A hair-darkening composition containing plant exosomes, its preparation method and application

Abstract

This invention provides a hair-darkening composition containing plant exosomes, its preparation method, and its application, belonging to the field of cosmetic technology. The hair-darkening composition of this invention is made from the following raw materials: Polygonum multiflorum extract, Ginkgo biloba leaf extract, Salvia miltiorrhiza root extract, Nasturtium extract, ginger extract, Ligustrum lucidum fruit extract, ginseng root extract, black mulberry fruit extract, Platycladus orientalis leaf extract, Raspberry extract, black sesame extract, a mixture of plant exosomes, dandelion extract, alligator polypeptide, calcium pantothenate, tyrosine, borneol, water, butylene glycol, glycerin, and preservatives. This invention combines natural Chinese herbal medicine ingredients with plant exosomes, utilizing the small size and strong tissue penetration of plant exosomes to allow them to carry active ingredients directly to cells. By activating tyrosinase activity, increasing melanocyte activity, increasing melanin content, and resisting ROS damage, it more safely and effectively turns gray hair back to black.

Description

Technical Field

[0001] This invention relates to the field of cosmetic technology, and in particular to a hair-darkening composition containing plant exosomes, its preparation method, and its application. Background Technology

[0002] Melanocytes are cells that form and secrete melanin in the body and are an important component of skin cells. Tyrosinase is a key enzyme in melanin production. In melanocytes, tyrosine is converted into dopa, dopaquinone, and finally eumelanin under the action of tyrosinase. Eumelanin is related to hair color and is the key substance for black hair; abnormal melanin production will lead to gray hair. In addition, antioxidants are an effective way to prevent hair from turning gray. ROS damage to melanocytes will cause hair to lose melanin, appearing white or gray.

[0003] Exosomes are extracellular vesicles secreted by eukaryotic cells, ranging in diameter from 40 to 100 nm. Exosomes derived from medicinal plants are rich in various bioactive lipids, proteins, RNA, and other components, exhibiting significant regulatory effects in skin repair, immune modulation, anti-inflammatory and anti-infective activities, and regenerative medicine. Furthermore, plant exosomes possess advantages such as small size and strong tissue penetration, maintaining good physicochemical stability under varying pH and temperature conditions. Plant exosomes can serve as highly efficient and safe carriers, effectively delivering bioactive substances into cells, mediating intercellular communication, and thus achieving more precise targeted therapy. They are also easily mass-produced, have low immunogenicity, and can reduce adverse reactions and side effects.

[0004] Hair-darkening products on the market generally fall into two categories. One type contains hair dye, which uses chemical dyeing agents to color the hair and achieve a blackening effect. These products don't last long, requiring repeated dyeing, and long-term use can have significant side effects, damaging the hair, making it increasingly dry and brittle, and also making the scalp sensitive and fragile, even causing scalp inflammation, thus exacerbating the gray hair problem. Some hair dye products claiming to have natural ingredients are plant-based dyes made from extracts of natural pigments. These generally don't damage the hair structure or destroy the stored pigments, but the blackening effect is usually mediocre and not long-lasting. The other type contains no hair dye. These products have a larger molecular weight, resulting in poor scalp absorption. They generally require about 4-6 months to see results, and the blackening effect is often unsatisfactory. Therefore, developing a safe and effective hair-darkening product that can improve insufficient melanin and fundamentally solve the problem of gray hair is essential. Summary of the Invention

[0005] The purpose of this invention is to provide a hair-darkening composition containing plant exosomes, its preparation method, and its application. This invention combines natural Chinese herbal ingredients with plant exosomes, utilizing the small size and strong tissue penetration of plant exosomes to allow them to carry active ingredients directly to cells. By activating tyrosinase activity, increasing melanocyte activity, increasing melanin content, and resisting ROS damage, it more safely and effectively and gradually turns gray hair back to black.

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

[0007] This invention provides a hair-darkening composition containing plant exosomes, made from raw materials comprising the following percentages by weight: 0.001%–10% Polygonum multiflorum extract, 0.01%–5% Ginkgo biloba leaf extract, 0.01%–5% Salvia miltiorrhiza root extract, 0.01%–5% Nasturtium extract, 0.01%–5% Zingiber officinale extract, 0.01%–5% Ligustrum lucidum fruit extract, 0.001%–5% Panax ginseng root extract, 0.01%–5% Mulberry fruit extract, and 0.01% Platycladus orientalis leaf extract. ~3%, raspberry extract 0.01%~3%, black sesame extract 0.01%~3%, plant exosome mixture 0.0001%~1%, dandelion extract 0.001%~1%, crocodile polypeptide 0.001%~2%, calcium pantothenate 0.001%~5%, tyrosine 0.001%~1%, borneol 0.0001%~0.5%, water 70%~95%, butylene glycol 0.01%~5%, glycerin 0.01%~5%, preservatives 0.01%~0.5%.

[0008] Preferably, the preservative includes sodium benzoate, methylparaben, and phenoxyethanol.

[0009] Preferably, the plant exosome mixture is made from the following raw materials in parts by weight: 0.0001-0.2 parts of Polygonum multiflorum, 0.0001-0.5 parts of Salvia miltiorrhiza, 0.0001-0.5 parts of ginger, and 0.0001-0.2 parts of Ginkgo biloba leaves.

[0010] Preferably, the method for preparing the plant exosome mixture includes the following steps:

[0011] (1) Mix Polygonum multiflorum with PBS buffer, break it up, and collect the filtrate;

[0012] (2) Centrifuge the filtrate at 250-350g for 8-12 minutes and collect the supernatant;

[0013] (3) Centrifuge the supernatant obtained in step (2) at 1500-3000g for 8-12 minutes and take the supernatant;

[0014] (4) Centrifuge the supernatant obtained in step (3) at 8000-15000g for 8-12 minutes and take the supernatant;

[0015] (5) Centrifuge the supernatant obtained in step (4) at 80,000 to 120,000 g for 80 to 100 min and collect the precipitate;

[0016] (6) The precipitate is mixed with water to obtain Polygonum multiflorum exosomes;

[0017] (7) Treat the salvia miltiorrhiza, ginger and ginkgo leaves according to steps (1) to (6) to obtain salvia miltiorrhiza exosomes, ginger exosomes and ginkgo leaf exosomes; mix the Polygonum multiflorum exosomes, salvia miltiorrhiza exosomes, ginger exosomes and ginkgo leaf exosomes to obtain the plant exosome mixture.

[0018] Preferably, the amount of PBS buffer added is (0.5 to 2) times the volume of Polygonum multiflorum.

[0019] Preferably, the concentration of the Polygonum multiflorum exosomes is 250–350 mg / mL.

[0020] The present invention also provides a method for preparing the above-mentioned hair-darkening composition, comprising the following steps:

[0021] 1) Divide the water into two parts. Mix the first part of the water with butanediol to obtain pre-prepared solution A;

[0022] 2) Mix the borneol with the second part of water to obtain pre-prepared solution B;

[0023] 3) Take the following extracts: Polygonum multiflorum extract, Ginkgo biloba extract, Salvia miltiorrhiza root extract, Nasturtium extract, ginger extract, Ligustrum lucidum fruit extract, ginseng root extract, black mulberry fruit extract, Platycladus orientalis leaf extract, raspberry extract, black sesame extract, plant exosome mixture, dandelion extract, crocodile polypeptide, and glycerin, and add them to pre-prepared solution A to obtain pre-prepared solution C.

[0024] 4) Mix the pre-prepared solution C with pre-prepared solution B, calcium pantothenate and tyrosine to obtain pre-prepared solution D;

[0025] 5) Add preservative to the pre-prepared liquid D and stir evenly to obtain the hair-darkening composition.

[0026] Preferably, the mass ratio of the first part of water to the second part of water is (80-95):(5-20).

[0027] Preferably, the mixing temperature in step 2) is 50–65°C.

[0028] The present invention also provides the application of the above-mentioned hair-darkening composition in the preparation of hair-darkening shampoos, conditioners, hair masks, sprays, and gels.

[0029] This invention provides a hair-darkening composition containing plant exosomes, its preparation method, and its applications. This invention combines effective active ingredients with different mechanisms of action for turning gray hair black, plant exosomes, and key components for melanin synthesis. Following the principles of traditional Chinese medicine and combining modern biotechnology, it increases melanin production by activating tyrosinase activity and enhancing melanocyte activity. It also reduces ROS damage to melanocytes by activating antioxidant defenses, stimulating melanin deposition in the hair and thus gradually turning gray hair back to black. Simultaneously, it repairs hair follicle cells, regulates the scalp microenvironment, improves scalp inflammation and sensitivity, bringing the scalp to a healthy state and preventing melanin loss from the hair.

[0030] In this invention, Polygonum multiflorum extract can nourish the liver and kidneys, replenish essence and blood, darken hair, strengthen muscles and bones, and reduce turbidity and lipids. It is used for blood deficiency and chlorosis, dizziness and tinnitus, premature graying of hair, soreness and weakness of the waist and knees, numbness of limbs, metrorrhagia and leukorrhea, and hyperlipidemia. Ginkgo biloba extract can clear deficiency heat and eliminate infantile malnutrition heat. It is used for yin deficiency fever and bone steaming fever. Salvia miltiorrhiza root extract can invigorate blood and remove blood stasis, regulate menstruation and relieve pain, clear the heart and relieve irritability, cool blood and eliminate carbuncles. Nasturtium extract can cool blood and stop bleeding, nourish the kidneys and replenish yin, darken hair, benefit kidney yin, and improve antioxidant properties. It can reduce ROS damage to melanocytes by activating antioxidant defense. Ginger extract contains gingerol, ginger oil and other components, which can normalize blood circulation in the scalp, promote scalp metabolism, and help drugs reach the lesion. Privet fruit extract nourishes the liver and kidneys, improves eyesight and darkens hair. It is used for liver and kidney yin deficiency, dizziness and tinnitus, soreness and weakness of the lower back and knees, premature graying of hair, blurred vision, internal heat and thirst, and night sweats. Ginseng root extract greatly replenishes vital energy, restores pulse and consolidates the body, strengthens the spleen and lungs, generates fluids and nourishes blood, calms the mind and improves intelligence. It is used for weakness and collapse, cold limbs and weak pulse, spleen deficiency and poor appetite, lung deficiency and cough, thirst due to fluid depletion, internal heat and thirst, deficiency of qi and blood, chronic illness and emaciation, palpitations and insomnia, impotence and cold uterus. Ginseng root extract also has strong antioxidant properties, preventing the oxidation of melanocytes in hair follicles, thus protecting cells and slowing down the aging process. Black mulberry fruit extract nourishes yin and blood, generates fluids and moistens dryness. It is used for liver and kidney yin deficiency, dizziness and tinnitus, palpitations and insomnia, premature graying of hair, thirst due to fluid depletion, internal heat and thirst, and constipation due to intestinal dryness. Arborvitae leaf extract has the effects of cooling blood and stopping bleeding, resolving phlegm and relieving cough, and promoting hair growth and darkening hair. It can also treat cough due to lung heat, hair loss due to blood heat, and premature graying of hair. Raspberry extract enters the liver, kidney, and bladder meridians; it is rich in flavonoids, which can effectively enhance the elasticity of the scalp. Black sesame extract can nourish the liver and kidneys, replenish essence and blood, and moisten dryness of the intestines. It is used for deficiency of essence and blood, dizziness, tinnitus, deafness, premature graying of hair, hair loss after illness, and constipation due to intestinal dryness. Dandelion extract has the effects of clearing heat and detoxifying, reducing swelling and dissipating nodules, and promoting diuresis. It is also rich in flavonoids, phenolic acids, triterpenoids, polysaccharides, etc., and has certain anti-inflammatory, antibacterial, antioxidant, and free radical scavenging effects. Crocodile polypeptide, a small molecule polypeptide extracted from crocodiles, is rich in 18 kinds of amino acids and various trace elements, which can supplement hair follicle nutrition and increase scalp elasticity. Calcium pantothenate, a B vitamin, is a component of coenzyme A and participates in the metabolism of carbohydrates, fats, and proteins. It is an essential trace element for maintaining normal physiological functions in humans and animals. Tyrosine is a key substance in melanin synthesis and can catalyze the oxidation of tyrosine to dopa, which in turn is oxidized to dopaquinone.

[0031] The hair-darkening composition of this invention does not contain hair dye ingredients, will not damage the hair or scalp, and can fundamentally solve the problem of insufficient melanin production, achieving the effect of turning gray hair black. The specially added plant exosomes have advantages such as small size and strong tissue penetration, and maintain good physicochemical stability under different pH levels and temperatures. At the same time, plant exosomes are highly efficient and safe carriers, effectively delivering bioactive substances into cells, mediating intercellular communication, thereby achieving more precise targeted therapy. They are also easy to mass-produce, have low immunogenicity, and can reduce adverse reactions and side effects. The hair-darkening composition of this invention improves the effectiveness of turning gray hair black, and the treatment time is reduced to 20 days for noticeable results. Attached Figure Description

[0032] Figure 1 The results are shown in the figure. Examples 1-4 show the effect of the hair-darkening compositions on the melanin content in melanoma cells.

[0033] Figure 2 The graph shows the effect of the hair-darkening compositions of Examples 1-4 on the activity of melanoma cells.

[0034] Figure 3 The graph shows the effect of the hair-darkening compositions of Examples 1-4 on tyrosinase activity.

[0035] Figure 4 The graph shows the effect of the hair-darkening compositions of Example 4 and the comparative example on the melanin content in melanoma cells. Detailed Implementation

[0036] This invention provides a hair-darkening composition containing plant exosomes, made from raw materials comprising the following percentages by weight: 0.001%–10% Polygonum multiflorum extract, 0.01%–5% Ginkgo biloba leaf extract, 0.01%–5% Salvia miltiorrhiza root extract, 0.01%–5% Nasturtium extract, 0.01%–5% Zingiber officinale extract, 0.01%–5% Ligustrum lucidum fruit extract, 0.001%–5% Panax ginseng root extract, 0.01%–5% Mulberry fruit extract, and 0.01% Platycladus orientalis leaf extract. ~3%, raspberry extract 0.01%~3%, black sesame extract 0.01%~3%, plant exosome mixture 0.0001%~1%, dandelion extract 0.001%~1%, crocodile polypeptide 0.001%~2%, calcium pantothenate 0.001%~5%, tyrosine 0.001%~1%, borneol 0.0001%~0.5%, water 70%~95%, butylene glycol 0.01%~5%, glycerin 0.01%~5%, preservatives 0.01%~0.5%.

[0037] In this invention, the hair-darkening composition is preferably made from raw materials comprising the following mass percentages: 2%–10% Polygonum multiflorum extract, 1%–5% Ginkgo biloba extract, 1%–5% Salvia miltiorrhiza root extract, 1%–5% Nasturtium extract, 1%–5% Zingiber officinale extract, 1%–5% Ligustrum lucidum fruit extract, 1%–5% Panax ginseng root extract, 0.05%–3% Mulberry fruit extract, 0.05%–2% Platycladus orientalis leaf extract, and Raspberry extract. 0.05%–3% of the following ingredients: black sesame extract 0.05%–3%; plant exosome mixture 0.05%–1%; dandelion extract 0.05%–1%; crocodile polypeptide 0.05%–2%; calcium pantothenate 0.05%–3%; tyrosine 0.05%–1%; borneol 0.05%–0.5%; water 73%–90%; butylene glycol 1%–5%; glycerin 0.05%–3%; preservatives 0.05%–0.5%.

[0038] Further preferred formulations are made from raw materials comprising the following percentages by weight: 2%–5% Polygonum multiflorum extract, 1%–3% Ginkgo biloba leaf extract, 1%–3% Salvia miltiorrhiza root extract, 1%–2% Nasturtium extract, 1%–2% ginger extract, 1%–2% Ligustrum lucidum fruit extract, 1%–2% ginseng root extract, 0.1%–1% black mulberry fruit extract, 0.1%–1% Platycladus orientalis leaf extract, and 0.1%–1% raspberry extract. Black sesame extract 0.1%–1%, plant exosome mixture 0.05%–0.5%, dandelion extract 0.05%–0.5%, crocodile polypeptide 0.1%–1%, calcium pantothenate 0.1%–0.5%, tyrosine 0.05%–0.5%, borneol 0.1%–0.2%, water 75%–85%, butylene glycol 2%–4%, glycerin 0.01%–0.5%, preservative 0.1%–0.3%;

[0039] Further preferred formulations are made from the following ingredients in the indicated weight percentages: 3% Polygonum multiflorum extract, 2% Ginkgo biloba leaf extract, 2% Salvia miltiorrhiza root extract, 1.2% Nasturtium extract, 1.2% Zingiber officinale extract, 1.2% Ligustrum lucidum fruit extract, 1.2% Panax ginseng root extract, 0.2% Mulberry fruit extract, 0.2% Platycladus orientalis leaf extract, 0.2% Raspberry extract, 0.15% Black sesame extract, 0.1% plant exosome mixture, 0.1% Taraxacum mongolicum extract, 0.2% Crocodile polypeptide, 0.2% Calcium pantothenate, 0.12% Tyrosine, 0.12% Borneol, 83.71% Water, 2.5% Butylene glycol, 0.2% Glycerin, and 0.2% Preservative.

[0040] In this invention, the preservative preferably includes sodium benzoate, methylparaben, and phenoxyethanol, and more preferably sodium benzoate.

[0041] In this invention, the plant exosome mixture is preferably made from raw materials comprising the following parts by weight: 0.0001-0.2 parts of Polygonum multiflorum exosomes, 0.0001-0.5 parts of Salvia miltiorrhiza exosomes, 0.0001-0.5 parts of ginger exosomes, and 0.0001-0.2 parts of Ginkgo biloba leaf exosomes; more preferably, it is made from raw materials comprising the following parts by weight: 0.0002-0.05 parts of Polygonum multiflorum exosomes, 0.0002-0.02 parts of Salvia miltiorrhiza exosomes, 0.003-0.2 parts of ginger exosomes, and 0.001-0.1 parts of Ginkgo biloba leaf exosomes; and even more preferably, it comprises 0.02 parts of Polygonum multiflorum exosomes, 0.01 parts of Salvia miltiorrhiza exosomes, 0.1 parts of ginger exosomes, and 0.05 parts of Ginkgo biloba leaf exosomes.

[0042] In this invention, the method for preparing the plant exosome mixture preferably includes the following steps:

[0043] (1) Mix Polygonum multiflorum with PBS buffer, break it up, and collect the filtrate;

[0044] (2) Centrifuge the filtrate at 250-350g for 8-12 minutes and collect the supernatant;

[0045] (3) Centrifuge the supernatant obtained in step (2) at 1500-3000g for 8-12 minutes and take the supernatant;

[0046] (4) Centrifuge the supernatant obtained in step (3) at 8000-15000g for 8-12 minutes and take the supernatant;

[0047] (5) Centrifuge the supernatant obtained in step (4) at 80,000 to 120,000 g for 80 to 100 min and collect the precipitate;

[0048] (6) The precipitate is mixed with water to obtain Polygonum multiflorum exosomes;

[0049] (7) Treat the salvia miltiorrhiza, ginger and ginkgo leaves according to steps (1) to (6) to obtain salvia miltiorrhiza exosomes, ginger exosomes and ginkgo leaf exosomes; mix the Polygonum multiflorum exosomes, salvia miltiorrhiza exosomes, ginger exosomes and ginkgo leaf exosomes to obtain the plant exosome mixture.

[0050] In this invention, before mixing, it is preferable to wash and cut the Polygonum multiflorum, Salvia miltiorrhiza, ginger, and Ginkgo biloba leaves into pieces.

[0051] In this invention, the particle size of the cut Polygonum multiflorum, Salvia miltiorrhiza, ginger, and Ginkgo biloba leaves is preferably 1-2 cm, and more preferably 1.5 cm.

[0052] In this invention, the amount of PBS buffer added is preferably (0.5 to 2) times the volume of Polygonum multiflorum, and more preferably 1 times the volume of Polygonum multiflorum.

[0053] In this invention, the amount of PBS buffer added is preferably (0.5 to 2) times the volume of the danshen, and more preferably 1 times the volume of the danshen.

[0054] In this invention, the amount of PBS buffer added is preferably (0.5 to 2) times the volume of ginger, and more preferably 1 times the volume of ginger.

[0055] In this invention, the amount of PBS buffer added is preferably (0.5 to 2) times the volume of the ginkgo leaf, and more preferably 1 times the volume of the ginkgo leaf.

[0056] In this invention, the concentration of the Polygonum multiflorum exosomes is preferably 250-350 mg / mL, and more preferably 300 mg / mL.

[0057] In this invention, the concentration of the tanshinone exosomes is preferably 250-350 mg / mL, and more preferably 300 mg / mL.

[0058] In this invention, the concentration of ginger exosomes is preferably 250-350 mg / mL, and more preferably 300 mg / mL.

[0059] In this invention, the concentration of the Ginkgo biloba exosomes is preferably 250-350 mg / mL, and more preferably 300 mg / mL.

[0060] The present invention also provides a method for preparing the above-mentioned hair-darkening composition, comprising the following steps:

[0061] 1) Divide the water into two parts. Mix the first part of the water with butanediol to obtain pre-prepared solution A;

[0062] 2) Mix the borneol with the second part of water to obtain pre-prepared solution B;

[0063] 3) Take the following extracts: Polygonum multiflorum extract, Ginkgo biloba extract, Salvia miltiorrhiza root extract, Nasturtium extract, ginger extract, Ligustrum lucidum fruit extract, ginseng root extract, black mulberry fruit extract, Platycladus orientalis leaf extract, raspberry extract, black sesame extract, plant exosome mixture, dandelion extract, crocodile polypeptide, and glycerin, and add them to pre-prepared solution A to obtain pre-prepared solution C.

[0064] 4) Mix the pre-prepared solution C with pre-prepared solution B, calcium pantothenate and tyrosine to obtain pre-prepared solution D;

[0065] 5) Add preservative to the pre-prepared liquid D and stir evenly to obtain the hair-darkening composition.

[0066] In this invention, the mass ratio of the first part of water to the second part of water is preferably (80-95):(5-20), and more preferably 95:5.

[0067] In this invention, the mixing temperature in step 2) is preferably 50-65°C, and more preferably 60°C.

[0068] The present invention also provides the application of the above-mentioned hair-darkening composition in the preparation of hair-darkening shampoos, conditioners, hair masks, sprays, and gels.

[0069] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0070] The extracts of Polygonum multiflorum, Ginkgo biloba, Salvia miltiorrhiza, Nasturtium, Zingiber officinale, Ligustrum lucidum, Panax ginseng, Morus alba, Platycladus orientalis, Rubus idaeus, Sesame, and Taraxacum mongolicum used in the following examples were purchased from Guangzhou Meiyi Biotechnology Co., Ltd.; Crocodile polypeptide was purchased from Shenzhen Jufang Cosmetics Co., Ltd.; Calcium pantothenate and tyrosine were purchased from Shenzhen Xingkaiyue Biotechnology Co., Ltd.; Borneol was purchased from Jiangxi Xinsen Natural Plant Oil Co., Ltd.; Butylene glycol was purchased from Guangzhou Meiyi Biotechnology Co., Ltd.; Glycerin was purchased from Wilmar Oils & Fats Technology Co., Ltd.; and Sodium benzoate was purchased from Beijing Sampu Biochemical Technology Co., Ltd.

[0071] Example 1

[0072] This embodiment provides a hair-darkening composition containing plant exosomes, comprising 5% Polygonum multiflorum extract, 0.5% Ginkgo biloba extract, 0.5% Salvia miltiorrhiza root extract, 0.5% Nasturtium extract, 2% ginger extract, 0.5% Ligustrum lucidum fruit extract, 0.5% ginseng root extract, 0.5% black mulberry fruit extract, 1.2% Platycladus orientalis leaf extract, 0.1% raspberry extract, 0.2% black sesame extract, 0.5% dandelion extract, 0.2% plant exosome mixture, 0.2% crocodile polypeptide, 0.5% calcium pantothenate, 1.2% tyrosine, 0.2% borneol, 79.5% purified water, 4% butylene glycol, 2% glycerin, and 0.2% sodium benzoate.

[0073] The specific preparation process is as follows:

[0074] 1) After cleaning the Polygonum multiflorum, Salvia miltiorrhiza, ginger, and Ginkgo biloba leaves, cut them into 2cm pieces, add an equal volume of PBS buffer, break them up, and filter them through a filter gauze and funnel to obtain Polygonum multiflorum filtrate, Salvia miltiorrhiza filtrate, ginger filtrate, and Ginkgo biloba leaf filtrate. Transfer each filtrate to a centrifuge tube, centrifuge at 300g for 10min, collect the supernatant, centrifuge the supernatant at 2000g for 10min, collect the supernatant, continue centrifuging at 10000g for 10min, collect the supernatant, and finally centrifuge at 100000g for 90min, remove the supernatant to obtain Polygonum multiflorum precipitate, Salvia miltiorrhiza precipitate, ginger precipitate, and Ginkgo biloba leaf precipitate. Resuspend each precipitate in pure water to 300mg / mL to obtain Polygonum multiflorum exosomes, Salvia miltiorrhiza exosomes, ginger exosomes, and Ginkgo biloba leaf exosomes. Take 0.03 parts of Polygonum multiflorum exosomes, 0.02 parts of Salvia miltiorrhiza exosomes, 0.2 parts of ginger exosomes, and 0.01 parts of Ginkgo biloba exosomes, mix them, and obtain a plant exosome mixture.

[0075] 2) Divide the water into two parts by mass ratio of 95:5. Mix the first part of the water with butanediol and stir well to obtain pre-prepared liquid A.

[0076] 3) Mix borneol with the second part of water, heat to 65°C, stir until dissolved and transparent, to obtain pre-prepared liquid B.

[0077] 4) Take the following extracts: Polygonum multiflorum extract, Ginkgo biloba extract, Salvia miltiorrhiza root extract, Nasturtium extract, ginger extract, Ligustrum lucidum fruit extract, ginseng root extract, black mulberry fruit extract, Platycladus orientalis leaf extract, raspberry extract, black sesame extract, plant exosome mixture, dandelion extract, crocodile polypeptide, and glycerin. Add them to pre-prepared solution A and stir well to obtain pre-prepared solution C.

[0078] 5) Mix the pre-prepared solution C with pre-prepared solution B, calcium pantothenate and tyrosine, and stir until homogeneous to obtain pre-prepared solution D.

[0079] 6) Add preservative to the pre-prepared liquid D and stir evenly to obtain the hair-darkening composition.

[0080] Example 2

[0081] This embodiment provides a hair-darkening composition containing plant exosomes, comprising 3.5% Polygonum multiflorum extract, 1% Ginkgo biloba leaf extract, 4% Salvia miltiorrhiza root extract, 2.5% Nasturtium extract, 1% ginger extract, 1.25% Ligustrum lucidum fruit extract, 1.25% ginseng root extract, 0.12% black mulberry fruit extract, 1.5% Platycladus orientalis leaf extract, 1% raspberry extract, 0.8% black sesame extract, 0.2% dandelion extract, 0.5% plant exosome mixture, 0.4% crocodile polypeptide, 1% calcium pantothenate, 0.1% tyrosine, 0.2% borneol, 76.48% purified water, 2% butylene glycol, 1% glycerin, and 0.2% sodium benzoate.

[0082] The specific preparation process is as follows:

[0083] 1) After cleaning the Polygonum multiflorum, Salvia miltiorrhiza, ginger, and Ginkgo biloba leaves, cut them into 2cm pieces, add an equal volume of PBS buffer, break them up, and filter them through a filter gauze and funnel to obtain Polygonum multiflorum filtrate, Salvia miltiorrhiza filtrate, ginger filtrate, and Ginkgo biloba leaf filtrate. Transfer each filtrate to a centrifuge tube, centrifuge at 300g for 10min, collect the supernatant, centrifuge the supernatant at 2000g for 10min, collect the supernatant, continue centrifuging at 10000g for 10min, collect the supernatant, and finally centrifuge at 100000g for 90min, remove the supernatant to obtain Polygonum multiflorum precipitate, Salvia miltiorrhiza precipitate, ginger precipitate, and Ginkgo biloba leaf precipitate. Resuspend each precipitate in pure water to 300mg / mL to obtain Polygonum multiflorum exosomes, Salvia miltiorrhiza exosomes, ginger exosomes, and Ginkgo biloba leaf exosomes. Take 0.01 parts of Polygonum multiflorum exosomes, 0.01 parts of Salvia miltiorrhiza exosomes, 0.05 parts of ginger exosomes, and 0.02 parts of Ginkgo biloba exosomes, mix them, and obtain a plant exosome mixture.

[0084] 2) Divide the water into two parts by mass ratio of 90:10. Mix the first part of the water with butanediol and stir well to obtain pre-prepared liquid A.

[0085] 3) Mix borneol with the second part of water, heat to 60°C, stir and dissolve until transparent to obtain pre-prepared solution B.

[0086] 4) Take the following extracts: Polygonum multiflorum extract, Ginkgo biloba extract, Salvia miltiorrhiza root extract, Nasturtium extract, ginger extract, Ligustrum lucidum fruit extract, ginseng root extract, black mulberry fruit extract, Platycladus orientalis leaf extract, raspberry extract, black sesame extract, plant exosome mixture, dandelion extract, crocodile polypeptide, and glycerin. Add them to pre-prepared solution A and stir well to obtain pre-prepared solution C.

[0087] 5) Mix the pre-prepared solution C with pre-prepared solution B, calcium pantothenate and tyrosine, and stir until homogeneous to obtain pre-prepared solution D.

[0088] 6) Add preservative to the pre-prepared liquid D and stir evenly to obtain the hair-darkening composition.

[0089] Example 3

[0090] This embodiment provides a hair-darkening composition containing plant exosomes, comprising 1.5% Polygonum multiflorum extract, 1% Ginkgo biloba leaf extract, 1.2% Salvia miltiorrhiza root extract, 0.5% Nasturtium extract, 3% ginger extract, 0.5% Ligustrum lucidum fruit extract, 0.5% ginseng root extract, 0.2% black mulberry fruit extract, 0.5% Platycladus orientalis leaf extract, 0.5% raspberry extract, 0.5% black sesame extract, 1% dandelion extract, 1% plant exosome mixture, 2% crocodile polypeptide, 1.5% calcium pantothenate, 1.2% tyrosine, 1.5% borneol, 74.7% purified water, 5% butylene glycol, 2% glycerin, and 0.2% sodium benzoate.

[0091] The specific preparation process is as follows:

[0092] 1) After cleaning the Polygonum multiflorum, Salvia miltiorrhiza, ginger, and Ginkgo biloba leaves, cut them into 2cm pieces, add an equal volume of PBS buffer, break them up, and filter them through a filter gauze and funnel to obtain Polygonum multiflorum filtrate, Salvia miltiorrhiza filtrate, ginger filtrate, and Ginkgo biloba leaf filtrate. Transfer each filtrate to a centrifuge tube, centrifuge at 300g for 10min, collect the supernatant, centrifuge the supernatant at 2000g for 10min, collect the supernatant, continue centrifuging at 10000g for 10min, collect the supernatant, and finally centrifuge at 100000g for 90min, remove the supernatant to obtain Polygonum multiflorum precipitate, Salvia miltiorrhiza precipitate, ginger precipitate, and Ginkgo biloba leaf precipitate. Resuspend each precipitate in pure water to 300mg / mL to obtain Polygonum multiflorum exosomes, Salvia miltiorrhiza exosomes, ginger exosomes, and Ginkgo biloba leaf exosomes. Take 0.05 parts of Polygonum multiflorum exosomes, 0.015 parts of Salvia miltiorrhiza exosomes, 0.15 parts of ginger exosomes, and 0.1 parts of Ginkgo biloba leaf exosomes, mix them, and obtain a plant exosome mixture.

[0093] 2) Divide the water into two parts by mass ratio of 93:7. Mix the first part of the water with butanediol and stir well to obtain pre-prepared liquid A.

[0094] 3) Mix borneol with the second part of water, heat to 55°C, stir until dissolved and transparent, to obtain pre-prepared liquid B.

[0095] 4) Take the following extracts: Polygonum multiflorum extract, Ginkgo biloba extract, Salvia miltiorrhiza root extract, Nasturtium extract, ginger extract, Ligustrum lucidum fruit extract, ginseng root extract, black mulberry fruit extract, Platycladus orientalis leaf extract, raspberry extract, black sesame extract, plant exosome mixture, dandelion extract, crocodile polypeptide, and glycerin. Add them to pre-prepared solution A and stir well to obtain pre-prepared solution C.

[0096] 5) Mix the pre-prepared solution C with pre-prepared solution B, calcium pantothenate and tyrosine, and stir until homogeneous to obtain pre-prepared solution D.

[0097] 6) Add preservative to the pre-prepared liquid D and stir evenly to obtain the hair-darkening composition.

[0098] Example 4

[0099] This embodiment provides a hair-darkening composition containing plant exosomes, comprising 3% Polygonum multiflorum extract, 2% Ginkgo biloba leaf extract, 2% Salvia miltiorrhiza root extract, 1.2% Nasturtium extract, 1.2% ginger extract, 1.2% Ligustrum lucidum fruit extract, 1.2% ginseng root extract, 0.2% black mulberry fruit extract, 0.2% Platycladus orientalis leaf extract, 0.2% raspberry extract, 0.15% black sesame extract, 0.1% dandelion extract, 0.7% plant exosome mixture, 0.2% crocodile polypeptide, 0.2% calcium pantothenate, 0.12% tyrosine, 0.12% borneol, 83.11% purified water, 2.5% butylene glycol, 0.2% glycerin, and 0.2% sodium benzoate.

[0100] The specific preparation process is as follows:

[0101] 1) After cleaning the Polygonum multiflorum, Salvia miltiorrhiza, ginger, and Ginkgo biloba leaves, cut them into 2cm pieces, add an equal volume of PBS buffer, break them up, and filter them through a filter gauze and funnel to obtain Polygonum multiflorum filtrate, Salvia miltiorrhiza filtrate, ginger filtrate, and Ginkgo biloba leaf filtrate. Transfer each filtrate to a centrifuge tube, centrifuge at 300g for 10min, collect the supernatant, centrifuge the supernatant at 2000g for 10min, collect the supernatant, continue centrifuging at 10000g for 10min, collect the supernatant, and finally centrifuge at 100000g for 90min, remove the supernatant to obtain Polygonum multiflorum precipitate, Salvia miltiorrhiza precipitate, ginger precipitate, and Ginkgo biloba leaf precipitate. Resuspend each precipitate in pure water to 300mg / mL to obtain Polygonum multiflorum exosomes, Salvia miltiorrhiza exosomes, ginger exosomes, and Ginkgo biloba leaf exosomes. Take 0.02 parts of Polygonum multiflorum exosomes, 0.01 parts of Salvia miltiorrhiza exosomes, 0.1 parts of ginger exosomes, and 0.05 parts of Ginkgo biloba exosomes, mix them, and obtain a plant exosome mixture.

[0102] 2) Divide the water into two parts by mass ratio of 95:5. Mix the first part of the water with butanediol and stir well to obtain pre-prepared liquid A.

[0103] 3) Mix borneol with the second part of water, heat to 60°C, stir and dissolve until transparent to obtain pre-prepared solution B.

[0104] 4) Take the following extracts: Polygonum multiflorum extract, Ginkgo biloba extract, Salvia miltiorrhiza root extract, Nasturtium extract, ginger extract, Ligustrum lucidum fruit extract, ginseng root extract, black mulberry fruit extract, Platycladus orientalis leaf extract, raspberry extract, black sesame extract, plant exosome mixture, dandelion extract, crocodile polypeptide, and glycerin. Add them to pre-prepared solution A and stir well to obtain pre-prepared solution C.

[0105] 5) Mix the pre-prepared solution C with pre-prepared solution B, calcium pantothenate and tyrosine, and stir until homogeneous to obtain pre-prepared solution D.

[0106] 6) Add preservative to the pre-prepared liquid D and stir evenly to obtain the hair-darkening composition.

[0107] Experimental Example 1

[0108] This experiment measured the efficacy of the hair-darkening compositions prepared in Examples 1-4 from three aspects: melanin content in melanoma cells, melanoma cell activity, and tyrosinase activity. The experimental procedure is as follows:

[0109] 1. Melanoma cell culture

[0110] Melanoma cells were seeded in high-glucose DMEM medium (containing 100 U / mL penicillin and 100 μg / mL streptomycin) with 10% FBS and cultured at 37°C in a 5% CO2 incubator for 12 h. When the cells reached near-confluence, they were digested with 0.25% trypsin and 0.02% EDTA, centrifuged, and resuspended in the same medium. The cell density was adjusted to 1 × 10⁶ cells / mL. 5 / mL, seeded into 96-well plates (for cell viability / toxicity assays) and 6-well plates (for melanin content assays), respectively.

[0111] 2. Melanin content detection

[0112] Melanoma cells seeded in 6-well plates were cultured at 37°C and 5% CO2 for 24 hours. The culture medium was then aspirated and replaced with fresh high-glucose DMEM medium (containing 100 U / mL penicillin and 100 μg / mL streptomycin) at 10% FBS, 2000 μL per well. Cells were divided into a blank group and experimental groups, including those from Examples 1, 2, 3, and 4. The blank group received 10 μL of culture medium, while the other experimental groups received 10 μL of the hair-darkening composition prepared according to the corresponding examples. After culturing for 24, 48, and 72 hours, the culture medium was aspirated, the cells were washed with PBS, digested with trypsin, and the digestion was terminated. The digestion solution was collected. The digestion solution from each well was transferred to a 1.5 mL EP tube, centrifuged, and the supernatant was discarded. The absorbance at 490 nm was immediately measured using a microplate reader. The absorbance value represents the melanin content. The results for each group are shown below. Figure 1 As shown.

[0113] from Figure 1 It can be seen that, compared with the blank control, at 48h and 72h, the hair-darkening composition of the present invention can promote melanin synthesis in melanoma cells and increase melanin content. Among them, the amount of melanin synthesized in the cells treated with the hair-darkening composition of Example 4 is higher than that in the other three examples.

[0114] 3. Cell viability detection - toxicity detection

[0115] Melanoma cells seeded in 96-well plates were pre-cultured at 37°C and 5% CO2 for 24 hours. The cells were then divided into a blank control group and experimental groups, including those from Examples 1, 2, 3, and 4. The blank control group received 10 μL of culture medium, while the other experimental groups received 10 μL of the hair-darkening composition prepared according to the corresponding examples. Cells were cultured in each group for 24, 48, and 72 hours. Cells were then collected, and 10 μL of CCK-8 solution was added to each well. The plates were incubated at 37°C for 3 hours, the supernatant was discarded, and the plates were incubated for 4 hours. The absorbance of each well was then measured at 450 nm using a microplate reader. The absorbance value represents cell viability, i.e., cytotoxicity. The results are as follows: Figure 2 As shown.

[0116] from Figure 2 As can be seen, compared with the blank control, the hair-darkening composition of the present invention showed a significant promoting effect on melanoma cell activity at 48h and 72h; wherein, the higher the OD value, the higher the cell activity and the lower the toxicity. The melanoma cells treated with the hair-darkening composition in Example 4 showed higher activity than those in the other three examples.

[0117] 4. Tyrosinase activity detection

[0118] Tyrosinase activity was detected using a tyrosinase ELISA kit (purchased from Hangzhou Xincheng Biotechnology Co., Ltd., batch number: L190200838). Blank wells (blank control wells without sample or enzyme-labeled reagent, all other steps were the same), positive control wells, and wells containing the hair-darkening composition sample to be tested were included.

[0119] Accurately add 50 μL of standard to the positive control well of the enzyme-labeled plate. Add 40 μL of sample diluent to the sample well, followed by 10 μL of the sample to be tested (the hair-darkening compositions prepared in Examples 1-4, with a final dilution of 5-fold). When adding the sample, place it at the bottom of the well, avoiding contact with the well walls, and gently shake to mix. Seal the plate with sealing film and incubate at 37°C for 30 minutes.

[0120] Dilute the 20x concentrated wash buffer 20 times with distilled water and set aside. Carefully remove the sealing film, discard the liquid, and shake dry. Fill each well with the wash buffer, let stand for 30 seconds, then discard. Repeat this process 5 times, and pat dry. Add 50 μL of enzyme-labeled reagent to each well, except for the blank wells. Repeat the previous incubation and washing steps. Add 50 μL of chromogenic reagent A to each well, followed by 50 μL of chromogenic reagent B. Gently vortex to mix, and incubate at 37°C in the dark for 15 min. Add 50 μL of stop solution to each well to stop the reaction (the blue color will immediately turn yellow). Zero the plate with a blank and measure the absorbance (OD value) of each well sequentially at a wavelength of 450 nm. Express the tyrosinase activity using the absorbance value. The detection results are as follows: Figure 3 As shown.

[0121] from Figure 3 As can be seen, compared with the blank control, the hair-darkening composition of the present invention has a promoting effect on tyrosinase activity, and the difference is statistically significant (P < 0.05). Among them, the hair-darkening composition of Example 4 has a higher promoting rate of tyrosinase activity than the other three examples.

[0122] Experimental Example 2

[0123] This experimental example, by setting up a comparative example, verified the effect of plant exosomes on the efficacy of the hair-darkening composition. The specific process is as follows:

[0124] 1. Preparation of comparative hair-darkening compositions

[0125] This comparative example provides a hair-darkening composition free of plant exosomes, comprising 3% Polygonum multiflorum extract, 2% Ginkgo biloba leaf extract, 2% Salvia miltiorrhiza root extract, 1.2% Nasturtium extract, 1.2% Zingiber officinale extract, 1.2% Ligustrum lucidum fruit extract, 1.2% Panax ginseng root extract, 0.2% Mulberry fruit extract, 0.2% Platycladus orientalis leaf extract, 0.2% Raspberry extract, 0.15% Sesame extract, 0.1% Taraxacum mongolicum extract, 0.2% Crocodile polypeptide, 0.2% Calcium pantothenate, 0.12% Tyrosine, 0.12% Borneol, 83.81% purified water, 2.5% Butylene glycol, 0.2% Glycerin, and 0.2% Sodium benzoate.

[0126] The specific preparation process is as follows:

[0127] 1) Divide the water into two parts by mass ratio of 95:5. Mix the first part of the water with butanediol and stir well to obtain pre-prepared liquid A.

[0128] 2) Mix borneol with the second part of water, heat to 60°C, stir and dissolve until transparent to obtain pre-prepared solution B.

[0129] 3) Take the extracts of Polygonum multiflorum, Ginkgo biloba, Salvia miltiorrhiza root, Nasturtium, ginger, Ligustrum lucidum, ginseng root, black mulberry, Platycladus orientalis leaf, raspberry, black sesame, dandelion, crocodile polypeptide, and glycerin, add them to pre-prepared solution A, stir well to obtain pre-prepared solution C.

[0130] 4) Mix the pre-prepared liquid C with pre-prepared liquid B, calcium pantothenate and tyrosine, and stir evenly to obtain pre-prepared liquid D.

[0131] 5) Add preservative to the pre-prepared liquid D and stir evenly to obtain the hair-darkening composition.

[0132] 2. Melanin content detection

[0133] Following the steps of melanoma cell culture and melanin content detection in Example 1, the hair-darkening compositions of Example 4 and the comparative example were used as samples, with a blank group set up. The melanin content of each hair-darkening composition was detected after 24h, 48h, and 72h of treatment. The detection results are as follows: Figure 4 As shown.

[0134] from Figure 4 It can be seen that, by measuring the melanin content of melanoma cells treated in the comparative example and Example 4, at 48h and 72h, the hair-darkening composition with added plant exosomes was more effective in promoting melanin synthesis and increasing melanin content in melanoma cells than the hair-darkening composition without added exosomes.

[0135] Experimental Example 3

[0136] This experiment used the hair-darkening composition prepared in Example 4 as the sample for a human trial. Thirty participants (healthy men or women, aged 18–45 years) were randomly selected. For statistical convenience, all participants had gray hair, with the number of gray hairs ≤1000. Participants used the hair-darkening composition prepared in Example 4 continuously for 20 days, and the effectiveness was calculated as a gray hair-to-blackening rate >70%. The statistical results are shown in Table 1.

[0137] Table 1. Experimental Results

[0138]

[0139] As shown in Table 1, after using the hair-darkening composition of the present invention for a period of time, the gray hair of 28 people was effectively turned black, with a cure rate of 93.3%. The gray hair-turning rate of the other two people was lower, but still reached over 50%. This indicates that the hair-darkening composition of the present invention can effectively reduce the amount of gray hair.

[0140] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A hair-darkening composition containing plant exosomes, characterized in that, It is made from the following raw materials in the indicated weight percentages: Polygonum multiflorum extract 0.001%–10%, Ginkgo biloba leaf extract 0.01%–5%, Salvia miltiorrhiza root extract 0.01%–5%, Nasturtium extract 0.01%–5%, Zingiber officinale extract 0.01%–5%, Ligustrum lucidum fruit extract 0.01%–5%, Panax ginseng root extract 0.001%–5%, Mulberry fruit extract 0.01%–5%, Platycladus orientalis leaf extract 0.01%–3%, Rubus idaeus extract 0.01%–3%, Sesame extract 0.01%–3%, a mixture of plant exosomes 0.0001%–1%, and Taraxacum mongolicum extract. The mixture contains 0.001%–1% of the following ingredients: 0.001%–2% of crocodile polypeptide, 0.001%–5% of calcium pantothenate, 0.001%–1% of tyrosine, 0.0001%–0.5% of borneol, 70%–95% of water, 0.01%–5% of butylene glycol, 0.01%–5% of glycerol, and 0.01%–0.5% of preservatives. The plant exosome mixture is made from the following raw materials in parts by weight: 0.0001–0.2 parts of Polygonum multiflorum exosomes, 0.0001–0.5 parts of Salvia miltiorrhiza exosomes, 0.0001–0.5 parts of ginger exosomes, and 0.0001–0.2 parts of Ginkgo biloba leaf exosomes.

2. The hair-darkening composition according to claim 1, characterized in that, The preservatives include sodium benzoate, methylparaben, and phenoxyethanol.

3. The hair-darkening composition according to claim 2, characterized in that, The method for preparing the plant exosome mixture includes the following steps: (1) Mix Polygonum multiflorum with PBS buffer, break it up, and collect the filtrate; (2) Centrifuge the filtrate at 250-350g for 8-12 minutes and collect the supernatant; (3) Centrifuge the supernatant obtained in step (2) at 1500-3000g for 8-12 minutes and take the supernatant; (4) Centrifuge the supernatant obtained in step (3) at 8000-15000g for 8-12 minutes and take the supernatant; (5) Centrifuge the supernatant obtained in step (4) at 80,000 to 120,000 g for 80 to 100 min and collect the precipitate; (6) The precipitate is mixed with water to obtain Polygonum multiflorum exosomes; (7) Treat the salvia miltiorrhiza, ginger and ginkgo leaves according to steps (1) to (6) to obtain salvia miltiorrhiza exosomes, ginger exosomes and ginkgo leaf exosomes; mix the Polygonum multiflorum exosomes, salvia miltiorrhiza exosomes, ginger exosomes and ginkgo leaf exosomes to obtain the plant exosome mixture.

4. The hair-darkening composition according to claim 3, characterized in that, The amount of PBS buffer added is (0.5 to 2) times the volume of the Polygonum multiflorum.

5. The hair-darkening composition according to claim 4, characterized in that, The concentration of the Polygonum multiflorum exosomes was 250–350 mg / mL.

6. A method for preparing the hair-darkening composition according to any one of claims 1 to 5, characterized in that, Includes the following steps: 1) Divide the water into two parts. Mix the first part of the water with butanediol to obtain pre-prepared solution A; 2) Mix the borneol with the second part of water to obtain pre-prepared solution B; 3) Take the following extracts: Polygonum multiflorum extract, Ginkgo biloba extract, Salvia miltiorrhiza root extract, Nasturtium extract, ginger extract, Ligustrum lucidum fruit extract, ginseng root extract, black mulberry fruit extract, Platycladus orientalis leaf extract, raspberry extract, black sesame extract, plant exosome mixture, dandelion extract, crocodile polypeptide, and glycerin, and add them to pre-prepared solution A to obtain pre-prepared solution C. 4) Mix the pre-prepared solution C with pre-prepared solution B, calcium pantothenate and tyrosine to obtain pre-prepared solution D; 5) Add preservative to the pre-prepared liquid D and stir evenly to obtain the hair-darkening composition.

7. The preparation method according to claim 6, characterized in that, The mass ratio of the first part of water to the second part of water is (80-95):(5-20).

8. The preparation method according to claim 7, characterized in that, The mixing temperature described in step 2) is 50–65°C.

9. The use of a hair-darkening composition according to any one of claims 1 to 5 or a hair-darkening composition prepared by any one of claims 6 to 8 in the preparation of hair-darkening shampoos, conditioners, hair masks, sprays, and gels.

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