Oak leaf extract as well as extraction method and application thereof

Abstract

The invention discloses an oak leaf extract as well as an extraction method and application thereof, and belongs to the technical field of oak leaf extracts. The extraction method comprises the following steps: crushing oak leaves, dissolving the crushed oak leaves in ethanol, and carrying out heating reflux extraction to obtain an extracting solution; concentrating the extracting solution to obtain a concentrated solution; extracting the concentrated solution with n-hexane, diethyl ether, ethyl acetate and water-saturated n-butyl alcohol in sequence to obtain a water-saturated n-butyl alcohol extract; and concentrating and drying the extract to obtain the oak leaf extract. Active substances are extracted from oak leaves, active ingredients capable of inhibiting prostaglandin D synthetase are screened out, the oak leaf extract can inhibit the prostaglandin D synthetase and inhibit generation of prostaglandin D2 in the body, has good anti-hair-loss and hair-growing effects, and can be used for finding out active ingredients which are reasonable in price, stable in curative effect and low in cost. The product for treating alopecia can also increase the income of a silkworm farm and improve the utilization rate of the silkworm farm.

Description

Technical Field

[0001] The invention belongs to the technical field of oak leaf extracts, and in particular relates to an oak leaf extract and an extraction method and application thereof. Background Art

[0002] Oak (Quercus) is a plant of the genus Quercus in the Fagaceae family. Oak leaves contain water, protein, carbohydrates, fat, ash, cellulose and other ingredients, and oak silkworms like to eat them. In addition to using its leaves to feed oak silkworms, the wood of oak is strong and rot-resistant, and is widely used in construction. It can also be processed to make furniture and burn charcoal. Oaks contain a lot of starch and can be used to make oak wine, alcohol, starch, rubber oil, etc., and can also be used as feed. Tannin extracted from oak bark, leaves, cupules, and oaks is a necessary material for the leather industry, printing and dyeing industry, and fisheries; the cork bark is thicker and can be used as industrial cork material; oak can also be used to cultivate a variety of edible fungi such as black fungus, shiitake mushrooms, and cordyceps militaris.

[0003] Hair loss, or alopecia, is a common hair disease. There are many types of hair loss, such as congenital alopecia, acquired alopecia, scarring alopecia, alopecia areata and seborrheic alopecia. The most common type is seborrheic alopecia, followed by alopecia areata. Medically speaking, hair loss is a relatively common skin disease. Although it does not endanger the life and survival of patients, it seriously affects the appearance of patients, causing excessive psychological burden on patients, making them in a very disadvantageous position in the fierce social competition and talent competition. At present, the main drugs for the treatment of hair loss in clinical practice are finasteride and minoxidil. However, it is reported that finasteride can cause side effects such as loss of libido and impotence, while the use of minoxidil is associated with irritant dermatitis, allergic contact dermatitis, headache and hypotension. Natural active ingredients are regarded as substances with broad application prospects because of their low cost, safety and small side effects. Therefore, it is particularly important to provide a safe, effective and low-cost natural active ingredient that can effectively promote hair growth and has good anti-hair loss and hair growth effects. Summary of the invention

[0004] In view of this, the object of the present invention is to provide a method for extracting an oak leaf extract, wherein the oak leaf extract obtained can inhibit the production of prostaglandin D2 and has good anti-hair loss and hair growth effects.

[0005] Another object of the present invention is to provide an oak leaf extract obtained by extracting the oak leaf extract using the above-mentioned method.

[0006] Another object of the present invention is to provide a use of the oak leaf extract in the preparation of a product for promoting hair growth, promoting hair regeneration or preventing and / or treating hair loss.

[0007] Another object of the present invention is to provide a preparation for promoting hair growth.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:

[0009] The present invention provides a method for extracting an oak leaf extract, comprising the following steps:

[0010] The oak leaves are crushed, dissolved in ethanol, and extracted by heating under reflux to obtain an extract; the extract is concentrated to obtain a concentrated solution; the concentrated solution is extracted with n-hexane, ether, ethyl acetate and water-saturated n-butanol in sequence to obtain a water-saturated n-butanol extract; the extract is concentrated and dried to obtain an oak leaf extract.

[0011] Preferably, the ethanol is an ethanol solution with a volume fraction of 60% to 80%.

[0012] Preferably, the mass volume ratio of the oak leaves and ethanol is 3g:5-15mL.

[0013] Preferably, the heating reflux extraction conditions are: heating reflux at 60-70° C. for 1-5 h.

[0014] Preferably, the volume ratio of the concentrated solution to n-hexane is 1:0.5-2; the volume ratio of the concentrated solution to ether is 1:0.5-2; the volume ratio of the concentrated solution to ethyl acetate is 1:0.5-2; the volume ratio of the concentrated solution to water-saturated n-butanol is 1:0.5-2.

[0015] The invention also provides an oak leaf extract obtained by the extraction method.

[0016] The present invention also provides an application of the oak leaf extract in a product for promoting hair growth and / or promoting hair regeneration and / or preventing and / or treating hair loss.

[0017] Preferably, the oak leaf extract promotes hair growth, hair regeneration or prevents and / or treats hair loss by inhibiting the production of prostaglandin D2.

[0018] Preferably, the hair loss comprises androgenic alopecia.

[0019] The invention also provides a preparation for promoting hair growth, which comprises the oak leaf extract.

[0020] Preferably, the dosage form of the preparation includes solution, ointment, plaster, cream or patch.

[0021] Beneficial effects of the present invention:

[0022] The invention uses ethanol to extract oak leaves, and uses n-hexane, ether, ethyl acetate and water-saturated n-butanol to extract in sequence, obtains a water-saturated n-butanol extract, and obtains an oak leaf extract after concentration and drying. The oak leaf extract obtained by the invention has low cytotoxicity and can promote the proliferation of human hair papilla cells (HHDPCs). At the same time, prostaglandin D2 (PGD2) is generated by the isomerization of unstable intermediate prostaglandin H2 (PGH2) catalyzed by prostaglandin D synthase (PGDS). The oak leaf extract of the invention can inhibit the expression of prostaglandin synthase LPGDS and G protein coupled receptor 44 (GPR44) genes, thereby reducing the release of PGD2 in HHDPCs cells; the oak leaf extract of the invention can also promote the expression of cyclin D1 protein, inhibit the expression of p27 protein, and promote the growth and proliferation of HHDPCs cells by regulating the expression of cell cycle-related proteins, thereby playing an important role in hair growth and hair regeneration, and having a good effect on preventing and treating hair loss.

[0023] Silkworm farms are rich in resources, green and pollution-free. Oak trees, oak silkworms and silk all have biological significance. It is very necessary to develop and utilize silkworm farm resources to develop products. The present invention extracts active substances from oak leaves, screens out active ingredients that can inhibit PGDS, and these natural active ingredients can inhibit prostaglandin D synthase and inhibit the generation of prostaglandin D2 in the body, and have good anti-hair loss and hair growth effects. Not only can we find hair loss products with reasonable prices, stable efficacy and significant effects, but we can also increase the income of silkworm farms and improve the utilization rate of silkworm farms. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 The effect of oak leaf extract on cell activity in Experimental Example 1;

[0025] Figure 2 The effect of oak leaf extract on the release of PGD2 in HHDPCs cells in Experimental Example 1;

[0026] Figure 3 The effect of oak leaf extract QML4 on the gene expression of P27, Cyclin D1, HPGDS, LPGDS and GPR44 in HHDPCs cells in Experimental Example 1;

[0027] Figure 4 The effect of oak leaf extract QML4 on HHDPCs cell growth in Experimental Example 1;

[0028] Figure 5 The scratch healing efficiency of HHDPCs cells after being treated with the oak leaf extract QML4 in Experimental Example 1;

[0029] Figure 6 The results and quantification of Western blot of P27, Cyclin D1, HPGDS, LPGDS and GPR44 protein expression in HHDPCs cells after treatment with the oak leaf extract QML4 in Experimental Example 1;

[0030] Figure 7 The effect of the oak leaf extract QML4 on hair growth in mice in Experimental Example 1;

[0031] Figure 8 It is the cross-section of the mouse skin in Experimental Example 1 and the hair growth effect of the mouse under a stereo microscope; a and b are the cross-section of the mouse skin, and c and d are the hair growth effect of the mouse under a stereo microscope. DETAILED DESCRIPTION

[0032] The present invention provides a method for extracting an oak leaf extract, comprising the following steps:

[0033] The oak leaves are crushed, dissolved in ethanol, and extracted by heating under reflux to obtain an extract; the extract is concentrated to obtain a concentrated solution; the concentrated solution is extracted with n-hexane, ether, ethyl acetate and water-saturated n-butanol in sequence to obtain a water-saturated n-butanol extract; the extract is concentrated and dried to obtain an oak leaf extract.

[0034] In the present invention, oak leaves are crushed, dissolved in ethanol, and heated under reflux extraction to obtain an extract. The present invention does not specifically limit the source of the oak leaves, which can be conventionally selected according to actual needs. Before the oak leaves are crushed, they can be dried; the drying method is a conventional method in the field. As an implementable method, natural drying can be selected for drying. The method and particle size of the oak leaves are not specifically limited, which can be conventionally selected according to actual needs. The ethanol is preferably an ethanol solution with a volume fraction of 60% to 80%, more preferably an ethanol solution with a volume fraction of 70% or 65% to 70% or 70% to 75%. The mass volume ratio of the oak leaves and ethanol is preferably 3g:5 to 15mL, more preferably 3g:7mL or 3g:6 to 7mL or 3g:7 to 10mL. In the present invention, as an embodiment, before the heating reflux extraction, cold soaking treatment is also included; the temperature of the cold soaking treatment is preferably 15-25°C, more preferably 20°C or 18-20°C or 20-22°C, and the time of the cold soaking treatment is preferably 12-24h, more preferably 12-16h or 16-18h. The temperature of the heating reflux extraction is preferably 60-70°C, more preferably 65°C or 63-65°C or 65-67°C; the time of the heating reflux extraction is preferably 1-5h, more preferably 3h or 2-3h or 3-4h. As an embodiment, after the heating reflux extraction, the extract is filtered under reduced pressure to obtain the extract. In the present invention, in order to extract the active ingredients in the oak leaves to the maximum extent, the above operation can be repeated for multiple extractions according to actual needs, and the number of repetitions is preferably 2-4 times, more preferably 3 times, and the extracts obtained each time are combined.

[0035] In the present invention, the extract is concentrated to obtain a concentrated solution. The concentration method can be a conventional concentration method. As an optional embodiment, the extract is concentrated under reduced pressure to obtain a concentrated solution. The concentrated volume is preferably 1 / 5 to 1 / 2 of the volume of the extract, and more preferably 1 / 3.

[0036] In the present invention, the concentrate is extracted with n-hexane, ether, ethyl acetate and water-saturated n-butanol in sequence to obtain a water-saturated n-butanol extract; the extract is concentrated and dried to obtain an oak leaf extract. The volume ratio of the concentrate to n-hexane is preferably 1:0.5-2, more preferably 1:1 or 1:0.8-1 or 1:1-1.5; the volume ratio of the concentrate to ether is preferably 1:0.5-2, more preferably 1:1 or 1:0.8-1 or 1:1-1.5; the volume ratio of the concentrate to ethyl acetate is preferably 1:0.5-2, more preferably 1:1 or 1:0.8-1 or 1:1-1.5; the volume ratio of the concentrate to water-saturated n-butanol is preferably 1:0.5-2, more preferably 1:1 or 1:0.8-1 or 1:1-1.5. As an implementation method, n-hexane is added to the concentrated solution for extraction, and after removing the n-hexane layer, ether is added to the remaining water layer for extraction, and after removing the ether layer, ethyl acetate is added to the remaining water layer for extraction, and after removing the ethyl acetate layer, water-saturated n-butanol is added to the remaining water layer for extraction to obtain a water-saturated n-butanol extract. The concentration and drying method can be conventionally selected according to actual needs.

[0037] The invention also provides an oak leaf extract obtained by the extraction method.

[0038] The present invention uses ethanol heating reflux extraction, and uses n-hexane, ether, and ethyl acetate for extraction in sequence, removes the components soluble in n-hexane, ether, and ethyl acetate, and then uses water-saturated n-butanol for extraction, and the obtained extract is concentrated and dried to obtain an oak leaf extract. The oak leaf extract extracted by the extraction method of the present invention has low cytotoxicity, can promote the proliferation of HHDPCs cells, and can inhibit the expression of prostaglandin synthase LPGDS and receptor gene GPR44, thereby reducing the generation of PGD2 in HHDPCs cells; the oak leaf extract of the present invention can also promote the expression of Cyclin D1 protein, inhibit the expression of p27 protein, and promote the growth and proliferation of HHDPCs cells by regulating the expression of cell cycle-related proteins, thereby playing an important role in hair growth and hair regeneration, and having a good effect on preventing and treating hair loss.

[0039] The present invention also provides an application of the oak leaf extract in preparing a product for promoting hair growth, promoting hair regeneration or preventing and / or treating hair loss.

[0040] Studies have shown that prostaglandins (PG) can regulate hair growth in mice; in contrast, prostaglandin D2 (PGD2) inhibits hair growth in humans and mice. PGD2 is elevated in the scalp of patients with androgenic alopecia and functionally inhibits hair elongation through its receptor GPR44. In addition, exogenous PGD2 inhibits hair growth in mouse and human hair follicle explants through the PGD2-GPR44 signaling pathway. These results suggest that there is a regulatory network in hair follicles and that PGD2 can inhibit hair follicle function. PGD2 is produced by the isomerization of the unstable intermediate prostaglandin H2 (PGH2) catalyzed by prostaglandin D synthase (PGDS). In other words, inhibiting prostaglandin D synthase can inhibit the production of PGD2, and inhibiting the production of prostaglandin D2 can prevent hair loss and promote hair growth. Brain-type PGDS (LPGDS), also known as glutathione-independent PGDS, is a small molecular weight secretory protein that is widely present in the extracellular fluid of the body. The oak leaf extract of the present invention can inhibit the expression of prostaglandin synthase LPGDS and GPR44 genes, thereby reducing the generation of PGD2, thereby achieving the effect of promoting hair growth and hair regeneration, and has a good effect on preventing or treating hair loss. The oak leaf extract of the present invention plays the role of promoting hair growth, hair regeneration or preventing and / or treating hair loss by inhibiting the generation of prostaglandin D2. In the present invention, the hair loss preferably includes androgenic alopecia.

[0041] In the present invention, there is no special limitation on the type of product, including but not limited to medicines, cosmetics, etc.; the product can be in any form, including solutions, oils, essences, creams, ointments, gels, lotions, shampoos, conditioners, cleansers, moisturizers, sprays, etc.

[0042] The invention also provides a preparation for promoting hair growth, which comprises the oak leaf extract.

[0043] In the present invention, the preparation may contain only one active ingredient of the oak leaf extract, or may include other active ingredients. The present invention does not specifically limit the dosage form of the preparation, and may be conventionally selected according to actual needs, including but not limited to solutions, ointments, plasters, creams or patches. The preparation may also include various carriers added according to the dosage form requirements of the preparation; the carriers include but are not limited to diluents, lubricants, adhesives, disintegrants, colorants, antioxidants, preservatives, solubilizers, solvents, suspending agents, surfactants, penetration enhancers or combinations thereof.

[0044] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0045] In the following embodiments, unless otherwise specified, all of them are conventional methods.

[0046] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0047] Example 1

[0048] A method for extracting oak leaf extract:

[0049] After the oak leaves are dried naturally, they are crushed and dissolved in 70% ethanol. The mass volume ratio of oak leaves to ethanol is 3g:7mL. They are cold soaked at 20℃ for 12h, heated to 65℃ for reflux extraction for 3h, and the extract is filtered under reduced pressure. Repeat the above operation 3 times, mix the extracts, and concentrate under reduced pressure to obtain a concentrated solution, which is concentrated to 1 / 3 of the volume of the extract. The concentrated solution is extracted with equal volumes of n-hexane, ether, ethyl acetate and water-saturated n-butanol in turn to obtain a water-saturated n-butanol extract. The extract is concentrated and dried to obtain an oak leaf extract.

[0050] Example 2

[0051] A method for extracting oak leaf extract:

[0052] After the oak leaves are dried naturally, they are crushed and dissolved in 60% ethanol. The mass volume ratio of oak leaves to ethanol is 3g:15mL. They are cold soaked at 25℃ for 12h, heated to 60℃ for reflux extraction for 5h, and the extract is filtered under reduced pressure. Repeat the above operation twice, mix the extracts, and concentrate under reduced pressure to obtain a concentrated solution, which is concentrated to 1 / 5 of the volume of the extract. The concentrated solution is extracted with 2 volumes of n-hexane, ether, ethyl acetate and water-saturated n-butanol in turn to obtain a water-saturated n-butanol extract. The extract is concentrated and dried to obtain an oak leaf extract.

[0053] Example 3

[0054] A method for extracting oak leaf extract:

[0055] After the oak leaves are dried naturally, they are crushed and dissolved in 80% ethanol. The mass volume ratio of oak leaves to ethanol is 3g:5mL. They are cold soaked at 15℃ for 24h, heated to reflux extraction at 70℃ for 1h, and the extract is filtered under reduced pressure. Repeat the above operation 4 times, mix the extracts, and concentrate under reduced pressure to obtain a concentrated solution, which is concentrated to 1 / 2 of the volume of the extract. The concentrated solution is extracted with 0.5 times the volume of n-hexane, ether, ethyl acetate and water-saturated n-butanol in turn to obtain a water-saturated n-butanol extract. The extract is concentrated and dried to obtain an oak leaf extract.

[0056] Example 4

[0057] A method for extracting oak leaf extract:

[0058] After the oak leaves are dried naturally, they are crushed and dissolved in 65% ethanol. The mass volume ratio of oak leaves to ethanol is 3g:10mL. They are cold soaked at 22℃ for 16h, heated to reflux at 63℃ for 4h, and the extract is filtered under reduced pressure. Repeat the above operation twice, mix the extracts, and concentrate under reduced pressure to obtain a concentrated solution, which is concentrated to 1 / 4 of the volume of the extract. The concentrated solution is extracted with 1.5 times the volume of n-hexane, ether, ethyl acetate and water-saturated n-butanol in turn to obtain a water-saturated n-butanol extract. The extract is concentrated and dried to obtain an oak leaf extract.

[0059] Example 5

[0060] A method for extracting oak leaf extract:

[0061] After the oak leaves are dried naturally, they are crushed and dissolved in 75% ethanol. The mass volume ratio of oak leaves to ethanol is 3g:6mL. They are cold soaked at 18℃ for 18h, heated under reflux at 67℃ for 2h, and the extract is filtered under reduced pressure. Repeat the above operation 3 times, mix the extracts, and concentrate under reduced pressure to obtain a concentrated solution, which is concentrated to 1 / 3 of the volume of the extract. The concentrated solution is extracted with 0.8 times the volume of n-hexane, ether, ethyl acetate and water-saturated n-butanol in turn to obtain a water-saturated n-butanol extract. The extract is concentrated and dried to obtain an oak leaf extract.

[0062] Test Example 1

[0063] 1. Separation and extraction of oak leaf extract

[0064] After the oak leaves are naturally dried in a ventilated place, 300g of them are taken, crushed into powder by a grinder, dissolved in 700mL of 70% ethanol, cold soaked at 20℃ for 12h, heated to reflux at 65℃ for 3h, and the extract is filtered under reduced pressure using a Buchner funnel. Repeat the above operation three times, mix the extracts, and concentrate under reduced pressure to 1 / 3 of the volume of the extract. Extract with equal volumes of n-hexane, ether, ethyl acetate, and water-saturated n-butanol in turn, and concentrate to dryness. The remaining water layer after extraction is also concentrated and dried. The five oak leaf extracts obtained are numbered QML1-5, where QML1 is the n-hexane layer, QML2 is the ether layer, QML3 is the ethyl acetate layer, QML4 is the water-saturated n-butanol layer, and QML5 is the water layer.

[0065] 2. Cytotoxicity and cell activity experiments of five oak leaf extracts on HHDPCs

[0066] In a 96-well plate, 5 × 10 4 / mL seeding density, inoculate each well with 90μL of human hair follicle derived dermal papilla cells (HHDPCs), and place the culture plate in a 37℃ incubator with 5% CO2 for pre-culture for 18 hours. Dissolve the above five kinds of oak leaf extracts in DMSO organic solvent at a concentration of 500mg / mL, and dilute them in a gradient with cell culture medium to obtain five kinds of oak leaf extracts with different concentrations. Add 10μL of five kinds of oak leaf extracts with different concentrations to the culture plate, so that the final concentration gradient of each oak leaf extract is 500μg / mL, 100μg / mL, 10μg / mL, 1μg / mL, and 0.1μg / mL. Place the culture plate in a 37℃ incubator with 5% CO2 and incubate for 24 hours. 10 μL of CCK-8 solution was added to each well, and the culture plate was placed in an incubator for 3 h. A blank group was set up with only 10 μL of cell culture medium added without adding oak leaf extract, and the absorbance at 450 nm was measured with an ELISA reader. The inhibitory concentration IC 50 and cell proliferation rate. The results are shown in Table 1 and Figure 1 shown.

[0067] Table 1 Cytotoxicity of oak leaf extracts

[0068]

[0069]

[0070] From the results in Table 1, it can be seen that the inhibitory concentrations of the five oak leaf extracts QML1, QML2, QML3, QML4, and QML5 for cell growth were IC 50 The concentrations were 156.78μg / mL, 82.49μg / mL, 281.61μg / mL, 236.14μg / mL and 182.56μg / mL, respectively. The cytotoxicity of QML3 and QML4 was relatively small.

[0071] Depend on Figure 1 It can be seen that compared with the blank group, the proliferation effects of oak leaf extracts QML2 and QML3 were not obvious, while QML4 had a cell proliferation effect at concentrations of 0.1, 1, and 10 μg / mL, and the cell proliferation effect was best at 10 μg / mL.

[0072] 3. Effects of five oak leaf extracts on the release of PGD2 in HHDPCs cells

[0073] In a 6-well plate, 2.5 × 10 5 / mL inoculation density, 2mL of HHDPCs cells were inoculated in each well and incubated in a 37℃ incubator with 5% CO2 for 18 hours. Five kinds of oak leaf extracts were added to make the final concentration of 10μg / mL. No oak leaf extract was added as a blank control. After incubation in a 37℃ incubator with 5% CO2 for 12 hours, the supernatant was collected. The PGD2 release amount was detected using a PGD2 kit. The results are shown in Figure 2 shown.

[0074] Depend on Figure 2 It can be seen that after treatment with QML3 or QML4, the content of PGD2 released from HHDPCs cells was significantly reduced compared with the blank group, while after treatment with QML1, QML2 or QML5, the content of PGD2 released from cells was increased compared with the blank group. This shows that oak leaf extracts QML3 and QML4 can reduce the content of PGD2 released from HHDPCs cells and have an inhibitory effect on the release of PGD2 from HHDPCs cells, while oak leaf extracts QML1, QML2 and QML5 do not have this effect.

[0075] 4. Effect of QML4 on the expression of HPGDS, LPGDS, CyclinD1, P27 and GPR44 genes in HHDPCs cells

[0076] HPGDS, LPGDS, CyclinD1, P27 and GPR44 genes are genes related to hair growth. Fluorescence quantitative PCR was used to evaluate the effects of oak leaf extract QML4 on the expression of these five genes.

[0077] (1) Cell culture

[0078] HHDPCs cells were cultured in DMEM containing 10% fetal bovine serum and 1% double-antibody (penicillin-streptomycin mixture) in a 5% CO2 incubator at 37°C. The cells were observed to be adherent cells under a microscope. 3×10 5 2 mL of HHDPCs cells with a concentration of 1 μg / mL were incubated in a 37°C incubator with 5% CO2 for 18 hours. QML4, an extract of oak leaves, was added to make the final concentration 1 μg / mL, and a blank control without QML4 was used. After incubation in a 37°C incubator with 5% CO2 for 12 hours, the cells were collected.

[0079] (2) RNA extraction

[0080] 1) Add 1 mL of TRIzol reagent to the cells and centrifuge at 12000 g for 10 minutes at 4°C to remove the cells.

[0081] 2) Place the homogenate at room temperature for 5 minutes to allow nucleic acids and proteins to fully dissociate. Add 0.2 mL of chloroform to each 1 mL of TRIzol reagent, cover the tube tightly, shake vigorously by hand for 15 seconds, and then place at room temperature for 2-3 minutes.

[0082] 3) Centrifuge at 10,000 g for 10 minutes at 4°C.

[0083] 4) Carefully pipette the upper aqueous phase (colorless) into a new test tube, and calculate the volume of the aqueous phase you pipette. Do not pipette too much to prevent the inhalation of DNA and protein impurities (middle layer, white).

[0084] 5) Add an equal volume of pre-cooled isopropanol to the absorbed water, cap the tube tightly, and shake gently.

[0085] 6) Let stand at room temperature for 10 minutes to allow the RNA to fully precipitate.

[0086] 7) Centrifuge at 10,000 g for 10 minutes at 4°C.

[0087] 8) Discard the supernatant (be careful not to discard the precipitate), add 1 mL of 75% ethanol to each tube for rinsing, cover the tube tightly, and gently shake the tube to remove residual isopropanol and salt. Centrifuge at 7500g at 4℃ for 5 minutes.

[0088] 9) Discard the supernatant (be careful not to discard the precipitate), open the tube cap, and dry the RNA precipitate (evaporate at room temperature or vacuum dry). Note that the RNA precipitate cannot be completely dried, otherwise it will be difficult to dissolve.

[0089] 10) Dissolve the RNA precipitate in an appropriate amount (20-50 μL) of RNase-free water. If the RNA precipitate is not completely dissolved, OD260 / OD280 will be ≤1.6. This RNA solution can be used for further experiments or stored at -70°C.

[0090] (3) Fluorescence quantitative PCR

[0091] Randomly take samples for preliminary experiments, and then determine the reaction conditions as follows:

[0092] 1) Preparation of reverse transcription reaction system, as shown in Table 2:

[0093] Table 2 Reverse transcription reaction system (total volume 20 μL)

[0094] Reagents Dosage 2×RT buffer 10μL Reverse transcription primer (100 pmol / μL) 1μL RT-mix 1μL Template (RNA) 5μL DEPC water 3μL

[0095] 2) Reverse transcription reaction conditions: 25°C for 5 minutes, 42°C for 50 minutes, and 85°C for 5 minutes;

[0096] 3) Preparation of fluorescent quantitative PCR reaction system, as shown in Table 3:

[0097] Table 3 Fluorescence quantitative PCR reaction system (total volume 50 μL)

[0098]

[0099]

[0100] 4) Fluorescence quantitative PCR amplification conditions were set as follows: 94°C for 4 minutes; 94°C for 20 seconds, 60°C for 25 seconds, 72°C for 30 seconds, 35 cycles; 72°C for signal detection. Figure 3 shown.

[0101] Depend on Figure 3 It can be seen that after QML4 treatment, the expression of P27 gene decreased compared with the blank group, the expression of CyclinD1 gene increased compared with the blank group, and the expression of HPGDS gene was not significantly different from the blank group, but the expression of prostaglandin synthase LPGDS gene decreased compared with the blank group, and the expression of GPR44 gene decreased compared with the blank group. This indicates that the reduction in PGD2 content in HHPDCs cells is because the oak leaf extract QML4 inhibits the expression of prostaglandin synthase LPGDS and receptor gene GPR44, thereby reducing the extracellular PGD2 content.

[0102] 5. Scratch effect of QML4 on HHDPCs cells

[0103] (1) Cell culture

[0104] HHDPCs cells were cultured in DMEM containing 10% fetal bovine serum and 1% double-antibody (penicillin-streptomycin mixture) in an incubator at 37° C. and 5% CO 2 . The cells were observed to be adherent cells under a microscope.

[0105] (2) Cell scratch

[0106] In order to study the effect of oak leaf extract QML4 on the growth of HHDPCs cells, scratches were made in HHDPCs cells, with oak leaf extract QML4 treatment as the experimental group and TGF-β1 treatment as the control group, and photos were taken at 0 and 24 hours after scratching. The specific process is as follows:

[0107] 1) Digest and count the cells and take 8×10 5 35mm 2 Incubate overnight in a Petri dish;

[0108] 2) Draw a line on the bottom of the dish with a marker pen as a mark, aspirate the culture medium, and use a 10 μL pipette tip to scratch the cells in the dish perpendicular to the marker pen mark;

[0109] 3) Wash with PBS to remove the scratched cells and perform experimental treatment. Each group has 2 replicate wells. The experimental groups are as follows:

[0110] Blank group (Control): add normal serum-free culture medium;

[0111] Control group (TGF-β1): serum-free culture medium containing 1 ng / mL TGF-β1 was added;

[0112] Experimental group (QML4): serum-free culture medium containing 10 μg / mL QML4 was added;

[0113] 4) Place in a 37°C incubator for culture;

[0114] 5) Take photos at each time point at 0h / 24h after scratching. When taking photos, select the intersection of the line drawn by the marker and the cell scratch as the observation point for fixed-point observation. Figure 4 The scratch healing efficiency of HHDPCs cells in each group was calculated according to the following formula. The results are shown in Figure 5 shown.

[0115] Scratch efficiency = (scratch distance of sample at 0 hours - scratch distance of sample at 24 hours) / (scratch distance of blank at 0 hours - scratch distance of blank at 24 hours)

[0116] Depend on Figure 4 It can be seen that the cell growth effect of the control group and the experimental group is significantly better than that of the blank group. The cell growth effect of the experimental group is the best compared with the blank group and the control group. Figure 5 It can be seen that after 24 hours, compared with the blank group, the cell scratch healing efficiency of the control group reached 171%, and the cell scratch healing efficiency of the experimental group reached 196%, indicating that the oak leaf extract QML4 can promote the growth of HHDPCs cells.

[0117] 6. Western blot experiment

[0118] In order to reveal the molecular mechanism by which oak leaf extract QML4 causes HHDPCs cell growth and proliferation, Western blotting was used to observe the expression of cell cycle proteins. The specific process is as follows:

[0119] (1) Cell culture

[0120] HHDPCs cells were cultured in DMEM containing 10% fetal bovine serum and 1% double-antibody (penicillin-streptomycin mixture) at 37°C and 5% CO2 in an incubator. The cells were observed to be adherent cells under a microscope. 2.5×10 5 / mL HHDPCs cells 2mL, 5% CO2, 37℃ incubated for 18 hours. Added oak leaf extract QML4 with a final concentration of 1μg / mL, with no oak leaf extract QML4 added as a blank control (Control), and minoxidil with a final concentration of 10μM added as a positive control (Minoxidil), 5% CO2, 37℃ incubated for 24 hours, and then the cells were collected.

[0121] (2) Western blotting

[0122] After the cells were collected, they were washed twice with 0.01M PBS. 100 μL RIPA lysis buffer (1 mMol / LPMSF, broad-spectrum protease inhibitors and phosphatase inhibitors were added to the cell pellet) was added to each group of cells. The centrifuge tube was placed on ice and sonicated three times at 150W for 5 seconds each time, with a 30-second pause in between. Then the cells were lysed on ice for 2 hours. Note that no bubbles should be generated during the sonication process. The cells were centrifuged at 12000g at 4°C, and the supernatant was aspirated for protein quantification. The proteins were separated on a 12% SDS-polyacrylamide gel and then transferred to a PVDF membrane (Biosharp, Beijing, China). The blot was blocked in a TBST solution (containing 1×TBS and 0.1% Tween20) dissolved with 5% skim milk powder at 37°C for 1 hour, washed with TBST, and incubated with the primary antibody (Table 4) at 4°C overnight. After washing, the membrane was reacted with the diluted secondary antibody (1:5000) at room temperature for 1 hour. The immunostained bands were visualized by enhanced chemiluminescence. Figure 6 shown.

[0123] Table 4 Western blot antibodies and dilution factors

[0124]

[0125] Cell proliferation is closely related to the cell cycle, which consists of G0 / G1, S and G2 / M phases. The cell cycle is affected by increased cyclin D1 protein, increased pRB phosphorylation, decreased p27, etc. Among these cyclins, Cyclin D1 is a protein that increases as the cell cycle progresses from G0 / G1 phase to S phase, and is a target gene of Wnt / β-catenin protein signaling. Wnt / β-catenin protein signaling plays an important role in hair growth and hair regeneration.

[0126] Depend on Figure 6It can be seen that compared with the blank group and the positive control group, the expression of Cyclin D1 protein increased after treatment with oak leaf extract QML4. Compared with the blank group, the expression of P27 protein decreased; and the expression of LPGDS and GPR44 proteins related to hair growth decreased, and the expression of HPGDS was not much different from the blank group, but the positive control minoxidil increased the expression of HPGDS protein compared with the blank group. This further proves that oak leaf extract QML4 can inhibit the expression of prostaglandin synthase LPGDS and GPR44 proteins, thereby reducing the extracellular PGD2 content. It also shows that oak leaf extract QML4 can promote the expression of Cyclin D1 protein, inhibit the expression of p27 protein, and promote the growth and proliferation of HHDPCs cells by regulating the expression of cell cycle-related proteins.

[0127] 7. Hair growth animal model experiment

[0128] To evaluate the hair growth effect of Quercus sylvestris leaf extract QML4, a hair growth model was established using C57BL / 6 mice.

[0129] (1) Animal grouping: Sixteen C57BL / 6 mice (7-week-old, male mice) were acclimated to the environment for 3 days and then randomly divided into two groups, 8 mice in each group, namely a blank group and a sample group.

[0130] (2) Drug preparation of sample group: Dissolve the oak leaf extract QML4 in propylene glycol to make the oak leaf extract QML4 concentration 30 μg / mL;

[0131] (3) Animal administration: After 3 days of adaptation, all animals were shaved with an electric shaver and depilatory cream. Starting from the next day, the sample group took 0.5 mL of the drug with a pipette and applied it twice a day on the back skin of C57BL / 6 mice. The second application was applied after the first application was completely dry. After the administration was completed, it was covered with vaseline to help drug absorption. The administration was once in the morning and evening for 14 days. The blank group was given 0.5 mL of propylene glycol twice and then applied with vaseline. The administration was once in the morning and evening for 14 days.

[0132] (4) Detection indicators: All mice were photographed on the day of shaving, and on the 7th, 9th, and 11th days of drug administration. On the 11th day of drug administration, two mice in each group were killed and the skin surface was photographed under a stereomicroscope. At the same time, the back skin was removed of excess tissue such as fat, embedded in paraffin, sliced, and the cross-section of the skin was photographed. The results are as follows Figure 7-Figure 8 shown.

[0133] Depend on Figure 7 It can be seen that there was no obvious effect in the blank group and the sample group on the 7th day, but on the 11th day, the hair growth of the mice in the QML4 sample group was significantly stronger than that in the blank group. Figure 8 As can be seen from c and d, the hair growth of the QML4 sample group is faster and more luxuriant than that of the blank group. Figure 8 As can be seen from a and b, the hair growth in the QML4 sample group was significantly stronger than that in the blank group.

[0134] It can be seen from the above test results that the oak leaf extract obtained by the present invention has low cytotoxicity, can promote the proliferation of HHDPCs cells, and can inhibit the expression of prostaglandin synthase LPGDS and receptor gene GPR44, thereby reducing the release of PGD2 in HHDPCs cells; promote the expression of Cyclin D1 protein, inhibit the expression of p27 protein, and promote the growth and proliferation of HHDPCs cells by regulating the expression of cell cycle-related proteins, thereby playing an important role in promoting hair growth and hair regeneration, and having a good effect on preventing and treating hair loss. It shows that the oak leaf extract obtained by the extraction method of the present invention can promote hair growth and hair regeneration, and has a good effect on preventing and treating hair loss.

[0135] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for extracting an oak leaf extract, characterized in that: The extraction method comprises the following steps: The oak leaves are crushed, dissolved in ethanol, and extracted by heating under reflux to obtain an extract; the extract is concentrated to obtain a concentrated solution; the concentrated solution is extracted with n-hexane, ether, ethyl acetate and water-saturated n-butanol in sequence to obtain a water-saturated n-butanol extract; the extract is concentrated and dried to obtain an oak leaf extract.

2. The extraction method according to claim 1, characterized in that The ethanol is an ethanol solution with a volume fraction of 60% to 80%.

3. The extraction method according to claim 1, characterized in that The mass volume ratio of the oak leaves and ethanol is 3g:5-15mL.

4. The extraction method according to claim 1, characterized in that The conditions for the heating reflux extraction are: heating reflux at 60-70° C. for 1-5 hours.

5. The extraction method according to claim 1, characterized in that The volume ratio of the concentrated solution to n-hexane is 1:0.5-2; the volume ratio of the concentrated solution to ether is 1:0.5-2; the volume ratio of the concentrated solution to ethyl acetate is 1:0.5-2; the volume ratio of the concentrated solution to water-saturated n-butanol is 1:0.5-2.

6. The oak leaf extract obtained by the extraction method according to any one of claims 1 to 5.

7. Use of the oak leaf extract according to claim 6 in the preparation of products for promoting hair growth, promoting hair regeneration or preventing and / or treating hair loss.

8. The use according to claim 7, characterized in that: The oak leaf extract has the effects of promoting hair growth, hair regeneration or preventing and / or treating hair loss by inhibiting the generation of prostaglandin D2.

9. A preparation for promoting hair growth, characterized in that The preparation comprises the oak leaf extract according to claim 6.

10. The preparation according to claim 9, characterized in that The dosage forms of the preparation include solution, ointment, plaster, cream or patch.

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