was measured by NaOH lysis, and the samples were tested in the same manner as in example 19. B16F10 cells were sampled at 1X 10⁵The cells were inoculated into 6-well plates at a density of 2mL per well, incubated for 24 hours, then the test samples were added, incubated for 48 hours, the supernatant was discarded, washed 3 times with PBS, 300. mu.L of a 1mol/L NaOH solution (containing 10% DMSO) was added per well, the cells were lysed thoroughly at 80 ℃ for 2 hours, the absorbance (A) of each well was measured at a wavelength of 405nm, and the relative melanin content was calculated, and the results of the experiment are shown in FIG. 6.

As can be seen from fig. 6, the nano-compositions prepared in examples 4, 5 and 6 all significantly increased the melanin content secreted by B16F10 cells, and the relative melanin contents were 62.4%, 68.2% and 75.3%, respectively, which indicates that the nano-compositions prepared in the present application have excellent hair-blackening effect, can induce the increase of tyrosinase activity, promote melanogenesis, optimize melanosome maturation and transfer to keratinocytes; compared with the 52.6% relative melanin content of the free polypeptide (sample No. 9), the relative melanin content of the nano composition (sample No. 8) prepared in example 6 has a significant difference, which shows that the hair-blackening peptide can promote melanin secretion of melanocytes and accelerate melanosome maturation after being encapsulated by the nano composition, and has a stronger hair-blackening effect.

Example 22

A C57BL/6 mouse is adopted to construct an androgenetic alopecia mouse model, and the in-vitro hair growth effect of the nano composition is evaluated. Grouping is performed first, group a: blank control group; group B: testosterone propionate group; group C: testosterone propionate + control 1 hair restorer group; group D: testosterone propionate + comparative example 2 nanocomposite hair growth water group; group E: testosterone propionate + comparative example 3 common hair restorer group. Each group of mice was treated with a depilatory cream by pricking the back of the mice with a depilatory knife, and the remaining hair was removed to confirm that the mice had rested (pink skin). B. C, D, E Each group of mice was injected with testosterone propionate suspension with a certain concentration into the skin of the back hairless area at multiple points every day, C, D, E groups of mice were applied with the same volume of hair growth promoting water every day in the back hairless area, continuously applied for 30 days and photographed for observation. The experimental results are shown in FIG. 7.

From A, B in FIG. 7, it was found that the hair grew normally in mice in group A and did not grow in mice in group B, indicating that the model of androgenetic alopecia mice was successfully modeled. After the hair growth lotion blank, the nano composite hair growth lotion and the common hair growth lotion are continuously smeared for 30 days, the hair growth effect of the hair growth lotion blank in the group C is basically not generated, the hair growth effect of the nano composite hair growth lotion in the group D is obvious, and the nano composite hair growth lotion is equivalent to the hair growth lotion A in the blank control group. Compared with the E group of common hair restorers, the D group of nano composite hair restorers has better hair restoring effect, and further proves that the anti-hair loss active substances can increase the solubility after being nano-encapsulated, so that the anti-hair loss active substances can be more easily and deeply permeated into hair follicles and are retained in the hair follicles for a long time, the bioavailability is obviously improved, and the anti-hair loss and hair restoring effects are enhanced.

The foregoing is merely a preferred embodiment of the invention and is not intended to limit the invention in any manner. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be construed as the protection scope of the present invention.

Claims

1. The nanometer composition with the functions of preventing hair loss, growing hair, fixing hair and blackening hair is characterized by comprising the following components in percentage by mass:

2. The nano-composition of claim 1, wherein the germinal peptide comprises one or more of oligopeptide-54, decapeptide-18, octapeptide-2, myristoyl pentapeptide-17, myristoyl pentapeptide-16, myristoyl pentapeptide-7, myristoyl pentapeptide-4, myristoyl hexapeptide-16, myristoyl tetrapeptide-12, palmitoyl hexapeptide-25, and tripeptide-2.

3. The nano-composition of claim 1, wherein the hair-fixing peptide comprises one or more of acetyl tetrapeptide-3, biotin tripeptide-1, palmitoyl tripeptide-1, and decapeptide-10.

4. The nano-composition of claim 1, wherein the hair blackening peptide comprises one or more of copper peptide, acetyl hexapeptide-1 and palmitoyl tetrapeptide-10.

5. The nano-composition of claim 1, wherein the cell penetrating agent comprises one or more of hydroxypropyl trimethyl ammonium chloride hydrolyzed soy protein, lauryl dimethyl ammonium hydroxypropyl hydrolyzed soy protein, soy oleyl trimethyl ammonium chloride, polyquaternium-51, polyquaternium-37, polyquaternium-11, polyquaternium-22, polyquaternium-39, behenyl trimethyl ammonium chloride, dicetyl dimethyl ammonium chloride, and hydroxypropyl guar hydroxypropyl trimethyl ammonium chloride.

6. The nanocomposite of claim 1, wherein the phospholipid comprises one or more of soy lecithin, egg yolk lecithin, cephalin, hydrogenated soy lecithin, hydrogenated egg yolk lecithin, and dilauroyl phosphatidylcholine.

7. The nanocomposite of claim 1, wherein the polyol comprises one or more of glycerol, butylene glycol, propylene glycol, dipropylene glycol, 1, 2-pentanediol, and 1, 2-hexanediol.

8. The nanocomposite as claimed in any one of claims 1 to 7, wherein the nanocomposite has a particle size of 10 to 300nm and a Zeta potential of 0 to +60 mV.

9. A method for preparing a nano-composition according to any one of claims 1 to 8, comprising the steps of:

There is no chronological definition between the step (1) and the step (2).

10. Use of the nano composition of any one of claims 1 to 8 in the preparation of cosmetics for preventing and growing hair, fixing hair and blackening hair and in the preparation of medicaments for treating androgenetic alopecia, alopecia areata, telogen effluvium and anagen phase hair loss syndrome.