Psoralea corylifolia total flavone extract and preparation method and application thereof in promoting hair growth

Abstract

The invention discloses a fructus psoraleae total flavone extract as well as a preparation method and application thereof in promoting hair growth. According to the preparation method of the fructus psoraleae total flavone extract, the specific eutectic solvent is adopted, the ultrasonic technology is combined for extraction, the content of total flavone in the fructus psoraleae extract can be remarkably increased, and the fructus psoraleae total flavone can be efficiently extracted; and the adopted extraction solvent has the advantages of greenness, environmental protection, efficient process, low cost, high safety and the like, and can solve the problems of the existing extraction process. The malaytea scurfpea fruit total flavone extract obtained by the method has the effect of promoting hair growth and can remarkably promote hair follicle cell proliferation and prolong the hair growth period, the prepared external gel product has the hair growth effect, the preparation method is simple, and external use is convenient. The malaytea scurfpea fruit total flavone extract provided by the invention can be used for developing more cosmetics and medical products for preventing alopecia and promoting hair growth, and a new scheme is provided for green extraction of natural active ingredients and treatment of alopecia.

Description

Technical Field

[0001] The present invention belongs to the field of natural product extraction technology and biomedicine technology. More specifically, it relates to a total flavonoid extract of Psoralea corylifolia and its preparation method and application in promoting hair growth. Background Art

[0002] In recent years, the global problem of hair loss has become increasingly prevalent and younger. Data from the World Health Organization shows that approximately 30% of adult men and 10% of women suffer from androgenic alopecia, while factors such as stress and environmental pollution further exacerbate hair follicle atrophy and disrupt the hair growth cycle. Current mainstream treatments, such as minoxidil and finasteride, have side effects (such as scalp irritation and sexual dysfunction) and limited efficacy. Natural plant-based active ingredients have become a research hotspot due to their multi-target and high safety profile. The total flavonoids in Psoralea corylifolia (such as psoralen and isopsoralen) have been shown to promote hair follicle stem cell proliferation by activating the Wnt / β-catenin signaling pathway and reduce dihydrotestosterone (DHT) production by inhibiting 5α-reductase activity, demonstrating the potential for multi-pathway synergistic hair growth.

[0003] Psoralea corylifolia is a traditional Chinese medicinal material containing a variety of chemical components, including coumarins, flavonoids, monoterpenoids, and volatile oils. Total flavonoids are one of its main active ingredients, exhibiting antioxidant, anti-inflammatory, and hair follicle cell proliferation-promoting activities, and have significant potential in the field of hair growth. Due to the different extraction methods and reagents used, the active ingredients extracted vary significantly. Existing methods for extracting total flavonoids from Psoralea corylifolia mainly include traditional organic solvent extraction, ultrasound-assisted extraction, and microwave-assisted extraction. However, these methods have several drawbacks. Traditional organic solvent extraction, which typically uses organic solvents such as ethanol and methanol, has the disadvantages of high solvent consumption, the need for high-temperature removal after extraction, a high risk of residues, and environmental pollution. Furthermore, extraction times are long and yields are low. Ultrasonic-assisted extraction and microwave-assisted extraction, while able to improve extraction efficiency to a certain extent, still suffer from expensive equipment, high energy consumption, and high sample requirements. Furthermore, flavonoids vary greatly in polarity, resulting in poor selectivity of conventional solvents, low extraction yields, and the need for multiple extraction and concentration steps, which is energy-intensive and easily degrades the flavonoid structure. The content of total flavonoids in the currently prepared total flavonoid extracts of Psoralea corylifolia still needs to be improved.

[0004] Therefore, developing more green extraction processes that can efficiently extract total flavonoids from Psoralea corylifolia and prepare high-efficiency transdermal hair growth products are of great significance to solving the problem of hair loss treatment. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to overcome the defects and shortcomings of the existing extraction of total flavonoids from Psoralea corylifolia, and to provide a total flavonoids extract from Psoralea corylifolia and a preparation method thereof and an application thereof in promoting hair growth.

[0006] The present invention aims to provide a method for preparing a psoralea corylifolia total flavonoids extract.

[0007] Another object of the present invention is to provide a total flavonoid extract of Psoralea corylifolia.

[0008] Another object of the present invention is to provide an application of the total flavonoids extract of Psoralea corylifolia.

[0009] Another object of the present invention is to provide a product.

[0010] The above-mentioned purpose of the present invention is achieved through the following technical solutions: The invention provides a preparation method of a total flavonoid extract of psoralea corylifolia. The method comprises the following steps: using a hydrogen bond donor and a hydrogen bond acceptor to prepare a deep eutectic solvent, mixing crushed psoralea corylifolia with the deep eutectic solvent at a material-liquid ratio of 1:5-20 g / mL, performing ultrasound-assisted extraction, separating the extract, and purifying the supernatant, followed by concentration and drying to obtain the total flavonoid extract of psoralea corylifolia.

[0011] The present invention uses deep eutectic solvents (DESs) as a medium to extract total flavonoids from Psoralea corylifolia. Using a specific deep eutectic solvent as the extraction solvent, combined with ultrasonic technology, can significantly increase the total flavonoid content of Psoralea corylifolia. The total flavonoid extract of Psoralea corylifolia obtained by this method has the effect of promoting hair growth, significantly promoting the proliferation of hair follicle cells, and prolonging the hair growth period. The extraction solvent used in the present invention has the advantages of being green and environmentally friendly, highly efficient, low-cost, and highly safe, and can solve the problems existing in existing extraction processes. DES is a green solvent formed by a hydrogen bond donor (HBD) and a hydrogen bond acceptor (HBA). It is a new type of green solvent with the advantages of low melting point, low volatility, good solubility, and biodegradability, which perfectly solves the drawbacks of traditional total flavonoid extraction methods.

[0012] Preferably, the hydrogen bond donor is selected from one of oxalic acid, malic acid, glucose, citric acid, and glycerol, the hydrogen bond acceptor is selected from one of choline chloride and betaine, and the molar ratio of the hydrogen bond donor to the hydrogen bond acceptor is 1:1-3.

[0013] More preferably, the hydrogen bond donor is selected from one of oxalic acid, malic acid, glucose, citric acid, and glycerol, and the hydrogen bond acceptor is choline chloride.

[0014] More preferably, the molar ratio of the hydrogen bond donor to the hydrogen bond acceptor is 1:3.

[0015] Preferably, the preparation method of the deep eutectic solvent is: select a hydrogen bond donor and a hydrogen bond acceptor, mix them in a molar ratio of 1:1-3, place them at 40-80°C, and continue stirring until they are completely dissolved to form a uniform and stable deep eutectic solvent.

[0016] More preferably, choline chloride and malic acid are mixed in a ratio of 1:3 and placed at 80° C. with continuous stirring until completely dissolved.

[0017] Furthermore, dry and high-quality Psoralea corylifolia is selected and passed through a 40-100 mesh sieve to obtain Psoralea corylifolia powder.

[0018] Preferably, the psoralea corylifolia is passed through a 50-mesh sieve to obtain psoralea corylifolia powder.

[0019] Preferably, the conditions for the ultrasonic-assisted extraction are: extraction temperature 30-70° C., extraction power 300-600 W, and extraction time 15-60 minutes.

[0020] More preferably, the crushed Psoralea corylifolia powder is mixed with the prepared deep eutectic solvent at a solid-liquid ratio of 1:10 g / mL, and extracted under an ultrasonic extraction temperature of 60° C. for 60 minutes.

[0021] Preferably, the purification method comprises the following steps: after extraction, the extract is transferred to a centrifuge tube and centrifuged at 5000 rpm for 10 minutes. The supernatant is then slowly passed through an AB-8 macroporous resin column for adsorption. The resin column is first rinsed with a large amount of deionized water to remove impurities, and then eluted with 70% ethanol. The eluate is collected, concentrated using a vacuum concentrator, and finally dried to obtain a total flavonoid extract of Psoralea corylifolia.

[0022] The present invention provides a psoralea corylifolia total flavonoids extract prepared by the method.

[0023] The invention provides application of a total flavonoid extract of psoralea corylifolia in promoting hair growth.

[0024] The invention also provides a product containing the total flavonoids extract of Psoralea corylifolia.

[0025] At the same time, the total flavonoid extract of Psoralea corylifolia prepared by the present invention is further prepared into a gel for external use. Compared with other dosage forms, the gel has good spreadability and adhesion, and can form a uniform covering layer on the scalp surface, which is conducive to the continuous release and penetration of effective ingredients, realizing the integrated function of "liquid application-body temperature gelation-sustained release and long-term effect", better playing the role of promoting hair growth, and can be used to develop more cosmetics and pharmaceutical products that prevent hair loss and promote hair growth, providing a new solution for the green extraction of natural active ingredients and hair loss treatment.

[0026] Preferably, the product is a gel and further contains hydroxyethyl cellulose, glycerin and water.

[0027] More preferably, the product contains 5-20 parts of psoralea corylifolia total flavonoids extract, 5-8 parts of hydroxyethyl cellulose, 5-8 parts of glycerin, and 20-30 parts of water, by weight.

[0028] The present invention also provides a method for preparing the gel of the above-mentioned product, comprising slowly adding hydroxyethyl cellulose to water in proportion and stirring evenly to obtain a gel matrix A; then dissolving the total flavonoid extract of Psoralea corylifolia in glycerin and stirring evenly to obtain a mixed liquid B; and finally slowly adding the mixed liquid B to the gel matrix A and stirring evenly to obtain a gel for external use.

[0029] In addition, the present invention also provides the use of the above product in promoting hair growth.

[0030] The present invention has the following beneficial effects: The present invention provides a method for efficiently extracting total flavonoids from Psoralea corylifolia using a deep eutectic solvent (DES), and its application in hair growth. To address the issues of high solvent toxicity, low extraction efficiency, high energy consumption, and insufficient product purity in current traditional total flavonoid extraction processes, the present invention innovatively utilizes a deep eutectic solvent prepared from oxalic acid, malic acid, glucose, citric acid, glycerol, and choline chloride as an extraction solvent. Combined with ultrasound technology, this method significantly increases the total flavonoid content of Psoralea corylifolia. Furthermore, the extraction solvent employed in the present invention is environmentally friendly (the solvent is recyclable), highly efficient, cost-effective, and safe, addressing the challenges of existing extraction processes. The Psoralea corylifolia total flavonoid extract obtained by the present invention promotes hair growth, significantly promoting hair follicle cell proliferation and prolonging the anagen phase. The topical gel product prepared from the extract exhibits significant hair growth efficacy, is simple to prepare, and is convenient for topical application. The Psoralea corylifolia total flavonoid extract provided by the present invention can be used to develop a wider range of cosmetic and pharmaceutical products for preventing and promoting hair loss, providing a new approach to the green extraction of natural active ingredients and the treatment of hair loss. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 This is the standard curve of rutin.

[0032] Figure 2 This is the growth condition of mice.

[0033] Figure 3 The results are hematoxylin-eosin staining.

[0034] Figure 4 The results of toluidine blue staining. DETAILED DESCRIPTION

[0035] The present invention will be further described below with reference to the accompanying drawings and specific examples, but the examples do not limit the present invention in any way. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0036] Unless otherwise specified, the reagents and materials used in the following examples were commercially available.

[0037] Example 1 Extraction of total flavonoids from Psoralea corylifolia using deep eutectic solvent Preparation of deep eutectic solvent: Choline chloride and malic acid were placed in a clean round-bottom flask at a molar ratio of 1:2. The round-bottom flask was placed in a constant temperature oil bath at 80°C and stirred continuously with a magnetic stirrer until a homogeneous and transparent deep eutectic solvent was formed.

[0038] Extraction: Accurately weigh 10g of Psoralea corylifolia powder and add it to 100mL of the deep eutectic solvent prepared above. Place the mixture in an ultrasonic machine at 40°C and extract at 600W for 30 minutes.

[0039] Isolation and Purification: After extraction, transfer the extract to a centrifuge tube and centrifuge at 5000 rpm for 10 minutes. The supernatant is then slowly passed through an AB-8 macroporous resin column for adsorption. The column is rinsed with copious amounts of deionized water to remove impurities, followed by elution with 70% ethanol. The eluate is collected, concentrated using a vacuum concentrator, and finally dried to obtain the total flavonoid extract of Psoralea corylifolia.

[0040] Example 2 Extraction of total flavonoids from Psoralea corylifolia using deep eutectic solvent Preparation of deep eutectic solvent: Choline chloride and oxalic acid were placed in a round-bottom flask at a molar ratio of 1:2. The round-bottom flask was placed in a constant temperature oil bath at 80°C and stirred continuously with a magnetic stirrer until a homogeneous and transparent deep eutectic solvent was formed.

[0041] Extraction: Accurately weigh 10g of Psoralea corylifolia powder and add it to 100mL of the deep eutectic solvent prepared above. Ultrasonicate the mixture at 40°C and 600W for 30 minutes.

[0042] Separation and purification: The separation and purification steps are the same as those in Example 1 to obtain the total flavonoid extract of Psoralea corylifolia.

[0043] Example 3 Extraction of total flavonoids from Psoralea corylifolia using deep eutectic solvent Preparation of deep eutectic solvent: Choline chloride and glucose were placed in a round-bottom flask at a molar ratio of 1:2. The round-bottom flask was placed in a constant temperature oil bath at 80°C and stirred continuously with a magnetic stirrer until a homogeneous and transparent deep eutectic solvent was formed.

[0044] Extraction: Accurately weigh 10g of Psoralea corylifolia powder and add it to 100mL of the deep eutectic solvent prepared above. Ultrasonicate the mixture at 40°C and 600W for 30 minutes.

[0045] Separation and purification: The separation and purification steps are the same as those in Example 1 to obtain the total flavonoid extract of Psoralea corylifolia.

[0046] Example 4 Extraction of total flavonoids from Psoralea corylifolia using deep eutectic solvent Preparation of deep eutectic solvent: Choline chloride and citric acid were placed in a round-bottom flask at a molar ratio of 1:2. The round-bottom flask was placed in a constant temperature oil bath at 80°C and stirred continuously with a magnetic stirrer until a homogeneous and transparent deep eutectic solvent was formed.

[0047] Extraction: Accurately weigh 10g of Psoralea corylifolia powder and add it to 100mL of the deep eutectic solvent prepared above. Ultrasonicate the mixture at 40°C and 600W for 30 minutes.

[0048] Separation and purification: The separation and purification steps are the same as those in Example 1 to obtain the total flavonoid extract of Psoralea corylifolia.

[0049] Example 5 Extraction of total flavonoids from Psoralea corylifolia using deep eutectic solvent Preparation of deep eutectic solvent: Choline chloride and glycerol were placed in a round-bottom flask at a molar ratio of 1:2. The round-bottom flask was placed in a constant temperature oil bath at 80°C and stirred continuously with a magnetic stirrer until a homogeneous and transparent deep eutectic solvent was formed.

[0050] Extraction: Accurately weigh 10g of Psoralea corylifolia powder and add it to 100mL of the deep eutectic solvent prepared above. Ultrasonicate the mixture at 40°C and 600W for 30 minutes.

[0051] Separation and purification: The separation and purification steps are the same as those in Example 1 to obtain the total flavonoid extract of Psoralea corylifolia.

[0052] Example 6 Preparation of external gel Preparation of gel matrix: Accurately weigh 2 g of hydroxyethyl cellulose and slowly add it to 100 mL of deionized water. Stir continuously with a stirrer until the hydroxyethyl cellulose is fully swollen and a uniform gel matrix is ​​formed.

[0053] Drug addition: Accurately weigh 0.6 g of the total flavonoid extract of Psoralea corylifolia prepared in Example 1 and dissolve it in 10 mL of glycerol. Then, slowly add the drug-glycerol solution to the gel matrix and stir thoroughly to obtain a topical gel.

[0054] Example 7 Preparation of external gel Preparation of gel matrix: Accurately weigh 3 g of hydroxyethyl cellulose, add it to 120 mL of deionized water, and stir until fully swollen to prepare the gel matrix.

[0055] Drug addition: Accurately weigh 0.8 g of the total flavonoid extract of Psoralea corylifolia from Example 2 and dissolve it in 15 mL of glycerol. Add the solution to the gel matrix and stir thoroughly to obtain a topical gel.

[0056] Comparative Example 1 Extraction: Accurately weigh 10g of Psoralea corylifolia powder and add 100mL of 95% ethanol. Place the mixture in an oil bath at 70°C and reflux for 2 hours.

[0057] Isolation and Purification: After extraction, filter the extract while hot. Transfer the filtrate to a rotary evaporator and concentrate under reduced pressure. The concentrated product is purified by silica gel column chromatography to obtain a total flavonoid extract from Psoralea corylifolia.

[0058] Comparative Example 2 Preparation of deep eutectic solvent: Betaine and glycerol were placed in a round-bottom flask at a molar ratio of 1:2. The round-bottom flask was placed in a constant temperature oil bath at 80°C and stirred continuously with a magnetic stirrer until a homogeneous and transparent deep eutectic solvent was formed.

[0059] Extraction: Accurately weigh 10g of Psoralea corylifolia powder and add it to 100mL of the deep eutectic solvent prepared above. Ultrasonicate the mixture at 40°C and 600W for 30 minutes.

[0060] Separation and purification: The separation and purification steps are the same as those in Example 1 to obtain the total flavonoid extract of Psoralea corylifolia.

[0061] Comparative Example 3 An external gel was prepared according to the method of Example 6, except that the total flavonoid extract of Psoralea corylifolia prepared in Example 1 was not added.

[0062] Comparative Example 4 An external gel was prepared according to the method of Example 6, except that the total flavonoids extract of Psoralea corylifolia prepared in Comparative Example 1 was used.

[0063] Test Example 1 Determination of total flavonoid content The total flavonoid content was determined using the sodium nitrite (NaNO2)-aluminum nitrate (Al(NO3)3)3) colorimetric method. To 1 mL of the sample solution, add 0.3 mL of a 5% sodium nitrite solution, shake well, and incubate for 6 minutes. Then, add 0.3 mL of a 10% aluminum nitrate solution, shake well, and incubate for 6 minutes. Then, add 4 mL of a 4% sodium hydroxide solution. Finally, the volume was adjusted to 10 mL with 50% ethanol. After shaking well for 15 minutes, the absorbance was measured at 510 nm. Rutin standard solutions with concentrations of 0.00, 0.01, 0.02, 0.04, 0.06, and 0.08 mg / mL were prepared, and a standard curve was constructed. The rutin method is based on the formation of a stable red complex between flavonoids and aluminum ions under alkaline conditions. This complex has a maximum absorption near 510 nm. The total flavonoid content (calculated as rutin) is calculated by colorimetric absorbance measurement. The relative content of total flavonoids was calculated by measuring the absorbance value of rutin in the sample, and the result was expressed as rutin equivalent per gram of psoralea corylifolia powder (mg / g).

[0064] The rutin standard curve was established as follows Figure 1 As shown, the total flavonoid content in the obtained total flavonoid extract of Psoralea corylifolia is shown in Table 1. The results show that Examples 1 to 5 can all effectively extract total flavonoids of Psoralea corylifolia, among which Example 1 has the best effect. Compared with the ethanol extraction in Comparative Example 1, the deep eutectic solvent can more effectively extract total flavonoids of Psoralea corylifolia.

[0065] Table 1 Total flavonoids content of Psoralea corylifolia extract

[0066] Test Example 2 Tyrosinase Activation Rate and Melanin Content Determination 1. Tyrosinase activation rate A 0.5 mg / mL L-tyrosine solution (solvent: phosphate buffer) was prepared, a 35 mg / mL mushroom tyrosinase solution (solvent: phosphate buffer) was prepared, and a 1 mg / mL test sample solution was prepared using propylene glycol as a solvent (Examples 1 to 5 were tested separately, with Comparative Examples 1 to 2 as a comparative test group).

[0067] Prepare four groups of solutions according to the following ratios: A: 0.7 mL of L-tyrosine solution + 0.7 mL of pure propylene glycol + 0.6 mL of phosphate buffer; B: 0.7 mL of pure propylene glycol + 1.3 mL of phosphate buffer; C: 0.7 mL of L-tyrosine solution + 0.7 mL of the test sample in propylene glycol + 0.6 mL of phosphate buffer; D: 0.7 mL of the test sample in propylene glycol + 0.6 mL of phosphate buffer. After the above four groups of solutions were kept constant at 37°C in a water bath for 10 minutes, 0.3 mL of tyrosinase solution was added to each solution. After reacting for 5 minutes, the solution was quickly transferred to a cuvette and the absorbance (OD value) was measured at 475 nm. The OD value was recorded and calculated according to the following formula: Tyrosinase activation rate = 100% × ((OD C- OD D )-(OD A -OD B )) / (OD A -OD B ) Among them, (OD A -OD B ) represents the change in OD value of the reaction solution without activator, (OD C -OD D ) represents the change in OD value of the reaction solution in the presence of an activator (Examples 1 to 5, Comparative Examples 1 to 2).

[0068] 2. Melanin content The density of the B16 melanoma cell suspension was adjusted to 1×10 51 mL was inoculated into each well of a 12-well plate to a cell density of 5 × 10 4 Each well was incubated in an incubator (37°C, 5% CO2) for 24 hours. The 12-well plate was removed, the culture medium removed, and 1 mL of culture medium containing the test sample (samples from Examples 1-5 and Comparative Examples 1-2, at a concentration of 1 mg / mL) was added. The blank control and zero-adjustment groups were incubated with 1 mL of culture medium without the test sample. The plates were incubated in the incubator for another 12 hours, after which the culture medium was removed. 1 mL of a 1 μM α-MSH solution prepared in RPMI 1640 medium was added to each well of the experimental and zero-adjustment groups. The blank control group was incubated with 1 mL of culture medium without α-MSH. The plates were incubated in an incubator for 36 hours. Each well was washed twice with 1 mL of PBS. 100 μL of a 1N NaOH solution containing 10% DMSO (prepared in PBS) was added to each well. After incubation in an 80°C water bath for 1 hour, the cell lysate from each group in the 12-well plate was transferred to a 96-well plate, and the absorbance (OD) value of each well was measured at 475 nm using a microplate reader.

[0069] The formula for calculating the intracellular melanin content is: Melanin content = 100% × (OD1-OD3) / (OD2-OD3) Among them, OD1 is the absorbance value of the experimental group, OD2 is the absorbance value of the blank control group, and OD3 is the absorbance value of the zero-adjustment group.

[0070] According to the above experimental method, the effects of the total flavonoids of Psoralea corylifolia in Examples 1 to 5 and Comparative Examples 1 to 2 on the tyrosinase activation rate and melanin content were tested respectively. The results are shown in Table 2, which shows that the total flavonoids of Psoralea corylifolia in Examples 1 to 5 of the present invention can activate tyrosinase by 21.45% to 23.12%, and increase the melanin content in B16 melanoma cells to 7.65% to 7.92%. Among them, Example 1 has the best effect, which proves that it has a significant promoting effect on the production of melanin, that is, it has a good hair blackening effect.

[0071] Table 2 Tyrosinase activation rate and melanin content

[0072] Test Example 3 Mouse Hair Growth Experiment 1. Construction of model mice After 1 week of adaptive feeding, 12 KM mice were randomly divided into 4 groups of 3 mice each: a blank group, Example 1 group, Example 6 group, and Comparative Example 4 group. On the 8th day, the hair on the back of the mice was shaved with a pet shaver, with the shaved area being 4 cm × 4 cm. After shaving, 200 μL / mouse of the drug from each group was applied to the shaved area on the back of the mice. The hair growth of each group of mice on the first and third days after hair removal was observed. One week later, the mice were sacrificed and the skin tissue at the doping site on the back of the mice was excised, wrapped in tin foil, and placed in a 5 mL EP tube containing 4% paraformaldehyde tissue fixative for pathological studies.

[0073] Hair growth results Figure 2 As shown, starting from the 3rd day, compared with the blank group and comparative example 4, the application of Example 1 and the comparative example 6 group can effectively promote the growth of hair on the back skin of mice, and the effect of Example 6 is better, indicating that the total flavonoids extracted from Psoralea corylifolia of the present invention can promote hair growth, and the prepared external gel has a better effect.

[0074] 2. Hematoxylin-eosin staining Paraffin sections embedded in mouse skin tissue were thoroughly dewaxed by immersing them in xylene I and xylene II for 15 minutes each. The sections were then rehydrated in graded ethanol (100%, 95%, 75%, and 50%) for 5 minutes each. Finally, the sections were transferred to double-distilled water and immersed for 3 minutes to completely hydrate. The hydrated sections were stained in hematoxylin solution for 1 minute and 30 seconds, followed by immediate rinsing with tap water to terminate the staining. After replacing the water, the sections were rinsed again to ensure uniform staining. The sections were differentiated by immersing them in 0.5% hydrochloric acid alcohol for several seconds (the specific time was adjusted according to the depth of the hematoxylin staining) and then immediately rinsed with tap water to terminate the differentiation. After replacing the water, the sections were rinsed under slowly running tap water for 30 to 60 minutes to promote blueing. After bluing, the sections were immersed in eosin stain for 1 minute and 20 seconds. Dehydrated through a gradient of ethanol (70%, 80%, 95% I, 95% II, 100% I, 100% II), with the specific time adjusted according to the intensity of the eosin stain. Dehydrated sections were then immersed in xylene I and then xylene II for 5 minutes each to clear them. After removing the sections, an appropriate amount of neutral gum was applied to the tissue area and the sections were covered with a coverslip, ensuring that the sections were flat and free of bubbles. After the resin dried, the location and number of hair follicles in the mice were observed under a microscope.

[0075] By HE staining the skin tissue sections, the hair follicle structure can be clearly distinguished under a microscope, thereby evaluating the hair follicle density and morphology. Figure 3As shown, starting from the 3rd day, compared with the blank group and comparative example 4, the application of Example 1 and Example 6 can effectively increase the number of hair follicles in the back skin tissue of mice, and the effect of Example 6 is better, indicating that the total flavonoids extract of Psoralea corylifolia and its gel product extracted by the present invention can promote hair growth by increasing the number of hair follicles.

[0076] 3. Toluidine blue staining Paraffin sections embedded with mouse skin tissue were immersed in xylene I and xylene II for 20 minutes each to complete the dewaxing step. Subsequently, the sections were transferred to anhydrous ethanol I and anhydrous ethanol II for 5 minutes each for further dehydration. Next, the sections were immersed in 75% alcohol for 5 minutes and finally rinsed with tap water. During the staining stage, the sections were immersed in toluidine blue dye for 2 to 5 minutes and then rinsed with tap water. Brief differentiation was performed using 0.1% glacial acetic acid, and the reaction was terminated by rinsing with tap water. The degree of differentiation was observed and controlled under a microscope to ensure appropriate staining. After differentiation was completed, the sections were rinsed again with tap water and dried in an oven. Finally, the sections were immersed in clean xylene for 10 minutes to enhance tissue transparency. After clearing, the sections were mounted with neutral gum. After the resin dried, changes in the number of mast cells in the mouse skin were observed under a microscope.

[0077] Mast cells are an important component of the skin immune system and are mainly distributed in the dermis. Their activation will release inflammatory mediators and degrade collagen, exacerbating skin inflammation, oxidative stress and aging. The number of mast cells in mouse skin can be observed by toluidine blue staining. Figure 4 As shown, starting from the 3rd day, compared with the blank group and comparative example 4, the application of Example 1 and Example 6 can effectively reduce the number of mast cells in the back skin tissue of mice, indicating that the total flavonoids extract of Psoralea corylifolia and its gel product extracted by the present invention can promote hair growth by reducing the number of mast cells and reducing inflammation.

[0078] In summary, the present invention provides a method for efficiently extracting total flavonoids from Psoralea corylifolia using deep eutectic solvents (DESs) and its application in hair growth. To address the issues of high solvent toxicity, low extraction efficiency, and high energy consumption in current traditional total flavonoid extraction processes, the present invention innovatively utilizes a deep eutectic solvent prepared from choline chloride, betaine, malic acid, oxalic acid, citric acid, glucose, and glycerol. Combined with ultrasound technology, this method significantly increases the total flavonoid content of Psoralea corylifolia. Furthermore, the extraction solvent employed in the present invention is environmentally friendly, highly efficient, cost-effective, and safe, addressing the challenges of existing extraction processes. The Psoralea corylifolia total flavonoid extract obtained by the present method promotes hair growth, significantly promoting hair follicle cell proliferation and prolonging the anagen phase. The topical gel product prepared using the extract exhibits significant hair growth efficacy. The Psoralea corylifolia total flavonoid extract provided by the present invention can be used to develop a wider range of cosmetic and pharmaceutical products for preventing and promoting hair loss, providing a new approach to the green extraction of natural active ingredients and hair loss treatment.

[0079] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. A method for preparing a total flavonoid extract of Psoralea corylifolia, characterized in that: A low eutectic solvent was prepared using a hydrogen bond donor and a hydrogen bond acceptor. The crushed psoralea corylifolia was mixed with the low eutectic solvent at a material-liquid ratio of 1:5~20 g / mL. Ultrasonic-assisted extraction was performed, and the extract was separated. The supernatant was purified, concentrated, and dried to obtain the total flavonoid extract of psoralea corylifolia.

2. The method according to claim 1, characterized in that The hydrogen bond donor is selected from one of oxalic acid, malic acid, glucose, citric acid, and glycerol, the hydrogen bond acceptor is selected from one of choline chloride and betaine, and the molar ratio of the hydrogen bond donor to the hydrogen bond acceptor is 1:1-3.

3. The method according to claim 2, characterized in that The hydrogen bond donor is selected from one of oxalic acid, malic acid, glucose, citric acid and glycerol, and the hydrogen bond acceptor is choline chloride.

4. The method according to claim 1, characterized in that The conditions for the ultrasonic-assisted extraction are: extraction temperature of 30-70° C., extraction power of 300-600 W, and extraction time of 15-60 minutes.

5. A total flavonoids extract of Psoralea corylifolia prepared by the method according to any one of claims 1 to 4.

6. Use of the total flavonoids extract of Psoralea corylifolia according to claim 5 in promoting hair growth or in preparing products promoting hair growth.

7. A product, characterized in that Contains the psoralea corylifolia total flavonoids extract according to claim 5.

8. The product according to claim 7, characterized in that: The product is a gel and also contains hydroxyethyl cellulose, glycerin and water.

9. The product according to claim 8, characterized in that The product contains, by weight, 5 to 20 parts of the total flavonoid extract of Psoralea corylifolia according to claim 5, 5 to 8 parts of hydroxyethyl cellulose, 5 to 8 parts of glycerol, and 20 to 30 parts of water.

10. The method for preparing the product according to claim 9, characterized in that: Slowly add hydroxyethyl cellulose to water in proportion and stir evenly to obtain gel matrix A; then dissolve the total flavonoid extract of Psoralea corylifolia in glycerin and stir evenly to obtain mixed liquid B; finally, slowly add the mixed liquid B to the gel matrix A and stir evenly to obtain a gel for external use.

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