A hair loss prevention and nourishing essence containing hirudin and its preparation method and application

Abstract

The present invention proposes an anti-hair loss nourishing essence containing hirudin and its preparation method and application, which belongs to the technical field of scalp care. It is prepared by the following raw materials in parts by weight: 1-5 parts of hirudin-Zn chelate complex, 20-25 parts of plant extract, 0.5-2 parts of pyridoxine hydrochloride, 3-5 parts of moisturizer, 0.3-0.6 parts of preservative, 0.5-1 parts of thickener, 0.05-0.5 parts of adenosine, and 60-80 parts of deionized water. The anti-hair loss nourishing essence containing hirudin prepared by the present invention can promote scalp blood circulation, stimulate hair follicle growth, reduce inflammation, remove dandruff, clean the scalp, relieve scalp itching, prevent hair loss, promote black hair growth, have the effect of nourishing and repairing the scalp, the preparation method is simple, and has broad application prospects.

Description

Technical Field

[0001] The invention relates to the technical field of scalp care, and in particular to an anti-hair loss nourishing essence containing hirudin, and a preparation method and application thereof. Background Art

[0002] The scalp is the main physiological attachment of hair. The physiological state of the scalp will significantly affect the state of hair. Insufficient nutrition of the scalp will lead to insufficient nutrition of hair follicles, further leading to problems such as accelerated hair loss, split ends, and hair breakage. In addition, the sebum secretion of the scalp is significantly higher than that of the facial skin. The rapid secretion of oil will lead to the growth of Malassezia, further leading to problems such as odor, dandruff, inflammation and itching. At the same time, the scalp ages faster than the facial and body skin. The scalp ages 6 times faster than the facial skin and 12 times faster than the body skin. The metabolic cycle of facial skin is 28 days, while the cell metabolism cycle of the scalp is only 14-21 days. Scalp aging leads to aging of scalp hair follicle cells, which leads to more scalp problems, such as dandruff, oily scalp, itchy scalp, inflammation and hair loss.

[0003] Hair loss refers to the phenomenon of hair loss. Normally lost hair is in the catagen and telogen phases. Since the hair entering the catagen and the hair entering the new growth phase are constantly in a dynamic balance, the normal amount of hair can be maintained. Pathological hair loss refers to abnormal or excessive hair loss. With the increasing social pressure and the accelerated pace of life, the continuous deterioration of the environment, and the poor eating habits, our unhealthy and sub-healthy conditions are also increasing day by day, and the number of hair loss patients in China is increasing.

[0004] Relevant studies have shown that hair loss is mainly caused by the degeneration and atrophy of hair follicle cells in the scalp, which results in insufficient nutrition for the hair. Therefore, to effectively solve the problem of hair loss, providing sufficient nutrition for the hair follicle cells is the key. At the same time, with appropriate hair follicle growth factors, it can not only effectively prevent hair loss, but also promote the regrowth of dormant hair follicles and grow new hair, thus completely solving the problem of hair loss. At present, there are many studies on anti-hair loss and hair thickening products, but their effects are average. Therefore, it is urgent to develop a more effective anti-hair loss nourishing essence. Summary of the invention

[0005] The purpose of the present invention is to provide an anti-hair loss nourishing essence containing hirudin and a preparation method and application thereof, which can promote scalp blood circulation, stimulate hair follicle growth, reduce inflammation, remove dandruff, clean the scalp, relieve scalp itching, prevent hair loss, promote black hair growth, and has the effect of nourishing and repairing the scalp. The preparation method is simple and has broad application prospects.

[0006] The technical solution of the present invention is achieved in this way:

[0007] The invention provides an anti-hair loss nourishing essence containing hirudin, which is prepared from the following raw materials in parts by weight: 1-5 parts of hirudin-Zn chelate complex, 20-25 parts of plant extract, 0.5-2 parts of pyridoxine hydrochloride, 3-5 parts of moisturizer, 0.3-0.6 parts of preservative, 0.5-1 parts of thickener, 0.05-0.5 parts of adenosine and 60-80 parts of deionized water.

[0008] As a further improvement of the present invention, the preparation method of the hirudin-Zn chelate complex is as follows:

[0009] S1. Dissolve vitamin B3, vitamin B5, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and N-hydroxysuccinimide in water, activate the reaction, add hirudin, and heat the reaction to obtain modified hirudin;

[0010] S2. Add the modified hirudin obtained in step S1 into water, add Zn salt, stir to react, dialyze, and dry to obtain a hirudin-Zn chelate complex.

[0011] As a further improvement of the present invention, the mass ratio of vitamin B3, vitamin B5, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide: hirudin in step S1 is 1-1.5:1.5-1.8:3.6-4:2.5-3:5-7, the temperature of the activation reaction is 0°C, the time is 20-30 min, the temperature of the heating reaction is 35-40°C, and the time is 5-7 h; the mass ratio of modified hirudin, water, and Zn salt in step S2 is 10-12:100:3-5, and the Zn salt is selected from at least one of zinc chloride, zinc sulfate, and zinc nitrate.

[0012] As a further improvement of the present invention, the preparation method of the plant extract is as follows:

[0013] T1. Ginseng fermentation: Ginseng is crushed, added to water, sterilized, inoculated with fermentation bacteria seed liquid, fermented, filtered, the solid is dried and retained, the filtrate is concentrated, and purified by macroporous resin to obtain a rare ginsenoside mixture;

[0014] T2. deep eutectic solvent enzymatic hydrolysis: crush Polygonum multiflorum, Platycladus orientalis leaves, and ginger, mix with the solid in step T1 to obtain a mixture, add to an aqueous solution containing a deep eutectic solvent, add a composite enzyme, enzymatically hydrolyze, inactivate the enzyme, and obtain an enzymatic hydrolysis product;

[0015] T3 fermentation: adding water to the enzymatic hydrolyzate in step T2, sterilizing, inoculating Bifidobacterium breve, Bifidobacterium infantis seed solution, fermentation culture, filtering, washing the solid, collecting the eluate and concentrating the bacterial solution, freeze-drying the liquid to obtain a fermentation product;

[0016] T4. Preparation of bifid yeast lysate: Freeze the bacterial solution obtained in step T3 with liquid nitrogen, dissolve at room temperature, centrifuge, collect the supernatant, and freeze-dry to obtain bifid yeast lysate;

[0017] T5. Preparation of plant extract: The rare ginsenoside mixture prepared in step T1, the fermentation product prepared in step T3, the bifida yeast lysate prepared in step T4, and vitamin C are uniformly mixed to prepare a plant extract.

[0018] As a further improvement of the present invention, the mass ratio of ginseng to water in step T1 is 1:10-15, the fermentation bacteria is Bacillus subtilis or Bacillus licheniformis, the inoculation amount of the fermentation bacteria seed liquid is 2-3v / v%, and the bacterial content is 10 8 -10 9 cfu / mL, the fermentation culture conditions are 45-55°C, 70-120r / min, and the fermentation culture is 18-24h, and the macroporous resin is selected from at least one of XDA-2, NKA-9, SP-825, D101, HPD600, X-5, LSA-10, NKI-9 and AB-8; in step T2, the mass ratio of Polygonum multiflorum, Platycladus orientalis leaves, ginger, and solid is 7-10:3-5:10-15:5-7 The mass ratio of the mixture, the complex enzyme, and the aqueous solution containing a deep eutectic solvent is 10:0.5-1:80-100, the water content in the aqueous solution containing a deep eutectic solvent is 40-60wt%, the deep eutectic solvent is prepared by mixing choline chloride and glucose in a molar ratio of 1:1, the complex enzyme is composed of a mixture of cellulase and pectinase in a mass ratio of 10-15:3-5, the enzymatic hydrolysis temperature is 40-50°C, and the time is 2-4h.

[0019] As a further improvement of the present invention, the mass ratio of the enzymatic hydrolysate to water in step T3 is 1:0.8-1.2, and the bacterial content of the bacterial seed liquid is 10 8 -10 9 cfu / mL, the inoculation amounts of the breve Bifidobacterium and infants Bifidobacterium species seed solutions are 1-3v / v% and 2-3v / v%, respectively, the fermentation culture conditions are anaerobic conditions, 36-39°C, 150-200r / min, and the fermentation culture is 36-48h; the mass ratio of the rare ginsenoside mixture, fermentation product, bifid yeast lysate, and vitamin C in step T5 is 2-4:15-20:5-7:0.5-1.

[0020] As a further improvement of the present invention, the moisturizer is selected from at least one of glycerin, butylene glycol, propylene glycol, sodium hyaluronate, and hexylene glycol; the thickener is selected from at least one of polyacrylate cross-linked polymer-6, sodium polyacryloyldimethyl taurate, and xanthan gum; and the preservative is a mixture of phenoxyethanol and ethylhexylglycerin, with a mass ratio of 8-10:1.

[0021] The present invention further protects a method for preparing the above-mentioned anti-hair loss nourishing essence containing hirudin, comprising the following steps:

[0022] Add moisturizer, adenosine and thickener into deionized water, heat to 70-80°C, stir at 300-500r / min for 20-30min, cool to 35-40°C, add hirudin-Zn chelate complex, pyridoxine hydrochloride, preservative and plant extract, stir for 10-15min, filter and obtain anti-hair loss nourishing essence containing hirudin.

[0023] The present invention further protects the use of the anti-hair loss nourishing essence containing hirudin in scalp care, hair growth promotion and hair loss prevention.

[0024] The invention has the following beneficial effects: the invention prepares a hirudin-Zn chelate complex, and two vitamins with carboxyl groups (vitamin B3 and vitamin B5) undergo a dehydration condensation reaction with free amino groups on hirudin under the activation of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide. Modified hirudin is prepared, and the hirudin part can accelerate cell metabolism, dredge capillaries, improve scalp microcirculation, and promote hair growth. Vitamin B3 can promote the metabolism of skin cells, enhance the protective function of the skin, reduce skin dryness, itching and other problems, promote hair growth, and relieve greasy scalp. Vitamin B5 can promote hair growth and prevent hair loss, and promote scalp blood circulation. The modified hirudin further chelates with zinc ions to form a hirudin-Zn chelate complex. Zinc ions are components of many metal enzymes or enzyme activators in the body, participate in the synthesis of DNA, and indirectly affect the synthesis of RNA and protein. It replenishes the zinc element in the scalp in time to avoid problems such as incomplete scalp keratinization, desquamation, dermatitis and hair loss caused by zinc deficiency. It also avoids cell apoptosis caused by zinc deficiency, prevents the influx of calcium ions, and avoids the occurrence of hair loss.

[0025] The plant extract prepared by the present invention uses ginseng, polygonum multiflorum, arborvitae leaves and ginger as raw materials. The ginseng is fermented by bacillus subtilis or bacillus licheniformis, which, on the one hand, causes the ginseng plant cell wall to rupture and the active components to dissolve, and on the other hand, promotes the conversion of common ginsenosides into rare saponins, thereby greatly increasing the content of rare saponins in ginseng. The rare saponins include Rh2, Rg3, aPPD, Rk2, Rh3, Rk1, Rg5, Rk3, Rh1, Rh3, Rh4 and the like, which can replenish blood and qi to the scalp, promote the development of hair follicles, strengthen fragile hair roots, improve and prevent hair loss, replenish essence and blood, ensure sufficient kidney qi, and promote the growth of black hair.

[0026] Polygonum multiflorum, Platycladus orientalis leaves and ginger are enzymatically hydrolyzed by deep eutectic solvents, using enzymes as catalysts to hydrolyze plant cell walls, promoting the diffusion of effective ingredients from the cell to the extraction medium, greatly improving the extraction of water-insoluble compounds, and having the advantages of low toxicity and greenness, and high solubility for compounds. Glucose is used as one of the raw materials in the deep eutectic solvent of the present invention, which also provides a carbon source for subsequent fermentation.

[0027] The present invention uses Bifidobacterium breve and Bifidobacterium infantis for fermentation, and the fermented product contains rich trace elements and nutrients, such as iron, zinc, selenium, vitamin B, etc., which can effectively nourish hair and make hair healthy and shiny; at the same time, it can also improve the dissolution of active components in the raw materials, so that the fermented product contains rich flavonoids, organic acids, terpenoids, polysaccharides, saponins and other substances. The fermentation of the two strains also has a synergistic effect.

[0028] Polygonum multiflorum extract has the effect of improving hair quality and blackening hair; Platycladus orientalis leaf extract can promote blood circulation in the scalp and stimulate the growth of hair follicles. Topical application can reduce inflammation, remove dandruff, clean the scalp, relieve scalp itching, and prevent hair loss; ginger extract can stimulate the scalp, promote the active growth of hair follicle cells, assist in hair growth, promote blood circulation on the surface of the scalp, and assist in preventing hair loss.

[0029] On the one hand, the fermentation process produces abundant fermentation by-products, and on the other hand, it promotes the massive proliferation of bifidobacteria. After liquid nitrogen freezing-room temperature thawing treatment, the bifidobacterium bacterial wall is broken and the contents are dissolved. The obtained bifidobacterium lysate can help increase the moisture of the scalp, improve the dandruff problem to a certain extent, and has the effect of nourishing and repairing the scalp.

[0030] In addition, the pyridoxine hydrochloride added in the present invention acts as a hair conditioning agent, a skin conditioning agent and an antistatic agent.

[0031] The anti-hair loss nourishing essence containing hirudin prepared by the present invention can promote scalp blood circulation, stimulate hair follicle growth, reduce inflammation, remove dandruff, clean the scalp, relieve scalp itching, prevent hair loss, promote black hair growth, has the effect of nourishing and repairing the scalp, has a simple preparation method, and has broad application prospects. DETAILED DESCRIPTION

[0032] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0033] Bacillus subtilis, 100 billion cfu / g, and Bacillus licheniformis, 100 billion cfu / g, were purchased from Guangzhou Zhenwei Microbiology Technology Co., Ltd.

[0034] Preparation method of Bacillus subtilis or Bacillus licheniformis seed solution: inoculate the strain into Gao's medium, activate and culture at 50℃, 100r / min for 18h, and obtain a medium with a bacterial content of 10 8 -10 9 cfu / mL of bacterial seed solution.

[0035] Bifidobacterium breve, 10 billion cfu / g, was purchased from Shaanxi Saien Biotechnology Co., Ltd.; Bifidobacterium infantis, 10 billion cfu / g, was purchased from Hubei Mingtuo Biotechnology Co., Ltd.

[0036] Preparation method of Bifidobacterium breve or Bifidobacterium infantis seed solution: inoculate the strain into Gao's medium, activate and culture at 37°C, 100r / min for 24h, and obtain a medium with a bacterial content of 10 8 -10 9 cfu / mL of bacterial seed solution.

[0037] Cellulase, 11,000 U / g, and pectinase, 60,000 U / g, were purchased from Xiasheng (Beijing) Biotechnology Development Co., Ltd.

[0038] Preparation Example 1 Preparation method of plant extract

[0039] The following steps are involved:

[0040] T1. Ginseng fermentation: 10 parts by weight of ginseng were crushed, added to 100 parts by weight of water, sterilized, inoculated with Bacillus licheniformis seed liquid, the inoculation amount was 2v / v%, 45°C, 70r / min, fermented and cultured for 18h, filtered, the solid was dried and retained, the filtrate was concentrated, purified by macroporous resin SP-825, the filtrate was decompressed to remove the solvent, diluted with 50wt% ethanol and loaded, wet column loading, the specifications of the chromatography column were 15mm×500mm, the column volume was 50mL, the eluate with 50wt% ethanol concentration was collected, the ethanol was removed under reduced pressure, and dried to obtain a rare ginsenoside mixture;

[0041] T2. Deep eutectic solvent hydrolysis: 7 parts by weight of Polygonum multiflorum, 3 parts by weight of Platycladus orientalis leaves, and 10 parts by weight of ginger were crushed and mixed with 5 parts by weight of the solid in step T1 to obtain a mixture, 10 parts by weight of the mixture were added to 80 parts by weight of an aqueous solution containing a deep eutectic solvent, 0.5 parts by weight of a composite enzyme was added, and the hydrolysis was performed at 40°C for 2h, and the enzyme was inactivated to obtain an enzymatic product;

[0042] The water content in the aqueous solution containing the deep eutectic solvent is 40 wt %, and the deep eutectic solvent is prepared by mixing choline chloride and glucose in a molar ratio of 1:1;

[0043] The complex enzyme is composed of a mixture of cellulase and pectinase, with a mass ratio of 10:3;

[0044] T3 fermentation: to 100 parts by weight of the enzymatic hydrolysate in step T2 was added 80 parts by weight of water, sterilized, inoculated with Bifidobacterium breve and Bifidobacterium infantis seed solution, the inoculation amounts were 1v / v% and 2v / v%, respectively, under anaerobic conditions, 36 ° C, 150r / min, fermentation culture 36h, filtered, the solid was washed, the eluate was collected and concentrated to obtain a bacterial solution, the liquid was freeze-dried to obtain a fermentation product;

[0045] T4. Preparation of bifid yeast lysate: Freeze the bacterial solution obtained in step T3 with liquid nitrogen, dissolve at room temperature, centrifuge, collect the supernatant, and freeze-dry to obtain bifid yeast lysate;

[0046] T5. Preparation of plant extract: 2 parts by weight of the rare ginsenoside mixture prepared in step T1, 15 parts by weight of the fermentation product prepared in step T3, 5 parts by weight of the bifida yeast lysate prepared in step T4, and 0.5 parts by weight of vitamin C were stirred and mixed for 30 minutes to prepare a plant extract.

[0047] Preparation Example 2 Preparation method of plant extract

[0048] The following steps are involved:

[0049] T1. Ginseng fermentation: 10 parts by weight of ginseng were crushed, added to 150 parts by weight of water, sterilized, inoculated with Bacillus subtilis seed liquid, the inoculation amount was 3v / v%, 55°C, 120r / min, fermented for 24h, filtered, the solid was dried and retained, the filtrate was concentrated, purified by macroporous resin SP-825, the filtrate was decompressed to remove the solvent, diluted with 60wt% ethanol, wet loaded, the chromatography column had a specification of 15mm×500mm, and a column volume of 50mL, and the eluate with 60wt% ethanol concentration was collected, the ethanol was removed under reduced pressure, and dried to obtain a rare ginsenoside mixture;

[0050] T2. Deep eutectic solvent hydrolysis: 10 parts by weight of Polygonum multiflorum, 5 parts by weight of Platycladus orientalis leaves, and 15 parts by weight of ginger were crushed and mixed with 7 parts by weight of the solid in step T1 to obtain a mixture, 10 parts by weight of the mixture were added to 100 parts by weight of an aqueous solution containing a deep eutectic solvent, 1 part by weight of a composite enzyme was added, and the mixture was hydrolyzed at 50°C for 4h, and the enzyme was inactivated to obtain an enzymatic product;

[0051] The water content in the aqueous solution containing the deep eutectic solvent is 60 wt %, and the deep eutectic solvent is prepared by mixing choline chloride and glucose in a molar ratio of 1:1;

[0052] The complex enzyme is composed of a mixture of cellulase and pectinase, with a mass ratio of 15:5;

[0053] T3 fermentation: to 100 parts by weight of the enzymatic hydrolysate in step T2 was added 120 parts by weight of water, sterilized, inoculated with Bifidobacterium breve and Bifidobacterium infantis seed solution, the inoculation amounts were 3v / v% and 3v / v%, respectively, under anaerobic conditions, 39 ° C, 200r / min, fermentation culture 48h, filtered, the solid was washed, the eluate was collected and concentrated to obtain a bacterial solution, the liquid was freeze-dried to obtain a fermentation product;

[0054] T4. Preparation of bifid yeast lysate: Freeze the bacterial solution obtained in step T3 with liquid nitrogen, dissolve at room temperature, centrifuge, collect the supernatant, and freeze-dry to obtain bifid yeast lysate;

[0055] T5. Preparation of plant extract: 4 parts by weight of the rare ginsenoside mixture prepared in step T1, 20 parts by weight of the fermentation product prepared in step T3, 7 parts by weight of the bifida yeast lysate prepared in step T4, and 1 part by weight of vitamin C were stirred and mixed for 30 minutes to prepare a plant extract.

[0056] Preparation Example 3 Preparation method of plant extract

[0057] The following steps are involved:

[0058] T1. Ginseng fermentation: 10 parts by weight of ginseng were crushed, added to 125 parts by weight of water, sterilized, inoculated with Bacillus subtilis seed liquid, the inoculation amount was 2.5v / v%, 50°C, 100r / min, fermented for 21h, filtered, the solid was dried and retained, the filtrate was concentrated, purified by macroporous resin SP-825, the filtrate was decompressed to remove the solvent, diluted with 55wt% ethanol, wet packed, the size of the chromatography column was 15mm×500mm, the column volume was 50mL, the eluate with 55wt% ethanol concentration was collected, the ethanol was removed under reduced pressure, and dried to obtain a rare ginsenoside mixture;

[0059] T2. Deep eutectic solvent hydrolysis: 8 parts by weight of Polygonum multiflorum, 4 parts by weight of Platycladus orientalis leaves, and 12 parts by weight of ginger were crushed and mixed with 6 parts by weight of the solid in step T1 to obtain a mixture, 10 parts by weight of the mixture were added to 90 parts by weight of an aqueous solution containing a deep eutectic solvent, 0.7 parts by weight of a composite enzyme was added, and the hydrolysis was performed at 45°C for 3h, and the enzyme was inactivated to obtain an enzymatic product;

[0060] The water content in the aqueous solution containing the deep eutectic solvent is 50 wt %, and the deep eutectic solvent is prepared by mixing choline chloride and glucose in a molar ratio of 1:1;

[0061] The complex enzyme is composed of a mixture of cellulase and pectinase, with a mass ratio of 12:4;

[0062] T3. Fermentation: adding 100 parts by weight of the hydrolyzate in step T2 to 100 parts by weight of water, sterilizing, inoculating Bifidobacterium breve and Bifidobacterium infantis seed solution, inoculation amounts were 2v / v% and 2.2v / v%, respectively, under anaerobic conditions, 37°C, 170r / min, fermentation culture for 42h, filtering, washing the solid, collecting the eluate and concentrating the bacterial solution, and freeze-drying the liquid to obtain a fermentation product;

[0063] T4. Preparation of bifid yeast lysate: Freeze the bacterial solution obtained in step T3 with liquid nitrogen, dissolve at room temperature, centrifuge, collect the supernatant, and freeze-dry to obtain bifid yeast lysate;

[0064] T5. Preparation of plant extract: 3 parts by weight of the rare ginsenoside mixture prepared in step T1, 17 parts by weight of the fermentation product prepared in step T3, 6 parts by weight of the bifida yeast lysate prepared in step T4, and 0.7 parts by weight of vitamin C were stirred and mixed for 30 minutes to prepare a plant extract.

[0065] Preparation Example 4

[0066] Compared with Preparation Example 3, the difference is that the composite enzyme is a single cellulase.

[0067] Preparation Example 5

[0068] Compared with Preparation Example 3, the difference is that the complex enzyme is a single pectinase.

[0069] Comparative Preparation Example 1

[0070] Compared with Preparation Example 3, the difference is that step T1 is not performed, and ginseng is directly added into step T2.

[0071] Comparative Preparation Example 2

[0072] Compared with Preparation Example 3, the difference is that no complex enzyme is added in step T2.

[0073] Comparative Preparation Example 3

[0074] Compared with Preparation Example 3, the difference is that the seed liquid of Bifidobacterium breve is not inoculated in step T3.

[0075] Comparative Preparation Example 4

[0076] Compared with Preparation Example 3, the difference is that the seed liquid of Bifidobacterium infantis is not inoculated in step T3.

[0077] Comparative Preparation Example 5

[0078] Compared with Preparation Example 3, the difference is that step T3 is not performed.

[0079] Comparative Preparation Example 6

[0080] Compared with Preparation Example 3, the difference is that no rare ginsenoside mixture is added in step T5.

[0081] Comparative Preparation Example 7

[0082] Compared with Preparation Example 3, the difference is that no fermentation product is added in step T5.

[0083] Comparative Preparation Example 8

[0084] The difference compared with Preparation Example 3 is that no bifida yeast lysate is added in step T5.

[0085] Preparation Example 6 Preparation of Hirudin-Zn Chelate Complex

[0086] Here’s how:

[0087] S1. 10 parts by weight of vitamin B3, 15 parts by weight of vitamin B5, 36 parts by weight of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 25 parts by weight of N-hydroxysuccinimide were dissolved in 1000 parts by weight of water, activated at 0°C for 20 min, 50 parts by weight of hirudin were added, heated to 35°C, stirred for 7 h, and modified hirudin was obtained;

[0088] S2. Add 10 parts by weight of the modified hirudin obtained in step S1 to 100 parts by weight of water, add 3 parts by weight of zinc chloride, stir and react for 30 minutes, dialyze, and dry to obtain a hirudin-Zn chelate complex.

[0089] Preparation Example 7 Preparation of Hirudin-Zn Chelate Complex

[0090] Here’s how:

[0091] S1. 15 parts by weight of vitamin B3, 18 parts by weight of vitamin B5, 42 parts by weight of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 30 parts by weight of N-hydroxysuccinimide were dissolved in 1000 parts by weight of water, activated at 0°C for 30 min, 70 parts by weight of hirudin were added, heated to 40°C, stirred for 7 h, and modified hirudin was obtained;

[0092] S2. Add 12 parts by weight of the modified hirudin obtained in step S1 to 100 parts by weight of water, add 5 parts by weight of zinc nitrate, stir and react for 30 minutes, dialyze, and dry to obtain a hirudin-Zn chelate complex.

[0093] Preparation Example 8 Preparation of Hirudin-Zn Chelate Complex

[0094] Here’s how:

[0095] S1. 12 parts by weight of vitamin B3, 16 parts by weight of vitamin B5, 39 parts by weight of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, and 27 parts by weight of N-hydroxysuccinimide were dissolved in 1000 parts by weight of water, activated at 0°C for 25 min, 60 parts by weight of hirudin were added, heated to 37°C, stirred for 6 h, and modified hirudin was obtained;

[0096] S2. Add 11 parts by weight of the modified hirudin obtained in step S1 to 100 parts by weight of water, add 4 parts by weight of zinc sulfate, stir and react for 30 minutes, dialyze, and dry to obtain a hirudin-Zn chelate complex.

[0097] Comparative Preparation Example 9

[0098] Compared with Preparation Example 8, the difference is that vitamin B3 is not added in step S1.

[0099] Comparative Preparation Example 10

[0100] Compared with Preparation Example 8, the difference is that vitamin B5 is not added in step S1.

[0101] Comparative Preparation Example 11

[0102] Compared with Preparation Example 8, the difference is that hirudin is not added in step S1.

[0103] Comparative Preparation Example 12

[0104] Compared with Preparation Example 8, the difference is that step S2 is not performed.

[0105] Example 1

[0106] This embodiment provides a method for preparing an anti-hair loss nourishing essence containing hirudin, which is characterized by comprising the following steps:

[0107] 3 parts by weight of butanediol, 0.05 parts by weight of adenosine, and 0.5 parts by weight of polyacrylate cross-linked polymer-6 were added to 60 parts by weight of deionized water, heated to 70°C, stirred at 300 r / min for 20 minutes, cooled to 35°C, and 1 part by weight of the hirudin-Zn chelate complex prepared in Preparation Example 6, 0.5 parts by weight of pyridoxine hydrochloride, 0.3 parts by weight of a preservative, and 20 parts by weight of the plant extract prepared in Preparation Example 1 were added, stirred for 10 minutes, and filtered to prepare an anti-hair loss nourishing essence containing hirudin;

[0108] The preservative is a mixture of phenoxyethanol and ethylhexylglycerin with a mass ratio of 8:1.

[0109] Example 2

[0110] This embodiment provides a method for preparing an anti-hair loss nourishing essence containing hirudin, which is characterized by comprising the following steps:

[0111] 5 parts by weight of glycerol, 0.5 parts by weight of adenosine, and 1 part by weight of xanthan gum were added to 80 parts by weight of deionized water, heated to 80° C., stirred at 500 r / min for 30 minutes, cooled to 40° C., 5 parts by weight of the hirudin-Zn chelate complex prepared in Preparation Example 7, 2 parts by weight of pyridoxine hydrochloride, 0.6 parts by weight of a preservative, and 25 parts by weight of the plant extract prepared in Preparation Example 2 were added, stirred for 15 minutes, and filtered to prepare an anti-hair loss nourishing essence containing hirudin;

[0112] The preservative is a mixture of phenoxyethanol and ethylhexylglycerin with a mass ratio of 10:1.

[0113] Example 3

[0114] This embodiment provides a method for preparing an anti-hair loss nourishing essence containing hirudin, which is characterized by comprising the following steps:

[0115] 4 parts by weight of sodium hyaluronate, 0.3 parts by weight of adenosine, and 0.7 parts by weight of xanthan gum were added to 70 parts by weight of deionized water, heated to 75° C., stirred at 400 r / min for 25 minutes, cooled to 37° C., 3 parts by weight of the hirudin-Zn chelate complex prepared in Preparation Example 8, 1 part by weight of pyridoxine hydrochloride, 0.45 parts by weight of a preservative, and 22 parts by weight of the plant extract prepared in Preparation Example 3 were added, stirred for 12 minutes, and filtered to prepare an anti-hair loss nourishing essence containing hirudin;

[0116] The preservative is a mixture of phenoxyethanol and ethylhexylglycerin with a mass ratio of 9:1.

[0117] Example 4

[0118] Compared with Example 3, the difference is that the plant extract is prepared by Preparation Example 4.

[0119] Example 5

[0120] Compared with Example 3, the difference is that the plant extract is prepared by Preparation Example 5.

[0121] Comparative Example 1

[0122] Compared with Example 3, the difference is that the plant extract is prepared by Comparative Preparation Example 1.

[0123] Comparative Example 2

[0124] Compared with Example 3, the difference is that the plant extract is prepared by Comparative Preparation Example 2.

[0125] Comparative Example 3

[0126] Compared with Example 3, the difference is that the plant extract is prepared by Comparative Preparation Example 3.

[0127] Comparative Example 4

[0128] Compared with Example 3, the difference is that the plant extract is prepared by Comparative Preparation Example 4.

[0129] Comparative Example 5

[0130] Compared with Example 3, the difference is that the plant extract is prepared by Comparative Preparation Example 5.

[0131] Comparative Example 6

[0132] Compared with Example 3, the difference is that the plant extract is prepared by Comparative Preparation Example 6.

[0133] Comparative Example 7

[0134] Compared with Example 3, the difference is that the plant extract is prepared by Comparative Preparation Example 7.

[0135] Comparative Example 8

[0136] Compared with Example 3, the difference is that the plant extract is prepared by Comparative Preparation Example 8.

[0137] Comparative Example 9

[0138] Compared with Example 3, the difference is that the hirudin-Zn chelate complex is prepared by Comparative Preparation Example 9.

[0139] Comparative Example 10

[0140] Compared with Example 3, the difference is that the hirudin-Zn chelate complex is prepared by Comparative Preparation Example 10.

[0141] Comparative Example 11

[0142] Compared with Example 3, the difference is that the hirudin-Zn chelate complex is prepared by Comparative Preparation Example 11.

[0143] Comparative Example 12

[0144] Compared with Example 3, the difference is that the hirudin-Zn chelate complex is prepared by Comparative Preparation Example 12.

[0145] Comparative Example 13

[0146] Compared with Example 3, the difference is that no plant extract is added.

[0147] Comparative Example 14

[0148] Compared with Example 3, the difference is that no hirudin-Zn chelate complex is added.

[0149] Comparative Example 15

[0150] Compared with Example 3, the difference is that pyridoxine hydrochloride is not added.

[0151] Test Example 1 Safety Evaluation

[0152] The anti-hair loss nourishing essence containing hirudin prepared in Examples 1-5 of the present invention and Comparative Examples 1-15 was evaluated for safety. The results are as follows:

[0153] Hygienic chemistry and microbiological indicators: lead content is less than 10mg / kg, mercury is less than 0.05mg / kg, arsenic is less than 1mg / kg; total bacteria is less than 10cfu / mL, and fecal coliform group, Staphylococcus aureus, and Pseudomonas aeruginosa are not detected. The above indicators are in compliance with the provisions of the national "Cosmetic Hygiene Standard" GB7916-87.

[0154] In the acute oral toxicity test of mice, the acute oral toxicity LD50 values ​​of female and male mice were both greater than 5000 mg / kg body weight. According to GB7919-87 Chemical Acute Toxicity Classification Standard, the anti-hair loss nourishing essence containing hirudin prepared in Examples 1-5 and Comparative Examples 1-15 belongs to the practically non-toxic grade.

[0155] In the skin multiple irritation test, the skin irritation score was <0.4, and the pathological histology score was <4. According to the national "Cosmetic Hygiene Standard" GB7916-87 chemical substance skin irritation intensity evaluation standard, the anti-hair loss nourishing essence containing hirudin prepared in Examples 1-5 and Comparative Examples 1-15 is a non-irritating substance.

[0156] Test Example 2: 2 - The clearance rate

[0157] The anti-hair loss nourishing essence containing hirudin prepared in Examples 1-5 and Comparative Examples 1-15 was diluted with deionized water to 1 mg / mL as a sample solution. Vitamin C was used as a control group.

[0158] Take 5 mL of Tris-HCl buffer solution with pH = 8.2 in a test tube, preheat it in a 25℃ water bath for 20 min, then add 2 mL of sample solution and 5.00×10 -3 mol / L pyrogallol solution 0.5mL (using 1.00×10 -2 mol / L HCl solution), shake well immediately and react in a 25℃ water bath for 4 min, and immediately measure its absorbance A at 320 nm. i ; 1.00×10 -2 0.5 mL of 1 mol / L HCl solution was used to replace the pyrogallol solution to measure the absorbance A j ; Use 2mL of pure water instead of sample solution to measure absorbance A 0 Calculate the value of ·O according to the formula 2 - The clearance rate (%).

[0159] ·O 2 - The clearance rate (%) = [1-(A i -A j ) / A 0 ]×100%

[0160] The results are shown in Table 1.

[0161] Table 1

[0162] Group <![CDATA[·O 2 - Clearance rate (%)]]> Control group 34.2 Example 1 77.5 Example 2 76.4 Example 3 78.0 Example 4 74.5 Example 5 75.1 Comparative Example 1 73.2 Comparative Example 2 72.9 Comparative Example 3 72.5 Comparative Example 4 71.6 Comparative Example 5 69.7 Comparative Example 6 70.4 Comparative Example 7 65.8 Comparative Example 8 71.9 Comparative Example 9 72.3 Comparative Example 10 70.9 Comparative Example 11 66.8 Comparative Example 12 73.1 Comparative Example 13 62.2 Comparative Example 14 64.5 Comparative Example 15 76.1

[0163] It can be seen from the above table that the anti-hair loss nourishing essence containing hirudin prepared in Examples 1-3 of the present invention has good antioxidant activity.

[0164] Test Example 3 Anti-hair loss experiment

[0165] Suitable volunteers were selected for the experiment and randomly divided into Example 1-5 and Comparative Example 1-15 groups, with 10 people in each group.

[0166] Inclusion criteria: ① Age 25-52 years old; ② Obvious thinning hair / baldness / conscious severe hair loss; ③ No congenital hair thinning or hair loss symptoms; ④ No hair dyeing or perming in the past year; ⑤ No allergic constitution; ⑥ No serious systemic disease, immunodeficiency or autoimmune disease; ⑦ The test site has not participated in other clinical tests in the past 3 months.

[0167] Exclusion criteria: ① Termination of the test due to personal reasons; ② Failure to use the product in accordance with the test requirements; ③ Adverse / uncomfortable reactions occurred and the test could not be continued.

[0168] Test method: The subjects use the anti-hair loss nourishing essence containing hirudin every night and apply it to the entire scalp. Use it continuously for 8 weeks. It should not be used with other similar products during the test period. Test once at 0 and 8 weeks. Wash your hair the day before the test. Sit still for 30 minutes in a laboratory with a temperature of (25±1)℃ and a humidity of 45%±5% RH on the day of the test. Use a stratum corneum hydration tester and a grease tester to measure a fixed area of ​​the subject's scalp, record the stratum corneum moisture content value and grease content value, and collect the hair that falls out of the pool when the subject washes his hair the night before the test. After drying, record the change in the number of hair and calculate the improvement rate.

[0169] Improvement rate (%) = |(test data of week 8 - test data of week 0)| / test data of week 0 × 100%. The improvement rate here is the absolute value to ensure that the value of the improvement rate is always a positive number. In actual testing, after using the essence, the moisture content of the stratum corneum increased, the oil content decreased, and the amount of hair loss decreased.

[0170] The results are shown in Table 2.

[0171] Table 2

[0172]

[0173]

[0174] It can be seen from the above table that the anti-hair loss nourishing essence containing hirudin prepared in Examples 1-3 of the present invention has a good effect of improving the moisture content of the scalp, reducing the oil content, and reducing hair loss.

[0175] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. An anti-hair loss nourishing essence containing hirudin, characterized in that: The invention is prepared from the following raw materials in parts by weight: 1-5 parts of hirudin-Zn chelate complex, 20-25 parts of plant extract, 0.5-2 parts of pyridoxine hydrochloride, 3-5 parts of moisturizer, 0.3-0.6 parts of preservative, 0.5-1 parts of thickener, 0.05-0.5 parts of adenosine and 60-80 parts of deionized water; The preparation method of the hirudin-Zn chelate complex is as follows: S1. Dissolving vitamin B3, vitamin B5, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide in water, activating the reaction, adding hirudin, and heating the reaction to obtain modified hirudin; S2. The modified hirudin prepared in step S1 is added to water, a Zn salt is added, the reaction is stirred, dialyzed, and dried to obtain a hirudin-Zn chelate complex; The preparation method of the plant extract is as follows: T1. Ginseng fermentation: Ginseng is crushed, added to water, sterilized, inoculated with fermentation bacteria seed liquid, fermented, filtered, the solid is dried and retained, the filtrate is concentrated, and purified by macroporous resin to obtain a rare ginsenoside mixture; the macroporous resin is selected from at least one of XDA-2, NKA-9, SP-825, D101, HPD600, X-5, LSA-10, NKI-9 and AB-8; the fermentation bacteria is Bacillus subtilis or Bacillus licheniformis; T2. Deep eutectic solvent enzymatic hydrolysis: Grind Polygonum multiflorum, Platycladus orientalis leaves and ginger, mix with the solid in step T1 to obtain a mixture, add to an aqueous solution containing a deep eutectic solvent, add a composite enzyme, perform enzymatic hydrolysis, inactivate the enzyme, and obtain an enzymatic hydrolysis product; the water content in the aqueous solution containing the deep eutectic solvent is 40-60wt%, the deep eutectic solvent is prepared by mixing choline chloride and glucose in a molar ratio of 1:1, and the composite enzyme is composed of a mixture of cellulase and pectinase in a mass ratio of 10-15:3-5; T3 fermentation: adding water to the enzymatic hydrolysate in step T2, sterilizing, inoculating Bifidobacterium breve and Bifidobacterium infantis seed solution, fermentation culture, filtering, washing the solid, collecting the eluate and concentrating the bacterial solution, freeze-drying the liquid to obtain a fermentation product; T4 preparation of bifid yeast lysate: the bacterial solution obtained in step T3 was frozen in liquid nitrogen, dissolved at room temperature, centrifuged, the supernatant was collected, and freeze-dried to obtain a bifid yeast lysate; T5. Preparation of plant extract: The rare ginsenoside mixture prepared in step T1, the fermentation product prepared in step T3, the bifida yeast lysate prepared in step T4 and vitamin C are uniformly mixed to prepare a plant extract.

2. The anti-hair loss nourishing essence containing hirudin according to claim 1, characterized in that: The mass ratio of vitamin B3, vitamin B5, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide and hirudin in step S1 is 1-1.5:1.5-1.8:3.6-4:2.5-3:5-7, the temperature of the activation reaction is 0°C, the time is 20-30min, the temperature of the heating reaction is 35-40°C, and the time is 5-7h; the mass ratio of modified hirudin, water and Zn salt in step S2 is 10-12:100:3-5, and the Zn salt is selected from at least one of zinc chloride, zinc sulfate and zinc nitrate.

3. The anti-hair loss nourishing essence containing hirudin according to claim 1, characterized in that: In step T1, the mass ratio of ginseng to water is 1:10-15, the inoculation amount of the fermentation bacteria seed liquid is 2-3v / v%, and the bacterial content is 10 8 -10 9 cfu / mL, the fermentation culture conditions are 45-55°C, 70-120r / min, and the fermentation culture is 18-24h. In step T2, the mass ratio of Polygonum multiflorum, Platycladus orientalis leaves, ginger, and the solid in step T1 is 7-10:3-5:10-15:5-7, the mass ratio of the mixture, the complex enzyme, and the aqueous solution containing the deep eutectic solvent is 10:0.5-1:80-100, and the enzymatic hydrolysis temperature is 40-50°C, and the time is 2-4h.

4. The anti-hair loss nourishing essence containing hirudin according to claim 1, characterized in that: The mass ratio of the enzymatic hydrolysate to water in step T3 is 1:0.8-1.2, and the bacterial content of the bacterial seed solution is 10 8 -10 9 cfu / mL, the inoculation amounts of the breve and infant Bifidobacterium species seed solutions are 1-3v / v% and 2-3v / v%, respectively, and the fermentation culture conditions are anaerobic conditions, 36-39°C, 150-200r / min, and the fermentation culture is 36-48h; the mass ratio of the rare ginsenoside mixture, fermentation product, bifid yeast lysate, and vitamin C in step T5 is 2-4:15-20:5-7:0.5-1.

5. The anti-hair loss nourishing essence containing hirudin according to claim 1, characterized in that: The moisturizer is selected from at least one of glycerin, butylene glycol, propylene glycol, sodium hyaluronate, and hexylene glycol; the thickener is selected from at least one of polyacrylate cross-linked polymer-6, sodium polyacryloyldimethyl taurate, and xanthan gum; the preservative is a mixture of phenoxyethanol and ethylhexylglycerin, with a mass ratio of 8-10:

1.

6. A method for preparing the anti-hair loss nourishing essence containing hirudin as claimed in any one of claims 1 to 5, characterized in that: The following steps are involved: Add moisturizer, adenosine and thickener into deionized water, heat to 70-80°C, stir at 300-500r / min for 20-30min, cool to 35-40°C, add hirudin-Zn chelate complex, pyridoxine hydrochloride, preservative and plant extract, stir for 10-15min, filter, and obtain anti-hair loss nourishing essence containing hirudin.

7. Use of the anti-hair loss nourishing essence containing hirudin as claimed in any one of claims 1 to 5 in the preparation of scalp care, hair growth promotion or anti-hair loss products.

8. A plant extract, characterized in that The preparation method comprises the following steps: T1. Ginseng fermentation: 10 parts by weight of ginseng crushed, added to 100-150 parts by weight of water, sterilized, inoculated with 10 8 -10 9 cfu / mL of Bacillus subtilis or Bacillus licheniformis seed solution, inoculation amount of 2-3v / v%, 45-55°C, 70-120r / min, fermentation culture for 18-24h, filtration, solid drying and retention, filtrate concentration, purification through macroporous resin SP-825, decompression of the filtrate to remove the solvent, dilute with 50-60wt% ethanol and load, wet column loading, column volume of 45-55mL, collect 50-60wt% ethanol concentration of eluate, decompression to remove ethanol, dry, to obtain a rare ginsenoside mixture; T2. Deep eutectic solvent hydrolysis: 7-10 parts by weight of Polygonum multiflorum, 3-5 parts by weight of Platycladus orientalis leaves and 10-15 parts by weight of ginger are crushed and mixed with 5-7 parts by weight of the solid in step T1 to obtain a mixture, 10 parts by weight of the mixture are added to 80-100 parts by weight of an aqueous solution containing a deep eutectic solvent, 0.5-1 parts by weight of a composite enzyme is added, and enzymolysis is performed at 40-50° C. for 2-4 hours, and the enzyme is inactivated to obtain an enzymolysis product; The water content in the aqueous solution containing the deep eutectic solvent is 40-60 wt %, and the deep eutectic solvent is prepared by mixing choline chloride and glucose in a molar ratio of 1:1; The complex enzyme is composed of a mixture of cellulase and pectinase, with a mass ratio of 10-15:3-5; T3 fermentation: adding 80-120 parts by weight of water to 100 parts by weight of the enzymatic hydrolysate in step T2, sterilizing, inoculating Bifidobacterium breve and Bifidobacterium infantis seed solution, inoculation amounts were 1-3v / v% and 2-3v / v%, respectively, under anaerobic conditions, 36-39 ° C, 150-200r / min, fermentation culture 36-48h, filtered, washed with solids, the eluate was collected and concentrated to obtain a bacterial solution, the liquid was freeze-dried to obtain a fermentation product; The bacterial content of the bacterial seed liquid is 10 8 -10 9 cfu / mL; T4 preparation of bifid yeast lysate: the bacterial solution obtained in step T3 was frozen in liquid nitrogen, dissolved at room temperature, centrifuged, the supernatant was collected, and freeze-dried to obtain a bifid yeast lysate; T5. Preparation of plant extract: 2-4 parts by weight of the rare ginsenoside mixture prepared in step T1, 15-20 parts by weight of the fermentation product prepared in step T3, 5-7 parts by weight of the bifida yeast lysate prepared in step T4 and 0.5-1 parts by weight of vitamin C are mixed uniformly to prepare a plant extract.

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