Use of an extract of an effective part of periplaneta americana in the preparation of a hair growth product

Abstract

This invention discloses the application of an effective component extract from the American cockroach (Periplaneta americana) in the preparation of products or functional compound products for hair growth, hair darkening, and hair care, belonging to the field of pharmaceutical application technology. Using modern separation and purification processes, the degreased extract of the American cockroach was processed to obtain component samples. A mouse model was established by injecting dihydrotestosterone (DHT), and the effective component extract with good hair growth activity was successfully screened. This extract can significantly shorten the time for hair to grow out of the skin in the bald area of ​​mice, prolong the hair growth cycle, promote the production of subcutaneous melanocytes in mice, significantly increase the content of superoxide dismutase and glutathione peroxidase in the skin tissue of the bald area, while decreasing the malondialdehyde content in the bald area tissue, and regulating the levels of testosterone and estradiol in mouse serum. It has a hair growth promoting effect and can be used to prepare products for treating or preventing hair loss or darkening hair, and can also be used in other compound products with corresponding hair growth efficacy.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical application technology, specifically relating to the application of an extract of the effective parts of the American cockroach in the preparation of hair growth, hair darkening and hair care products or functional compound products. Background Technology

[0002] In today's high-pressure, fast-paced society, hair loss has become a common skin condition that seriously affects people's self-confidence and self-esteem. Hair loss can be categorized into two types: scarring alopecia and non-scarring alopecia. Among these, androgenetic alopecia (AGA), a type of non-scarring alopecia, is the most common, accounting for over 90% of cases. Androgenetic alopecia, also known as seborrheic alopecia, is a progressive hair loss disease that begins in adolescence or late adolescence, characterized by the miniaturization of hair follicles. According to the "Guidelines for the Diagnosis and Treatment of Androgenetic Alopecia in Chinese People," the current prevalence of androgenetic alopecia in Chinese men is 21.3%, and in women it is 6.0%, meaning there are nearly 200 million people with hair loss in my country, and the number of patients is increasing annually and is trending towards affecting younger people. Besides androgenetic alopecia, alopecia areata (AA) is another typical non-scarring type of hair loss, commonly known as "spot baldness." Its incidence in the general population is approximately 0.1-0.2%. Clinically, it often manifests as sudden, well-defined patches of hair loss. The causes are complex, but are generally believed to be related to factors such as autoimmunity, genetics, emotional stress, and endocrine disorders. While hair loss does not affect physical health, it significantly impacts mental health and quality of life.

[0003] Currently, the main treatments for hair loss include topical medications, oral medications, plasma-rich protein (PRP) injections, and hair transplantation surgery. Among these, only two drugs, minoxidil and finasteride, approved by the U.S. Food and Drug Administration (FDA) for treating alopecia areata (AGA), are internationally recognized. However, recent studies have reported certain side effects in male AGA patients, including loss of libido, erectile dysfunction, and ejaculation problems. Although these side effects gradually disappear after discontinuation of the medication, a new round of hair loss often occurs, failing to achieve the desired therapeutic effect. Therefore, finding and discovering highly effective, safe, and non-toxic drugs for treating hair loss is of significant practical importance.

[0004] my country is a major country in terms of traditional Chinese medicine resources, and traditional Chinese medicine has a long history of treating hair loss. It is renowned for its multi-component, multi-target, and multi-pathway effects. Therefore, with the hair loss population expanding and increasingly affecting younger people, finding highly effective, safe, and non-toxic hair loss treatments from my country's traditional Chinese medicine resources has become a hot topic of widespread interest among researchers.

[0005] The American cockroach (Periplaneta americana), belonging to the family Blattodea and the genus Periplaneta, is commonly known as a "cockroach." Its medicinal use was first recorded in the *Shennong Bencao Jing* (Shennong's Classic of Materia Medica), and is also documented in ancient texts such as the *Tang Bencao* (Tang Dynasty Materia Medica) and the *Bencao Gangmu* (Compendium of Materia Medica). Modern separation and analytical techniques have shown that the American cockroach contains a rich array of beneficial compounds, including common proteins, lipids, amino acids, and sugars, as well as abundant small-molecule bioactive peptides, nucleosides, and various types of small organic molecules. These substances possess a wide range of biological activities, including antioxidant, anti-inflammatory, antibacterial, antiviral, antitumor, anti-fibrotic, and tissue repair-promoting effects.

[0006] Existing technologies utilize extracts from American cockroaches to prepare antimyelosuppressive drugs or related products, but have not developed or utilized their application in hair growth, hair darkening, and hair care products. Summary of the Invention

[0007] The purpose of this invention is to provide an application of effective extracts from the American cockroach (Periplaneta americana) in the preparation of hair growth, hair darkening, and hair care products or functional compound products. This involves using modern separation and purification processes to separate and purify the defatted extract of the American cockroach alcohol to obtain sample parts. Then, a "androgenetic alopecia" model was established in C57BL / 6J mice by injecting them with dihydrotestosterone, and a "patchy alopecia" model was established by sodium sulfide hair removal. The hair growth effects of the obtained sample parts were screened, and effective extracts with good hair growth activity were successfully identified and applied in the fields of hair growth, hair darkening, and hair care. This efficient extraction and utilization of chemically active ingredients solves the technical problems of insufficient research on the American cockroach in existing products, slow efficacy of conventional hair growth products using only medicinal formulations, and difficulties in quality control.

[0008] This invention is achieved through the following technical solution:

[0009] The application of an extract of an effective component of the American cockroach in the preparation of hair growth, hair darkening and hair care products or functional compound products.

[0010] Preferably, the method for preparing the extract of the effective component of the American cockroach includes the following steps:

[0011] The American cockroach extract was obtained by dissolving the defatted extract in water-containing alcohol and then heating and flocculating it, followed by low-temperature storage and precipitation, filtration, concentration and drying of the filtrate to obtain the effective fraction extract of the American cockroach.

[0012] Preferably, the method for preparing the defatted extract of American cockroach is as follows: the dried body of the American cockroach is extracted with alcohol and then defatted.

[0013] During the alcohol extraction, a methanol solution or ethanol solution with an alcohol mass fraction of not less than 70 wt% is used.

[0014] Preferably, the mass fraction of alcohol in the aqueous alcohol is 40wt%-50wt%;

[0015] The mass ratio of the aqueous alcohol to the defatted extract of American cockroach alcohol is 6-10:1;

[0016] The alcohol is methanol or ethanol.

[0017] Preferably, the mass ratio of the aqueous alcohol to the defatted extract of American cockroach is 8:1.

[0018] The above-described preparation method, which involves dissolving the defatted extract of American cockroaches in aqueous alcohol to obtain a suspension, followed by heating flocculation and low-temperature cold storage and sedimentation, firstly solves the problems of unclear extract, difficulty in filtration, and severe emulsification during defatting caused by the extraction of large amounts of proteins, peptides, sugars, and lipids when directly extracting American cockroaches with alcohol of this concentration. Secondly, "heating flocculation" enables rapid flocculation and suspension of insoluble, inactive components in the emulsion obtained after reconstitution of the extract at a low cost, quickly clarifying the solution. "Low-temperature cold storage and sedimentation" successfully enriches the effective components with germination-promoting effects in the extract at a low cost, while simultaneously causing the previously difficult-to-filter flocculent, inactive, insoluble matter to aggregate at the bottom, making it easier to remove by filtration.

[0019] Preferably, the heating flocculation involves heating the suspension at 60-70°C for 15-30 minutes, then removing it and allowing it to cool naturally to room temperature.

[0020] Preferably, the process of low-temperature cold storage, precipitation, filtration, concentration and drying of the filtrate is as follows: after heating and flocculation treatment, the solution is transferred to a 0-10℃ cold storage environment for 8-10 hours, filtered to remove the insoluble precipitate at the bottom, and the filtrate is concentrated under reduced pressure at 60-80℃ to an appropriate amount and then freeze-dried.

[0021] Preferably, the dosage form of the product or functional compound product includes any one of the following: ointment, spray, lotion, paste, liniment, and gel.

[0022] Compared with the prior art, the present invention has at least the following technical effects:

[0023] This invention provides the application of an effective component extract from the American cockroach (Periplaneta americana) in the preparation of hair growth, hair darkening, and hair care products or functional compound products. The American cockroach is rich in various chemical groups and exhibits a wide range of biological activities. In this method, modern separation and purification processes are used to separate and purify the defatted extract of the American cockroach alcohol to obtain component samples. Then, a "androgenic alopecia" model is established using C57BL / 6J mice injected with dihydrotestosterone, and a "patchy alopecia" model is established using sodium sulfide hair removal. The hair growth effects of the obtained component samples are screened, and effective component extracts with good hair growth activity are successfully selected.

[0024] Meanwhile, experimental results show that this product can significantly shorten the time for hair to grow out of the skin in the bald area of ​​mice, prolong the hair growth cycle in mice, promote the production of subcutaneous melanocytes in mice, significantly increase the content of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in the skin tissue of the bald area of ​​mice, while reducing the content of malondialdehyde (MDA) in the tissue of the bald area, and regulating the content of testosterone and estradiol in mouse serum. Many of its indicators are comparable to those of the positive control drug "minoxidil" group, indicating that this product has a good effect on promoting hair growth. It can be used to prepare products for the treatment or prevention of "androgenetic alopecia" and "alopecia areata", as well as hair darkening products. It can also be used in other functional compound products with hair growth and hair darkening effects. Attached Figure Description

[0025] Figure 1 This is a schematic diagram illustrating the effect of the invention on hair growth in mice in Experiment Example 3.

[0026] Figure 2 This is a schematic diagram of the timeline of the experiment in Experiment Example 10 of the present invention;

[0027] Figure 3 This is a schematic diagram illustrating the effect of the experiment on hair growth in male mice in Experiment Example 10 of the present invention;

[0028] Figure 4 This is a schematic diagram illustrating the effect of the experiment on hair growth in female mice in Experiment Example 10 of the present invention;

[0029] Figure 5 This is a schematic diagram showing the comparison of serum testosterone levels in different groups of male mice in Experiment 10 of the present invention.

[0030] Figure 6 This is a schematic diagram showing the comparison of serum testosterone levels in different groups of female mice in Experiment 10 of the present invention.

[0031] Figure 7 This is a schematic diagram showing the comparison of serum estradiol levels in different groups of male mice in Experiment 10 of the present invention.

[0032] Figure 8This is a schematic diagram showing the comparison of serum estradiol levels in different groups of female mice in Experiment 10 of the present invention.

[0033] Figure 9 This is a schematic diagram comparing the MDA content in the skin tissue of different groups of male mice in Experiment 10 of the present invention;

[0034] Figure 10 This is a schematic diagram comparing the MDA content in the skin tissue of different groups of female mice in Experiment 10 of the present invention;

[0035] Figure 11 This is a schematic diagram comparing the SOD content in the skin tissue of different groups of male mice in Experiment 10 of the present invention;

[0036] Figure 12 This is a schematic diagram comparing the SOD content in the skin tissue of different groups of female mice in Experiment 10 of the present invention;

[0037] Figure 13 This is a schematic diagram comparing the GSH-Px content in the skin tissue of different groups of male mice in Experiment 10 of the present invention.

[0038] Figure 14 This is a schematic diagram comparing the GSH-Px content in the skin tissue of different groups of female mice in Experiment 10 of the present invention.

[0039] Figure 15 HE-stained images of skin tissue sections from the hairless areas of male mice in Experiment 10 of this invention, with a scale bar of 200 μm.

[0040] Figure 16 This is a schematic diagram illustrating the effect of the sample in Experiment 11 of the present invention on hair regrowth in Na2S-treated mice;

[0041] Figure 17 The diagram shows the sample preparation process in Experiment Examples 7-9 of this invention. Detailed Implementation

[0042] The following will describe the implementation scheme of the present invention in detail with reference to experimental examples. However, those skilled in the art will understand that the following experimental examples are only used to illustrate the present invention and should not be regarded as limiting the scope of the present invention. Specific conditions not specified in the experimental examples shall be carried out according to conventional conditions or conditions recommended by the manufacturer. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0043] Experimental Example 1:

[0044] 1. Screening of suitable alcohol concentrations for extraction from American cockroaches: Based on the fact that American cockroaches are animal (insect) medicinal materials, rich in proteins, peptides, amino acids, nucleosides and other small molecule components, the alcohol concentrations used for extraction were investigated in order to obtain the optimal extraction conditions.

[0045] 2. Extraction: Weigh 500g of dried American cockroach body powder (passed through a 10-mesh sieve) into 6 round-bottom flasks (3000mL each), and add 2000mL of aqueous ethanol solution with mass fractions of 40%, 50%, 60%, 70%, 80%, and 90%, respectively. After reflux extraction in a water bath at 80℃ for 2.0h, filter and collect the filtrate. Repeat the extraction once with an equal volume of ethanol solution of the same mass fraction. Combine the two extracts and concentrate under reduced pressure at 60℃, then dry to obtain the American cockroach alcohol extract of each mass fraction.

[0046] 3. Degreasing: Add 6 times the weight of the obtained alcohol extracts to 80°C deionized water and stir until completely dissolved. Let stand and cool naturally for 10 hours to remove the upper layer of oil, filter out the insoluble matter at the bottom, concentrate the middle aqueous layer under reduced pressure at 60°C, and dry to obtain the defatted alcohol extract of American cockroach. The parameters during the preparation process are shown in Table 1.

[0047] Table 1. Parameters during the preparation of defatted extract

[0048]

[0049] 4. Conclusion: As shown in Table 1, when the same amount of medicinal materials are extracted with aqueous ethanol solutions of different mass fractions, the lower the mass fraction of alcohol in the aqueous ethanol solution, the less clear the extract is, the more difficult it is to filter, and the higher the yield of alcohol extract. However, after defatting the alcohol extract, the amount of defatted extract obtained is actually lower.

[0050] The reason is that when using low-concentration alcohol for extraction, a large amount of fat and protein components in the insect body are extracted, resulting in an emulsion. The reason the yield is lower after defatting is that the addition of hot water and stirring during defatting causes severe emulsification due to the presence of large amounts of fat and protein. Under the same settling time, the emulsion layer cannot dissipate, resulting in a small water layer and ultimately a lower yield. Therefore, considering factors such as the color of the extract, the ease of filtration, the emulsification during defatting, and the final yield of the defatted extract, when extracting from American cockroaches, the alcohol concentration should be greater than 70%, and even better results are achieved when the alcohol concentration is above 80%.

[0051] Experimental Example 2:

[0052] 1. Sample preparation for hair regrowth activity screening experiment:

[0053] (1) Weigh 10g of dried American cockroach alcohol-degreased extract for activity screening, and name it PA-010 for subsequent description.

[0054] (2) Weigh 50g of defatted extract of American cockroach and add 400mL of pre-diluted aqueous ethanol with a mass fraction of 45%. Stir and dissolve completely at room temperature to obtain a suspension of defatted extract of American cockroach in aqueous ethanol. Heat the suspension at 60℃ for 20min, remove it and let it cool naturally to room temperature. Store it in a 4℃ refrigerator for 10h. Carefully remove it and filter it to remove the insoluble precipitates at the bottom. Concentrate the filtrate under reduced pressure at 60℃ to a minimum volume and freeze dry to obtain 33.7g of sample for activity screening, with a yield of 67.4%. For ease of subsequent description, name it PA-011.

[0055] (3) Four macroporous adsorption resins of the types DM130, ADS-7, DA201 and X-5 were selected. After passing through a 60-mesh sieve, 500g of each type was weighed and pretreated. The samples were then prepared according to the following steps.

[0056] Pretreatment method: Soak the resin bed in 95% ethanol for 24 hours to allow it to fully swell. Then rinse with fresh 95% ethanol solution until the ethanol solution is clear when mixed with an equal amount of water and no absorption peak is observed when the ethanol solution is scanned with UV spectroscopy.

[0057] A. The pretreated four macroporous adsorption resins were packed into columns with 95% ethanol (column bed volume approximately 0.6L). The packed columns were washed with deionized water until no alcohol was present in the effluent at the bottom of the column. When the water at the top of the resin bed was tangent to the resin at the top of the column, the stopcock at the bottom of each resin column was closed for later use (column bed volume shrank to approximately 0.5L).

[0058] B. Weigh 4 portions of 50g each of the defatted extract of American cockroach alcohol and dissolve them in 150mL of deionized water to obtain an aqueous suspension of the defatted extract of American cockroach alcohol.

[0059] C. With the piston at the bottom of the resin column open, slowly add the above-mentioned aqueous suspension from the top of each resin column. When the suspension flows to the point where it is tangent to the top of the resin column bed, close the piston at the bottom of each resin column and maintain natural adsorption at room temperature for 12 hours.

[0060] D. Each column is first eluted with 0.5L of deionized water, which is one column bed volume of resin, at a flow rate of about 1 / 30 of the resin bed volume per minute, to wash away the non-adsorbed substances.

[0061] E. After washing the resin columns with water, each column was eluted with 1.5 L of 80% aqueous ethanol (3 times the volume of the resin column) at a flow rate of 1 / 30 of the resin column volume per minute. The aqueous ethanol eluent from each column was collected and concentrated under reduced pressure to approximately 100 mL at 60 °C. The eluents were then dried using a freeze dryer and weighed. The relevant parameters are shown in Table 2.

[0062] Table 2 Enrichment yields of defatted extracts of Periplaneta americana by macroporous adsorption resins of different models

[0063] Resin type polar / nonpolar Average pore size (nm) Amount (g) Yield (%) Named DM130 weak polarity 9～10 10.2 20.4 PA-012 ADS-7 polarity 25～30 11.6 23.2 PA-013 DA201 polarity 23～25 8.4 16.8 PA-014 X-5 nonpolar 28～30 7.6 15.2 PA-015

[0064] 2. Conclusion: The results in Table 2 show that among the four selected resins, the two resins of models DM130 and ADS-7 have relatively good enrichment effects on the defatted extract, and the yields are greater than 20%. If the results of activity screening are good, there is a possibility of industrial production. For ease of subsequent description, the product treated with resin model DA201 is named PA-014, and the product treated with resin model X-5 is named PA-015.

[0065] Therefore, two samples named PA-012 and PA-013, which are enriched by using the two resins DM130 and ADS-7, are selected for the hair growth activity screening experiment.

[0066] Experimental Example 3: Hair growth activity screening of each sample for "androgenic alopecia"

[0067] Sample information for activity screening: See Table 3.

[0068] Table 3 Information table of samples for activity screening

[0069] Serial Number Sample naming Sample preparation method 1 PA-010 American cockroach defatted extract 2 PA-011 The defatted extract of American cockroaches was prepared by reconstitution with 45% aqueous alcohol followed by cold storage and coagulation. 3 PA-012 American cockroach defatted extract was prepared by enrichment with DM130 resin. 4 PA-013 American cockroach defatted extract was prepared by enrichment with ADS-7 resin. 5 Minoxidil Purchased from Aladdin Reagents

[0070] Main reagents and consumables: See Table 4.

[0071] Table 4 Main reagents and consumables

[0072] Names of reagents and consumables Manufacturer batch number Dihydrotestosterone (DHT) Solarium 521-18-6 Minoxidil Aladdin 8304-91-3 corn oil Aladdin 8001-30-7 Propylene glycol Sinopharm Group - Anhydrous ethanol Sinopharm Group -

[0073] Experimental animals: SPF-grade C57BL / 6J mice, 6 weeks old, male, body weight (18 - 20) g, a total of 50 mice. Purchased from Hunan Slack Jingda Experimental Animal Co., Ltd., license number: SCXK(Xiang) 2019-0004; experimental unit use license number: SYXK(Yun) 2018-0002; experimental animal quality certificate number: No. 430727220101569153.

[0074] Preparation of samples and reagents: Weigh 10 g each of PA-010, PA-011, PA-012, PA-013 and minoxidil, and add 200 mL of a solution with water: ethanol: propylene glycol = 1:4:5 as the matrix solution to dissolve them, and prepare each solution with a content of 5% for standby. Dihydrotestosterone (DHT) is dissolved in corn oil to prepare a 0.5 mg / mL solution for standby.

[0075] Modeling: C57BL / 6J mice were acclimatized for 5 days at an ambient temperature of 22-25℃ and a relative humidity of 50-70%. Eight mice were randomly selected as the blank control group. The other mice were injected subcutaneously with 5 mg / kg DHT daily in the neck and back until the end of the experiment. During this period, on the 6th day after DHT injection, all mice (including the blank control group mice) were anesthetized with 50 mg / kg sodium pentobarbital. After the mice were anesthetized, a mixture of hot-melted paraffin and rosin in a 1:1.5 ratio was evenly applied to the back of the mice. After cooling, the mixture was carefully peeled off, and the back hair of the mice was removed.

[0076] Grouping and Drug Administration: After hair removal, mice with smooth, undamaged skin were selected and divided into 7 groups of 6 mice each. Starting from the second day after hair removal, the appropriate reagents or drugs were applied to the hair removal area twice daily (8:00-9:00 AM and 8:00-9:00 PM). The blank control group and model group received a base solution applied to the hair removal area; the control group received 5% minoxidil solution; and the other 4 sample groups received 5% of their respective sample solutions applied to the hair removal area. See Table 5 for mouse grouping and drug administration details.

[0077] Table 5. Mouse grouping and administration

[0078]

[0079] Observation indicators: Observe the time it takes for the hair to grow out of the body surface and the hair growth status of mice in each group, and take pictures for recording.

[0080] Experimental results: such as Figure 1 The diagram shown illustrates the effect of the drug on hair growth in mice.

[0081] During the experiment, except for the model group, the skin of the hair-removed area of ​​mice in other groups began to turn from pink to gray on the 6th day of hair removal, while the model group did not start to turn from pink to gray until the 7th day. This indicates that the selected samples could improve the hair retardation phenomenon in mice caused by DHT to varying degrees. By the 14th day of hair removal, the difference in hair growth among the groups was quite obvious. Compared with the model group, the hair growth of mice in each sample group and the control group was better. Among the four sample groups, the hair growth of mice in the PA-011 group was comparable to that of the blank group and the minoxidil group, indicating that PA-011 had the best effect among the selected samples.

[0082] Experiment Example 4:

[0083] 1. Investigation on the effect of cold storage temperature on the yield during PA-011 sample preparation

[0084] Based on the screening results showing that sample PA-011 performed best, the effect of cold storage temperature on sample yield was investigated. Six 20g portions of defatted extract were weighed into 250mL beakers, and 160mL of 45% ethanol (eight times the mass of the extract) was added to each beaker. The beakers were stored at -5, 0, 5, 10, 15, and 20℃ for 10 hours, filtered, and the filtrates were concentrated under reduced pressure at 60℃, dried, and weighed. The results are shown in Table 6.

[0085] Table 6. Effect of cold storage temperature on the yield of PA-011 fraction

[0086] Cold storage temperature (°C) -5 0 5 10 15 20 PA-011 yield (g) 11.85 13.26 13.52 13.84 14.95 16.26 PA-011 yield (%) 59.25 66.30 67.6 69.20 74.75 81.30

[0087] 2. Conclusion: The results in the table show that the yield of PA-011 fraction does not change significantly when the storage temperature is between 0 and 10℃. However, the yield decreases significantly when the temperature is below 0℃, while the yield increases significantly when the temperature is above 10℃. Since the PA-011 sample was prepared in a 4℃ refrigerator during the activity screening, combined with the yield changes in the table, it indicates that the storage temperature between 0 and 10℃ has little impact on the yield and will not significantly affect the activity. Therefore, the storage temperature can be between 0 and 10℃, with the optimal storage temperature being around 5℃.

[0088] Experimental Example 5:

[0089] 1. Investigation on the effect of the amount of alcohol used to dissolve the defatted extract during the preparation of PA-011 sample on its yield.

[0090] Six 20g portions of extract were weighed and placed in 250mL beakers. Ethanol solution with a mass fraction of 45% was added to each portion at different ratios of the extract mass. The mixture was stored at 5℃ for 10 hours to allow it to precipitate. After filtration, the filtrate was concentrated under reduced pressure at 60℃, dried, and weighed. The results are shown in Table 7.

[0091] Table 7. Effect of alcohol content in extract dissolution on the yield of PA-011 fraction.

[0092] Amount of alcohol (g) used to dissolve the extract 100 120 140 160 180 200 The amount of alcohol is a multiple of the extract. 5 times 6 times 7 times 8 times 9 times 10 times PA-011 yield (g) 12.85 13.10 13.28 13.45 13.66 14.02 PA-011 yield (%) 64.25 65.50 66.40 67.25 68.3 70.10

[0093] 2. Conclusion: The table shows that the amount of alcohol used to dissolve the extract does not significantly affect the yield of the PA-011 fraction. However, during the experiment, it was found that when the amount of alcohol used was less than 6 times the mass of the extract, the cold storage and precipitation effect was poor, and it was difficult to filter and remove the precipitate. Excessive alcohol usage, on the other hand, leads to excessive dissolution of inactive components, increases costs, and prolongs concentration time. Therefore, the appropriate amount of alcohol used to dissolve the extract is between 6 and 10 times the mass of the extract, with 8 times being optimal.

[0094] Experimental Example 6:

[0095] 1. Investigation on the effect of alcohol concentration used for extract dissolution on the yield during PA-011 sample preparation.

[0096] Six portions of extract, each weighing 20g, were placed in 250mL beakers. Eight times the mass of the extract was added to each portion of aqueous ethanol of different mass fractions to dissolve them. The mixtures were then stored at 5℃ for 10 hours to allow to precipitate. After filtration, the filtrates were concentrated under reduced pressure at 60℃, dried, and weighed. The results are shown in Table 8.

[0097] Table 8. Effect of alcohol concentration used for extract dissolution on the yield of PA-011

[0098] Alcohol mass fraction (%) 30 35 40 45 50 55 60 PA-011 yield (g) 11.52 12.50 13.22 13.49 13.84 14.52 15.56 PA-011 yield (%) 57.60 62.50 66.10 67.45 69.20 72.60 76.8

[0099] 2. Conclusion: As can be seen from the table, the higher the mass fraction of alcohol used to dissolve the extract, the higher the yield of PA-011 fraction. However, based on the sample yield used in the activity screening, which was about 67%, from the perspective of maintaining activity, the mass fraction of alcohol in the alcohol solution for dissolving the extract is more suitable between 40% and 50%, with 45% being the most ideal.

[0100] like Figure 17 The diagram shown is a flowchart illustrating the preparation process of PA-011 in Experiment 7-9.

[0101] Experimental Example 7: Preparation of PA-011, an extract of the effective hair growth component of the American cockroach.

[0102] (1) Redissolution of extract: Weigh 50g of defatted extract of American cockroach and add 300mL of pre-diluted ethanol with a mass fraction of 50%. Stir slowly until completely dissolved to obtain a suspension of defatted extract of American cockroach in ethanol.

[0103] (2) Heating and flocculation: The defatted extract of American cockroaches obtained in (1) containing aqueous ethanol reconstituted suspension was placed in a water bath at 60°C for 15 minutes, and then taken out and allowed to cool naturally to room temperature;

[0104] (3) Cold storage and precipitation: The solution obtained in (2) was transferred to a cold storage room at 0°C and allowed to stand for 10 hours. The precipitate was removed by filtration to obtain an aqueous ethanol solution containing the effective germinal part of the American cockroach.

[0105] (4) Obtaining the effective hair growth component: The aqueous ethanol solution containing the effective hair growth component of American cockroach obtained in (3) was concentrated under reduced pressure at 60°C and freeze-dried to obtain the extract PA-011 of the effective hair growth component of American cockroach, with a mass of 33.4g and a yield of 66.8%.

[0106] Experimental Example 8: Preparation of PA-011, an extract of the effective hair growth component of the American cockroach.

[0107] (1) Resolution of extract: Weigh 50g of defatted extract of American cockroach and add 400mL of pre-diluted ethanol with a mass fraction of 45%. Stir slowly until completely dissolved to obtain a suspension of defatted extract of American cockroach in ethanol.

[0108] (2) Heating and flocculation: The defatted extract of American cockroaches obtained in (1) and the aqueous ethanol reconstituted suspension were placed in a water bath at 60°C for 15 minutes and then naturally cooled to room temperature.

[0109] (3) Cold storage and precipitation: The solution obtained in (2) was transferred to a 5°C freezer and allowed to stand for 10 hours. The precipitate was then removed by filtration to obtain an aqueous ethanol solution containing the effective germinal part of the American cockroach.

[0110] (4) Obtaining the effective hair growth component: The aqueous ethanol solution containing the effective hair growth component of American cockroach obtained in (3) was concentrated under reduced pressure at 60°C and freeze-dried to obtain the effective hair growth component of American cockroach PA-011, with a mass of 33.9g and a yield of 67.8%.

[0111] Experimental Example 9: Preparation of PA-011, an extract of the effective hair growth component of the American cockroach.

[0112] (1) Resolution of extract: Weigh 50g of defatted extract of American cockroach and add 500mL of pre-diluted ethanol with a mass fraction of 40%. Stir slowly to dissolve completely and prepare a suspension of defatted extract of American cockroach in ethanol.

[0113] (2) Heating and flocculation: The defatted extract of American cockroaches obtained in (1) containing aqueous ethanol reconstituted suspension was placed in a water bath at 60°C for 15-30 minutes and then naturally cooled to room temperature;

[0114] (3) Cold storage and precipitation: The solution obtained in (2) was transferred to a cold storage room at 10°C and allowed to stand for 10 hours. The precipitate was removed by filtration to obtain an aqueous ethanol solution containing the effective germinal part of the American cockroach.

[0115] (4) Obtaining the effective hair growth component: The aqueous ethanol solution containing the effective hair growth component of American cockroach obtained in (3) was concentrated under reduced pressure at 60°C and freeze-dried to obtain the effective hair growth component of American cockroach PA-011, with a mass of 34.2g and a yield of 68.4%.

[0116] Experimental Example 10: Verification of the hair regrowth effect of PA-011 sample on androgenetic alopecia.

[0117] Based on the screening results of sample activity, the PA-011 sample had the best effect and was comparable to the positive control minoxidil. Therefore, to further confirm the reliability of its hair growth effect, male and female mice were separately experimented, and the hair growth effect was verified under the condition of increasing the dose of the modeling agent dihydrotestosterone.

[0118] 1. Experimental sample: PA-011 sample; 2. Control sample: Minoxidil; 3. Main reagents and consumables: See Table 9.

[0119] Table 9 Main Reagents and Consumables

[0120] Names of reagents and consumables Manufacturer batch number Dihydrotestosterone (DHT) Solarium 521-18-6 Minoxidil Aladdin 8304-91-3 corn oil Aladdin 8001-30-7 Propylene glycol, anhydrous ethanol Sinopharm Group - Mouse estradiol (E2) enzyme-linked immunosorbent assay kit Nanjing was built 20220808 Mouse testosterone (T) enzyme-linked immunosorbent assay kit Nanjing was built 20220808 Malondialdehyde (MDA) content assay kit Nanjing was built 20220816 Superoxide dismutase (SOD) assay kit Nanjing was built 20220816 Glutathione peroxidase (GSH-Px) assay kit Nanjing was built 20220816

[0121] 4. Experimental animals: SPF-grade C57BL / 6J mice, male, 6 weeks old, body weight (18 - 20) g, 70 mice; female, 7 weeks old, body weight (18 - 20) g, 50 mice. All were purchased from Beijing Speefoo Biotechnology Co., Ltd., license number: SCXK(Beijing)2019 - 0010; the license number for the experimental unit's use: SYXK(Yunnan)2018 - 0002; the quality certificate number for experimental animals: No.110324220107238063.

[0122] 5. Preparation of samples and reagents: PA-011 was prepared into 2% and 5% solutions with the matrix solution used in Experimental Example 3, and minoxidil was prepared into 2% and 5% solutions with the same matrix solution. Dihydrotestosterone was dissolved in corn oil to prepare a 10 mg / mL solution.

[0123] 6. Modeling: C57BL / 6J mice were adaptively raised for 5 days at an environmental temperature of 22 - 25°C and a relative humidity of 60 - 70%. Then, 12 male mice and 10 female mice were randomly selected and used as the blank groups for males and females respectively. The other mice were subcutaneously injected with dihydrotestosterone (DHT) at a dose of 1 mg / time / animal / 2 days on the nape of the neck until the end of the 21-day experiment of hair loss. During this period, 1 day after the 3rd injection of DHT, all mice were anesthetized with sodium pentobarbital at a dose of 50 mg / kg according to their body weight. After the mice were anesthetized, paraffin and rosin melted at a ratio of 1:1.5 were evenly applied to the back of the mice, and after cooling, it was carefully peeled off to remove the back hair of the mice.

[0124] 7. Grouping and Administration: After hair removal, all mice with smooth, undamaged skin were selected and divided into 5 groups of males and 5 groups of females. Starting from the second day after hair removal, each group received the corresponding drug or reagent intervention in the hair removal area twice daily (8:00-9:00 AM and 8:00-9:00 PM). The blank control group and model group of both male and female mice received a base solution applied to the hair removal area; the male control group received 5% minoxidil solution, and the female control group received 2% minoxidil solution; the high-dose and low-dose sample groups of both sexes received 5% and 2% PA-011 solution, respectively. See Table 10 for experimental grouping and administration details.

[0125] Table 10 Experimental Groups and Dosing Table

[0126]

[0127]

[0128] 8. Observation and detection indicators

[0129] (1) Hair growth observation: Before applying the medicine each day, observe the hair growth of the mouse skin and take photos as appropriate, and record the time when the mouse skin turns gray and black and the time when the hair grows out of the epidermis.

[0130] (2) Determination of hormone levels in serum: Mice in each group were sacrificed 1 hour after applying the medication on the 21st day of hair removal, and blood was collected to measure the levels of testosterone and estradiol in the serum of each group of mice.

[0131] (3) Skin tissue in the hair-removed area: After the mice were sacrificed, the skin in the hair-removed area was homogenized and centrifuged, and the contents of malondialdehyde (MDA), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were detected.

[0132] (4) Skin histological observation (HE staining): Take 1cm×1cm skin tissue from the hairless area of ​​the euthanized mouse, attach it tightly to a cardboard, fix it with 4% paraformaldehyde, prepare a routine HE pathological section, and observe and photograph it under a microscope.

[0133] 9. Experimental Results:

[0134] (1) Hair growth in mice:

[0135] like Figure 3 The diagram shown illustrates the effect of this treatment on hair growth in male mice. Figure 4 The diagram shown illustrates the effect of the treatment on hair growth in female mice.

[0136] During the experiment, on day 7 of hair removal, the skin in the hair-removed areas of mice in the control group, minoxidil group, and PA-011 high- and low-dose groups began to change from pink to gray. In contrast, the skin in the hair-removed areas of mice in the model group remained pink until day 8, when it began to gradually turn gray. By day 13, it was evident that the hair in the two sample groups and the minoxidil group had completely covered the hair-removed areas, with hair growth similar to the control group, while some skin in the hair-removed areas of the model group remained uncovered.

[0137] Apparently, both high- and low-dose PA-011 significantly improved DHT-induced hair retardation in both male and female mice. In male mice, both high- and low-dose PA-011 groups were comparable to the control group treated with 5% minoxidil. Figure 3 In female mice, both high- and low-dose PA-011 groups were superior to the control group treated with 2% minoxidil. Figure 4 ).

[0138] Therefore, the experimental results show that PA-011 still has a significant effect on promoting hair growth even when the dihydrotestosterone content is extremely high.

[0139] (2) Changes in serum testosterone and estradiol levels in mice

[0140] like Figure 5-8 This represents a variation in the levels of testosterone and estradiol in mouse serum. Figure 5 A schematic diagram comparing serum testosterone levels in different groups of male mice; Figure 6 A schematic diagram comparing serum testosterone levels in different groups of female mice;

[0141] Figure 7 A schematic diagram comparing serum estradiol levels in different groups of male mice; Figure 8 This is a schematic diagram comparing serum estradiol levels in different groups of female mice.

[0142] Experimental results showed that, regardless of whether the mice were male or female, the application of PA-011 to the hair removal area significantly regulated the DHT-induced imbalance of serum testosterone and estradiol. Its effect was similar to that of the positive control, minoxidil.

[0143] (3) Changes in the levels of MDA, SOD and GSH-Px in the skin tissue of the hairless area of ​​mice

[0144] like Figure 9-14 This diagram illustrates the changes in MDA, SOD, and GSH-Px levels in the shaved skin tissue of mice. Figure 9 This is a schematic diagram comparing the MDA content in the skin tissue of different groups of male mice. Figure 10 A schematic diagram comparing the MDA content in the skin tissue of different groups of female mice; Figure 11Schematic diagram of comparison of SOD content in skin tissues of male mice in each group; Figure 12 Schematic diagram of comparison of SOD content in skin tissues of female mice in each group; Figure 13 Schematic diagram of comparison of GSH-Px content in skin tissues of male mice in each group; Figure 14 Schematic diagram of comparison of GSH-Px content in skin tissues of female mice in each group.

[0145] The experimental results showed that, whether male or female mice, after applying PA-011 to the depilated skin area, it could improve the increase of MDA in the skin and the decrease of antioxidant enzymes SOD and GSH-Px caused by injecting DHT, and its effect was comparable to that of the positive control minoxidil.

[0146] (4) Observation of HE staining of skin tissue sections in the depilated area of mice

[0147] As Figure 15 shown, it is a photo of HE staining of skin tissue sections in the depilated area of male mice in each group, and the scale bar is 200 μm. After sacrificing male mice in each group on the 21st day of hair removal, the staining results of skin sections in the depilated area.

[0148] It can be seen from the figure that the hair follicles in the skin of mice in the model group have significantly shrunk and become smaller, while the hair follicles of mice in other groups are still growing normally without atrophy. It shows that the sample PA-011 has a significant effect of prolonging the hair growth cycle. In addition, there is an obvious increase in melanocytes in the hair follicles of mice in the high-dose group of PA-011, indicating its certain hair-darkening effect.

[0149] Experimental Example 11: Hair growth-promoting effect of PA-011 sample on the sodium sulfide-induced "alopecia areata" model

[0150] 1. Based on the verification of the hair growth-promoting effect of the PA-011 sample on "androgenetic alopecia", it shows that the PA-011 sample indeed has a good hair growth-promoting effect on androgenetic alopecia. Therefore, further experiments were conducted to investigate the hair growth-promoting effect of PA-011 on "alopecia areata". 2. Experimental sample: PA-011. 3. Control sample: Minoxidil.

[0151] 4. Experimental animals: SPF-grade C57BL / 6J mice, 6 weeks old, male, body weight (18 - 20) g, 30 mice, purchased from Hunan Slack Jingda Experimental Animal Co., Ltd., license number: SCXK(Xiang)2019-0004; experimental unit use license number: SYXK(Yun)2018-0002; experimental animal quality certificate number: No.43072720102070828.

[0152] 5. Sample preparation: Both PA-011 sample and minoxidil were prepared using a matrix solution; the matrix solution was a solution of water:ethanol:propylene glycol = 1:4:5.

[0153] 6. Preparation of sodium sulfide solution for hair removal: Weigh 8g of sodium sulfide (Na2S) and dissolve it in 60mL of water, then add 40mL of 75% ethanol and mix well to obtain 8% Na2S ethanol solution.

[0154] 7. Modeling: SPF-grade C57BL / 6J mice were acclimatized for 5 days at an ambient temperature of 22-25℃ and a relative humidity of 50-70%. On the 6th day, sodium sulfide solution was applied to the area on the back of the mice to be shaved. After 3 minutes of application, the area was gently wiped with a cloth to remove hair. Then, the residual Na2S in the shaved area was wiped away with a soft, damp towel to establish a Na2S-induced alopecia areata model.

[0155] 8. Grouping and Administration: Mice with smooth, undamaged skin were randomly divided into 4 groups of 6 mice each. Administration began on the second day after hair removal, twice daily (8:00-9:00 AM and 8:00-9:00 PM). The blank control group received a base solution applied to the hair removal area; the control group received a 5% minoxidil solution applied to the hair removal area; the low-dose sample group received a 2% PA-011 solution applied to the hair removal area; and the high-dose sample group received a 5% PA-011 solution applied to the hair removal area. The experimental grouping and administration details are shown in Table 11.

[0156] Table 11 Experimental Groups and Drug Administration

[0157] Serial Number Animal grouping Number of animals (n) Dosage route of administration Dosage frequency 1 Blank group 6 matrix solution Apply to hair removal area 2 times / day 2 control group 6 5% minoxidil solution Apply to hair removal area 2 times / day 3 PA-011 Low Group 6 2% PA-011 solution Apply to hair removal area 2 times / day 4 PA-011 High Group 6 5% PA-011 solution Apply to hair removal area 2 times / day

[0158] 9. Observation indicators: Observe the skin color change, hair growth time and overall hair growth of mice in each group, and take photos and record them as appropriate.

[0159] 10. Results: such as Figure 16 The image shows a schematic diagram illustrating the effect of the sample on hair regrowth in Na2S-treated mice.

[0160] Mice in both sample groups and the minoxidil group showed patchy graying of the shaved area on their backs on day 10 after hair removal, while some mice in the model group showed patchy graying of the shaved area on their backs only on day 12. By day 14, some mice in the model group had started to grow patchy hair on their backs, while the back hair of mice in the two sample groups and the minoxidil group had grown relatively dense by this time. By day 21, the back hair of mice in the two sample groups and the minoxidil group had basically grown back, but most mice in the model group still had large areas of baldness on their backs. This indicates that sample PA-011 has a good hair growth promoting effect on the alopecia areata model established by sodium sulfide hair removal, and its effect is visually comparable to that of the positive control minoxidil group.

[0161] Finally, it should be noted that the above description is merely a preferred experimental example of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The application of an extract of an effective component of the American cockroach in the preparation of products for hair growth, hair darkening, and hair care, characterized in that, The preparation method of the effective component extract of the American cockroach includes the following steps: After dissolving the defatted extract of American cockroach in water-containing alcohol, a suspension was obtained. The suspension was then heated to flocculate, subjected to low-temperature cold storage and precipitation, filtered, and the filtrate was concentrated and dried to obtain the effective fraction extract of American cockroach. The method for preparing the defatted extract of American cockroach alcohol is as follows: the dried body of the American cockroach is extracted with alcohol and then defatted. During the alcohol extraction, a methanol solution or ethanol solution with an alcohol mass fraction of not less than 70 wt% is used. The mass fraction of alcohol in the aqueous alcohol is 40 wt%-50 wt%; The alcohol is methanol or ethanol; The mass ratio of the aqueous alcohol to the defatted extract of American cockroach alcohol is 8:1; The heating flocculation process involves heating the suspension at 60-70°C for 15-30 minutes, then removing it and allowing it to cool naturally to room temperature. The process of low-temperature cold storage, precipitation, filtration, concentration and drying of the filtrate is as follows: after heating and flocculation treatment, the solution is transferred to a 0-10℃ cold storage environment for 8-10 hours, filtered to remove the insoluble precipitate at the bottom, and the filtrate is concentrated under reduced pressure at 60-80℃ to an appropriate amount and then freeze-dried.

2. The application of the extract of the effective components of the American cockroach according to claim 1 in the preparation of products for hair growth, hair darkening, and hair care, characterized in that, The dosage form of the product includes any one of the following: coating, spray, lotion, ointment, liniment, and gel.

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