CN116370589A - Preparation method and application of hair-loss prevention hair-growing composition - Google Patents
Preparation method and application of hair-loss prevention hair-growing composition Download PDFInfo
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- CN116370589A CN116370589A CN202310151948.6A CN202310151948A CN116370589A CN 116370589 A CN116370589 A CN 116370589A CN 202310151948 A CN202310151948 A CN 202310151948A CN 116370589 A CN116370589 A CN 116370589A
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Abstract
The invention discloses a preparation method and application of an anti-hair-loss hair-growing composition, which uses supercritical CO 2 Removing 6-gingerol contained in rhizoma Dioscoreae Zingiberensis by extraction technique, and simultaneously providing a supermolecule NaDES solvent for extracting alkaloid, polysaccharide and flavonoids from rhizoma Dioscoreae Zingiberensis, folium Platycladi, rhizoma Drynariae and coffee seed, and preparing the four plant extractsThe supermolecule NaDES solvent has good characteristics such as no toxicity, easy synthesis, low cost, biodegradability and the like, can efficiently extract active ingredients of natural products, is widely accepted as a traditional solvent substitute, and the hair loss prevention composition finally obtained through supermolecule NaDES solvent extraction has excellent hair loss prevention and hair growth effects.
Description
Technical Field
The invention relates to the technical field of cosmetic raw materials, in particular to a preparation method and application of an anti-hair-loss hair-growing composition.
Background
Alopecia is classified into physiological alopecia and pathological alopecia. The pathological alopecia includes alopecia areata, psychogenic alopecia, endocrine dyscrasia alopecia, seborrheic alopecia, etc. The cause of alopecia is generally due to excessive levels of androgens; sebum secretion is vigorous; bacterial proliferative infections. From the perspective of traditional Chinese medicine, alopecia is related to the liver, kidney, spleen and blood and qi of five internal organs of the human body. Most of them have deficiency of qi and blood, large pores and pathogenic wind, invasion, wind-blood dryness or stagnation of qi, blood stasis, and failure of blood to rise from the hair, resulting in deficiency and abscission of hair roots. Washing and caring products using natural plant components as raw materials are increasingly favored in the market. Research shows that the natural plant active ingredient has various effects of nourishing and protecting hair, improving hair quality, keeping hair healthy and lustrous, and the like. The ancient medical literature describes anti-hair loss Chinese herbal medicines which mainly promote blood circulation of the scalp, improve the growth and development of hair follicles and inhibit bacteria.
Ginger is traditionally used in east asia to treat patients with baldness and hair loss. Some companies produce shampoos containing ginger extracts, which are said to have anti-hair loss and hair growth promoting effects. For example, the Korean jasmine flag lower Lv anti-hair loss washing and protecting series uses ginger extract as main anti-hair loss and hair-growing active ingredient. Research shows that the external ginger extract can achieve the effect of promoting the local blood circulation of the scalp, thereby stimulating the hair follicle to open and regenerating the hair. In addition, the pharmacological action of the ginger extract slows down or inhibits the propagation of bacteria and mould on the scalp, thereby achieving the effects of resisting bacteria, diminishing inflammation, relieving itching and removing dandruff.
Chinese patent CN 106727167A discloses a ginger hair-growing composition. A composition for promoting hair growth is prepared from ginger, eclipta, american ginseng, gynostemma pentaphyllum, etc. and features improving blood circulation and immunity of head skin, promoting hair growth and development and promoting hair follicle growth.
However, it is reported that the active substance 6-gingerol contained in ginger inhibits hair growth through inhibition and pro-apoptosis of hair follicle cells as shown by the results of in vitro hair follicle cell experiments. Thus, 6-gingerol is not a hair growth-stimulating agent, but a potential hair growth-inhibiting agent. Based on this, this patent contemplates the use of supercritical CO 2 The extraction technology removes 6-gingerol contained in ginger.
The main chemical components of the biota orientalis leaves with the function of growing and blackening hair are total flavonoid components. The hair growing and blackening mechanism can promote blood circulation of scalp parts by activating hair mother cells, repair degenerated hair follicles and promote blood circulation around the hair follicles, so that nutrition supply of head skin is increased to achieve the purposes of growing and blackening hair.
Rhizoma Drynariae is bitter and warm in nature, has the effect of activating blood and mainly treats alopecia areata. Studies show that the external application of the drynaria acid extract to the skin of an affected part can improve and promote microcirculation of skin and hair of the affected part, excite peripheral nerves, increase the activity of hair bulb cells, promote medicine, nutrition absorption and regeneration of hair follicle germ, strengthen the activity of hair follicles, and further promote the growth of hair.
Chinese patent CN 104225147B discloses a hair tonic. A hair-growing composition prepared from arborvitae twig, drynaria rhizome, fleece-flower root, etc. features that it can quickly penetrate deep layer of scalp and hair follicle, activate hair follicle, promote metabolism and supplement nutrients for hair growth.
Caffeine, an alias methylxanthine alkaloid, is one of the main active ingredients in coffee extracts. The caffeine can dredge hair follicles, solve the problem of the obstruction of testosterone to nutrient substance transportation, enable the hair to obtain the certain nutrient energy, and simultaneously can prolong the active period of hair roots and effectively protect the hair roots; in addition, the caffeine has high penetration rate, and only needs two minutes to penetrate into the scalp, when the scalp is washed, the hair follicle is opened under the action of hot water, and the caffeine can rapidly penetrate into the scalp, so that the negative influence of testosterone on hair growth is reduced, and the growth cycle of the hair follicle is activated.
Aiming at the problem of pathological alopecia, surgical operation, oral medicine or local smearing medicine can be used for treatment at present, but the problems of large wound, high operation cost, large side effect, poor compliance and the like exist. The current research shows that the application of the plant extract to the washing and caring product can effectively solve the problem of pathological alopecia.
The traditional natural product extraction method has the problems of low biocompatibility, large chemical residue, low extraction efficiency, long production period, low equipment utilization rate and the like, so that the finally obtained natural extract has low active ingredient content, poor efficacy performance and residual organic solvent, thereby limiting the wide application of the natural extract.
Disclosure of Invention
The invention aims to overcome the defects of the traditional extraction solvent, and provides a preparation method and application of an anti-hair-loss hair-growing composition, which utilize supercritical CO 2 The extraction technology removes 6-gingerol substances contained in the yellow ginger, provides a supermolecule L-carnitine tartaric acid NaDES solvent for extracting alkaloid, polysaccharide and flavonoid compounds from the yellow ginger, cacumen biotae, rhizoma drynariae and coffee seeds, combines and composites the four extracts to obtain the preparation method of the hair-loss prevention and hair-growing composition, and the hair-loss prevention and hair-growing composition has excellent hair-loss prevention and hair-growing functionsThe effect is achieved.
The deep eutectic solvent is a green and safe liquid solvent formed by the intermolecular hydrogen bond interactions of Hydrogen Bond Acceptors (HBA) and Hydrogen Bond Donors (HBD). The deep eutectic solvent has the characteristics of degradability, designability, low cost, simple synthesis process, good safety, biocompatibility and the like, and is widely applied to the extraction of natural products.
Supercritical CO 2 The method can control the temperature of the whole extraction process within the room temperature range, and has the advantages of simple separation process, no organic solvent residue, high extraction rate, obvious selectivity, short time, energy conservation, environmental protection and the like. Compared with the prior art, the extraction rate of active substances is improved, and the method becomes a means for extracting the effective components of plants.
The invention combines the L-carnitine and the tartaric acid according to a specific molar ratio to form the supermolecule NaDES solvent, and the two raw materials are common safe components of cosmetics. The L-carnitine has strong water absorption and strong moisturizing effect on skin; meanwhile, the L-carnitine serving as beta-hydroxy acid can effectively accelerate the update of epidermis, and has good repairing effect on skin. The epidermis is updated faster, so that the basal layer has less chance of carrying melanin from melanocytes to epidermis, and the whitening effect is achieved. Tartaric acid is used as pH regulator, humectant and buffer in cosmetics.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
the preparation method and application of the hair-loss prevention hair-growing composition are characterized by comprising the following steps of:
(1) Drying rhizoma Dioscoreae Zingiberensis, folium Platycladi, rhizoma Drynariae, and coffee seed at low temperature, pulverizing, and sieving;
(2) Supercritical CO 2 Extracting the small yellow ginger powder to obtain small yellow ginger extraction residues, and drying for later use;
(3) Preparation of supermolecule L-carnitine tartrate NaDES solvent: adding L-carnitine and tartaric acid into a reactor according to a certain molar ratio, heating and stirring for a certain time to obtain uniform transparent clear viscous liquid, fully mixing the viscous liquid with a certain amount of ultrapure water, and preparing to obtain a supermolecule NaDES solvent for plant extraction;
(4) Respectively adding the yellow crow rhizome extraction residues, the biota orientalis leaf powder, the drynaria rhizome powder and the coffee seed powder into a supermolecule NaDES solvent, and carrying out constant-temperature water bath extraction to respectively obtain four plant extract solutions, wherein the constant-temperature water bath temperature is 35-75 ℃ and the water bath time is 0.5-2 hours;
(5) Respectively centrifuging and passing the four plant extracts through a membrane to obtain a clear and transparent plant extract for standby, wherein the centrifugal parameters are as follows: the centrifugal speed is 3000-11000 r/min, the centrifugal time is 10-30 min, and the aperture of the membrane equipment is 0.01-5 mu m;
(6) Mixing the clear and transparent four plant extracts with water according to a certain proportion to obtain the hair loss preventing and growing composition.
Preferably, in the step (1), the low-temperature drying time is 2-9 h, the drying temperature is 25-75 ℃, and the crushed raw materials need to be subjected to 100-mesh sieving operation.
Preferably, in step (2), supercritical CO 2 Extraction conditions: extraction temperature: 25-60 ℃ and extraction pressure: 10-45 Kpa, separation temperature: 25-80 ℃ and separating pressure: 5-15 Kpa.
Preferably, in the step (2), the low-temperature drying time is 2-9 hours, and the drying temperature is 25-75 ℃.
Preferably, in the step (3), the molar ratio of the L-carnitine to the tartaric acid is 1:1 to 5; heating time is 4-8 h, heating temperature is 30-80 ℃.
Preferably, in the step (3), the supermolecule L-carnitine tartrate NaDES solvent is mixed with a certain amount of ultrapure water, and the volume fraction of the supermolecule L-carnitine tartrate NaDES solvent is 5% -90%.
Preferably, in the step (4), the feed liquid ratio (g/mL) of the small yellow ginger extract residue, the biota orientalis powder, the drynaria powder, the coffee seed powder and the supermolecule L-carnitine tartrate NaDES solvent is 1:5 to 60.
Preferably, in step (5), the centrifugation parameters are: the centrifugal speed is 3000-11000 r/min, the centrifugal time is 10-30 min, and the aperture of the membrane equipment is 0.01-5 mu m.
Preferably, in the step (6), the hair loss preventing and growing composition comprises extracts of yellow ginger, biota orientalis, drynaria rhizome and coffee seeds, and the content of each plant extract is as follows in percentage by weight: 2-30% of yellow ginger extract, 2-30% of cacumen biotae extract, 2-30% of rhizoma drynariae extract and 2-30% of coffee seed extract.
Application of hair-growing composition with alopecia preventing effect by supercritical CO 2 The extraction technology removes 6-gingerol contained in the small yellow ginger, so that the effective extraction of the anti-hair loss and hair-growing active ingredients of the small Huang Jiangzhong is realized.
The supermolecular solvent of the hair-loss prevention and hair-growth composition has the advantages of designability, low cost, simple synthesis process and good biocompatibility, can be used as a novel green solvent, can efficiently extract active ingredients of natural products, and can be finally obtained to play excellent hair-loss prevention and hair-growth effects when applied to the field of cosmetics.
The application of the composition with the hair loss prevention and hair growth promotion effects in cosmetics can inhibit the activity of I I type 5 alpha-reductase, promote the hair follicle to be regenerated, shorten the time of the hair follicle entering the growing period, and promote the hair growth, so that the hair loss prevention and hair growth promotion effects are achieved.
The invention has the beneficial effects that: the technical proposal of the invention flexibly uses the supercritical CO 2 The extraction technology removes 6-gingerol substances of small Huang Jiangzhong, and skillfully proposes that the supermolecule L-carnitine tartaric acid NaDES solvent is applied to the extraction technology of natural products, and the hair-loss preventing and growing composition has the effects of hair-loss preventing and growing, and has the following specific advantages:
1. by means of supercritical CO 2 The extraction technique removes the active substance 6-gingerol which is Huang Jiangzhong and inhibits hair growth. This allows for subsequent efficient extraction of the small Huang Jiangzhong anti-hair loss hair growth promoting active ingredient.
2. Extracting natural product by supermolecular technology. Compared with the traditional solvent, the supermolecular technology can extract the plant active ingredients more efficiently; meanwhile, the supermolecule L-carnitine tartrate NaDES solvent does not need to be separated, can be used as a safety component in a product, and can play roles in preventing hair loss and developing in cooperation with other active substances.
3. The hair loss prevention and hair growth promotion composition can promote the hair follicle to be regenerated by inhibiting the activity of I I type 5α -reductase, shortens the time of the hair follicle entering the growing period, and plays a role in promoting the hair growth, thereby achieving the effect of hair loss prevention and hair growth promotion.
Drawings
FIG. 1 is a flow chart of a method of preparing the anti-hair-growing composition of the present invention;
FIG. 2 is a graph showing the variation of 6-gingerol content before and after treatment with small Huang Jiangchao critical CO2 in accordance with the present invention;
FIG. 3 is a graph showing the variation of polysaccharide content of Curcuma rhizome before and after critical CO2 treatment of small Huang Jiangchao in the embodiment of the invention;
FIG. 4 is a graph showing the comparison of the active substance content of the supramolecular solvent and the aqueous solvent extracts of the examples of the present invention;
FIG. 5 is a graph of testosterone peak area for a 5α -reductase inhibition assay of the test example of the present invention;
FIG. 6 shows the 5. Alpha. -reductase inhibition rate of the test example of the present invention;
FIG. 7 is a graph showing the growth of hair follicles in an in vitro culture test of a test example of the present invention;
FIG. 8 is a graph showing the relative rate of hair follicle growth in an in vitro culture assay for hair follicles according to the test example of the present invention;
FIG. 9 is a graph showing the length of tentacle hair follicles in an in vitro culture test of hair follicles according to the test example of the present invention;
FIG. 10 is a graph showing the change in back hair of mice in the negative control group tested for hair growth efficacy by the in vivo model of the test case of the present invention;
FIG. 11 is a graph showing the change in back hair of mice in the positive control group tested for hair growth efficacy by the in vivo model of the test case of the present invention;
FIG. 12 is a graph of the change in back hair of a sample group of mice tested for hair growth efficacy in an in vivo model of a test case of the present invention;
FIG. 13 is a graph of HE staining of skin tissue of mice in an in vivo model hair growth efficacy test of the test examples of the invention.
Detailed Description
The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention. The raw material reagent adopted in the embodiment of the invention is a conventionally purchased raw material reagent.
As shown in fig. 1 to 13, the present invention provides a preparation method and application of a supramolecular composition with repairing and soothing effects, and specific examples are as follows:
example 1:
(1) Drying rhizoma Dioscoreae Zingiberensis, folium Platycladi, rhizoma Drynariae and coffee seed at low temperature for 6 hr, pulverizing, and sieving with 100 mesh sieve;
(2) Supercritical CO 2 Extracting rhizoma Dioscoreae Zingiberensis powder, collecting rhizoma Dioscoreae Zingiberensis extract residue, and oven drying;
(3) Preparation of supermolecule L-carnitine tartrate NaDES solvent: the L-carnitine tartaric acid is prepared by the following steps of: 1, putting the mixture into a reactor, heating and stirring the mixture at 70 ℃ for 6 hours to obtain uniform and clear viscous liquid; adding a certain amount of ultrapure water into the uniform and clarified viscous liquid to obtain a supermolecule L-carnitine tartrate NaDES solvent with the water content of 50%;
(4) Adding rhizoma Dioscoreae Zingiberensis extract residue, folium Platycladi powder, rhizoma Drynariae powder and coffee seed powder into NaDES solvent of supramolecular L-carnitine tartrate, respectively, and extracting in constant temperature water bath. The feed liquid ratio (g/mL) of the plant powder to the solvent is 1:25, the water bath time is 1.5h, and the water bath temperature is 65 ℃;
(5) And (3) respectively centrifuging and membrane filtering the related extracting solution to obtain a clarified plant extracting solution. The centrifugation speed is 10000 r/min, the centrifugation time is 10 min, and the pore diameter of the membrane equipment is 0.45 mu m;
(6) Mixing the four obtained plant extracts with water according to a certain proportion, and compounding to obtain the hair loss preventing hair growth promoting composition. The content of each plant extract is as follows by weight percent: 2% of yellow ginger extract, 4% of cacumen biotae extract, 3% of drynaria extract and 2% of coffee seed extract.
The detection basis is as follows:
determination of crude polysaccharide in SN/T4260-2015 export plant food phenol-sulfuric acid method.
Determination of total flavonoids in SN/T4592-2016 exit foods.
Liquid chromatography for measuring caffeine in cosmetics imported and exported from SN/T1781-2006.
Conclusion:
1. as can be seen from FIG. 2, the yellow ginger passes through supercritical CO 2 After the extraction technology treatment, the content of 6-gingerol is obviously reduced and is close to zero. The results show that supercritical CO is utilized 2 The extraction technique can effectively remove 6-gingerol substances with the size of Huang Jiangzhong for inhibiting hair growth. As can be seen from FIG. 3, the supercritical CO is passed through 2 After the extraction technology treatment, the polysaccharide content in the yellow ginger extract is obviously improved, which indicates that the supercritical CO 2 The extraction technology not only effectively removes 6-gingerol substances contained in the yellow croaker, but also improves the extraction efficiency of other active substances of the yellow croaker.
2. As shown in FIG. 4, the extraction efficiency of the supermolecule L-carnitine tartrate NaDES solvent and the traditional extraction solvent was evaluated by using the detection indexes of the yellow-ginger polysaccharide, the biota flavone, the drynaria flavone and the caffeine. As shown in fig. 5, compared with the traditional solvent, the extraction efficiency of the supramolecular l-carnitine tartaric acid NaDES solvent on the yellow ginger polysaccharide, the biota orientalis flavone, the drynaria flavone and the caffeine is obviously improved.
Example 2:
(1) Drying rhizoma Dioscoreae Zingiberensis, folium Platycladi, rhizoma Drynariae and coffee seed at low temperature for 6 hr, pulverizing, and sieving with 100 mesh sieve;
(2) Supercritical CO 2 Extracting rhizoma Dioscoreae Zingiberensis powder, collecting rhizoma Dioscoreae Zingiberensis extract residue, and oven drying;
(3) Preparation of supermolecule L-carnitine tartrate NaDES solvent: the L-carnitine tartaric acid is prepared by the following steps of: 5, putting the mixture into a reactor, heating and stirring the mixture at 70 ℃ for 6 hours to obtain uniform and clear viscous liquid; adding a certain amount of ultrapure water into the uniform and clarified viscous liquid to obtain a supermolecule L-carnitine tartrate NaDES solvent with the water content of 50%;
(4) Adding rhizoma Dioscoreae Zingiberensis extract residue, folium Platycladi powder, rhizoma Drynariae powder and coffee seed powder into NaDES solvent of supramolecular L-carnitine tartrate, respectively, and extracting in constant temperature water bath. The feed liquid ratio (g/mL) of the plant powder to the solvent is 1:50, the water bath time is 1.5h, and the water bath temperature is 55 ℃;
(5) And (3) respectively centrifuging and membrane filtering the related extracting solution to obtain a clarified plant extracting solution. The centrifugation speed is 10000 r/min, the centrifugation time is 10 min, and the pore diameter of the membrane equipment is 0.45 mu m;
(6) Mixing the four obtained plant extracts with water according to a certain proportion, and compounding to obtain the hair loss preventing hair growth promoting composition. The content of each plant extract is as follows by weight percent: 4% of yellow ginger extract, 4% of cacumen biotae extract, 2% of drynaria extract and 2% of coffee seed extract.
In order to prove that the hair loss preventing and hair growing composition provided by the invention has the hair loss preventing and hair growing effects, the following provides relevant efficacy tests. The hair-loss preventing and hair-growing composition obtained in example 1 was used as a test sample, and was subjected to a hair-loss preventing and hair-growing efficacy test.
Test example 1:5 alpha-reductase inhibition assay
Detection purpose and principle: androgenic alopecia (AGA) is caused by the conversion of testosterone (T) to more active Dihydrotestosterone (DHT) by 5 a-reductase, which is sensitive to hair follicles and affects hair follicle development, resulting in hair loss. Whether the test substance can inhibit 5α -reductase, thereby inhibiting DHT production, can be used as one of the basis for determining whether the hair-growing efficacy is achieved.
The detection basis is as follows: standard protocol for CALT/TM/SOP281-01 5. Alpha. -reductase inhibition assay.
The detection step comprises:
1. positive Control (PC) 1. Mu.M finasteride solution
Sample preparation (TA) samples were diluted to 50%,10% (v/v), test concentrations 100%,50%,10% using PBS buffer
Blank control: phosphate Buffer (PBS)
Preparation of type I5 a-reductase: 10 male rats were taken, and after no water retention, they were sacrificed by cervical vertebra removal and the prostate was removed and minced on an ice table. Preparation of 1 in a glass homogenizer with pre-chilled buffer at 0 ℃): 5 homogenizing, centrifuging at 3000g for 10 min, collecting supernatant, centrifuging at 10000g for 30min, collecting supernatant, packaging into EP tubes with 1mL of enzyme extract, and storing in refrigerator at-80deg.C.
Type I5 a-reductase activity assay: the 2mL reaction system contains: phosphate buffer 0.25mL, enzyme extract 0.70mL, testosterone solution (0.5 mg/mL) 0.1mL, NADPH solution (0.8 mg/mL) 0.35mL (10% ethanol in complete reaction group, finasteride in positive control group, sample solution in sample group) 0.6mL. The control group replaces the enzyme extract with phosphate buffer and 2.0mL of dichloromethane is added to terminate the reaction; the complete reaction, positive and sample sets were reacted at 37℃for 30min, after the reaction was completed, 2.0mL of methylene chloride was added to stop the reaction, after 1 min shaking, 5000 r/min was centrifuged for 10 min, the upper aqueous phase was removed, about 1.0mL of the organic phase was removed and evaporated to dryness, and the residue was dissolved in 1.0mL of methanol, and the testosterone peak area (T) was measured by high performance liquid chromatography. Each reaction was set up with 3 parallel tubes.
In the formula (1), a T sample tube is the testosterone peak area of a sample group; the T reaction tube is the testosterone peak area of the complete reaction group; testosterone peak area of T blank tube as blank group
Detection conclusion: through statistical analysis, compared with a complete reaction group, the average peak area of testosterone (T) in a positive group is increased, the inhibition rate of 5 alpha-reductase II is 86.18 percent, and the testosterone (T) in the positive group has statistical difference (p < 0.05); the samples showed an increase in the average peak area of testosterone (T) at concentrations of 10%,50% and 100% (v/v), and a statistical difference (p < 0.05) in inhibition of type II 5α -reductase of 11.97%,57.20% and 81.48%, respectively, and the hair-growing composition was anti-hair-loss at concentrations of 10%,50% and 100% (v/v) under the present experimental conditions.
Test example 2: hair follicle in vitro culture test
Detection purpose and principle: hair follicles are meaningful structures that control the periodic growth of hair, consisting of an epithelial component and a dermal component. The periodic changes in hair follicles are regulated by a variety of factors (e.g., growth factors, cytokines, corticoids, surface-dermal interactions, drugs, etc.), and also determine hair growth. The in vitro culture and reconstruction of hair follicles are of great significance in elucidating hair follicle biology.
The detection basis is as follows: standard procedure for CALT/TM/SOP226-01 hair follicle in vitro culture assay
The detection step comprises:
1. test sample (TA): samples were formulated at concentrations of 0.01%,0.1% and 0.5% (v/v) using medium
Positive Control (PC) minoxidil liniment was formulated at a concentration of 10. Mu.M using culture medium
Negative Control (NC) Williams' E serum-free Medium (Gibco) containing 2mM L-Glutamine, 10. Mu.g/mL bovine insulin, 10ng/mL hydrocortisone, lOOU/mL penicillin, lOO. Mu.g/mL streptomycin
2. Hair follicles were isolated: the C57BL/6 mice were humanly sacrificed, double-sided beard was cut off, upper lips were sterilized 3 times with 75% ethanol, and double-sided beard pads were cut off and placed in a 1:1 HBSS: in PBS (100U/mL penicillin, 100. Mu.g/mL streptomycin in PBS) and with 1:1 HBSS: the hair follicle was gently removed from surrounding tissue by gentle forceps in the direction of the follicle, the distal end of the follicle was gently clamped, carefully plucked from the subcutaneous tissue, and the isolated follicle placed in a different one of 24-well plates.
3. Hair follicle culture: at 37℃with 5% CO 2 Incubation was carried out for 14 days with 0.5mL of medium per well.
4. Sample adding: and (3) adding samples on the next day of culture, adding culture mediums containing samples with different concentrations into corresponding holes, setting 8 compound holes in each group, placing 1-2 isolated hair follicles in each hole, and changing liquid once every 3 days.
5. Image analysis: the whisker follicle length was photographed on days 1 and 14, and morphological observation and measurement of the whisker follicle length.
In the formula (2): average hair follicle length of each group (D 14 ) For the 14 th day sampleAverage hair follicle length for group and positive control group administration; average hair follicle length of each group (D 0 ) Average hair follicle length for the sample and positive control groups not dosed; control group hair follicle length (D) 14 ) Hair follicle length for day 14 negative control group administration; control group hair follicle length (D) 0 ) Hair follicle length not administered to the negative control group.
Detection conclusion: from the statistical analysis, as can be seen from fig. 6, 7 and 8, there is a statistical difference (p < 0.05) in the relative growth rate of hair follicles in the positive group compared with the negative control group; the relative growth rates of hair follicles were statistically different (p < 0.05) for samples 0.01%,0.1% and 0.5% (v/v) compared to the negative control group, and the hair loss preventing hair growth promoting composition had a certain effect on promoting hair growth at concentrations of 0.01%,0.1% and 0.5% (v/v) under the present experimental conditions.
As shown in fig. 9: figures a, b are hair follicle lengths on days 1, 14 of NC group; panels c, d are hair follicle lengths on day 1, day 14 of the PC group; panels e, f are hair follicle lengths on days 1, 14 for sample TA (0.01%); figures g, h are hair follicle lengths on days 1, 14 for sample TA (0.1%); figures i, j are hair follicle lengths on days 1 and 14 for sample TA (0.5%).
Test example 3: in vivo model hair growth efficacy test
Detection purpose and principle: detecting the effect of the sample on hair; the hair growth effect of the samples was evaluated by detecting changes in the back hair growth of mice.
The detection step comprises:
table 1 smearing liquid for different sample groups
1. As shown in table 1, in vivo experiments were divided into 3 groups.
2. The experimental animals enter a feeding room after being quarantined for 3 days in a quarantine room, and the back hair of the mice is removed one day before the experiment, so that the skin is prevented from being damaged.
3. The mice were weighed and randomly divided into 3 groups, NC, PC, S, according to body weight.
4. According to the grouping, different substances were applied once a day, 29 consecutive days, 0.2mL each time, as evenly as possible to the back of the mice.
5. The skin on the back of the mice was observed daily, and the time for the skin to turn from pink to black was recorded as the time for the skin to grow from black to hair, each group expressed as mean ± standard deviation.
6. Mice were weighed on days 7, 14, 21, 29, respectively, and expressed as the mean soil standard deviation.
7. Photographs were taken on days 1, 7, 14, 21, 29, and the back skin condition of each group was compared and shown as a picture.
8. The humane was sacrificed 24 hours after the application on day 14, the same parts of the shaved area were sampled for skin, fixed, conventional flaking, HE staining, and the number of microscopic hair follicles expressed as mean ± standard deviation.
9. After the mice were dehaired, the skin appeared pink, suggesting that the hair entered telogen phase, and after the hair follicle entered anagen phase, the skin became grey, growing hair gradually. The total number of new hair follicles/follicles is used as an indicator of hair growth from stationary phase to anagen phase. The above evaluation index can be used as evidence for promoting hair growth.
Detection conclusion: in this experiment, after the mice were dehaired, the skin appeared pink, prompting the hair to enter resting stage, and the time required for observing the skin change and long hair of the mice every day was found, as can be seen from table 2, compared with the negative control group, the sample group and the positive control group shortened the entering growing stage, with statistical difference (p < 0.05); the positive control group shortened the time to hair growth compared to the negative control group, with statistical differences (p < 0.05).
TABLE 2 skin changes and time required for long hair in mice
Note that: results are expressed as mean ± standard deviation, P <0.05, with statistical differences compared to NC.
As can be seen from fig. 10, 11 and 12, the growth of skin and hair on the back of the mice was recorded every week and last day before and during the experiment, and the negative control group, the sample group and the positive control group showed different growth of skin and hair after administration, and 6 mice were grayed out in the negative control group at D14, 5 mice showed hair growth, 2 mice showed hair growth in the back 80% region and 3 mice showed hair growth in the back 90% region; the sample group showed 8 mice with greying skin in D14, 7 mice with hair growing, and 6 mice with hair growing in D21 at 90% of the back; in the positive control group, the skin of 8 mice in D14 turns grey, the hair of 8 mice grows out, and the hair of 6 mice grows out in the 90% area of the back of D21;
as can be seen from Table 3, the body weight change trend of the mice was substantially consistent with that of the negative control throughout the experiment, and the mice were free of abnormalities.
TABLE 3 weight variation in mice
| Group of | NC | S | PC |
| D1 | 21.7±1.2 | 21.9±1.5 | 21.1±0.9 |
| D7 | 23.1±1.4 | 23.5±1.4 | 23.5±0.7 |
| D14 | 24.7±1.9 | 24.8±1.8 | 23.7±1.2 |
| D21 | 25.2±2.4 | 25.4±2.1 | 24.7±1.5 |
| D29 | 26.1±2.8 | 26.1±1.6 | 24.4±1.7 |
Note that: results are expressed as mean.+ -. Standard deviation, D1, D7, D14, D21, D29 are body weights (g) of mice on day 1, day 7, day 14, day 21, day 29
TABLE 4 skin histological index of mice on day 14
Note that: results are expressed as mean ± standard deviation, P <0.05, with statistical differences compared to NC.
As can be seen from table 4, the skin histology of the back of D7 mice showed a significant increase in the total number of hair follicles compared to the negative control group, with a statistical difference (p < 0.05) between the sample group and the positive control group; the number of new hair follicles is compared with that of a negative control group, the number of the new hair follicles is obviously increased, and the number of the new hair follicles is obviously increased in the sample group and the positive control group, and the new hair follicles and the positive control group have statistical differences (p < 0.05); the neonatal coefficient was significantly increased in the percentage of induction during the growth phase for both the sample and positive control groups compared to the negative control group, with statistical differences (p < 0.05).
As shown in fig. 13: FIG. O is a histological section of the skin of the mice of the negative control group; panel P is a histological section of the skin of the mice of the positive control group; panel Q is a histological section of the skin of a sample group of mice.
As can be seen from the HE staining pattern shown in fig. 13, microscopic examination of the histological sections of the mouse skin revealed no abnormality in the skin of each group of mice.
In conclusion, the hair loss preventing and hair growing composition can promote hair follicle neogenesis, shorten the time for hair follicle to enter anagen phase, and promote hair growth.
The invention has the beneficial effects that: the technical proposal of the invention flexibly uses the supercritical CO 2 The extraction technology removes 6-gingerol substances of small Huang Jiangzhong, and skillfully proposes that the supermolecule L-carnitine tartaric acid NaDES solvent is applied to the extraction technology of natural products, and the hair-loss preventing and growing composition has the effects of hair-loss preventing and growing, and has the following specific advantages:
1. by means of supercritical CO 2 The extraction technique removes the active substance 6-gingerol which is Huang Jiangzhong and inhibits hair growth. This allows for subsequent efficient extraction of the small Huang Jiangzhong anti-hair loss hair growth promoting active ingredient.
2. Extracting natural product by supermolecular technology. Compared with the traditional solvent, the supermolecular technology can extract the plant active ingredients more efficiently; meanwhile, the supermolecule L-carnitine tartrate NaDES solvent does not need to be separated, can be used as a safety component in a product, and can play roles in preventing hair loss and developing in cooperation with other active substances.
3. The hair loss prevention and hair growth promotion composition can promote the hair follicle to be regenerated by inhibiting the activity of II type 5 alpha-reductase, shortens the time of the hair follicle entering the growing period, and plays a role in promoting the hair growth, thereby achieving the effect of hair loss prevention and hair growth promotion.
Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art will understand that; the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced with equivalents; such modifications and substitutions do not depart from the spirit of the invention, and are intended to be included within the scope of the appended claims and description.
Claims (9)
1. The preparation method of the hair-loss prevention hair-growing composition is characterized by comprising the following steps of:
(1) Drying rhizoma Dioscoreae Zingiberensis, folium Platycladi, rhizoma Drynariae, and coffee seed at low temperature, pulverizing, and sieving;
(2) Supercritical CO 2 Extracting the small yellow ginger powder to obtain small yellow ginger extraction residues, and drying for later use;
(3) Preparation of supermolecule L-carnitine tartrate NaDES solvent: adding L-carnitine and tartaric acid into a reactor according to a certain molar ratio, heating and stirring for a certain time to obtain uniform transparent clear viscous liquid, fully mixing the viscous liquid with a certain amount of ultrapure water, and preparing to obtain a supermolecule NaDES solvent for plant extraction;
(4) Respectively adding the yellow crow rhizome extraction residues, the biota orientalis leaf powder, the drynaria rhizome powder and the coffee seed powder into a supermolecule NaDES solvent, and carrying out constant-temperature water bath extraction to respectively obtain four plant extract solutions, wherein the constant-temperature water bath temperature is 35-75 ℃ and the water bath time is 0.5-2 hours;
(5) Respectively centrifuging and passing the four plant extracts through a membrane to obtain a clear and transparent plant extract for standby, wherein the centrifugal parameters are as follows: the centrifugal speed is 3000-11000 r/min, the centrifugal time is 10-30 min, and the aperture of the membrane equipment is 0.01-5 mu m;
(6) Mixing the clear and transparent four plant extracts with water according to the following proportion to obtain the hair loss preventing hair-growing composition, wherein the hair loss preventing hair-growing composition comprises the extracts of small yellow ginger, biota orientalis, drynaria rhizome and coffee seeds, and the content of each plant extract is as follows: 2-30% of yellow ginger extract, 2-30% of cacumen biotae extract, 2-30% of rhizoma drynariae extract and 2-30% of coffee seed extract.
2. The method for preparing hair-growing composition with hair loss prevention according to claim 1, wherein in the step (1), the low-temperature drying time is 2-9 h, the drying temperature is 25-75 ℃, and the crushed raw materials are subjected to 100-mesh sieving operation.
3. The method for preparing an anti-hair loss and hair growth composition according to claim 1, wherein in the step (2), supercritical CO is used 2 Extraction conditions: extraction temperature: 25-60 ℃ and extraction pressure: 10-45 Kpa, separation temperature: 25-80 ℃ and separating pressure: 5-15 Kpa.
4. The method for preparing hair-growing composition with hair loss prevention according to claim 1, wherein in the step (2), the low-temperature drying time is 2 to 9 hours and the drying temperature is 25 to 75 ℃.
5. The method of preparing a hair-growing composition with anti-hair loss according to claim 1, wherein in the step (3), the molar ratio of l-carnitine to tartaric acid is 1:1 to 5; heating time is 4-8 h, heating temperature is 30-80 ℃.
6. The method of preparing an anti-hair-growing composition according to claim 1, wherein in the step (3), the supramolecular l-carnitine tartrate NaDES solvent is mixed with a certain amount of ultra-pure water, and the volume fraction of the supramolecular l-carnitine tartrate NaDES solvent is 5% to 90%.
7. The method of preparing hair-growing composition with hair loss prevention according to claim 1, wherein in the step (4), the ratio (g/mL) of the extract residue of yellow ginger, the powder of biota orientalis, the powder of drynaria rhizome, the powder of coffee seeds to the solvent of supermolecular l-carnitine tartrate, naDES, is 1:5 to 60.
8. The application of the hair-growth composition with the hair loss prevention function is characterized in that the hair-growth composition with the hair loss prevention function adopts supercritical CO 2 The extraction technology removes 6-gingerol contained in the small yellow ginger, so that the effective extraction of the anti-hair loss and hair-growing active ingredients of the small Huang Jiangzhong is realized.
9. The use of the hair-growth composition according to claim 8, wherein the supermolecular solvent to which the hair-growth composition belongs is a novel green solvent, has the advantages of designability, low cost, simple synthesis process and good biocompatibility, can efficiently extract active ingredients of natural products, and can be finally obtained to play an excellent hair-growth effect when applied to the field of cosmetics.
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