Pericarpium citri reticulatae extract with hair loss preventing and hair growth promoting effects as well as preparation method and application of pericarpium citri reticulatae extract

Abstract

The invention discloses a pericarpium citri reticulatae extract with hair loss preventing and hair growth promoting effects as well as a preparation method and application of the pericarpium citri reticulatae extract. The pericarpium citri reticulatae is extracted by adopting a specific magnetic response type eutectic solvent system, the content of polymethoxylated flavonoid active substances in the prepared pericarpium citri reticulatae extract is relatively high, the pericarpium citri reticulatae extract has relatively good anti-hair-loss and hair-growing effects, the anti-inflammatory and anti-oxidation effects of the pericarpium citri reticulatae extract can be improved, and the pericarpium citri reticulatae extract has a relatively good anti-hair-loss and hair-growing effect. By inhibiting 5alpha-reductase and reducing expression of inflammatory factors such as TNF-alpha and IL-6, inflammatory reaction is relieved to relieve hair follicle damage, hair follicle cycle transformation is induced, and finally the anti-hair loss and hair growth effects are achieved. Meanwhile, under the action of an external magnetic field, the solvent and the extract can be rapidly separated without centrifugation and filtration, the preparation method is simple and convenient, and the pericarpium citri reticulatae extract can be used as an active ingredient for preventing hair loss and growing hair and can be used for preparing more anti-hair loss and hair growing products containing natural ingredients.

Description

Technical Field

[0001] The invention belongs to the technical field of daily necessities preparation, and more specifically, relates to a tangerine peel extract with the effect of preventing hair loss and promoting hair growth, and the preparation and application thereof. Background Art

[0002] Hair loss is becoming more and more serious among modern people, and is becoming younger. Hair loss can affect people's mental state, bring serious mental and psychological burdens, lead to a decline in quality of life and social communication barriers. At present, the treatment of hair loss can be divided into traditional Chinese medicine and Western medicine. Traditional Chinese medicine clinically summarizes the common causes of hair loss: essentially, it is mostly caused by internal dampness and heat, liver depression and blood deficiency, and liver and kidney deficiency. Traditional Chinese medicine can regulate the micro-molecular level by affecting the expression of inflammatory mediators, inhibiting cell apoptosis, regulating gene expression, regulating immune function, etc., control the occurrence and development of hair loss lesions, achieve a state of coordination and yin and yang balance in the body, thereby reducing hair follicle damage and ultimately achieving hair loss prevention and hair growth. Traditional Chinese medicine treatment often uses traditional Chinese medicine such as Polygonum multiflorum, Platycladus orientalis, Ligustrum lucidum, and ginger, mainly based on syndrome differentiation and treatment. In clinical practice, Western medicine often uses drugs such as finasteride and minoxidil to inhibit 5α-reductase and prevent and treat hair loss. In order to improve the effect of preventing hair loss and promoting hair growth, more and more studies are currently extracting Chinese medicinal plants that prevent hair loss and promote hair growth, preparing external anti-hair loss and hair growth shampoo products containing natural ingredients, and using them externally for daily anti-hair loss prevention to achieve long-term anti-hair loss and hair growth effects.

[0003] Tangerine peel is a commonly used Chinese medicinal material. It is the dried peel of citrus fruits with a fragrant taste and multiple medicinal values. At present, about 140 compounds have been extracted and identified from tangerine peel, including flavonoids, volatile oils and alkaloids, among which flavonoids are considered to be the most important bioactive ingredients. Tangerine peel extract is rich in polymethoxylated flavonoids, which have significant anti-inflammatory, antioxidant and anti-apoptotic activities, can promote skin metabolism, increase the thickness of the stratum corneum, and promote hair growth. Therefore, it has a certain auxiliary therapeutic effect on hair loss caused by insufficient qi and blood and excessive dandruff. Therefore, increasing the content of polymethoxylated flavonoids in tangerine peel is of great significance for the development of more Chinese medicinal active substances with anti-hair loss and hair growth effects.

[0004] Existing traditional Chinese medicine anti-hair loss products are directly prepared by compounding tangerine peel with other Chinese herbal ingredients, or using pure water or organic solvents for extraction. The extraction efficiency is low, resulting in the active substances in tangerine peel not fully exerting their effects, and the symptoms of improving hair loss are limited. The use of specific extraction methods and solvents can improve the extraction efficiency, and can also be used to specifically extract its active substances. For example, the use of specific low eutectic solvents can extract flavonoid compounds in tangerine peel, but after extraction with low eutectic solvents, there is a problem of high viscosity and difficulty in separation, easy to remain, and it will also affect the active effect of the extract. In addition, the active ingredients and contents obtained by different extraction methods and reagents are different. Therefore, it is urgent to establish an efficient, highly selective, green and environmentally friendly tangerine peel extract that is easy to operate, easy to separate, and has better anti-hair loss and hair growth effects, to provide support for the development of more anti-hair loss and hair growth products containing natural ingredients. Summary of the invention

[0005] The technical problem to be solved by the present invention is to overcome the defects of low content of polymethoxyflavonoid active substances and low extraction rate in existing tangerine peel extracts, as well as insufficient natural Chinese medicine extract products with anti-hair loss and hair growth effects, and to provide a tangerine peel extract with anti-hair loss and hair growth effects, as well as its preparation and application.

[0006] The first object of the present invention is to provide a method for preparing a tangerine peel extract having the effect of preventing hair loss and promoting hair growth.

[0007] The second object of the present invention is to provide an application of a method for preparing a tangerine peel extract having the efficacy of preventing hair loss and promoting hair growth.

[0008] The third object of the present invention is to provide a tangerine peel extract.

[0009] The fourth object of the present invention is to provide the application of tangerine peel extract.

[0010] A fifth object of the present invention is to provide a hair loss prevention and hair growth product.

[0011] The above-mentioned purpose of the present invention is achieved through the following technical solutions:

[0012] The present invention provides a method for preparing a tangerine peel extract having the effect of preventing hair loss and promoting hair growth, comprising the following steps:

[0013] S1. After crushing the tangerine peel sample, soak it in an organic solvent at a solid-liquid ratio of 1:3-10 (g / mL) for defatting, centrifuge until the upper liquid becomes colorless, and take the supernatant to obtain a sample solution;

[0014] S2. Preparation of a deep eutectic solvent DES: mixing a hydrogen bond acceptor and a hydrogen bond donor at a molar ratio of 1:2-4, and then heating and stirring to obtain a stable, clear, homogeneous liquid;

[0015] S3. The homogeneous liquid of S2 and the sample solution of S1 are mixed and then ultrasonically treated, and then a paramagnetic metal chloride is added to form a magnetic low eutectic solvent system, and the mixture is shaken for homogenization; the molar ratio of the metal chloride to the hydrogen bond acceptor and the hydrogen bond donor is (0.2-1):1:(2-4); after the extraction is completed, an external magnet is used for phase separation, and the separated liquid is the tangerine peel extract.

[0016] The present invention uses a magnetically responsive low eutectic solvent system to extract tangerine peel. The prepared tangerine peel extract has a high content of polymethoxyflavonoid active substances, has good anti-hair loss and hair growth effects, can enhance the anti-inflammatory and antioxidant effects of the tangerine peel extract, and can inhibit 5α-reductase, reduce the expression of inflammatory factors such as TNF-α and IL-6, thereby alleviating inflammatory reactions to reduce hair follicle damage, induce hair follicle cycle transformation, and ultimately achieve anti-hair loss and hair growth effects. At the same time, under the action of an external magnetic field, the tangerine peel extract can achieve rapid separation of solvent and extract without centrifugation or filtration. The preparation method is simple and convenient. As an active ingredient for anti-hair loss and hair growth, it can be used to prepare more anti-hair loss and hair growth products containing natural ingredients.

[0017] In particular, the present invention adopts a specific magnetically responsive low eutectic solvent system, which exhibits a strong response under the action of an external magnetic field by introducing magnetic components, and can achieve rapid separation of solvents and extracts without the need for centrifugation and other steps. The magnetic low eutectic solvent system prepared by the present invention forms strong hydrogen bonds and weak electrostatic interactions between hydrogen bond acceptors (HBA), hydrogen bond donors (HBD) and paramagnetic metal chloride (MC) components. The metal ions in the metal chloride can form strong interactions with the functional groups in HBA or HBD, thereby stabilizing the hydrogen bond structure and enhancing the hydrogen bonding effect. These hydrogen bonding effects can enhance the stability and solubility of the low eutectic solvent, thereby reducing the viscosity; at the same time, the metal chloride itself has magnetism, which can achieve rapid and efficient extraction and separation.

[0018] Preferably, the solid-liquid ratio of the tangerine peel to the organic solvent is 1:3-5 (g / mL).

[0019] More preferably, the organic solvent is hexane or acetone.

[0020] Preferably, the paramagnetic metal chloride is selected from FeCl3 or MnCl2.

[0021] More preferably, the metal chloride is FeCl3.

[0022] Furthermore, the water content in the low eutectic solvent is 20%-60%.

[0023] Preferably, the conditions for the ultrasonic extraction are: extraction temperature 60°C-80°C, ultrasonic power 200W-500W, and extraction time 1h-3h.

[0024] More preferably, the ultrasonic power is 250 W, the extraction temperature is 60° C., and the extraction time is 2 h.

[0025] Preferably, the centrifugation condition is 3800-6000 rpm, and the centrifugation time is 5-10 min.

[0026] Preferably, the tangerine peel is tangerine peel powder, which is obtained by grinding dried tangerine peel through a 200-mesh sieve.

[0027] The present invention provides application of the above method in preparing a product with the efficacy of preventing hair loss and promoting hair growth.

[0028] The invention provides a tangerine peel extract, which is prepared by the method.

[0029] The invention provides application of tangerine peel extract in preventing hair loss and promoting hair growth or in preparing products with the effects of preventing hair loss and promoting hair growth.

[0030] The invention provides a hair loss prevention and hair growth product containing tangerine peel extract.

[0031] Preferably, the product is a shampoo.

[0032] More preferably, the product contains 1%-15% of tangerine peel extract.

[0033] Further preferably, the product also contains the following percentages of components: 5%-25% surfactant, 0.1%-5% moisturizer, 0.1%-3% hair conditioner, 0.1%-0.8% emulsifier, 0.01%-1% emulsion stabilizer, 0.005%-0.5% fragrance, 0.01%-0.1% preservative, and the balance is water.

[0034] Preferably, the surfactant is cocamidopropyl betaine, sodium lauroamphoacetate and decyl glucoside.

[0035] Preferably, the hair conditioning agent is polyquaternium-10 and jojoba oil. Polyquaternium-10 can help hair become smoother, and jojoba oil can effectively promote the absorption of anti-hair loss ingredients by hair follicles.

[0036] Preferably, the humectant is glycerol.

[0037] Preferably, the preservative is potassium sorbate.

[0038] Preferably, the emulsifier is glycerol oleate.

[0039] Preferably, the emulsion stabilizer is sodium chloride.

[0040] In addition, the present invention provides a method for preparing the anti-hair loss and hair growth product as follows:

[0041] S1. The surfactant and hair conditioner are heated to 70-90 ° C and stirred for 20-60 min, mixed evenly, kept warm for 30 min and then cooled to 30-45 ° C to obtain a mixture A;

[0042] S2. The moisturizer, emulsifier, emulsion stabilizer and pure water are mixed and heated to 30-50°C, stirred for 20-45 minutes, and mixed evenly to obtain a mixture B;

[0043] S3. Evenly mix mixture A, mixture B, essence, preservative and the tangerine peel extract solution that has been left to stand to obtain an anti-hair loss and hair growth product.

[0044] The present invention has the following beneficial effects:

[0045] The present invention extracts and prepares tangerine peel by a magnetically responsive low eutectic solvent system, and the extraction rate of polymethoxyflavonoid active substances in the prepared tangerine peel extract is significantly improved, and its effect is better than that after extraction with traditional low eutectic solvents and alcohol solvents, and has good anti-hair loss and hair growth effects, and further improves the anti-inflammatory and antioxidant effects of the tangerine peel extract, and alleviates hair follicle damage by inhibiting 5α-reductase and reducing the expression of inflammatory factors such as TNF-α and IL-6, thereby alleviating inflammatory reactions, inducing hair follicle cycle transformation, and finally achieving anti-hair loss and hair growth effects; this extract can be used as an active ingredient to prepare more anti-hair loss and hair growth products. At the same time, a magnetic component is introduced into the extraction system, and a strong response is shown under the action of an external magnetic field. Without the steps of centrifugation and filtration, the solvent and the extract can be quickly separated, and the system is stable and reusable, reducing the extraction cost. The method provided by the present invention is simple to operate and provides a new idea for the rapid separation of tangerine peel extract. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 This is a graph showing the test results of the reusability performance of MDES.

[0047] Figure 2 This is a graph showing the results of a 5α-reductase inhibition experiment. DETAILED DESCRIPTION

[0048] The present invention is further described below in conjunction with the accompanying drawings and specific examples, but the examples do not limit the present invention in any form. Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0049] Unless otherwise specified, the reagents and materials used in the following examples are commercially available.

[0050] Example 1 Preparation of Tangerine Peel Extract

[0051] 1. Pretreatment of tangerine peel

[0052] 50 g of dried tangerine peel was crushed, ultrasonically treated in 150 mL of hexane for 5 min, and centrifuged at 6000 rpm for 5 min, which was repeated 3 times until the upper liquid was colorless, and then the supernatant (sample solution) was separated for further analysis.

[0053] 2. Preparation of magnetic deep eutectic solvent (MDES) and extraction of active substances from tangerine peel

[0054] DES was prepared by mixing acetic acid and benzalkonium bromide (dodecyl dimethyl benzyl ammonium bromide) in a molar ratio of 2:1 and heating at 80°C for 30 minutes under constant stirring to obtain a stable homogeneous liquid, which was sealed and stored. 10 mL of DES was added to the sample solution and ultrasonicated for 4 minutes to enrich the analyte in the DES phase. After adsorption, 0.3 g of ferric chloride was carefully weighed and added to the solution to form MDES in situ, and then shaken to homogenize for 5 minutes to form MDES1 (benzalkonium bromide / acetic acid [FeCl4]), and then the MDES adsorbed with the target substance was collected by centrifugation, and the DES phase was collected using an external magnet (neodymium magnet) to obtain the tangerine peel extract.

[0055] Example 2 Preparation of Tangerine Peel Extract

[0056] The steps for preparing the tangerine peel extract are the same as those of Example 1, with the only difference being that pentaerythritol is used as the hydrogen bond donor to obtain MDES2 (benzalkonium bromide / pentaerythritol [FeCl4]).

[0057] Example 3 Preparation of Tangerine Peel Extract

[0058] The steps for preparing the tangerine peel extract are the same as those in Example 1, except that tryptophan is used as the hydrogen bond donor to obtain MDES3 (benzalkonium bromide / tryptophan [FeCl4])

[0059] Example 4 Preparation of Anti-hair Loss Shampoo

[0060] S1. 25% of the surfactants cocamidopropyl betaine, sodium lauroamphoacetate and decyl glucoside (mass ratio 2:2:1) and 3% of the hair conditioning agent polyquaternium-10 and jojoba oil (mass ratio 2:1) were heated to 80°C and stirred for 60min, mixed evenly, kept warm for 30min and then cooled to 30°C to obtain a mixture A;

[0061] S2. 0.5% of humectant glycerol, 0.8% of emulsifier glyceryl oleate, 1% of emulsifier stabilizer sodium chloride and the remaining pure water solvent are mixed and heated to 30-50°C, stirred for 20-45min, and mixed evenly to obtain a mixture B;

[0062] S3. Mix mixture A, mixture B, 0.01% flavor, 0.1% preservative potassium sorbate and the 5% tangerine peel extract solution (Example 1) that has been allowed to stand, and obtain an anti-hair loss and hair growth shampoo product.

[0063] Comparative Example 1 Preparation of Tangerine Peel Extract Using Traditional Deep Eutectic Solvent

[0064] The steps for preparing the tangerine peel extract are the same as those in Example 1, except that FeCl3 is not added to form MDES.

[0065] Comparative Example 2 Preparation of Traditional Alcohol Extract of Tangerine Peel

[0066] Weigh dried tangerine peel, add 75% ethanol solution at a solid-liquid ratio of 1:10 (g / mL), heat in a water bath at 80°C for 2 h, extract twice, and then filter and rotary evaporate to obtain tangerine peel alcohol extract.

[0067] Test Example 1 Effect of different extraction methods on the extraction rate of polymethoxylated flavonoids in tangerine peel

[0068] The UPLC-Q-TOF-MS method was used to determine the content of polymethoxylated flavonoids in the tangerine peel extracts prepared in Examples 1 to 3 and Comparative Examples 1 to 2, and the sum of the contents of 9 polymethoxylated flavonoids PMFs (nobiletin, tangeretin, isocitronellal, tangeretin, 5,7,3,4'-tetramethoxyflavonoids, 5,6,7,4'-tetramethoxyflavonoids, 3,5,6,7,8,3,4'-heptamethoxyflavonoids, 5,7,4'-trimethoxyflavonoids, and 5-hydroxy-6,7,8,3',4'-pentamethoxyflavonoids) was taken as the total polymethoxylated flavonoids content (TPMF), and the extraction rate of polymethoxylated flavonoids was calculated.

[0069] Instrument condition setting: UPLC detection used ACQUITY UPLC BEH C18 column (2.1×100 mm, 1.7 μm, Waters, USA), with 0.1% formic acid aqueous solution (A) and methanol (B) as mobile phases, gradient elution, and separation conditions were as follows: 0-0.6 min, 10%-20% B; 0.6-5.0 min, 20%-70% B; 5.0-7.0 min, 70%-90% A; flow rate: 0.4 mL / min; column temperature 40°C; injection volume 1 μL; detection wavelength: 330 nm.

[0070] Mass spectrometry detection was carried out in the positive ionization mode of Q-TOF-MS / MS, and the mass spectrometry conditions were as follows: ionization mode was ESI, capillary voltage was 0.45 kV; cone voltage was 40 V; ion source temperature was 100 °C; desolvation gas temperature was 400 °C; cone gas flow rate was 50 L / h; desolvation gas flow rate was 600 L / h; low energy voltage was 6.0 V; high energy voltage was 20.0-40.0 V; scanning range was from 100 m / z to 1000 m / z; scanning time was 0.2 s.

[0071] The measurement results are shown in Table 1, which show that after using different extractants to extract tangerine peel, the content of its active substance polymethoxylated flavonoids has obvious differences. The effects of extraction using traditional low eutectic solvents (Comparative Example 1) and traditional alcohol solvents are not much different. However, after extraction using a magnetic low eutectic solvent system, the content and extraction rate of polymethoxylated flavonoids in tangerine peel can be significantly improved. At the same time, the introduction of magnetic components into the low eutectic solvent facilitates the recovery of the solvent, increases the effective content of the extract, and exhibits a strong response under the action of an external magnetic field. Rapid separation of the solvent and the extract can be achieved without the need for centrifugation, filtration, and other steps.

[0072] Table 1 Extraction results of polymethoxylated flavonoids in dried orange peel by different extractants

[0073]

[0074] Test Example 2 Effect of the Stoichiometry of Components in the Magnetic Deep Eutectic Solvent on the Extraction Rate

[0075] On the basis of Example 1, when preparing the magnetic deep eutectic solvent, different molar ratios of HBA, HBD and FeCl3 were set respectively for preparation, and the remaining preparation methods and steps were the same as those in Example 1. The extraction rate of polymethoxylated flavonoids was measured and counted with reference to the detection method of Test Example 1. The results are shown in Table 2, which shows that the molar ratio of HBA:HBD:MC in the magnetic deep eutectic solvent has a high polymethoxylated flavonoid extraction rate in the range of 2.32-2.96% in the range of 1:2-4:0.2-1.

[0076] Table 2 Effect of different molar ratios of components on the extraction rate of polymethoxyflavonoids

[0077] Group HBA:HBD:MC Extraction rate of polymethoxyflavonoids Case 1 1:2:0.2 2.32％ Case 2 1:2:0.4 2.41％ Case 3 1:2:0.6 2.76％ Case 4 1:2:0.8 2.96％ Case 5 1:2:1 2.68％ Case 6 1:3:0.8 2.86％ Case 7 1:4:0.8 2.56％

[0078] Test Example 3 Repeated Performance Test

[0079] The solvent MDES of the tangerine peel extract prepared in Example 2 was adsorbed and recovered through multiple recovery adsorption experiments and then repeatedly extracted. Then, the content of polymethoxylated flavonoids in the tangerine peel extract was determined. The determination method was the same as that in Test Example 1. 10 μL was injected under the same chromatographic conditions to verify its reusability.

[0080] The results are as follows Figure 1 As shown in Table 3, the recovery rate of MDES and the content of polymethoxyflavonoids extracted from tangerine peel did not decrease significantly after 5 repeated uses, and the monomer components in the extract did not show a significant downward trend as the number of repeated uses of MDES increased, indicating that the MDES prepared by the present invention is stable and reusable.

[0081] Table 3 Recovery adsorption results of solvent MDES

[0082] Cycle times PMFs recovery rate 1 81.23％ 2 80.86％ 3 76.62％ 4 74.02％ 5 69.82％

[0083] Test Example 4 Effect of Tangerine Peel Extract

[0084] 1. DPPH removal effect

[0085] On the same day, 0.2 mmol / L DPPH solution was prepared, 200 μL of the test solution (the tangerine peel extract prepared in Example 1-3 and Comparative Example 1-2) was added to 800 μL of DPPH solution, and the mixture was incubated at room temperature in the dark for 30 and 60 min, respectively. The absorbance (A value) was measured at a wavelength of 517 nm. The test was repeated 3 times, and the A value was counted and the DPPH radical scavenging rate was calculated by the following formula.

[0086] DPPH free radical scavenging rate = 1-(A sample-A blank) / A control × 100%

[0087] The test results are shown in Table 4, which show that the tangerine peel extract prepared in Examples 1-3 has better DPPH radical scavenging effect than that in Comparative Examples 1-2, and has better antioxidant properties, which corresponds to the change in the content of polymethoxylated flavonoids in the tangerine peel extract; the use of MDES has a better effect than the traditional DES and alcohol solvent extraction of tangerine peel extract, and improves the antioxidant effect of the tangerine peel extract.

[0088] Table 4 DPPH scavenging effect of different tangerine peel extracts

[0089] Group DPPH free radical scavenging rate Example 1 32.25％ Example 2 30.29％ Example 3 28.42％ Comparative Example 1 14.42％ Comparative Example 2 10.22％

[0090] 2. 5α-reductase inhibitory effect

[0091] Take 0.5mL phosphate buffer, 0.1mL test solution (Example 1-3, Comparative Example 1-2 prepared tangerine peel extract), 200μL testosterone solution (300mg / L) and 200μL NADPH solution (0.8g / L), add 0.5mL 5α-reductase extract, react at 37°C for 30min, add 2.5mL dichloromethane to stop the reaction and extract testosterone, then add 0.25mL propyl hydroxybenzoate (100mg / L) as internal standard, centrifuge at 3000r / min for 10min. Remove the upper aqueous phase, remove about 1mL of the organic phase and evaporate to dryness, dissolve the residue in 1.5mL methanol, and take 10μL to measure the residual testosterone with high performance liquid chromatography. At the same time, set the 5α-reductase reaction tube and the 5α-reductase blank tube, take 0.05mg / L finasteride as the positive control group, and calculate the 5α-reductase inhibition rate according to the following formula.

[0092] 5α-reductase inhibition rate = (R sample - R reaction) / (R blank - R reaction) × 100%

[0093] Where R is the ratio of the target peak area to the internal standard peak area.

[0094] The results of the test are as follows Figure 2 As shown in Table 5, 5α-reductase accelerates the conversion of androgen, causing hair follicle degeneration and hair loss on the head, while inhibiting 5α-reductase can prevent the progression of hair follicle shrinkage and hair loss by reducing the production of dihydrotestosterone (DHT), and has a certain anti-hair loss activity. The tangerine peel extract prepared in Example 1-3 has a better 5α-reductase inhibitory effect than that in Comparative Example 1-2, and has a better anti-hair loss activity.

[0095] Table 5 5α-reductase inhibition effect of different tangerine peel extracts

[0096]

[0097] Test Example 5 Inflammatory Cytokine Determination

[0098] The occurrence of inflammation can cause an immune response in the local skin, producing a series of cytokines and chemical mediators that can directly act on the hair follicles, interfering with the normal growth cycle of the hair, causing the hair to enter the resting phase or fall out prematurely. The active ingredients in tangerine peel also have anti-inflammatory effects, which can be determined by measuring the content of inflammatory cytokines TNF-α, IL-6, and IL-β.

[0099] The cells used in the experiment were RAW264.7 (mouse mononuclear macrophages). An appropriate amount of activated RAW264.7 cells was taken and 5×10 4The cells were inoculated in a 24-well plate at a density of cells / well, complete medium was added to the blank group, LPS with a final mass concentration of 1 μg / mL was added to the LPS group (model group), and tangerine peel extract (tangerine peel extract prepared in Example 1-3 and Comparative Example 1-2) with a final mass concentration of 10, 50, 250, 500, and 1000 μg / mL, 500 and 1000 μg / mL of polymethoxyflavonoids and 1 μg / mL of LPS were added to the drug-treated groups, respectively, so that the total volume of each group was 3 mL, and the cells were placed in a cell culture incubator and cultured for 24 hours, and then the supernatant was taken and Griess reagent was added, and the OD value was detected at a wavelength of 540 nm using a microplate reader, and the contents of TNF-α, IL-6, and IL-β in each group were detected by referring to the instructions of the ELISA kit and the transforming growth factor (TGF) kit.

[0100] The measurement results are shown in Table 6, which show that the tangerine peel extract prepared in Example 1-3 has a better effect of reducing the content of inflammatory factors TNF-α, IL-6, and IL-β than Comparative Example 1-2. Compared with the model group, it can significantly reduce the content of TNF-α, IL-6, and IL-β, and has a better anti-inflammatory effect.

[0101] Table 6 Contents of inflammatory factors TNF-α, IL-6, and IL-β

[0102] Group TNF-α(pg / ml) IL-6 (pg / ml) IL-β (pg / ml) Example 1 37.63±3.75 80.93±8.62 57.48±8.69 Example 2 46.89±5.27 89.59±4.26 79.85±4.86 Example 3 49.93±3.53 92.66±9.36 88.93±6.76 Comparative Example 1 57.93±4.98 107.33±9.75 106.94±3.68 Comparative Example 2 59.03±6.79 127.93±8.66 117.36±8.85 Blank group 9.89±2.26 29.36±6.86 30.56±8.29 Model Group 69.03±6.42 158.24±6.12 201.56±6.89

[0103] Test Example 6 Mouse hair growth test

[0104] The anti-hair loss effect of the composite active substance was evaluated using a model C57BL / 6 mouse. C57BL / 6J male mice were divided into 4 groups, namely a blank group (distilled water), a positive control group (smeared with 5% minoxidil tincture solution), Example 1 (1000 μg / mL), and Comparative Example 1-2 (1000 μg / mL), with 6 mice in each group.

[0105] First, use an animal depilatory to shave both sides of the back of all mice, and then apply depilatory cream to remove the hair in an area of ​​about 2cm×3cm, so that the hair growth of C57BL / 6J mice is in the resting phase (the skin is pink), that is, the C57BL / 6J mouse hair loss model is established. The application amount is 200μL / mouse, twice a day (6h interval), and the skin and hair condition of the back of the mice are observed. The time when the skin starts to turn black from pink is recorded as T1, and the time when the skin starts to grow from black to hair is recorded as T2. The application is continued for 18 days.

[0106] The change of skin color of mice can reflect the different growth cycles of hair. When the skin is pink, the hair follicles are in the resting phase. When the hair follicles enter the growth phase from the resting phase, the melanocytes in the hair follicles secrete melanin to make the skin black. The statistical results of the time of hair growth on the skin of mice after different treatments are shown in Table 7. It can be seen from the results that the blank control group mice enter the hair follicle growth phase at about 12 days, and begin to grow hair at about 15 days; compared with the blank control group, the number of days for the positive control group, embodiment 1, comparative example 1, and comparative example 2 mice to enter the hair follicle growth phase is about 6 days, 7 days, 9 days and 10 days respectively, and the time for the mice in the comparative example to enter the hair follicle growth phase is longer. The time for the hair growth in the hair removal area of ​​the positive control group, embodiment 1, comparative example 1, and comparative example 2 mice is about 8 days, 9 days, 12 days and 13 days respectively, showing that the time for the hair growth in the hair removal area of ​​the embodiment 1 group is shorter than that of the comparative example, which has a better effect of promoting hair growth in mice.

[0107] Table 7 Statistics of hair growth time of mouse skin after different treatments

[0108] Group T1 Skin graying time / d T2 Skin hair growth time / d Blank control group 12.42±0.35 15.34±1.53 Positive control group 6.42±1.14 8.45±2.54 Example 1 7.35±0.56 9.12±2.45 Comparative Example 1 9.12±1.23 12.21±2.52 Comparative Example 2 10.52±2.73 13.33±1.58

[0109] In summary, the present invention uses a specific magnetically responsive low eutectic solvent system to extract tangerine peel, and the prepared tangerine peel extract has a high content of polymethoxyflavonoid active substances, has good anti-hair loss and hair growth effects, can enhance the anti-inflammatory and antioxidant effects of tangerine peel extract, and can inhibit 5α-reductase, reduce the expression of inflammatory factors such as TNF-α and IL-6, thereby alleviating inflammatory reactions to reduce hair follicle damage, induce hair follicle cycle changes, and ultimately achieve anti-hair loss and hair growth effects. At the same time, under the action of an external magnetic field, the prepared tangerine peel extract does not need centrifugation or filtration, and can achieve rapid separation of solvent and extract. The preparation method is simple and convenient. As an active ingredient for anti-hair loss and hair growth, it can be used to prepare more anti-hair loss and hair growth products containing natural ingredients.

[0110] 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 equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. A method for preparing a tangerine peel extract having the effect of preventing hair loss and promoting hair growth, characterized in that: The following steps are involved: S1. After crushing the tangerine peel sample, soak it in an organic solvent at a solid-liquid ratio of 1:3-10 (g / mL) for defatting, centrifuge until the upper liquid becomes colorless, and take the supernatant to obtain a sample solution; S2. Preparation of a deep eutectic solvent DES: mixing a hydrogen bond acceptor and a hydrogen bond donor at a molar ratio of 1:2-4, and then heating and stirring to obtain a stable, clear, homogeneous liquid; S3. The homogeneous liquid of S2 and the sample solution of S1 are mixed and then ultrasonically treated, and then a paramagnetic metal chloride is added to form a magnetic low eutectic solvent system, and the mixture is shaken for homogenization; the molar ratio of the metal chloride to the hydrogen bond acceptor and the hydrogen bond donor is (0.2-1):1:(2-4); after the extraction is completed, an external magnet is used for phase separation, and the separated liquid is the tangerine peel extract.

2. The method according to claim 1, characterized in that: The hydrogen bond acceptor is benzalkonium bromide (dodecyl dimethyl benzyl ammonium bromide), and the hydrogen bond donor is selected from one of tryptophan, pentaerythritol and acetic acid.

3. The method according to claim 1, characterized in that: The conditions of the ultrasonic extraction are: extraction temperature 60°C-80°C, ultrasonic power 200W-500W, and extraction time 1h-3h.

4. The method according to claim 1, characterized in that: The paramagnetic metal chloride is selected from one or more of FeCl3 and MnCl2.

5. Use of the method according to any one of claims 1 to 4 in preparing a product having the efficacy of preventing hair loss and promoting hair growth.

6. A tangerine peel extract, characterized in that The method is prepared by any one of claims 1 to 4.

7. Use of the tangerine peel extract according to claim 6 in preventing hair loss and promoting hair growth or in preparing products with the efficacy of preventing hair loss and promoting hair growth.

8. An anti-hair loss and hair growth product, characterized in that: Containing the tangerine peel extract according to claim 6.

9. The product according to claim 8, characterized in that: The product is a shampoo containing 1%-15% of the tangerine peel extract according to claim 6.

10. The product according to claim 9, characterized in that: The invention also contains the following components in percentages: 5%-30% surfactant, 0.1%-5% hair conditioner, 0.1%-1% emulsifier, 0.5%-2% emulsion stabilizer, 0.1%-0.5% humectant, 0.005%-0.5% fragrance, 0.01%-0.2% preservative, and the balance is water.

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