CN102028759A - Chinese medicine hair-darkening empirical formula and preparation method thereof - Google Patents
Chinese medicine hair-darkening empirical formula and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a Chinese medicine hair-darkening empirical formula which comprises the following Chinese medicine compositions in parts by weight: 1-40 parts of tuber fleeceflower root, 1-40 parts of black sesame, 1-40 parts of rhizoma drynariae, 1-40 parts of eclipta alba, 1-30 parts of fruit of glossy privet, 1-30 parts of dodder, 1-30 parts of selfheal, 1-30 parts of herba lycopi, 1-30 parts of mint, 1-30 parts of chuanxiong rhizome and 1-30 parts of rhizoma zingiberis. A lotion and a liniment/tincture as two preparation forms for external use can be prepared by adding a certain amount of water or ethanol into the Chinese medicine hair-darkening empirical formula for extraction. The Chinese medicine hair-darkening empirical formula has the main effects of nourishing liver and kidney, cooling the blood to stop bleeding, removing dampness to relieve itching, penetrating deeply into hair follicle to improve the activity of tyrosinase, promoting the proliferation of melanoma cells and generating more melanins to achieve the purpose of darkening the hair. A Chinese medicine compound is adopted by the invention, all the Chinese medicines are synergetic, and compared with a single Chinese medicine, the Chinese medicine hair-darkening empirical formula has the characteristics of multisystem, multi-target and multilevel exertion of all-round medicine actions and radically solves the problem that the black hair is whitened.
Description
Technical Field
The invention belongs to the field of cosmetics and discloses a traditional Chinese medicine hair-blacking empirical formula and a preparation method thereof.
Background
With the development of economy in China, the enhancement of consumption consciousness and the improvement of consumption level of people, hairdressing products gradually become daily consumption necessities from luxury goods. On one hand, the 21 st century enters an aging society, and the aged population of China reaches 1.3 hundred million; meanwhile, young people in the modern society have increased white hair due to intense competition and mental stress. The market demand of the hair dyeing and blackening cosmetics is gradually increased, and the hair dyeing and blackening cosmetic has huge development space and potential market. On the other hand, due to rapid progress of beauty science, people no longer satisfy the hair dyeing of hair dyeing cosmetics, but pursue that nutrient substances in the cosmetics directly participate in hair metabolism and pursue scientific care.
Hair dyes on the market at home and abroad undergo a series of changes from temporary hair dyeing to semi-permanent hair dyeing until the present permanent hair dyeing, and all of the hair dyes are added with a hair bleaching agent, namely, the hair is firstly decolorized by oxidizing and decomposing melanin in the hair so as to be dyed again.
In the process of implementing the invention, the inventor finds that the prior art has at least the following problems: the existing hair dye can damage hair in different degrees, and the newly generated hair is still in original color along with the continuous progress of metabolism of people, especially for people with white hair in the hair, the problem of blackening the hair is not fundamentally solved, so that from the safe and effective perspective, the market needs natural products to replace the existing hair dye, and the hair is blackened in the true sense.
Disclosure of Invention
The embodiment of the invention aims to overcome the defects of the prior art and provides a traditional Chinese medicine hair-blacking empirical formula capable of blacking hair.
Another objective of the embodiments of the present invention is to provide a pharmaceutical composition comprising an effective amount of the above-mentioned Chinese medicinal composition and a pharmaceutically acceptable carrier.
It is a further object of embodiments of the present invention to provide a process for the preparation of such pharmaceutical compositions.
In order to achieve the aim, the invention adopts the technical scheme that:
the traditional Chinese medicine hair-blacking empirical formula comprises the following traditional Chinese medicine composition in parts by weight:
1-40 parts of cacumen biotae, 1-40 parts of eclipta alba, 1-30 parts of glossy privet fruit, 1-30 parts of mulberry, 1-30 parts of scutellaria baicalensis and 1-30 parts of mint.
The invention provides a preferable technical scheme that: the traditional Chinese medicine hair-blacking empirical formula comprises the following traditional Chinese medicine composition in parts by weight:
10-30 parts of cacumen biotae, 10-30 parts of eclipta alba, 5-20 parts of glossy privet fruit, 5-20 parts of mulberry, 5-20 parts of scutellaria baicalensis and 5-20 parts of mint.
The invention provides a more preferable technical scheme that: the traditional Chinese medicine hair-blacking empirical formula comprises the following traditional Chinese medicine composition in parts by weight: 20 parts of cacumen biotae, 20 parts of eclipta alba, 10 parts of glossy privet fruit, 10 parts of mulberry, 10 parts of scutellaria baicalensis and 10 parts of mint.
The invention also provides a pharmaceutical composition, which contains the traditional Chinese medicine composition with effective dose and a medicinal carrier.
The preparation method of the pharmaceutical composition comprises the following steps:
the preparation method of the lotion comprises the following steps: mixing cacumen Platycladi, Ecliptae herba, fructus Ligustri Lucidi, Mori fructus, Scutellariae radix and herba Menthae, adding 5-7 times of distilled water into the mixed medicinal materials, cold soaking for 20-40min, heating, boiling, keeping boiling for 40-80 min, filtering to obtain filtrate, adding 3-5 times of distilled water into the administration residue, boiling, keeping boiling for 20-40min, filtering, mixing the filtrates, standing for 36-60h, collecting supernatant, and adjusting pH to 6.0-6.5 with citric acid to obtain lotion.
The preparation method of the liniment/tincture comprises the following steps: mixing folium Platycladi, Ecliptae herba, fructus Ligustri Lucidi, Mori fructus, Scutellariae radix and herba Menthae, adding 75% ethanol 5-7 times of the total weight of the medicinal materials into the mixed medicinal materials, soaking at room temperature for 20-40min under sealed condition, reflux-extracting at 85 deg.C twice, boiling for 20-60min after the first boiling, boiling the residue with 75% ethanol 3-5 times of the total weight of the mixed medicinal materials for 20-40min after the filtration, filtering, mixing filtrates, standing for 36-60h, and adjusting pH of the supernatant with citric acid to 6.0-6.5 to obtain liniment/tincture.
The embodiment of the invention has the beneficial effects that: the invention mainly aims at the condition that the hair is early white due to excessive blood heat and hair root malnutrition, and has the effects of nourishing liver and kidney, cooling blood to stop bleeding, removing dampness to relieve itching, improving the activity of tyrosinase by penetrating into hair follicles of the hair, promoting the proliferation of melanoma cells and generating more melanin so as to blacken the hair. The invention adopts a Chinese herbal compound, each Chinese herbal medicine has synergistic effect, compared with a single Chinese herbal medicine, the invention has the characteristics of multiple systems, multiple targets and multiple levels of full-range efficacy, and fundamentally solves the problem of the whitening of black hair.
The medicinal functions and effects of the medicines of the invention are as follows:
cacumen biotae: bitter and astringent taste, slightly pungent. Cool blood to stop bleeding, generate hair and blacken hair, and can be used for treating alopecia due to blood heat and premature graying of beard and hair. The book Rihuazi materia Medica records: "decoction, head coating, blacking and hair-blackening"; compendium of materia Medica: "blacken and moisten the hair on the temples".
Eclipta alba: sweet, sour, adult and cold in nature. Nourish liver and tonify kidney, cool blood and stop bleeding, blacken beard and hair. Ben Cao gang mu (compendium of materia Medica) "blacken beard and hair, tonify kidney yin"; ben Cao Jing Shu (Ben Cao Jing Shu) "snakehead intestine excels in cooling blood. If the hairs are white, the hairs are blackened, and the teeth are fixed . Therefore, the herbs that become white in ancient times and modern times should be the wins. "
Glossy privet fruit: sweet, bitter and mild in nature, has the functions of nourishing liver and kidney, improving eyesight and blackening hair. Compendium of materia Medica: strengthening yin, strengthening waist and knees, whitening hair and improving eyesight. Can proliferate hair follicle cells, promote hair growth, enhance tyrosinase activity, and increase melanin production, and can be used for treating premature gray hair.
Mulberry: sweet and cold in nature and taste. Tonify liver and kidney, nourish blood and promote fluid production, and is used for early whitening of beard and hair. The herbal Zhenzhen: 'removing heat and nourishing yin … … blackens beard and hair'.
Scutellaria baicalensis: bitter and cold in property, has the functions of clearing heat, eliminating dampness, detoxifying, inhibiting bacterial growth, removing dandruff and relieving itching, and has a strong function of resisting malassezia; experimental research shows that the scutellaria baicalensis extract has a strong activating effect on tyrosinase.
Mint: pungent and cool in nature and flavor, and has the effects of dispelling wind-heat, refreshing head and eyes and promoting eruption. Can be used for relieving scalp itching, hair dryness, excessive oil and fat, seborrheic alopecia, and tinea capitis. Modern studies have shown that mint extracts have a tyrosinase activity enhancing effect.
Detailed Description
The present invention is further illustrated by the following examples, which are not to be construed as limiting the invention.
Example 1
Weighing the following raw materials (g) in parts by weight:
20g of cacumen biotae, 20g of eclipta alba, 10g of glossy privet fruit, 10g of mulberry, 10g of scutellaria baicalensis and 10g of mint.
The preparation method of the lotion of the traditional Chinese medicine hair-blacking empirical formula comprises the following steps:
mixing 20g of cacumen biotae, 20g of eclipta alba, 10g of glossy privet fruit, 10g of mulberry, 10g of scutellaria baicalensis and 10g of mint, adding distilled water which is 6 times of the total weight of the mixed medicinal materials into the mixed medicinal materials, cold soaking for 30min, heating, boiling, keeping boiling for 1h, filtering, adding distilled water which is 4 times of the total weight of the mixed medicinal materials into administration residues, boiling, keeping boiling for 30min, filtering, combining filtrates of two times, standing for 48h, taking supernate, and adjusting the pH value to 6.0-6.5 by using citric acid to obtain the lotion.
The using method comprises the following steps: after a user cleans the hair, the boiled soup is evenly poured on the head to completely soak the hair, the scalp is massaged while pouring, after 15 minutes, the hair is wrapped by a towel, and after 3 hours, the hair is cleaned by clean water.
Example 2
Weighing the following raw materials (g) in parts by weight:
10g of cacumen biotae, 25g of eclipta alba, 15g of glossy privet fruit, 5g of mulberry, 15g of scutellaria baicalensis and 5g of mint.
The preparation method of the lotion of the traditional Chinese medicine hair-blacking empirical formula comprises the following steps:
mixing 10g of Chinese arborvitae twig, 25g of eclipta alba, 15g of glossy privet fruit, 5g of mulberry, 15g of scutellaria baicalensis and 5g of mint, adding distilled water which is 5 times of the total weight of the mixed medicinal materials into the mixed medicinal materials, cold soaking for 20min, heating, keeping boiling for 40min after boiling, filtering, adding distilled water which is 3 times of the total weight of the mixed medicinal materials into administration residues, keeping boiling for 20min after boiling, filtering, combining filtrates of two times, standing for 36h, taking supernate, and regulating the pH value to 6.0-6.5 by using citric acid to obtain the lotion.
The procedure was as in example 1.
Example 3
Weighing the following raw materials (g) in parts by weight:
35g of cacumen biotae, 10g of eclipta alba, 1g of glossy privet fruit, 25g of mulberry, 30g of scutellaria baicalensis and 1g of mint.
The preparation method of the lotion of the traditional Chinese medicine hair-blacking empirical formula comprises the following steps:
mixing 35g of cacumen biotae, 10g of eclipta alba, 1g of glossy privet fruit, 25g of mulberry, 30g of scutellaria baicalensis and 1g of mint, adding distilled water which is 7 times of the total weight of the mixed medicinal materials into the mixed medicinal materials, cold soaking for 40min, heating, boiling, keeping boiling for 80min, filtering, adding distilled water which is 5 times of the total weight of the mixed medicinal materials into administration residues, boiling, keeping boiling for 40min, filtering, combining filtrates of two times, standing for 60h, taking supernate, and adjusting the pH value to 6.0-6.5 by using citric acid to obtain the lotion.
The procedure was as in example 1.
Example 4
Weighing the following raw materials (g) in parts by weight:
30g of cacumen biotae, 15g of eclipta alba, 20g of glossy privet fruit, 20g of mulberry, 5g of scutellaria baicalensis and 15g of mint.
The preparation method of the liniment/tincture of the traditional Chinese medicine hair-blacking empirical formula comprises the following steps:
mixing folium Platycladi 30g, Ecliptae herba 15g, fructus Ligustri Lucidi 20g, Mori fructus 20g, Scutellariae radix 5g and herba Menthae 15g, adding 75% ethanol 6 times of the total weight of the medicinal materials into the mixed medicinal materials, soaking at room temperature for 30min under sealed condition, heating and reflux extracting at 85 deg.C for two times, boiling for 40min after the first boiling, filtering, boiling the residue with 75% ethanol 4 times of the total weight of the mixed medicinal materials, keeping boiling for 30min, filtering, mixing the filtrates, standing for 48h, collecting the supernatant, and adjusting pH to 6.0-6.5 with citric acid to obtain liniment/tincture.
The using method comprises the following steps: spraying the medicinal liquid on hair for 1-2 times per day, kneading while spraying, massaging each part of the hair with fingers for 3-5 min, and repeatedly combing hair with comb to expand capillary vessel under scalp, increase blood flow, and help medicinal liquid penetrate into hair root.
Example 5
Weighing the following raw materials (g) in parts by weight:
15g of cacumen biotae, 10g of eclipta alba, 5g of glossy privet fruit, 15g of mulberry, 20g of scutellaria baicalensis and 20g of mint.
The preparation method of the liniment/tincture of the traditional Chinese medicine hair-blacking empirical formula comprises the following steps:
mixing 15g of cacumen biotae, 10g of eclipta alba, 5g of glossy privet fruit, 15g of mulberry, 20g of scutellaria baicalensis and 20g of mint, adding 75% ethanol which is 5 times of the total weight of the medicinal materials into the mixed medicinal materials, hermetically soaking for 20min at room temperature, heating and refluxing at 85 ℃ for two times, keeping boiling for 20min after the first boiling, boiling the filtered dregs of a decoction with 75% ethanol which is 3 times of the total weight of the mixed medicinal materials, keeping boiling for 20min, filtering, combining the filtrates of the two times, standing for 36h, taking the supernatant, and adjusting the pH value to 6.0-6.5 by using citric acid to obtain the liniment/tincture.
The procedure was used as in example 4.
Example 6
Weighing the following raw materials (g) in parts by weight:
1g of cacumen biotae, 2g of eclipta alba, 30g of glossy privet fruit, 30g of mulberry, 1g of scutellaria baicalensis and 30g of mint.
The preparation method of the liniment/tincture of the traditional Chinese medicine hair-blacking empirical formula comprises the following steps:
mixing 1g of cacumen biotae, 2g of eclipta alba, 30g of glossy privet fruit, 30g of mulberry, 1g of scutellaria baicalensis and 30g of mint, adding 75% ethanol which is 7 times of the total weight of the medicinal materials into the mixed medicinal materials, hermetically soaking for 40min at room temperature, heating and refluxing at 85 ℃ for two times, keeping boiling for 1h after the first boiling, boiling the filtered dregs of a decoction with 75% ethanol which is 5 times of the total weight of the mixed medicinal materials, keeping boiling for 40min, filtering, combining the filtrates of the two times, standing for 60h, taking the supernatant, and adjusting the pH value to 6.0-6.5 by using citric acid to obtain the liniment/tincture.
The procedure was used as in example 4.
The invention has the following pharmacological actions: the traditional Chinese medicine hair-blacking proved recipe has the effects of activating tyrosinase, promoting cell proliferation and promoting melanin synthesis, and the pharmacological effects provide experimental basis for clinical application of the traditional Chinese medicine hair-blacking proved recipe in prevention and treatment of white hair.
The pharmacodynamic study of the water extract (hereinafter referred to as water extract) and the ethanol extract (hereinafter referred to as ethanol extract) of the traditional Chinese medicine hair-blackening empirical formula prepared by the invention is carried out, and the method and the result are as follows:
effects on tyrosinase Activity
1 materials and methods
1.1 formula of empirical formula for blackening hair with traditional Chinese medicine
20g of cacumen biotae, 10g of glossy privet fruit, 20g of eclipta alba, 10g of mulberry, 10g of scutellaria baicalensis and 10g of mint
1.2 preparation of extract of empirical formula of Chinese medicine for blackening hair
1.2.1 preparation of aqueous extract of empirical formula for blackening hair with traditional Chinese medicine
The traditional Chinese medicine hair-blacking empirical formula is prepared by cold soaking the traditional Chinese medicine hair-blacking materials in distilled water 6 times of the total weight of the materials for 30min, heating, boiling, keeping boiling for 1h, filtering, adding distilled water 4 times of the total weight of the materials, boiling, keeping boiling for 30min, filtering, combining two filtrates, evaporating a solvent to form a paste, dissolving the paste in a phosphate buffer solution with the pH value of 6.8 and the concentration of 0.05mol/L, and diluting to the concentration of 1g/ml for later use.
1.2.2 preparation of extract of empirical formula of Chinese medicine for blackening hair
The traditional Chinese medicine hair-blacking empirical formula is prepared by hermetically soaking 75% ethanol 6 times the total weight of medicinal materials at room temperature for 30min, heating and refluxing at 85 ℃ for two times, boiling for 40min after the first time, filtering, boiling medicinal residues with 75% ethanol 4 times the total weight of the medicinal materials, boiling for 30min, filtering, combining secondary filtrates, volatilizing ethanol to form paste, adding 5mL of dimethyl sulfoxide, and diluting with 0.05mol/L Phosphate Buffer Solution (PBS) with pH of 6.8 to a concentration of 1g/mL for later use.
1.3 reagents and instruments
Reagent: tyrosinase and levodopa are from sigma; disodium hydrogen phosphate (Tianjin, Daloco chemical Co., Ltd., batch No. 20090721); potassium dihydrogen phosphate (New chemical plant, Guangzhou, batch No. 20010526).
The instrument comprises the following steps: ELX800 type microplate reader (BIOTEK, USA); SW-CJ-IF model superclean bench (Wujiang City Conghua clean plant); BT224S model electronic analytical balance (Sartorius corporation); 110S electric heating constant temperature water bath (SHIPING company).
1.4 preparation of reagents
1.4.3 preparation of PBS solution
Liquid A: 0.05mol/L Na2HPO47.099g of disodium hydrogen phosphate was weighed out and distilled water was added to the weighed solution to 1000 ml.
B, liquid B: 0.05mol/L KH2PO46.803g of potassium dihydrogen phosphate was weighed out and distilled water was added to the weighed solution to 1000 ml.
And subpackaging the solution A and the solution B in brown bottles, storing in a refrigerator at 4 ℃, and mixing the solution A and the solution B according to the same proportion when in use to obtain the PBS solution with the pH value of 6.8.
1.4.1 preparation of tyrosinase solution
Accurately weighing 25ku of tyrosinase, dissolving in 125ml of PBS (200 u/ml of enzyme activity unit), shaking, and dissolving for later use.
1.4.2 preparation of Levodopa solution
And precisely weighing 20mg of levodopa 1h before experimental determination, dissolving the levodopa in 50ml of PBS (the concentration is 0.4 mg/ml), shaking, and fully dissolving for later use.
1.4.4 preparation of test drugs
The concentration of 2 Chinese medicine hair-blacking empirical formula concentrated solutions with the concentration of 1g/ml are respectively diluted into high, middle and low dosage concentrations with a freshly prepared PBS solution, wherein the dosage concentrations are respectively 1mg/ml, 0.5mg/ml and 0.25 mg/ml.
2 method of experiment
Mixing 100ul of the prepared Chinese medicinal hair-blackening empirical formula diluent with different concentrations with 50ul of tyrosinase, incubating in a constant-temperature water bath kettle at 36.8 ℃ for 5min, adding 50ul of L-dopa, continuing to incubate for 2min, and immediately measuring the optical density OD value at 490nm of an enzyme-labeling instrument. The measurement was repeated 6 times for each sample, and the activation rate of tyrosinase was calculated after averaging.
The tyrosinase activity activation rate was calculated according to the following formula: activation rate [ (% C-D) - (a-B) ]/(a-B) × 100
In the formula: A-A475-drug + enzyme
A 475-drug-enzyme
A475+ drug + enzyme
D ═ a475+ drug-enzymes
Wherein,
the A value is the OD value of 100ul of liquid medicine, 50ul of 500u/ml of tyrosine solution and 50ul of 0.4 percent L-dopa solution.
The B value is the OD value of 100ul of the liquid medicine, 50ul of the PBS solution and 50ul of the 0.4 percent L-dopa solution.
The C value is the OD value of 100ul PBS +50ul tyrosine solution of 500u/ml +50ul 0.4% L-dopa solution.
D is the OD of 100ul PBS +50ul 0.4% L-dopa solution.
3 results of the experiment
The results in table 1 show that the aqueous extract and the alcohol extract of the traditional Chinese medicine hair-blackening empirical formula both have the effect of activating tyrosinase, and the tyrosinase activity of each traditional Chinese medicine formula is gradually increased along with the increase of the concentration, so that a dose dependence relationship is presented. Wherein, the highest activation rate is that the activation rate of the aqueous extract to the tyrosinase reaches 32.43 percent at the level of 1 mg/ml.
TABLE 1 influence of aqueous and alcoholic extracts of the Chinese medicinal hair blackening empirical formula on tyrosinase
Second, Effect on B16 melanoma cell proliferation and melanogenesis
1 materials and methods
1.1 Primary reagents and instruments
Sources of cell lines: from Shanghai cell institute of Chinese academy of sciences.
Reagent: DMEM high-glucose medium (Hyclone, usa); fetal bovine serum (Hyclone, USA); trypsin (Sigma, usa); tetramethylazothiazole blue (MTT, Amersham Corp.); dimethyl sulfoxide (Guangdong Guanghua chemical Co., Ltd.).
The instrument comprises the following steps: ELX800 type microplate reader (BIOTEK, USA); BH2-RFL-T2 type inverted microscope (OLYMPUS Corp.); type 311 CO2Incubator (Thermo corporation); SW-CJ-IF model superclean bench (Wujiang City Conghua clean plant); model 3K18 high speed bench top centrifuge (Sigma, usa); BT224S model electronic analytical balance (Sartorius corporation); 110S type electric heating constant temperature water bath (SHIPING company).
1.2 prescription of empirical prescription for blackening hair
20g of cacumen biotae, 10g of glossy privet fruit, 20g of eclipta alba, 10g of mulberry, 10g of scutellaria baicalensis and 10g of mint.
1.3 preparation of extract of empirical formula of Chinese medicine for blackening hair
1.3.1 preparation of the aqueous extract
The traditional Chinese medicine hair-blacking proved recipe is added with distilled water 6 times of the total weight of the medicinal materials for cold soaking for 30min, heated and boiled, kept boiling for 1h, filtered, then added with distilled water 4 times of the total weight of the medicinal materials for boiling, kept boiling for 30min, filtered, combined with two filtrates, concentrated into an extract, dissolved and diluted to a concentration of 1g/ml by using a phosphate buffer solution with a pH value of 6.8 and a concentration of 0.05mol/L, and stored at 4 ℃ for later use.
1.3.2 preparation of alcohol extracts
The traditional Chinese medicine hair-blacking empirical formula is prepared by hermetically soaking 75% ethanol 6 times the total weight of medicinal materials at room temperature for 30min, heating and refluxing at 85 ℃ for two times, boiling for the first time, keeping boiling for 40min, filtering, boiling medicinal residues for the second time by 75% ethanol 4 times the total weight of the medicinal materials, filtering, combining the two filtrates, volatilizing ethanol to form a paste, dissolving and diluting by using Phosphate Buffer (PBS) with pH of 6.8 and 0.05mol/L until the concentration is 1g/ml, and storing at 4 ℃ for later use.
2 method of experiment
2.1 culture of mouse B16 melanoma cells
Mouse melanoma cells B16 were cultured in high-glucose DMEM medium containing 10% PBS (100U/ml penicillin, 100g/ml streptomycin), digested with 0.25% trypsin and 0.02% EDTA, centrifuged, cells were suspended in the above medium at an adjusted cell density of 1X 105/ml, and 96-well plates (for cell proliferation assay) and 6-well plates (for melanin content assay) were seeded, respectively. After 24h of cell inoculation, the medium was changed with fresh medium to which the drug was added, and the cells were incubated for additional 48h (cell proliferation assay) and 72h (melanin content assay) for measurement.
2.2 preparation of test solutions and Experimental groups
The water extract and the alcohol extract are respectively diluted by 100mg/ml by serum-free DMEM medium, filtered and sterilized, and then diluted by a multiple dilution method to 10mg/ml, 5mg/ml, 2.5mg/ml, 1.25mg/ml, 0.625mg/ml, 0.3125mg/ml, 0.15625mg/ml and 0.078125mg/ml, and the blank control group is a medium (pure water or ethanol) containing a corresponding solvent of the tested substance.
2.3 detection of cell proliferation by MTT method
B16 cells in logarithmic growth phase were seeded in 96-well plates at a density of 100 l/well (seeding density 1X 10)4Hole/bore). After 24h, the medium was aspirated, the medium was changed with fresh medium (10mg/ml, 5mg/ml, 2.5mg/ml, 1.25mg/ml, 0.625mg/ml, 0.1325mg/ml, 0.0613mg/ml, 0.0306mg/ml) containing different concentrations of the treatment factors, no drug addition blank was set, incubation was continued for 48h, the medium was aspirated, washed with PBS, fresh medium was added, 10l of 5g/LMTT solution was added to each well, the incubator was set for 4h, the medium was aspirated, 150l of DMSO was added to each well, and the cells were shaken well enoughShaking, and measuring the absorbance at 490nm of each well by using a microplate reader. Each experiment was repeated 4 times.
2.4 determination of the Melanin content
B16 cells in logarithmic growth phase were seeded in 6-well plates at 1000 l/well (seeding density 1X 10)5In ml). After 24h, the medium was aspirated, the medium was changed with fresh medium (5mg/ml, 2.5mg/ml, 1.25mg/ml, 0.625mg/ml, 0.3125mg/ml) containing different concentrations of the treatment factors, 2000l per well, without addition of a drug blank, after further culturing for 72h, the medium was aspirated, washed with PBS, trypsinized, digestion was stopped, and the digestion solution was collected. Transferring the digestion solution into 1.5ml EP tube, centrifuging, removing supernatant, adding 10% trichloroacetic acid, centrifuging, removing supernatant, adding 1N NaOH solution, dissolving in water bath at 80 deg.C, transferring into ELISA plate, and immediately measuring 490nm absorbance of each well with ELISA reader. Each experiment was repeated 4 times. Melanin increment rate (drug group A-blank control group A)/blank control group A x 100%.
2.5 statistical methods
All the measured data are usedShowing that the statistical process was done using the SPSS13.0 statistical software using a one-way analysis of variance test.
3 results of the experiment
3.1 determination of mouse B16 melanoma cell proliferation Activity
The results in table 2 show that compared with the blank group, the aqueous extract of the empirical formula for blackening hair of traditional Chinese medicine has the obvious effect of promoting the proliferation of melanocyte (P is less than 0.05) at the concentration (0.15625, 0.3125, 0.625 and 1.25mg/ml), and the increment rate is highest at the concentration of 0.625 mg/ml; the concentration of the compound is (5, 10mg/ml), the compound has obvious inhibition effect on cell proliferation; the concentration of 2.5mg/ml has no statistical difference compared with a blank control group (P is more than 0.05); the traditional Chinese medicine hair-blacking experience formula alcohol extract has obvious promotion effect (P is less than 0.05) on melanocyte proliferation at the concentration (0.15625, 0.3125mg/ml), and has higher proliferation rate at the concentration of 0.3125 mg/ml; the concentration of the compound shows obvious effect of inhibiting cell proliferation at 0.625, 1.25, 5 and 10mg/ml, and the inhibition rate of the concentration of 10mg/ml is the highest; the concentration of 0.078125mg/ml was not statistically different from the blank control group (P > 0.05).
TABLE 2 influence of aqueous and alcoholic extracts of the empirical formula for blackening hair of traditional Chinese medicine on the proliferative activity of B16 melanoma cells
Note: p is less than 0.05, which shows obvious promoting effect compared with blank group; # shows that P is less than 0.05, and has obvious inhibition effect compared with blank group.
3.2 determination of the melanin content in mouse B16 melanoma cells
The results in Table 3 show that compared with the blank control group, the concentrations of the aqueous extract of the traditional Chinese medicine hair-blackening empirical formula are statistically different from those of the blank control group, which is represented by that the melanin content is obviously increased (P is less than 0.05), and the melanin content is the highest when the concentration is 1.25 mg/ml. The statistics of each concentration group of the traditional Chinese medicine hair-blacking proved recipe alcohol extract is different from that of a blank control group, which shows that the melanin content is obviously increased (P is less than 0.05), and the melanin content is the highest when the concentration is 2.5 mg/ml.
TABLE 3 influence of aqueous and alcoholic extracts of the empirical formula for blackening hair of Chinese medicinal herbs on the synthesis of melanoma cells: (
Value A)
Note: p is less than 0.05, which shows obvious promoting effect compared with blank group; # shows that P is less than 0.05, and has obvious inhibition effect compared with blank group.
Third, conclusion
The experimental research shows that: the aqueous extract and the alcohol extract of the traditional Chinese medicine hair-blacking empirical formula have the effects of activating tyrosinase, promoting cell proliferation and promoting melanin synthesis.
The above-described embodiment is only one of the preferred embodiments of the present invention, and general changes and substitutions by those skilled in the art within the technical scope of the present invention are included in the protection scope of the present invention.
Claims (6)
1. The traditional Chinese medicine hair-blacking empirical formula comprises the following traditional Chinese medicine composition in parts by weight:
1-40 parts of cacumen biotae, 1-40 parts of eclipta alba, 1-30 parts of glossy privet fruit, 1-30 parts of mulberry, 1-30 parts of scutellaria baicalensis and 1-30 parts of mint.
2. The traditional Chinese medicine hair-blacking empirical formula according to claim 1, which is characterized in that: the traditional Chinese medicine composition comprises the following traditional Chinese medicine compositions in parts by weight:
10-30 parts of cacumen biotae, 10-30 parts of eclipta alba, 5-20 parts of glossy privet fruit, 5-20 parts of mulberry, 5-20 parts of scutellaria baicalensis and 5-20 parts of mint.
3. The traditional Chinese medicine hair-blacking empirical formula according to claim 1, which is characterized in that: the traditional Chinese medicine composition comprises the following traditional Chinese medicine compositions in parts by weight: 20 parts of cacumen biotae, 20 parts of eclipta alba, 10 parts of glossy privet fruit, 10 parts of mulberry, 10 parts of scutellaria baicalensis and 10 parts of mint.
4. A pharmaceutical composition comprising an effective amount of the composition of any one of claims 1 to 3 and a pharmaceutically acceptable carrier.
5. A process for preparing the pharmaceutical composition of claim 4, comprising the steps of:
the preparation method of the lotion comprises the following steps: mixing cacumen Platycladi, Ecliptae herba, fructus Ligustri Lucidi, Mori fructus, Scutellariae radix and herba Menthae, adding 5-7 times of distilled water into the mixed medicinal materials, cold soaking for 20-40min, heating, boiling, keeping boiling for 40-80 min, filtering to obtain filtrate, adding 3-5 times of distilled water into the administration residue, boiling, keeping boiling for 20-40min, filtering, mixing the filtrates, standing for 36-60h, collecting supernatant, and adjusting pH to 6.0-6.5 with citric acid to obtain lotion.
6. A process for preparing the pharmaceutical composition of claim 4, comprising the steps of:
the preparation method of the liniment/tincture comprises the following steps: mixing folium Platycladi, Ecliptae herba, fructus Ligustri Lucidi, Mori fructus, Scutellariae radix and herba Menthae, adding 75% ethanol 5-7 times of the total weight of the medicinal materials into the mixed medicinal materials, soaking at room temperature for 20-40min under sealed condition, reflux-extracting at 85 deg.C twice, boiling for 20-60min after the first boiling, boiling the residue with 75% ethanol 3-5 times of the total weight of the mixed medicinal materials for 20-40min after the filtration, filtering, mixing filtrates, standing for 36-60h, and adjusting pH of the supernatant with citric acid to 6.0-6.5 to obtain liniment/tincture.
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