A plant anti-drop composition, its preparation method and application

Abstract

The present application relates to the field of cosmetic technology, especially to IPC A61K8, more particularly to a kind of anti-shedding plant composition and its preparation method and application.The present application provides a kind of anti-shedding plant composition, its preparation raw material includes Radix Polygoni Multiflori, Biota orientalis, Radix Astragali, Salvia miltiorrhiza, Rhubarb, Eclipta prostrata L., safflower, Angelica sinensis, Ligusticum sinense, Selaginella tamariscina, kidney tea, gold cinchona tree.The present application provides a kind of anti-shedding plant composition, the effective components in plant are extracted using water extraction alcohol precipitation technology to obtain high concentration anti-shedding plant composition, the composition is safe and non-irritating, and has good anti-shedding effect.

Description

Technical Field

[0001] This invention relates to the field of cosmetic technology, and more particularly to IPC A61K8, and more specifically to an anti-hair loss plant composition, its preparation method, and its application. Background Technology

[0002] Hair, as part of the body structure, plays an important role in daily life, directly protecting the scalp, relieving external forces, reducing damage, and protecting the head. However, due to changes in people's lifestyles, frequent perming and dyeing can lead to seborrheic dermatitis, reduced subcutaneous fat layer, hair follicle atrophy, insufficient microcirculation in hair follicles, and prolonged dormancy phase, ultimately resulting in hair loss.

[0003] With the improvement of people's living standards and quality of life, the demand for scalp care products is increasing, and the requirements for product quality are also becoming higher. Because plant-based ingredients are gentle and offer both efficacy and nutrition, their application in scalp and hair cosmetics has gained widespread recognition in recent years, and research on their application in cosmetics is receiving increasing attention both domestically and internationally. Currently, commonly used plant sources for preventing hair loss include: Polygonum multiflorum, Platycladus orientalis leaves, Ligustrum lucidum, black sesame, ginger, ginseng, Rhodiola rosea, Angelica sinensis, Ligusticum chuanxiong, mulberry leaves, Artemisia capillaris, Astragalus membranaceus, Sophora flavescens, Zanthoxylum bungeanum, Drynaria fortunei, safflower, blister beetle, Clematis chinensis, Eclipta prostrata, Sophora japonica fruit, ginger, Sage, and mulberry leaves, etc.

[0004] Existing patent CN201610804465.1 discloses a hair care and anti-hair loss composition and its application for oily seborrheic alopecia. It uses Polygonum multiflorum, Dictamnus dasycarpus root bark, American ginseng, and black beans for fermentation to obtain a fermented product that can dispel wind, unblock meridians, promote qi and blood circulation, and enhance new hair growth. However, as a fermented liquid, its active ingredients are easily deactivated and have a short storage time.

[0005] Existing patent CN201811199596.7 discloses an oil-controlling and hair-preventing composition, its preparation method, and its application. It uses a combination of ginseng root extract, ginger root extract, forsythia fruit extract, and sophora flavescens extract, which has a good oil-controlling and hair-preventing effect. Although it reduces the irritation to the human body, it still has a certain irritating effect. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides a plant-based composition for preventing hair loss, the raw materials of which include the root of Polygonum multiflorum, the leaf of Platycladus orientalis, Astragalus membranaceus, Salvia miltiorrhiza, Rheum officinale, Eclipta prostrata, the flower of Carthamus tinctorius, the root of Angelica sinensis, the fruit of Ligustrum lucidum, Selaginella tamariscina, Orthosiphonaristatus, and Cinchona calisaya.

[0007] Preferably, the Astragalus includes Astragalus mongholicus or Astragalus capsulatus; more preferably, it is Astragalus capsulatus.

[0008] Preferably, the Astragalus membranaceus is the root of Astragalus membranaceus capsulatum.

[0009] Preferably, the safflower is the flower of the safflower.

[0010] Preferably, the danshen includes danshen root and rhizome.

[0011] Preferably, the rhubarb includes Rheum palmatum and Rheum palmatum for medicinal use; more preferably, it is Rheum palmatum for medicinal use.

[0012] Preferably, the rhubarb includes medicinal rhubarb root and rhizome.

[0013] Preferably, the cinchona tree comprises cinchona bark and cinchona seeds; more preferably, it comprises cinchona bark.

[0014] Preferably, the anti-hair loss plant composition, by weight, comprises 15-25 parts of Polygonum multiflorum root, 15-25 parts of Platycladus orientalis leaf, 10-15 parts of Astragalus membranaceus, 5-15 parts of Salvia miltiorrhiza, 2-10 parts of rhubarb, 2-10 parts of Eclipta prostrata, 2-10 parts of Carthamus tinctorius, 2-10 parts of Angelica sinensis root, 2-10 parts of Ligustrum lucidum fruit, 2-10 parts of Selaginella tamariscina, 2-10 parts of ginseng tea, and 1-5 parts of Cinchona barbata; more preferably, it comprises 20-25 parts of Polygonum multiflorum root, 20-25 parts of Platycladus orientalis leaf, 10-15 parts of Astragalus membranaceus, 10-12 parts of Salvia miltiorrhiza, 3-5 parts of rhubarb, 3-5 parts of Eclipta prostrata, 3-5 parts of Carthamus tinctorius, 3-5 parts of Angelica sinensis root, 3-5 parts of Ligustrum lucidum fruit, 3-5 parts of Selaginella tamariscina, 3-5 parts of ginseng tea, and 1-3 parts of Cinchona barbata.

[0015] In this invention, the stability of the anti-hair loss plant composition was improved by adding 10-15 parts Astragalus membranaceus, 3-5 parts Eclipta prostrata, and 3-5 parts Kidney tea. The inventors hypothesize that Astragalus membranaceus, Eclipta prostrata, and Kidney tea are rich in saponins. The saponins in these three ingredients act as surfactants throughout the extraction process, dissolving some of the fat-soluble substances in the plant extract in the aqueous system and also promoting better dissolution of some alkaloids and organic acids in the water-ethanol system.

[0016] Preferably, the mass ratio of Selaginella tamariscina, ginseng, and cinchona bark is 1:1:(0.7-1.2); more preferably, it is 1:1:1.

[0017] In this invention, by controlling the mass ratio of Selaginella tamariscina, ginseng, and cinchona bark to 1:1:(0.7-1.2), the composition of effective substances in the anti-hair loss plant composition is improved, as is the stability of the composition. The inventors found that when the amount of cinchona bark added to the composition is too small, the solid content of the anti-hair loss plant composition is significantly reduced; conversely, when the amount of cinchona bark added is too large, the solid content does not increase significantly, leading to increased costs. The inventors hypothesize that cinchona bark is rich in alkaloids, which can form salts, amides, ketones, and ethers with phenolic acids and ferulic acids in ginseng and Selaginella tamariscina, dissolving in water and ethanol solutions. This increases the solubility of organic matter in the plant composition in the water and ethanol solutions, thereby improving the composition of active substances and extraction efficiency in the anti-hair loss plant composition. Simultaneously, it reduces the waste of filter residue during the preparation of the anti-hair loss plant composition, lowering costs. In addition, alkaloids and organic acids form salts and amides, and the chemical bonds in these substances are stronger than the hydrogen bonds between organic acids and water, as well as between alkalis and water. This prevents the solubility of organic acids and alkaloids from decreasing under external conditions such as vibration and light, thus preventing the precipitation of these substances and improving the stability of the anti-hair loss composition.

[0018] In this invention, the compound of Polygonum multiflorum, Platycladus orientalis, Astragalus membranaceus, Salvia miltiorrhiza, Eclipta prostrata, Angelica sinensis, and Ligustrum lucidum can improve the microcirculation of blood in the scalp, inhibit hair follicle damage caused by excessive proliferation of scalp flora, and inhibit 5α-reductase. It has a good preventive and therapeutic effect on hair loss and acne caused by high androgen levels, and can also increase melanin secretion. However, when the plant composition obtained after extracting Polygonum multiflorum, Platycladus orientalis, Astragalus membranaceus, Salvia miltiorrhiza, Eclipta prostrata, Angelica sinensis, and Ligustrum lucidum was used as a shampoo, a small number of volunteers reported slight irritation after the shampoo accidentally got into their eyes, and some volunteers who scratched their scalp during shampooing reported a slight stinging sensation on their scalp after washing. However, after adding rhubarb, safflower, Selaginella tamariscina, ginseng, and cinchona bark to the anti-hair loss plant combination and adjusting their mass ratio, almost no volunteers reported any irritation. The applicant hypothesizes that adding only Polygonum multiflorum, Platycladus orientalis, Astragalus membranaceus, Salvia miltiorrhiza, Eclipta prostrata, Angelica sinensis, and Ligustrum lucidum, whose anthraquinone compounds, saponins, and phenolic acids can effectively act on the scalp, but they are also highly irritating. However, adding rhubarb, safflower, Selaginella tamariscina, ginseng, and cinchona bark may cause some of the added substances to combine with the previously added substances, or during the precipitation process, some of the more irritating compounds may be encapsulated and carried out, thus reducing the irritation.

[0019] A second aspect of the present invention provides a method for preparing an anti-hair loss plant composition, comprising the following steps:

[0020] S1: Take each type of plant from the anti-hair loss plant composition, select, wash, dry, pulverize to a suitable particle size, sieve to obtain fine powder, and set aside for use;

[0021] S2: Weigh the above fine powder according to the mass fraction of the plants in the anti-hair loss plant composition, mix them evenly, place them in an extraction tank, add water to soak, heat under reflux, filter to remove the dregs, and set the filtrate aside.

[0022] S3: Take the above-mentioned dregs, place them in an extraction tank, add water, heat and reflux, filter to remove the dregs, and set the filtrate aside;

[0023] S4: Combine the above filtrates, place them in a concentration tank, heat to boiling, concentrate under reduced pressure to 1 times the mass of the fine powder placed in the extraction tank in S2, let stand, cool to room temperature, and weigh.

[0024] S5: Slowly add the above concentrated solution to the ethanol aqueous solution while stirring. After the addition is complete, continue stirring for 5 to 10 minutes until the solution is uniform and let it stand for 6 to 8 hours.

[0025] S6: Filter the above liquid to obtain filtrate and treat the residue appropriately; place the liquid in a concentration tank and concentrate it under reduced pressure to 100 kg to obtain the anti-hair loss plant composition.

[0026] Preferably, the mass ratio of the anti-hair loss plant composition to water in S2 is 1:(6-8).

[0027] Preferably, the soaking time in S2 is 40-100 min; the heating reflux time is 100-150 min.

[0028] Preferably, the mass ratio of the anti-hair loss plant composition in S2 to the water in S3 is 1:(4-6).

[0029] Preferably, the heating reflux time in S3 is 1 to 2 hours.

[0030] Preferably, the mass ratio of the anti-hair loss plant composition in S2 to the ethanol aqueous solution in S5 is 1:(3-5).

[0031] Preferably, the mesh size of the filter screen used in S1 is 60-120 mesh; more preferably, it is 80-100 mesh.

[0032] Preferably, the mesh size of the gauze used for filtration in S2 and S3 is 300 to 500 mesh; more preferably, it is 400 mesh.

[0033] Preferably, the ethanol-water solution in S5 has a mass percentage of 75-90%; more preferably, it has a mass percentage of 80%.

[0034] In this invention, the plant-based anti-hair loss composition is first extracted twice by heating and reflux with water, and then purified with an 80wt% ethanol aqueous solution. This water extraction and alcohol precipitation method increases the concentration of effective substances in the composition, while reducing its viscosity and irritation, thus improving user experience. Furthermore, it also improves the stability of the composition. The inventors hypothesize that using a material-to-liquid ratio of 1:7 and 1:5 for two-stage water extraction extracts a large amount of water-soluble proteins, starches, polysaccharides, alkaloids, organic acids, and other compounds. Further purification with an 80wt% ethanol aqueous solution causes the precipitation of starch, proteins, mucilage, tannins, and other substances in the composition, thereby reducing its viscosity and preventing these substances from contacting the scalp, thus reducing irritation. Simultaneously, the reduction or even complete removal of proteins and starches further prevents them from forming flocculent precipitates that adhere to the scalp and hair, making them difficult to rinse, thus improving the user experience for volunteers. The removal of large amounts of proteins, starches, and other substances makes the anti-hair loss plant composition less prone to forming flocculent precipitates during transportation and shaking, thus forming an emulsified state.

[0035] A third aspect of the present invention provides an application of an anti-hair loss plant composition in the preparation of hair cosmetics; the hair cosmetics include shampoo, conditioner, and serum.

[0036] Beneficial effects

[0037] 1. In this invention, by controlling the mass ratio of Selaginella tamariscina, Leonurus japonicus, and Cinchona barbata to 1:1:(0.7-1.2), the composition of effective substances in the anti-hair loss plant composition is improved, and the stability of the anti-hair loss plant composition is also improved.

[0038] 2. In this invention, the extraction is first performed twice by heating and reflux with water, followed by purification with an 80wt% ethanol aqueous solution. This water extraction and alcohol precipitation method increases the content of effective substances in the anti-hair loss plant composition, while reducing the viscosity of the plant composition, decreasing its irritation, and improving the user experience. Furthermore, it also improves the stability of the plant composition.

[0039] 3. In this invention, the combination of Polygonum multiflorum, Platycladus orientalis, Astragalus membranaceus, Salvia miltiorrhiza, Eclipta prostrata, Angelica sinensis, and Ligustrum lucidum can improve the microcirculation of blood in the scalp, inhibit hair follicle damage caused by excessive proliferation of scalp flora, inhibit 5α-reductase, and have a good preventive and therapeutic effect on hair loss and acne caused by high androgen levels. At the same time, it can increase the secretion of melanin.

[0040] 4. In this invention, the stability of the anti-hair loss plant composition is improved by adding 10-15 parts of Astragalus membranaceus, 3-5 parts of Eclipta prostrata and 3-5 parts of Kidney Tea.

[0041] 5. This invention provides a plant composition for preventing hair loss. The effective components of the plant are extracted using water extraction and alcohol precipitation techniques to obtain a high concentration of the plant composition for preventing hair loss. This composition is safe and non-irritating, and has a good effect on preventing hair loss. Detailed Implementation

[0042] Example 1

[0043] This embodiment provides a plant composition for preventing hair loss, the raw materials of which, by weight, include 21 parts of Polygonum multiflorum root, 21 parts of Platycladus orientalis leaf, 12.4 parts of Astragalus membranaceus root, 10 parts of Salvia miltiorrhiza root and rhizome, 4.8 parts of Rheum officinale root and rhizome, 4.8 parts of Eclipta prostrata whole herb, 4.8 parts of Carthamus tinctorius flower, 4.8 parts of Angelica sinensis root, 4.8 parts of Ligustrum lucidum fruit, 4.8 parts of Selaginella tamariscina whole herb, 4.8 parts of Orthosiphonaristatus, and 2 parts of Cinchona calisaya bark.

[0044] The second aspect of this embodiment provides a method for preparing an anti-hair loss plant composition, comprising the following steps:

[0045] S1: Take each type of plant from the anti-hair loss plant composition, select, wash, dry, and pulverize them to a suitable particle size. Sieve them through a 100-mesh filter to obtain fine powder for later use.

[0046] S2: Weigh the above fine powder according to the mass fraction of the plant in the anti-hair loss plant composition, with a total mass of 100 kg, mix evenly, place in an extraction tank, add 700 kg of water to soak for 60 min, heat under reflux for 120 min, filter with 400 mesh gauze to remove the residue, and set the filtrate aside.

[0047] S3: Take the above-mentioned dregs, place them in an extraction tank, add 500 kg of water, heat under reflux for 1.5 hours, filter with 400 mesh gauze to remove the dregs, and set the filtrate aside.

[0048] S4: Combine the above filtrates, place them in a concentration tank, heat to boiling, concentrate under reduced pressure to 1 times the mass of the fine powder placed in the extraction tank in S2, let stand, cool to 25°C, and weigh.

[0049] S5: Slowly add the above concentrated solution to 400kg of 80wt% ethanol aqueous solution with constant stirring. After the addition is complete, continue stirring for 5 minutes until the solution is uniform and let it stand for 8 hours.

[0050] S6: Filter the above liquid to obtain filtrate and treat the residue appropriately; place the liquid in a concentration tank and concentrate it under reduced pressure to 100 kg to obtain the anti-hair loss plant composition.

[0051] The third aspect of this embodiment provides an application of an anti-hair loss plant composition in the preparation of hair cosmetics; the hair cosmetics include shampoo.

[0052] Example 2

[0053] The specific implementation method of Example 2 is the same as that of Example 1, except that the raw materials for preparing the anti-hair loss plant composition, by weight, include 25 parts of Polygonum multiflorum root, 25 parts of Platycladus orientalis leaf, 15 parts of Astragalus membranaceus root, 12 parts of Salvia miltiorrhiza root and rhizome, 5 parts of Rheum officinale root and rhizome, 5 parts of Eclipta prostrata whole herb, 5 parts of Carthamus tinctorius flower, 5 parts of Angelica sinensis root, 5 parts of Ligustrum lucidum fruit, 5 parts of Selaginella tamariscina whole herb, 5 parts of Orthosiphon aristatus, and 3 parts of Cinchona calisaya bark.

[0054] Example 3

[0055] The specific implementation method of Example 3 is the same as that of Example 1, except that the raw materials for preparing the anti-hair loss plant composition, by weight, include 20 parts of Polygonum multiflorum root, 20 parts of Platycladus orientalis leaf, 10 parts of Astragalus membranaceus root, 10 parts of Salvia miltiorrhiza root and rhizome, 3 parts of Rheum officinale root and rhizome, 3 parts of Eclipta prostrata whole herb, 3 parts of Carthamus tinctorius flower, 3 parts of Angelica sinensis root, 3 parts of Ligustrum lucidum fruit, 3 parts of Selaginella tamariscina whole herb, 3 parts of Orthosiphon aristatus, and 1 part of Cinchona calisaya bark.

[0056] Comparative Example 1

[0057] The specific implementation method of Comparative Example 1 is the same as that of the Example, except that the preparation method of the anti-hair loss plant composition includes the following steps:

[0058] S1: Take each type of plant from the anti-hair loss plant composition, select, wash, dry, and crush them to a suitable particle size in advance, and sieve them through a 100-mesh filter for later use.

[0059] S2: Weigh the above fine powder according to the mass fraction of the plants in the anti-hair loss plant composition, with a total mass of 100 kg, mix evenly; place in an extraction tank, add 250 kg of 40% ethanol aqueous solution, soak at room temperature for 48 hours, filter with 400 mesh gauze to obtain filtrate, and set aside separately;

[0060] S3: Take the filter residue, add 200 kg of 40% ethanol aqueous solution, soak for 48 hours, filter with 400 mesh gauze, and set aside the filtrate.

[0061] S4: Take the filter residue, add 150 kg of 40% ethanol aqueous solution, soak for 48 hours, filter with 400 mesh gauze, and set aside the filtrate.

[0062] S5: Combine the above filtrates, weigh them, and make up the mass to 600 kg with 40 wt% ethanol aqueous solution. Stir well, let stand for 48 hours to obtain the intermediate, which is ready for use.

[0063] S6: Place the liquid in a concentration tank and concentrate it under reduced pressure to 100kg to obtain the anti-hair loss plant composition.

[0064] Comparative Example 2

[0065] The specific implementation method of Comparative Example 2 is the same as that of Example 1, except that the mass fraction of Selaginella tamariscina in the anti-hair loss plant composition is 1.2 parts, the mass fraction of Kidney Tea is 1.2 parts, and the mass fraction of Cinchona barbata is 0.6 parts.

[0066] Comparative Example 3

[0067] The specific implementation method of Comparative Example 3 is the same as that of Example 1, except that 400 kg of 70 wt% ethanol aqueous solution was added in step S5.

[0068] Comparative Example 4

[0069] The specific implementation method of Comparative Example 4 is the same as that of Example 1, except that the raw materials for preparing the anti-hair loss plant composition, by weight, include 21 parts of Polygonum multiflorum root, 21 parts of Platycladus orientalis leaf, 12.4 parts of Astragalus membranaceus root, 10 parts of Salvia miltiorrhiza root and rhizome, 4.8 parts of Eclipta prostrata whole herb, 4.8 parts of Angelica sinensis root, and 4.8 parts of Ligustrum lucidum fruit.

[0070] Comparative Example 5

[0071] The specific implementation of Comparative Example 5 is the same as that of Example 1, except that the Astragalus membranaceus root in the anti-hair loss plant composition is 8 parts by weight.

[0072] The whole herb of Eclipta prostrata in the anti-hair loss plant composition is 2 parts by weight;

[0073] The kidney tea (Orthosiphon aristatus) in the anti-hair loss plant composition comprises 2 parts by weight.

[0074] Performance testing

[0075] 1. Take 2g of the anti-hair loss plant composition from Examples 1-3 and Comparative Examples 1 and 3, place it in an evaporating dish that has been dried to constant weight, weigh it accurately, evaporate it to dryness in a water bath, dry it at 105℃ for 3 hours, transfer it to a desiccator, cool it for 30 minutes, and quickly weigh it to obtain the mass of the solids. The results are shown in Table 1.

[0076] 2. Record the preparation time of the anti-hair loss plant composition in Example 1 and Comparative Example 1, as shown in Table 1.

[0077] 3. The anti-hair loss plant compositions obtained in Examples 1-3 and Comparative Examples 1 and 3 were added to the base formula of the shampoo at a rate of 1 wt% to prepare the shampoo of Example 1 and Comparative Example 1. Twelve female volunteers aged 25-35 with medium-length hair were selected and divided into four groups of three. A half-head experiment was conducted, with the left side using the shampoo of Example 1 and the right side using the shampoo of Example 2 (or Example 3, or Comparative Example 1, or Comparative Example 3). "+" indicates volunteer satisfaction, "++++" indicates high volunteer satisfaction, "++" or "+++" indicates moderate volunteer satisfaction, and "+" indicates low volunteer satisfaction. Volunteer evaluation forms were compiled, and the evaluations with the highest percentages were recorded in Table 2.

[0078] The basic formula is as follows: 13% ammonium lauryl ether sulfate, 7% sodium lauryl sulfate, 5% coconut oil diethanolamide, 7% cocamidopropyl betaine, 0.5% guar hydroxypropyltrimethylammonium chloride, 0.1% disodium EDTA, 2.0% polyquaternium-39, 0.5% pyridone ethanolamine salt, 3.0% panthenol, 0.5% aloe vera concentrate, 0.1% butylparaben, 0.2% Kathon, and the balance is water.

[0079] Table 1

[0080]

[0081] Table 2

[0082] User Reviews Example 1 ++++, no noticeable stickiness Example 2 ++++, no noticeable stickiness Example 3 ++++, no noticeable stickiness Comparative Example 1 +, My scalp feels sticky and needs to be rinsed with plenty of water. Comparative Example 3 ++, the scalp feels sticky and needs to be rinsed with plenty of water.

[0083] 4. Stability Test

[0084] 10 mL of the anti-hair loss plant composition prepared in Examples 1-3 and Comparative Examples 1-5 was placed in a 20 mL beaker, a stir bar was added, and the mixture was stirred at 1000 rpm for 30 min. After standing for 20 min, the mixture was observed, and the results were recorded in Table 3.

[0085] Table 4

[0086]

[0087]

[0088] 5. Stimulation test

[0089] 1) Acute eye irritation test

[0090] Sample Information

[0091] Test substance: The anti-hair loss plant composition prepared in Example 1; Appearance: Brown liquid; Preparation method: Dilute the concentration to one-twentieth of the original with deionized water and use directly.

[0092] Experimental animals and rearing environment

[0093] Experimental animals: New Zealand white rabbits; Grade: ordinary grade; Quantity: 3; Sex: male; Body weight: 2.0 kg - 3.0 kg;

[0094] Animal source: Chendun Experimental Animal Breeding Farm Co., Ltd., Songjiang District, Shanghai. Experimental animal production license number: SCXK(Shanghai)2022 - 0001, Quality certificate number: 20220001000088.

[0095] Rearing environment: Ordinary - grade animal room, Room number: Rabbit Room 321. The temperature in the rearing room is 16°C - 26°C, and the relative humidity is 40% - 70%.

[0096] Experimental animal use license number: SYX(Shanghai)2021 - 0023.

[0097] Feed source: Jiangsu Xietong Pharmaceutical Biotechnology Co., Ltd., Production license number: Su Feed License(2019)01008.

[0098] Instruments and equipment

[0099] Electronic counting scale model: ASC - 30, serial number: WPE - TL0055.

[0100] Test methods

[0101] Before the test, the animals were acclimated in the experimental animal room environment for 3 days.

[0102] Within 24 hours before the start of the test, the eyes of the animals were examined. Animals with eye irritation symptoms, corneal defects, and conjunctival injuries could not be used for the test. The animals were weighed before the test.

[0103] Gently pull down the lower eyelid of the left eye of the New Zealand white rabbit, and drop 0.1 mL of the test substance into the conjunctival sac, then make the upper and lower eyelids close passively for 1 s to prevent the loss of the test substance. The right eye was not treated as a self - control.

[0104] Do not rinse the eyes within 24 hours after dropping the test substance.

[0105] Clinical examination: The eyes of the New Zealand white rabbits were examined at 1 h, 24 h, 48 h, and 72 h after eye - dropping respectively. If no irritation reaction occurred within 72 h, the test was terminated. After the observation and recording at 24 h, the eyes of all animals were further examined with 2% sodium fluorescein solution.

[0106] Judgment criteria and scoring: According to the scoring criteria for eye damage in Table 1 of the relevant regulations of the "Technical Specifications for Cosmetics Safety" (2015 Edition), Chapter 6, 5 Acute Eye Irritation / Corrosion Test, the eye irritation response is scored. The evaluation is based on the highest integral mean of the irritation responses of the animal's cornea, iris or conjunctiva at the 24h, 48h and 72h observation time points after the test substance is administered and the recovery time. The irritation intensity of the test substance to the eye is determined according to the classification of the eye irritation response of the raw material.

[0107] Test results

[0108] After the New Zealand white rabbits were exposed to the test substance in the eyes, no other toxicities were observed.

[0109] The results of the acute eye irritation test of the test substance on New Zealand white rabbits are shown in Table 4 specifically.

[0110] The acute eye irritation of this sample to New Zealand white rabbits: Under the condition of not rinsing, it is non-irritating.

[0111] Table 4

[0112]

[0113] 2) Repeated skin irritation test

[0114] Sample information

[0115] Test substance: The anti-hair loss plant composition prepared in Example 1; Appearance: Brown liquid; Preparation method: Dilute the concentration to one-twentieth of the original with deionized water and use directly.

[0116] Experimental animals and breeding environment

[0117] Experimental animals: New Zealand white rabbits; Grade: Ordinary grade; Quantity: 4; Gender: Half male and half female; Body weight: 2.0 kg - 3.0 kg;

[0118] Animal source: Shanghai Songjiang Chendun Experimental Animal Breeding Farm Co., Ltd., Experimental Animal Production License Number: SCXK (Shanghai) 2022 - 0001, Quality Certificate Number: 20220001000088.

[0119] Breeding environment: Ordinary-grade animal house, Room number: Rabbit House 322, Breeding room temperature 16°C - 26°C, Relative humidity 40% - 70%,

[0120] Experimental Animal Use License Number: SYX (Shanghai) 2021 - 0023.

[0121] Feed source: Jiangsu Xietong Pharmaceutical Biotechnology Co., Ltd., Production License Number: Su Feed License (2019) 01008.

[0122] Instrument and equipment

[0123] Electronic counting scale model: ASC-30, serial number: WPE-TL0055.

[0124] Test methods

[0125] Animals were allowed 4 days to acclimatize to the experimental animal facility environment before the experiment to ensure that the animals entering the group were healthy and had no broken skin.

[0126] About 24 hours before the experiment, the hair on both sides of the spine on the back of the experimental animal was shaved off. The hair removal area was about 3cm × 3cm on the left and right sides, without damaging the skin, and the area covered by the shaving was 2.5cm × 2.5cm.

[0127] The following day, after weighing, 0.5 mL of the test substance was applied to the left side of the skin. The right side served as a blank control. The substance was applied once daily for 14 consecutive days.

[0128] Clinical observation: Starting from the second day, residual test substance was removed with warm water and the results were observed 1 hour later.

[0129] Judgment criteria and scoring: The results were evaluated according to the results of the skin irritation test 5.4.3 in Chapter 6, Section 4, Skin Irritation I Corrosion Test of the Cosmetic Safety Technical Specifications (2015 Edition). Multiple skin irritation reaction scores were conducted. Erythema and edema of the samples and controls were observed and scored. The average score of each animal per day was calculated to determine the intensity of skin irritation.

[0130] Test results

[0131] The sample showed no skin irritation on New Zealand white rabbits after repeated exposures. The results are shown in Table 5.

[0132] Table 5

[0133]

[0134] Note: Average score per animal per day (skin irritation index) = Total score of erythema and edema per animal over 14 days / (Number of test animals × Number of days of skin irritation in multiple skin tests)

[0135] 3) Skin allergy test

[0136] Sample Information

[0137] Test substance: The anti-hair loss plant composition prepared in Example 1; Appearance: Brown liquid; Preparation method: ① Induction concentration: Dilute the concentration to one-twentieth of the original concentration with deionized water and use directly; ② Activation concentration: Dilute the concentration to one-twentieth of the original concentration with deionized water and use directly.

[0138] Positive reagent: 2,4-dinitrochlorobenzene; Batch number: WT6CA-TL; Manufacturer: TCI; Solvent: Acetone;

[0139] Induction concentration: 0.8%, excitation concentration: 0.2%; dosage: both are 0.2 mL / animal.

[0140] Experimental animals and breeding environment

[0141] Experimental animals: albino guinea pigs; grade: ordinary grade; quantity: 20 in the test substance group and 10 in the negative control group; body weight: 200 g - 300 g.

[0142] Source of animals: Shanghai Chedun Experimental Animal良种 Farm Co., Ltd., production license number of experimental animals: SCXK(Shanghai)2022 - 0001, quality certificate number: 20220001000156.

[0143] Breeding environment: ordinary - grade animal house, room number: Guinea Pig Room 325, temperature in the breeding room: 18°C - 29°C, relative humidity: 40% - 70%, experimental animal use license number: SYXK(Shanghai)2021 - 0023.

[0144] Source of feed: Jiangsu Xietong Pharmaceutical and Biological Engineering Co., Ltd., production license number: Su Feed License(2019)01008.

[0145] Instrument and equipment

[0146] Model of electronic counting scale:: ASC - 30, serial number: WPE - TL0055

[0147] Test method

[0148] Before the test, the animals were acclimated in the experimental animal house environment for 5 days to ensure that the enrolled animals were healthy and had no skin damage.

[0149] Approximately 24 hours before the test, the hair on the left side of the back of albino guinea pigs was removed, and the hair - removal area was 4 cm 2 .

[0150] Induction contact: Take 0.2 mL of the test substance at the induction concentration and directly apply it to the 2 cm×2 cm hair - removed skin on the left side of the experimental animal, cover it with two layers of gauze and one layer of cellophane, and then seal and fix it with non - irritating adhesive tape for 6 hours. Repeat once on the 7th day and the 14th day in the same way.

[0151] 24 hours before the excitation contact, the hair on the right side of the back of albino guinea pigs was removed, and the hair - removal area was 4 cm 2

[0152] Excitation contact: 14 days after the last induction, take 0.2 mL of the test substance at the excitation concentration and directly apply it to the 2 cm×2 cm hair - removed skin on the right side of the experimental animal; then cover it with two layers of gauze and one layer of cellophane, and fix it with non - irritating adhesive tape for 6 hours. Observe the skin reaction 24 hours and 48 hours after the excitation contact.

[0153] Negative control group: Distilled water was used as a control during induction contact, and the test substance was applied during stimulation contact. The procedure for the negative control group was the same as that for the experimental group.

[0154] Positive control group: A 0.8% solution of 2,4-dinitrochlorophenylacetone was applied during induction contact, and a 0.2% solution of 2,4-dinitrochlorophenylacetone was applied during activation contact. The procedure for the positive control group was the same as that for the experimental group.

[0155] Clinical observation: Skin reactions were observed 24 hours and 48 hours after stimulation.

[0156] Judgment criteria and scoring: According to Chapter 6, Section 6, Skin Allergy Test, of the "Cosmetic Safety Technical Specifications" (2015 Edition).

[0157] The Buehler Test (BT) is used to evaluate skin allergic reactions. A reaction score ≥2 in the test substance group is considered a positive skin allergic reaction in the animal. The sensitization intensity is then graded according to a sensitization intensity table. A sensitization rate of 0% is considered as no skin allergic reaction.

[0158] Test results

[0159] The results of the skin allergy test on the sample in albino guinea pigs were as follows: no skin allergy was observed. The results are shown in Table 6.

[0160] Table 6

[0161]

[0162] Note: ① Starting weight and ending weight are expressed as mean ± SD.

[0163] 4) Repeated skin irritation tests

[0164] Sample Information

[0165] Test substance: The anti-hair loss plant composition prepared in Example 1; Appearance: Brown liquid; Preparation method: Dilute the concentration to one-twentieth of the original with deionized water and use directly.

[0166] Positive reagent: 8-methoxypsoralen; Batch number: 20210727; Manufacturer: Wokai; Solvent: anhydrous ethanol; Concentration used: 0.1%: Take 0.01g of sample and add anhydrous ethanol to make up to 10mL.

[0167] Laboratory animals and their living environment

[0168] Laboratory animals: Adult albino guinea pigs; Grade: Common; Quantity: 6; Sex: Half male and half female.

[0169] Animal source: Shanghai Songjiang Chendun Laboratory Animal Breeding Farm Co., Ltd., License number for experimental animal production: SCXK(Shanghai)2022-0001, Quality certificate number: 20220001000160.

[0170] Feeding environment: Ordinary-grade animal house, Room number: Guinea pig room 326, Temperature in the feeding room: 18°C - 29°C, Relative humidity: 40% - 70%, License number for using experimental animals: SYXK(Shanghai)2021-0023.

[0171] Feed source: Jiangsu Xietong Pharmaceutical Biotechnology Co., Ltd., Production license number: Su Feed License(2019)01008, Drinking water is not restricted.

[0172] Litter source: Jiangsu Xietong Pharmaceutical Biotechnology Co., Ltd.

[0173] Instruments and equipment

[0174] Electronic counting scale, Model: ASC-30, Serial number: WPE-TL0055

[0175] Light source

[0176] Ultraviolet light therapy instrument, Model: KN-4002A, Serial number: WPE-TL0080,

[0177] Manufacturer: Xuzhou Kenuo Medical Instrument and Equipment Co., Ltd.

[0178] Lamp tube model: TL-K 40W / 10R, Manufacturer: Philips. Irradiation meter main unit model: LS125, Serial number: WPE-TL0057.

[0179] UVA irradiation meter probe model: UVA-X1, Serial number: WPE-TL0059.

[0180] UVB irradiation meter probe model: UVB-X0, Serial number: WPE-TL0058.

[0181] Test method

[0182] Average light intensity and irradiation time: Use the irradiation meter to measure the light intensity at 6 points in the irradiation area on the back of the experimental animals. The average UVA(400nm) irradiation intensity is 6.73 mw / cm 2 and the average UVB irradiation intensity is 0.027 mw / cm 2

[0183] The UVB intensity < 1% of the UVA intensity, The UVA irradiation dose is 10 J / cm 2 and the irradiation time is 24 minutes and 48 seconds.

[0184] Test steps

[0185] Animals were allowed 4 days to acclimatize to the experimental animal facility environment before the experiment.

[0186] 24 hours before the formal experiment, remove the hair from the skin on both sides of the animal's spine. The skin at the test site must be intact, without damage or abnormalities. Prepare four hair-removed areas, each 2cm × 2cm. The left side is designated as areas 1 and 3, and the right side as areas 2 and 4.

[0187] The following day, after weighing, the animal was fixed in place, and 0.2 mL of the test substance was evenly applied to areas 1 and 2, while areas 3 and 4 were left untreated. After 30 minutes, areas 1 and 3 on the left side were covered with aluminum foil and secured with tape, while the right side was irradiated with UVA.

[0188] Clinical observation: Skin reactions were observed at 1h, 24h, 48h and 72h after irradiation, and the scores of each animal at different observation periods were recorded according to the skin irritation response scoring criteria.

[0189] Judgment criteria and scoring: Skin reaction scoring for skin phototoxicity testing shall be conducted in accordance with the relevant provisions of Chapter 6, Section 7 of the "Cosmetic Safety Technical Specifications" (2015 edition). If no skin reaction is observed in areas untouched by irradiation after applying the test substance, but one or more animals show skin reaction scores of 2 or higher in areas irradiated after applying the test substance, the test substance shall be judged to be phototoxic.

[0190] Test results

[0191] The results of the phototoxicity test on guinea pig skin for this sample were as follows: no skin phototoxicity was observed. The results of the phototoxicity test on guinea pig skin for the positive control are shown in Table 7; the results of the phototoxicity test on guinea pig skin for the test substance are shown in Table 8.

[0192] Table 7

[0193]

[0194] Note: In the table header, 1, 2, 3, and 4 represent different experimental treatments in four hair removal areas of the same animal; 1 is smeared with positive agent and not irradiated; 2 is smeared with positive agent and irradiated; 3 is not smeared with positive agent and not irradiated; 4 is not smeared with positive agent and irradiated.

[0195] Positive control group trial dates: August 26, 2022 to August 29, 2022

[0196] Table 8

[0197]

[0198] Note: In the table header, 1, 2, 3, and 4 represent different test treatments for four hair removal areas of the same animal; 1 means the test substance is applied but not irradiated; 2 means the test substance is applied and irradiated; 3 means the test substance is not applied and not irradiated; 4 means the test substance is not applied but irradiated.

[0199] Test dates for the test substance group: October 13, 2022 to October 16, 2022

[0200] 5) Bacterial reverse mutation sample

[0201] Experimental strains: TA97a, TA98, TA100, TA1535, WP2uvrA (pKM101)

[0202] Metabolite activation system: Liver microsomal enzyme mixture - S9 mixture

[0203] Positive samples: see Table 9

[0204] Table 9

[0205]

[0206] Note: All three strains mentioned above are derived from the Ames Genetic Toxicity Kit, supplied by Beijing Huizhi Taikang, kit batch number 22J001. Prior to the experiment, all five strains were identified by our laboratory, and their biological characteristics met the experimental requirements.

[0207] Test substance:

[0208] State of matter: semi-solid;

[0209] Solvent: Sterile water

[0210] Solubility: greater than 50 mg / mL;

[0211] Pretreatment (sterilization) method: membrane filtration; the plant anti-hair loss composition prepared in Example 1 was diluted to one-twentieth of its original concentration with deionized water, 2.5g was dissolved in 50mL of solvent, mixed well, and shaken at room temperature for 5min for detection.

[0212] Dosage design:

[0213] Based on the highest dose design principle considering the toxicity and solubility of the test substance, the highest dose determined in the preliminary experiment is:

[0214] -S9: 5000 μg / plate; +S9: 5000 μg / plate. Results are shown in Table 10.

[0215] Table 10

[0216]

[0217] The formal test doses were: -S9: 5000 μg / plate, 2500 μg / plate, 1250 μg / plate, 625 μg / plate;

[0218] +S9: 5000μg / dish, 2500μg / dish, 1250μg / dish, 625μg / dish.

[0219] Test methods

[0220] Add 0.1 mL of the test substance and 0.1 mL of bacterial enrichment solution (add 0.5 mL of 10% S9 mixture if activation is required) to 2 mL of top-layer culture medium kept at 45℃ in a water bath. Mix thoroughly and quickly pour into the bottom plate. Make three parallel plates for each dose. Place horizontally and wait for the culture medium to solidify before inverting in a 37℃ incubator and incubate for 48 h. Count the number of revertant colonies.

[0221] In addition to setting up each dose group of the test substance, blank control, solvent control, positive control and sterile control should also be set up in the experiment.

[0222] Criteria for interpreting positive results

[0223] The number of revertant colonies of test compound TA1535 is three times or more than three times the number of revertant colonies of the solvent control; the number of revertant colonies of test compounds TA97a, TA98, TA100, and WP2uvrA (pKM101) is two times or more than two times the number of revertant colonies of the solvent control, and the following conditions are observed:

[0224] (1) A dose-response relationship

[0225] (2) A positive reaction occurred and was repeatable under any dosage condition.

[0226] Test results

[0227] The sample was tested by five different bacterial strains, and all results were negative regardless of whether S9 was added or not, indicating that it was non-mutagenic. The results are shown in Table 11.

[0228] Table 11

[0229]

[0230] 6) Chromosomal aberrations in in vitro mammalian cells

[0231] Cell lines, metabolite post-processing system (liver microsomal enzyme mixture-S9 mixture), and positive samples were all derived from an in vitro chromosome aberration kit, supplied by Beijing Huizhi Taikang, kit batch number 20220505.

[0232] Test substance:

[0233] State of matter: semi-solid

[0234] Solvent: Sterile water

[0235] Solubility: greater than 50 mg / mL

[0236] Pretreatment and preparation method: direct sampling

[0237] Dosage design:

[0238] Based on the highest dose design principle considering the toxicity and solubility of the test substance, the highest dose determined in the preliminary experiment is:

[0239] -S9: 5000μg / mL; +S9: 5000μg / mL.

[0240] The formal test doses were: -S9: 5000 μg / mL, 2500 μg / mL, 1250 μg / mL;

[0241] +S9: 5000μg / mL, 2500μg / mL, 1250μg / mL.

[0242] Test method:

[0243] Preliminary experiment: Take 2.5g of test sample, add 5mL of water, mix with a shaker for 5min, observe its dissolution. If precipitation occurs, add more serum-free culture medium until the sample is completely dissolved, and record the solubility. Take logarithmic growth phase, well-adhered 96-well plate cells, add the test substance at a maximum concentration of 5000μg / mL, and perform 3-fold serial dilutions. Perform 4 groups, incubate for 24 days, and observe the cell coverage. Select the test substance concentration with a cell coverage greater than 50% as the highest test concentration, and perform 4-fold serial dilutions for the formal experiment.

[0244] Formal experiment: Remove the cells to be used from the incubator, aspirate the culture medium from the cell culture plate, add 3 groups of test substances and S9 mixture (10%) and complete culture medium without fetal bovine serum, and incubate in a carbon dioxide incubator for 24 hours.

[0245] Remove the cells after the treatment with the drug, aspirate the complete culture medium, and wash the cells three times with Hanks' solution. Add the complete culture medium and incubate in a CO2 incubator. After 20 hours of culture, add colchicine solution (final concentration 1 μg / mL) to block the cells from metaphase of mitosis. Continue culturing for another 4 hours before harvesting the cells.

[0246] Cell harvesting: Remove the cells to be harvested, aspirate the cell culture medium, add 2 mL of trypsin-EDTA solution to digest the cells, and wait for the cells to detach. Add culture medium and mix well to stop the trypsin action. Transfer to a 10 mL centrifuge tube, centrifuge (1000 r / min, 5 min), and discard the supernatant.

[0247] Hypotonic treatment: Add 2 mL of 0.075 mol / L potassium chloride solution, gently mix the cells with a dropper, and place in a 37°C cell culture incubator for 30 min of hypotonic treatment.

[0248] Fixation: Remove the hypotonic cells, add 5 mL of methanol / glacial acetic acid solution, fix for at least 5 min, centrifuge at 1000 r / min for 5 min, discard the supernatant (keep 500 μL, first blow the bottom cells evenly), add 5 mL of fixative, centrifuge at 1200 rpm for 10 min, repeat once, discard the supernatant (keep 500 μL, first blow the bottom cells evenly), add 500 μL of fixative.

[0249] Preparing the slide: Take a few drops of fresh fixative and mix well. Place the suspension onto a glass slide that has been pre-soaked in ice water and allow it to air dry.

[0250] Staining: Stain with Mussa's solution for 20 minutes, then remove and rinse off the stain with running water.

[0251] Observation: 100 well-dispersed metaphase cells from each group were selected for chromosome aberration analysis. For cosmetic raw materials, 100 cells were selected from each treatment group. Abnormalities in chromosome structure and number were observed and recorded.

[0252] Data processing and result evaluation:

[0253] Data processing

[0254] Data are listed by dosage, and indicators include the number of observed cells, the number of mutated cells, the chromosomal aberration rate, and the number and rate of different types of chromosomal aberrations in each dosage group and control group. Clefts should be recorded and reported separately, but are generally not included in the total aberration rate. The chromosomal aberration rate for each group is expressed as X. 2 The test results were statistically analyzed.

[0255] Results Evaluation

[0256] The following two conditions can be used to determine that the test substance is a positive result in this testing system:

[0257] a) The increase in the number of chromosomal structural aberrations induced by the test substance was statistically significant and dose-dependent;

[0258] b) The increase in the number of chromosomal structural aberrations caused by the test substance at any dosage condition is statistically significant and reproducible.

[0259] Test results

[0260] 1. Determination of the maximum dose of the test substance and test results: dissolution and cell viability, the results are shown in Table 12.

[0261] 2. Chromosomal aberration rates and statistical results for each treatment group and control group are shown in Table 13.

[0262] The sample tested negative regardless of whether S9 was added or not, and was therefore determined to be non-mutagenic.

[0263] Table 12

[0264]

[0265] Table 13

[0266]

[0267] Note 1. The positive control substance with added activation system is cyclophosphamide; the positive control substance without added activation system is mitomycin C.

[0268] 2. Use X 2 Statistical analysis was performed, and compared with the negative control group, *P<0.05.

Claims

1. A plant-based composition for preventing hair loss, characterized in that, The raw materials for its preparation, by weight, include 15-25 parts of Polygonum multiflorum root, 15-25 parts of Platycladus orientalis leaves, 10-15 parts of Astragalus membranaceus root, 5-15 parts of Salvia miltiorrhiza root and stem, 2-10 parts of Rheum palmatum root and stem, 2-10 parts of Eclipta prostrata whole herb, 2-10 parts of Carthamus tinctorius flower, 2-10 parts of Angelica sinensis root, 2-10 parts of Ligustrum lucidum fruit, 2-10 parts of Selaginella tamariscina whole herb, 2-10 parts of Celosia argentea, and 1-5 parts of Cinchona bark; the weight ratio of Selaginella tamariscina, Celosia argentea, and Cinchona bark is 1:1:(0.7-1.2). The preparation method of the anti-hair loss plant composition includes the following steps: S1: Take various plants, select, wash, dry, and then grind them into a fine powder of 60-120 mesh for later use; S2: Weigh the above fine powder according to the mass fraction, mix evenly and place in an extraction tank, add 6 to 8 times the mass of the fine powder in water, soak for 40 to 100 minutes, heat under reflux for 100 to 150 minutes, filter with 300 to 500 mesh gauze, and set the filtrate aside. S3: Take the filter residue after step S2, add 4 to 6 times the weight of the fine powder in water, heat under reflux for 1 to 2 hours, filter with 300 to 500 mesh gauze, and set the filtrate aside. S4: Combine the above filtrates, concentrate under reduced pressure to 1 times the mass of the fine powder, and let stand and cool to room temperature; S5: Slowly add the concentrate to an 80%~90% ethanol aqueous solution, with a mass ratio of concentrate to ethanol aqueous solution of 1:(3~5), stir for 5~10 minutes and then let stand for 6~8 hours; S6: Filter the static system of step S5, take the filtrate and concentrate it under reduced pressure to 100kg to obtain the anti-hair loss plant composition.

2. The anti-hair loss plant composition according to claim 1, characterized in that, By weight, it includes 20-25 parts of Polygonum multiflorum root, 20-25 parts of Platycladus orientalis leaves, 10-15 parts of Astragalus membranaceus root, 10-12 parts of Salvia miltiorrhiza root and stem, 3-5 parts of Rheum palmatum root and stem, 3-5 parts of Eclipta prostrata whole herb, 3-5 parts of Carthamus tinctorius flower, 3-5 parts of Angelica sinensis root, 3-5 parts of Ligustrum lucidum fruit, 3-5 parts of Selaginella tamariscina whole herb, 3-5 parts of Gynostemma pentaphyllum, and 1-3 parts of Cinchona bark.

3. The preparation method according to claim 1, characterized in that, The mesh size of the filter screen used in S1 is 80-100 mesh.

4. The preparation method according to claim 1, characterized in that, The mesh size of the gauze filtered in S2 and S3 is 400 mesh.

5. The application of the anti-hair loss plant composition according to claim 1 or 2, characterized in that, It is used in the preparation of hair cosmetics; the hair cosmetics include shampoo, conditioner, and serum; the hair cosmetics are free from acute eye irritation, repeated skin irritation, and skin allergic reactions.

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