CN104706836B - A kind of Chinese medicine composition of eliminating chloasma and its application and preparation method - Google Patents
A kind of Chinese medicine composition of eliminating chloasma and its application and preparation method Download PDFInfo
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Abstract
The present invention relates to the field of Chinese medicines, more particularly to a kind of Chinese medicine composition and its application and preparation method.The Chinese medicine composition, it is made by following raw material：Radix Angelicae Sinensis, eclipta, the fruit of glossy privet, rose and grape seed extract.The result organized by the more different sides of tearing open of the present invention finds that lack the effect of Radix Angelicae Sinensis, eclipta, the fruit of glossy privet, rose, grape seed extract for suppressing the melanoma cells of mouse B 16 activity, tyrosinase activity and melanin generation weakens successively, and Radix Angelicae Sinensis, whether the addition of eclipta, the fruit of glossy privet has for result significantly affects.Illustrate that Chinese medicine composition provided by the invention has rational formula, wherein each component is indispensable.
Description
Technical field
The present invention relates to the field of Chinese medicines, more particularly to a kind of Chinese medicine composition and its application and preparation method.
Background technology
Chloasma is commonly called as " butterfly macules ", " chloasma hepaticum " or " cyasma ", a kind of acquired pigmentation skin disease, shows as
Pigment symmetry is calm, occurs mainly in face, and based on cheekbone portion, cheek, nose, forehead, chin portion, the lighter is faint yellow or shallow brown
Color；Severe one is in dark brown or light/dark balance.Be mainly in young and middle-aged women, be it is clinically common and be difficult to the skin disease cured it
One.Epidemiology shows that chloasma increases occurred in women, and with the age, aggravation.And Most patients with
Endocrine and the vegetative nerve functional disturbances such as different degrees of menstrual disorder, insomnia, dysphoria, life and essence are brought to patient
Refreshing aspect is many worried and painful.
Traditional Chinese medicine thinks, the generation of chloasma is more due to the deficiency of the kidney yin, kidney water can not on hold, or liver depression is depressed, liver loses bar
Reach, strongly fragrant longization heat, cloudy blood of burning, cause face qi-blood disharmony and fall ill.Clinically often there is irregular menstruation, waist in patient with chloasma
The card of the deficiencies of the kidney yin such as knees soreness, or show personality depression, feelings the will not liver-qi depression symptom such as smooth, being irritable and getting angry easily.It is and modern
Medical science thinks that chloasma increases mainly due to melanin and caused in skin, and melanocytosis and melanin generation increase
It is two principal elements for causing melanin to increase mostly.The factors such as ultraviolet, endocrine, heredity, oxygen radical, microorganism are to draw
Play the factor that melanin increases.In addition, also studies have found that, the formation of chloasma is relevant with following factors：It is endocrinopathy, pregnant
It is pregnent estrogen and progesterone level, oral contraceptive, ovary and uterus disease, inherent cause, oxygen radical, ultraviolet irradiation, blood
Clear copper content, hepatitis a and b, cholecystitis, tyrosine dysfunction, cosmetics, phototoxic drug, antiepileptic and mood ripple
Move.
Therefore, it is vital energy regualting and blood circulation-promoting mostly from the angle of differentiation of tcm, soothing liver-qi stagnation reconciles the integral syndrome differentiations such as heart spleen
It is mainly tyrosinase from the generation enzyme of suppression melanin, regulation is internal from the angle of Modern medical therapy by this disease is treated
Estrogen and progestogen is horizontal, removes free radical and MDA etc. treatment.
That circulates in the market claims that the product category with effect of dispelling spots is various, both includes being positioned at beauty modification work
Cream, emulsion, the cosmetics of aqua kenel, health food for oral administration to be also used for including capsule, oral liquid etc., in addition, also
It for cutaneous lesions and caused spot is the topical drug for the purpose for the treatment of to have some.It is most of to include in these products
The composition of drug composition or Chinese medical extract.Whether Chinese medicine or Chinese medical extract compatibility rationally decide that these products can rise
It is effect to good treatment chloasma.Irrational formula can not only have the function that to treat chloasma, on the contrary may
Cause the exacerbation of chloasma.
Therefore, further exploitation compatibility is reasonable, and the Chinese medicine composition that can effectively treat chloasma is very necessary.
The content of the invention
In view of this, the technical problem to be solved in the present invention is to provide a kind of Chinese medicine group that can effectively treat chloasma
Compound and its using preparation method.
The invention provides a kind of Chinese medicine composition, is made by following raw material：Radix Angelicae Sinensis, eclipta, the fruit of glossy privet, rose and
Grape seed extract.
In an embodiment of the present invention, Chinese medicine composition provided by the invention is made by the raw material of following mass parts：Radix Angelicae Sinensis 1
Part~10 parts, 1 part~6 parts of eclipta, 1 part~6 parts of the fruit of glossy privet, 1 part~8 parts of rose, grape seed extract 0.1 part~1.0
Part.
In certain embodiments, Chinese medicine composition provided by the invention is made by the raw material of following mass parts：
4 parts~8 parts of Radix Angelicae Sinensis, 2 parts~4 parts of eclipta, 2 parts~4 parts of the fruit of glossy privet, 3 parts~5 parts of rose, grape pip extraction
0.1 part~0.4 part of thing.
In certain embodiments, Chinese medicine composition provided by the invention is made by the raw material of following mass parts：6 parts of Radix Angelicae Sinensis,
3 parts of eclipta, 3 parts of the fruit of glossy privet, 4 parts of rose, 0.2 part of grape seed extract.
In certain embodiments, Chinese medicine composition provided by the invention is made by the raw material of following mass parts：5 parts of Radix Angelicae Sinensis,
4 parts of eclipta, 2 parts of the fruit of glossy privet, 5 parts of rose, 0.3 part of grape seed extract.
In certain embodiments, Chinese medicine composition provided by the invention is made by the raw material of following mass parts：4 parts of Radix Angelicae Sinensis,
4 parts of eclipta, 4 parts of the fruit of glossy privet, 4 parts of rose, 0.2 part of grape seed extract.
The grape seed extract that the eclipta present invention uses can also be bought voluntarily to prepare by market, and its implementation all exists
Within protection scope of the present invention.
In an embodiment of the present invention, the preparation method of grape seed extract is：Grape pip is crushed, carried with alcohol reflux
It is concentrated, dry after taking, obtain grape seed extract.
Preferably, the water content of grape pip is not more than 5%.
Preferably, the quality of ethanol is 10 times of grape pip.
Preferably, the temperature of refluxing extraction is 90 DEG C~100 DEG C, extract 2 times, each 2h.
Preferably, concentration, using being concentrated in vacuo, it is 1.2g/cm to be concentrated into density3~1.5g/cm3。
Preferably, drying using spray drying, temperature is 170 DEG C~180 DEG C.
Proanthocyanidin from Grape-seed Extracts >=95% used in the embodiment of the present invention, moisture≤5%,
PH value is 2.5~4.5, ash content≤0.5%, arsenic≤10mg/kg, iron≤3mg/kg.
Melanin is a kind of biopolymer, is widely present in the skin, hair and eyes of humans and animals.Melanin
Generation is closely related with tyrosinase, and it is occurred mainly in melanocyte.The forming process of melanin approximately as：Body
Interior tyrosine forms 3,4- dihydroxyphenylalanines (i.e. DOPA) under tyrosinase catalysis, DOPA further aoxidize to be formed it is more
Bar quinone, DOPA quinone generate dopachrome, dopachrome again through a series of hydroxylation reactions, decarboxylation through more polymerisations and oxidation reaction
Reaction, oxidation reaction form 5,6- istains, then combine to form melanin with other intermediate products.Therefore, the formation of chloasma
It is closely related with melanocyte activity and internal existing tyrosinase activity, in consideration of it, drug effect of the present invention in correlation
Detection mouse B-16 melanoma cells activity, tyrosinase activity and Melanin productions are have selected in experimentation to inquire into
Invention composition dispels effect for chloasma.
The result that the present invention is organized by the more different sides of tearing open finds to lack Radix Angelicae Sinensis, eclipta, the fruit of glossy privet, rose, grape pip
Effect of the extract for suppressing mouse B-16 melanoma cells activity, tyrosinase activity and melanin generation subtracts successively
It is weak, and Radix Angelicae Sinensis, eclipta, the fruit of glossy privet addition whether for result have significantly affect.Illustrate, Chinese medicine group provided by the invention
Compound has rational formula, and wherein each component is indispensable.
Application of the Chinese medicine composition provided by the invention in the product of eliminating chloasma is prepared.
Test result indicates that：(1) present composition can stimulate the propagation of B16 cells in extremely low concentration；But when its is dense
When degree is between 50 μ g/ml-400 μ g/ml, increase with concentration, inhibitory action enhancing；When present composition concentration is more than 400 μ g/
During ml, the inhibitory action of B-16 melanoma cells propagation is tended to balance, no longer strengthened；(2) present composition is extremely low
During concentration, there is certain promotion to tyrosinase activity, but when its concentration is between 100 μ g/ml-300 μ g/ml, with concentration
Increase, inhibitory action enhancing；When present composition concentration is more than 400 μ g/ml, tyrosinase activity inhibitory action is tended to
Balance, no longer strengthens；(3) present composition has certain promotion to melanin generation in extremely low concentration, but when its is dense
When degree is between 50 μ g/ml-150 μ g/ml, increase with concentration, inhibitory action enhancing；When present composition concentration is more than 150 μ g/
During ml, melanin generation inhibitory action is tended to balance, no longer strengthened.
In an embodiment of the present invention, the product of functions of removing chloasma is Orally taken product.
Preferably, the formulation of Orally taken product is tablet, pill, capsule, drink or granule.
Preferably, the product of functions of removing chloasma is health products, food or medicine.
Present invention also offers a kind of product of eliminating chloasma, including Chinese medicine composition provided by the invention.
Preferably, the medicine of eliminating chloasma includes Chinese medicine composition provided by the invention and pharmaceutically acceptable auxiliary
Material.
Preferably, pharmaceutically acceptable auxiliary material is：Diluent, absorbent, adhesive, wetting agent, disintegrant, lubrication
Any or both combination above in agent, coating material or capsule material.
Preferably, the health products of eliminating chloasma include being subjected in Chinese medicine composition provided by the invention and health products
Auxiliary material.
Preferably, acceptable auxiliary material is in health products：Microcrystalline cellulose, silica, pregelatinized starch, stearic acid
Magnesium, Ac-Di-Sol, methyl-silicone oil, lactose, sucrose, mannitol, sorbierite, steviol glycoside, cornstarch, wheat
In starch, rice starch, farina, gelatin, tragacanth, polyvinylpyrrolidone or calcium phosphate it is any or both more than
Combination.
Preferably, the food of eliminating chloasma includes acceptable original in Chinese medicine composition provided by the invention and food
Material and auxiliary material.
Preferably, auxiliary material is citric acid, malic acid, potassium sorbate, farina, cornstarch, wheaten starch, crystallite
Cellulose, lactose, sucrose, mannitol or sorbierite, stevioside, hydroxypropylcellulose, sodium carboxymethylcellulose, gelatin, soybean
Oil, tragacanth, sodium chloride, potassium chloride or calcium phosphate.
Preferably, raw material is honey, royal jelly or propolis.
The preparation method of Chinese medicine composition provided by the invention, including：By Radix Angelicae Sinensis, eclipta, the fruit of glossy privet and rose wate
After decocting, filter to get filtrate, filtrate is concentrated, mixed after drying with grape seed extract, obtains Chinese medicine composition.
In an embodiment of the present invention, the temperature of decocting is 80 DEG C~100 DEG C, and number is 1 time~3 times, each decocting
60min~120min.
Preferably, the temperature of decocting is 100 DEG C, number is 3 times, each decocting 90min.
In an embodiment of the present invention, the quality of the water of the decocting is Radix Angelicae Sinensis, eclipta, the fruit of glossy privet and rose quality
8 times of sum~10 times.
Preferably, the quality of the water of the decocting is 9 times of Radix Angelicae Sinensis, eclipta, the fruit of glossy privet and rose quality sum.
Preferably, concentration, using being concentrated in vacuo, thickening temperature is 60 DEG C.
Preferably, it is 1.1g/mL to be concentrated into density.
Preferably, it is 150 DEG C~200 DEG C to dry as spray drying, drying temperature.
The invention provides a kind of Chinese medicine composition, is made by following raw material：Radix Angelicae Sinensis, eclipta, the fruit of glossy privet, rose and
Grape seed extract.The result that the present invention is organized by the more different sides of tearing open finds to lack Radix Angelicae Sinensis, eclipta, the fruit of glossy privet, rose, Portugal
Grape seed extract is for suppressing the effect of mouse B-16 melanoma cells activity, tyrosinase activity and melanin generation successively
Weaken, and Radix Angelicae Sinensis, eclipta, the fruit of glossy privet addition whether for result have significantly affect.Illustrate Chinese medicine provided by the invention
Composition has rational formula, and wherein each component is indispensable.
Embodiment
The invention provides a kind of Chinese medicine composition and its application and preparation method, those skilled in the art can use for reference this
Literary content, it is suitably modified technological parameter realization.In particular, all similar replacements and change are to art technology
It is it will be apparent that they are considered as being included in the present invention for personnel.The method of the present invention and application are by preferable
Embodiment is described, related personnel substantially can not depart from present invention, in spirit and scope to side as described herein
Method and application are modified or suitably changed with combining, to realize and using the technology of the present invention.
The raw material that the present invention uses is all common commercially available product, can all be bought by market.
Wherein, mouse B-16 melanoma cells strain, provided by Hua Shen groups；
ELIASA：Bio-Rad, Model 550；
Centrifuge：LDZ5-2, Beijing Medical Centrifugal Machine Factory；
Inverted microscope：Nikon DEAPHOT Fx-35DX；
Carbon dioxide cell incubator：BNA-311, ESPEC；
Precision electronic balance (precision 0.01g)：EB-3200D, Japanese Shimadzu；
Electronic balance (precision 0.001g)：JA1003A, Shanghai Jingtian Electronic Instrument Co., Ltd.；
Centrifugation device：80-2, Shanghai Surgical Operation Equipment Factory；
Continuous and quantitative adjustable moving liquid-transfering gun：10ul-100ul, 20ul-200ul, 100-1000ul, 1-5ml, Finland's thunder
Vigorous Finnpipette；
Quartz ultraviolet germicidal lamp：The sensible health equipment Co., Ltd in Yangzhou；
Intelligent vertical stainless steel sterilizer：LDZM-40KCS, Shanghai Shen An Medical Devices Co., Ltd.s；
SOD reagents：Bioengineering Research Institute's offer, lot number 20031225 are built up in Nanjing；
MDA reagents：Bioengineering Research Institute's offer, lot number 20031226 are built up in Nanjing；
Vulcanized sodium：500g/ bottles, chemical pure, winner chemical reagent factory, lot number 980923；
Physiological saline：0.9% sodium chloride injection, 500ml：4.5g.Chinese medicines quasi-word H51021158, the big medicine in Sichuan Cologne
Co., Ltd of factory, lot number 20030921-18；
RPMI-1640：GIBCO companies；
Hyclone, hyclone：HyClone companies；
L DOPA：U.S.'s Slpa Products, lot number D9628；
Trypsase：The magnificent biotech firm's product in Shanghai, lot number 2186；
MTT, SDS, ConA, agar：Sigma companies；
Separately there are glutamine, penicillin, gentamicin, Tris Base and other domestic AR.
With reference to embodiment, the present invention is expanded on further：
The preparation of the Chinese medicine composition of embodiment 1
After Radix Angelicae Sinensis 60g, eclipta 30g, fruit of glossy privet 30g, rose 40g are cleaned, 1280g drinking water, 80 DEG C are heated to,
Decocting three times, decocts 120 minutes, obtains filtered fluid every time；Filtrate is concentrated, thickening temperature is 60 DEG C, is concentrated into proportion 1.1g/
ml；Concentrate is spray-dried, spray drying temperature is 150~200 DEG C, obtains fine powder A.Fine powder A and 2g grape seed extracts are mixed
Close, obtain Chinese medicine composition.
The preparation of the Chinese medicine composition of embodiment 2
After Radix Angelicae Sinensis 80g, eclipta 40g, fruit of glossy privet 40g, rose 50g are cleaned, 2100g drinking water, 90 DEG C are heated to,
Decocting 3 times, decoct 90 minutes every time, obtain filtered fluid；Filtrate is concentrated, thickening temperature is 60 DEG C, is concentrated into proportion 1.1g/ml；
Concentrate is spray-dried, spray drying temperature is 150~200 DEG C, obtains fine powder A.Fine powder A is mixed with 4g grape seed extracts, obtained
To Chinese medicine composition.
The preparation of the Chinese medicine composition of embodiment 3
After Radix Angelicae Sinensis 40g, eclipta 20g, fruit of glossy privet 20g, rose 30g are cleaned, 1100g drinking water, 120 are heated to
DEG C, decocting 3 times, decoct 60 minutes every time, obtain filtered fluid；Filtrate is concentrated, thickening temperature is 60 DEG C, is concentrated into proportion 1.1g/
ml；Concentrate is spray-dried, spray drying temperature is 150~200 DEG C, obtains fine powder A.Fine powder A and 2g grape seed extracts are mixed
Close, obtain Chinese medicine composition.
The preparation of the Chinese medicine composition of embodiment 4
After Radix Angelicae Sinensis 10g, eclipta 10g, fruit of glossy privet 10g, rose 10g are cleaned, 400g drinking water, 120 DEG C are heated to,
Decocting 2 times, decoct 60 minutes every time, obtain filtered fluid；Filtrate is concentrated, thickening temperature is 60 DEG C, is concentrated into proportion 1.1g/ml；
Concentrate is spray-dried, spray drying temperature is 150~200 DEG C, obtains fine powder A.Fine powder A is mixed with 1g grape seed extracts, obtained
To Chinese medicine composition.
The preparation of the Chinese medicine composition of embodiment 5
After Radix Angelicae Sinensis 100g, eclipta 60g, fruit of glossy privet 60g, rose 80g are cleaned, 3000g drinking water, 90 are heated to
DEG C, decocting 1 time, decoct 120 minutes, obtain filtered fluid；Filtrate is concentrated, thickening temperature is 60 DEG C, is concentrated into proportion 1.1g/ml；
Concentrate is spray-dried, spray drying temperature is 150~200 DEG C, obtains fine powder A.Fine powder A is mixed with 10g grape seed extracts, obtained
To Chinese medicine composition.
The preparation of the side's of tearing open the Chinese medicine composition of comparative example 1
After eclipta 30g, fruit of glossy privet 30g, rose 40g are cleaned, 1000g drinking water, 80 DEG C are heated to, decocting three
It is secondary, decoct 120 minutes every time, obtain filtered fluid；Filtrate is concentrated, thickening temperature is 60 DEG C, is concentrated into proportion 1.1g/ml；Will be dense
Contracting liquid is spray-dried, and spray drying temperature is 150~200 DEG C, obtains fine powder A.Fine powder A is mixed with 2g grape seed extracts, in obtaining
Drug composition.
The preparation of the side's of tearing open the Chinese medicine composition of comparative example 2
After Radix Angelicae Sinensis 60g, fruit of glossy privet 30g, rose 40g are cleaned, 1300g drinking water is heated to 80 DEG C, decocting three times,
Decoct 120 minutes every time, obtain filtered fluid；Filtrate is concentrated, thickening temperature is 60 DEG C, is concentrated into proportion 1.1g/ml；By concentrate
Spray drying, spray drying temperature are 150~200 DEG C, obtain fine powder A.Fine powder A is mixed with 2g grape seed extracts, obtains Chinese medicine group
Compound.
The preparation of the side's of tearing open the Chinese medicine composition of comparative example 3
After Radix Angelicae Sinensis 60g, eclipta 30g, rose 40g are cleaned, 1300g drinking water is heated to 80 DEG C, decocting three times,
Decoct 120 minutes every time, obtain filtered fluid；Filtrate is concentrated, thickening temperature is 60 DEG C, is concentrated into proportion 1.1g/ml；By concentrate
Spray drying, spray drying temperature are 150~200 DEG C, obtain fine powder A.Fine powder A is mixed with 2g grape seed extracts, obtains Chinese medicine group
Compound.
The preparation of the side's of tearing open the Chinese medicine composition of comparative example 4
After Radix Angelicae Sinensis 60g, eclipta 30g, fruit of glossy privet 30g are cleaned, 1200g drinking water is heated to 80 DEG C, decocting three times,
Decoct 120 minutes every time, obtain filtered fluid；Filtrate is concentrated, thickening temperature is 60 DEG C, is concentrated into proportion 1.1g/ml；By concentrate
Spray drying, spray drying temperature are 150~200 DEG C, obtain fine powder A.Fine powder A is mixed with 2g grape seed extracts, obtains Chinese medicine group
Compound.
The preparation of the side's of tearing open the Chinese medicine composition of comparative example 5
After Radix Angelicae Sinensis 60g, eclipta 30g, fruit of glossy privet 30g, rose 40g are cleaned, 1600g drinking water, 80 DEG C are heated to,
Decocting three times, decocts 120 minutes, obtains filtered fluid every time；Filtrate is concentrated, thickening temperature is 60 DEG C, is concentrated into proportion 1.1g/
ml；Concentrate is spray-dried, spray drying temperature is 150~200 DEG C, obtains Chinese medicine composition.
The preparation of the side's of tearing open the Chinese medicine composition of comparative example 6
After fruit of glossy privet 30g, rose 40g are cleaned, 700g drinking water, 80 DEG C are heated to, decocting three times, decocts every time
120 minutes, obtain filtered fluid；Filtrate is concentrated, thickening temperature is 60 DEG C, is concentrated into proportion 1.1g/ml；Concentrate spraying is dry
Dry, spray drying temperature is 150~200 DEG C, obtains fine powder A.Fine powder A is mixed with 2g grape seed extracts, obtains Chinese medicine composition.
The preparation of the side's of tearing open the Chinese medicine composition of comparative example 7
After rose 40g is cleaned, 400g drinking water, 80 DEG C are heated to, decocting three times, is decocted 120 minutes, obtained every time
Filtered fluid；Filtrate is concentrated, thickening temperature is 60 DEG C, is concentrated into proportion 1.1g/ml；Concentrate is spray-dried, spray drying temperature
For 150~200 DEG C, fine powder A is obtained.Fine powder A is mixed with 2g grape seed extracts, obtains Chinese medicine composition.
The preparation of the side's of tearing open the Chinese medicine composition of comparative example 1
After Radix Angelicae Sinensis 60g, rose 40g are cleaned, 1000g drinking water, 80 DEG C are heated to, decocting three times, decocts 120 every time
Minute, obtain filtered fluid；Filtrate is concentrated, thickening temperature is 60 DEG C, is concentrated into proportion 1.1g/ml；Concentrate is spray-dried,
Spray drying temperature is 150~200 DEG C, obtains fine powder A.Fine powder A is mixed with 2g grape seed extracts, obtains Chinese medicine composition.
The preparation of the functions of removing chloasma health products of embodiment 6
Chinese medicine composition prepared by any one of Example 1~5, is mixed with auxiliary material, is pelletized, is obtained the health care of functions of removing chloasma
Product.
The preparation of the chloasma diminishing medicine of embodiment 7
Chinese medicine composition prepared by any one of Example 1~5, is mixed with microcrystalline cellulose, is pelletized, and obtains removing chloasma
Medicine.
The preparation of the functions of removing chloasma food of embodiment 8
Chinese medicine composition prepared by any one of Example 1~5, mixes with honey, obtains the food of functions of removing chloasma.
The Chinese medicine composition of embodiment 9 is acted on B16 cell inhibitory effects
The suppression for taking Chinese traditional medicine composition analyte detection made from the embodiment of the present invention 1~3 and comparative example 1~8 to breed B16 cells
Effect, experiment set the blank control for not adding Chinese medicine composition.Experimental method is：
1.1 nutrient solutions are prepared
(1) RPMI-1640 nutrient solutions (1000ml)：RPMI-1640 dry powder is dissolved in 300ml tri-distilled waters, bag is washed with water
Pack 2 times, pour into mergings, stir, add tri-distilled water at nearly 1000ml：Add NaHCO32g, supplement glutamine is to final concentration of
3mmol/L, add phenol red (final concentration of 5.3mg/L)；Add antibiotic a penicillin (100U/ml), b gentamicins (50U/
ml)；It is settled to 1000ml；With NaOH or HCl regulations PH to 7.2.Filtration sterilization, 4 DEG C of preservations.Draw the RPMI- prepared
1640 culture medium 2ml enters in 25ml glass Tissue Culture Flask, in 37 DEG C, 5%CO2With overnight incubation under complete damp condition,
Whether microbiological contamination is observed under inverted microscope.The nutrient solution of non-microbiological contamination can be tested.
The RMPI-1640 nutrient solutions (100ml) of (2) 10% calf serums：The RPMI-1640 nutrient solution 90ml prepared are taken,
10ml calf serums are added, with NaOH or HCl regulations PH to 7.2.Filtration sterilization, 4 DEG C of preservations.What absorption prepared contains 10%
The RPMI-1640 nutrient solutions 2ml of calf serum enters in 25ml glass Tissue Culture Flask, in 37 DEG C, 5%CO2With complete humidity
Under the conditions of overnight incubation, observe whether microbiological contamination under inverted microscope.The nutrient solution of non-microbiological contamination can be tested.
The RMPI-1640 nutrient solutions (100ml) of (3) 10% hyclones：The RPMI-1640 nutrient solution 80ml prepared are taken,
Add 10ml hyclones and 10mlHeper ' s (final concentration of 5mmol/L)；2- mercaptoethanols (2-Me) are added to final concentration
PH to 7.2 is adjusted with NaOH or HCI for 50 μm of ol/L.Filtration sterilization, 4 DEG C of preservations.What absorption prepared contains 10% hyclone
RPMI-1640 nutrient solutions 2ml enter in 25ml glass Tissue Culture Flask, in 37 DEG C, 5%CO2Trained with complete damp condition
Support overnight, microbiological contamination is seen whether under inverted microscope.The nutrient solution of non-microbiological contamination can be tested.
1.2 digestive juices are prepared
(1) mother liquor：With the trypsin solution that 0.25% is prepared without calcium and magnesium Hanks liquid.Filtration sterilization, 4 DEG C of guarantors
Deposit.0.04% EDTA, autoclaving, 4 DEG C of preservations are prepared with PBS.
(2) prepared using liquid：0.4ml 0.04% EDTA is taken, the trypsase of 0.4ml 0.25%, sterile PBS is added and arrives
2ml, 4 DEG C of preservations.
1.3 inorganic salt buffers are prepared
(1) PBS solution (1000ml)：8g NaCl (137mmol/L), 0.2g KCl (2.7mmol/L), 1.15g
Na2HPO4·7H2O (4.3mmol/L), 0.2g KH2PO4(1.4mmol/L) is dissolved in 1L distilled waters, adjusts PH to 7.2.High pressure
Sterilizing, 4 DEG C of preservations.
(2) 0.83%Tris-NH4Cl solution (500ml)：3.0275g Tris (0.05mol/L) are dissolved in 250ml distilled waters
In；4.15g NH4Cl (0.83%) is dissolved in 250ml distilled waters.Two kinds of solution mixing, add and 500ml are settled in distilled water.It is high
Pressure sterilizing, 4 DEG C of preservations.
1.4 observation Chinese medicine compositions are acted on B16 cell inhibitory effects
Take the logarithm the B16 cells in growth period, 0.25% Trypsin Induced, RMPI-1640 nutrient solutions are collected thin after digestion
Born of the same parents, 1000rpm centrifugations 3-5min.Discard supernatant liquor.Cell is dispelled with the RMPI-1640 nutrient solutions of 10% calf serum, will
It is diluted to 5 × 104Individual/ml.Take 0.1ml cell suspensions to add 96 porocyte culture plates, be placed on 37 DEG C, 5%CO2Carbon dioxide
Cultivated in incubator.
The Chinese medicine composition that embodiment 1~3 and comparative example 1~8 provide is configured to 2000 μ g/ml's with physiological saline
Mother liquor, 121 DEG C of sterilizing 20min.Mother liquor is diluted to final concentration with the RMPI-1640 nutrient solutions of 10% calf serum：0、25、
50th, 75,100,150,200,250,300,350,400,800,1200,1600 μ g/ml (totally 14 gradients) add adherent growth
It is 200 μ L per hole final volume, each concentration sets 4 multiple holes, averaged in 4h cell culture system.Experiment sets blank pair
According to blank control group does not add decoction.
In 37 DEG C, 5%CO2Cultivated 72 hours in CO2gas incubator.Terminate to abandon per hole for first 5 hours in cell culture
The μ l of culture supernatant 100 are removed, add the MTT10 μ l (using normal saline, filtration sterilization) that concentration is 5mg/ml.Culture terminates
Afterwards, add 10%SDS (to configure) 100 μ l vibrations with 0.01M HCl per hole to mix, 4 hours at 37 DEG C, with ELIASA in 570nm
Read 0D values in place.Statistical software uses DPS 7.05 editions, compares between processing and uses LSD using single factor analysis, Multiple range test
Method.
Wherein, the inhibitory action that the Chinese medicine composition that embodiment 1 provides is bred to B16 cells is as shown in table 1：
The embodiment 1 of table 1 provides the influence (n-4, x ± S) that Chinese medicine composition suppresses B-16 propagation in vitro
| Concentration (μ g/ml) | OD values | Concentration (μ g/ml) | OD values |
| 0 | 0.397±0.011a | 300 | 0.375±0.013b |
| 25 | 0.405±0.008a | 400 | 0.360±0.008b |
| 50 | 0.410±0.013a | 500 | 0.359±0.013b |
| 75 | 0.407±0.014a | 800 | 0.358±0.016b |
| 100 | 0.401±0.012a | 1000 | 0.359±0.011b |
| 150 | 0.400±0.010a | 1200 | 0.358±0.009b |
| 200 | 0.386±0.011a | 1400 | 0.357±0.015b |
| 250 | 0.390±0.013a | 1600 | 0.358±0.023b |
Note：A, b represents significant difference (P be present between different groups in same row<0.05).
From table 1, the influence bred with the mtt assay measure present composition to B-16 melanoma cells, with blank
Control, which is compared, obvious inhibitory action.In extremely low concentration, the present composition can stimulate the propagation of B16 cells；But work as it
When concentration is more than 50 μ g/ml, increase with concentration, then show inhibitory action, optimal inhibition concentration is 400 μ g/ml；Work as the present invention
When composition concentration is more than 400 μ g/ml, to B-16 melanoma cells propagation inhibitory action will not again with concentration increase and
Increase.The testing result of Chinese medicine composition is made similarly in the embodiment of the present invention 2~5.
Wherein, fixed concentration is 400 μ g/ml, and the Chinese medicine composition and comparative example 1~8 that comparing embodiment 1 provides provide
The inhibitory action bred to B16 cells of Chinese medicine composition it is as shown in table 2：
The present composition of table 2 (the difference side of tearing open group) suppresses the influence (n-4, x ± S) of B-16 propagation
Note：The selection that table 2 tests each drug concentration is carried out on the basis of table 1 is preferably rear, i.e., concentration is 400 μ g/
ml；
A, b, c represent significant difference (P between different groups with a line be present<0.05)
From table 2, determine what the present composition (the difference side of tearing open group) was bred to B-16 melanoma cells with mtt assay
Influence, compared with blank group, other groups have certain inhibitory action, and compared with control group, all sides of tearing open group suppresses
Effect is obvious to be reduced, in addition, the result that the more different sides of tearing open organize finds to lack Radix Angelicae Sinensis, eclipta, the fruit of glossy privet, rose, grape
Influence of the seed extract for result weakens successively, and whether Radix Angelicae Sinensis, the addition of eclipta, the fruit of glossy privet have significantly for result
Influence.The testing result of Chinese medicine composition is made similarly in the embodiment of the present invention 2~5.
It can be seen that the present composition in extremely low concentration, can stimulate the propagation of B16 cells；But when its concentration is between 50 μ g/
During ml-400 μ g/ml, increase with concentration, inhibitory action enhancing；When present composition concentration is more than 400 μ g/ml, to B-16
The inhibitory action of melanoma cells propagation tends to balance, and no longer strengthens.
Influence of the Chinese medicine composition of embodiment 10 to tyrosinase activity
Take suppression of the Chinese traditional medicine composition analyte detection to tyrosinase activity made from the embodiment of the present invention 1~3 and comparative example 1~8
Make and use, experiment sets the blank control for not adding Chinese medicine composition.The preparation of nutrient solution, digestive juice, inorganic salt buffer, mother liquor
Method is same as Example 9, and experimental method is：
Take the logarithm the B16 cells in growth period, 0.25% Trypsin Induced, RMPI-1640 nutrient solutions are collected thin after digestion
Born of the same parents, 1000rpm centrifugations 3-5min.Discard supernatant liquor.Cell is dispelled with the RMPI-1640 nutrient solutions of 10% calf serum, will
It is diluted to 8 × 104Individual/ml.0.1ml cell suspensions are taken to add 96 porocyte culture plates.It is placed on 37 DEG C, 5%CO2Saturation vapour
Cultivated in CO2gas incubator.
Mother liquor is diluted to final concentration with the RMPI-1640 nutrient solutions of 10% calf serum：0、25、50、 75、100、
150th, 200,250,300,350,400,800,1200,1600 μ g/ml (totally 14 gradients) add adherent growth 4h cell training
It is 200 μ L per hole final volume, each concentration sets 4 multiple holes in the system of supporting.In 37 DEG C, 5%CO2Trained in CO2gas incubator
Support 48 hours.Supernatant liquor is discarded, cell is washed 2 times with PH7.4 PBS, 50 μ l, 1%TritonX-100 are added per hole.Will
96 porocyte culture plates are put into rapidly -30 DEG C of refrigerators, are taken out after 30min, and naturally to thaw is so as to destroying cell at room temperature. 37℃
1%L- DOPA solution (10 μ l/ holes) is added after pre-temperature, 37 DEG C of reaction 2h, 0D values are read at 490nm with ELIASA.Count soft
Part uses DPS 7.05 editions, compares between processing and uses LSD methods using single factor analysis, Multiple range test.
Wherein, the Chinese medicine composition that embodiment 1 provides is as shown in table 3 to the inhibitory action of tyrosinase activity：
Influence (n-4, x ± S) of the present composition of table 3 to tyrosinase activity
| Concentration (ug/ml) | OD values | Concentration (ug/ml) | OD values |
| 0 | 0.208±0.015a | 300 | 0.142±0.022b |
| 25 | 0.215±0.010a | 400 | 0.100±0.017c |
| 50 | 0.212±0.018a | 600 | 0.099±0.015c |
| 75 | 0.213±0.015a | 800 | 0.098±0.014c |
| 100 | 0.209±0.014a | 1000 | 0.097±0.012c |
| 150 | 0.197±0.017a | 1200 | 0.098±0.014c |
| 200 | 0.160±0.014b | 1400 | 0.099±0.008c |
| 250 | 0.160±0.012b | 1600 | 0.099±0.009c |
Note：A, b, c represent significant difference (P be present between different groups in same row<0.05)
From table 3, mouse B-16 melanoma cells through the present composition after acting on 48 hours, this hair of low concentration
Bright composition (the μ g/ml of ＜ 50) has certain promotion to tyrosinase activity, but increases with concentration, then to tyrosinase activity
There is obvious inhibitory action.Present composition concentration can effectively suppress B-16 melanin in the range of 100-400 μ g/ml
The tyrosinase activity of tumor cell strain, and optimal inhibition concentration is 400 μ g/ml, when present composition concentration is more than 400 μ g/
During ml, the inhibitory action of tyrosinase activity will not be increased as concentration increases again.During the embodiment of the present invention 2~5 is made
The testing result of drug composition is similarly.
Wherein, fixed concentration is 400 μ g/ml, and the Chinese medicine composition and comparative example 1~8 that comparing embodiment 1 provides provide
Chinese medicine composition it is as shown in table 4 to the inhibitory action of tyrosinase activity：
The present composition of table 4 (the difference side of tearing open group) suppresses the influence (n-4, x ± S) of tyrosinase activity
Remarks：The selection that table 4 tests each drug concentration is carried out on the basis of table 3 is preferably rear, i.e. 400 μ g/ml；
A, b, c represent significant difference (P between different groups with a line be present<0.05).
From table 4, compared with blank group, control group and the different sides of tearing open group have necessarily for tyrosinase activity
Inhibitory action, and compared with control group, all sides of tearing open group inhibitory action substantially reduces, in addition, the more different side's of tearing open groups
Result find lack the influence of Radix Angelicae Sinensis, eclipta, the fruit of glossy privet, rose, grape seed extract for result weaken successively, and work as
Return, eclipta, the fruit of glossy privet addition whether for result have significantly affect.
It can be seen that the present composition in extremely low concentration, has certain promotion to tyrosinase activity, but work as its concentration
During between 100 μ g/ml-300 μ g/ml, increase with concentration, inhibitory action enhancing；When present composition concentration is more than 400 μ g/
During ml, tyrosinase activity inhibitory action is tended to balance, no longer strengthened.
The influence that the Chinese medicine composition of embodiment 11 generates to melanin
The suppression for taking Chinese traditional medicine composition analyte detection made from the embodiment of the present invention 1~3 and comparative example 1~8 to generate melanin
Effect, experiment set the blank control for not adding Chinese medicine composition.Nutrient solution, digestive juice, inorganic salt buffer, the preparation side of mother liquor
Method is same as Example 9, and experimental method is：
Take the logarithm the B16 cells in growth period, 0.25% Trypsin Induced, RMPI-1640 nutrient solutions are collected thin after digestion
Born of the same parents, 1000rpm centrifugations 3-5min.Discard supernatant liquor.Cell is dispelled with the RMPI-1640 nutrient solutions of 10% calf serum, will
It is diluted to 1 × 105Individual/ml.1ml cell suspensions are taken to add 24 porocyte culture plates.In 37 DEG C, 5%CO2Carbon dioxide culture
Cultivated in case.
Mother liquor is diluted to final concentration with the RMPI-1640 nutrient solutions of 10% calf serum：0、25、50、 75、100、
150th, 200,250,300,350,400,800,1200,1600 μ g/ml (totally 14 gradients) add the adherent growth cell of 4 hours
It is 200 μ L per hole final volume, each concentration sets 4 multiple holes, in 37 DEG C, 5%CO in cultivating system2In CO2gas incubator
Culture 96 hours.0.25% trypsin digestion and cell, 1 × 10 is taken per hole5Individual cell centrifugation discards supernatant liquor, adds 1ml
1mol/L NaOH, shake 5 minutes, 0D values are read at 490nm with ELIASA.Statistical software uses DPS 7.05 editions, place
Compare between reason and LSD methods are used using single factor analysis, Multiple range test.
Wherein, the inhibitory action that the Chinese medicine composition that embodiment 1 provides generates to melanin is as shown in table 5：
The influence (n-4, x ± S) that the present composition of table 5 generates to melanin
| Concentration (ug/ml) | OD values | Concentration (ug/ml) | OD values |
| 0 | 0.099±0.010a | 300 | 0.049±0.019c |
| 25 | 0.095±0.009a | 400 | 0.048±0.008c |
| 50 | 0.090±0.012a | 600 | 0.050±0.018c |
| 75 | 0.088±0.009a | 800 | 0.049±0.020c |
| 100 | 0.076±0.020b | 1000 | 0.051±0.013c |
| 150 | 0.069±0.015b | 1200 | 0.047±0.010c |
| 200 | 0.049±0.010c | 1400 | 0.049±0.009c |
| 250 | 0.049±0.010c | 1600 | 0.049±0.009c |
Note：A, b, c represent significant difference (P be present between different groups in same row<0.05).
From table 5, after the present composition acts on 4d to mouse B-16 melanoma cells, melanin content is determined,
Below 50 μ g/ml, increase with drug concentration, melanin content is continuously increased, and is illustrated at much lower concentrations, present invention combination medicine
Thing has the function that necessarily to promote melanin to generate；And when being more than 50 μ g/ml concentration, increase with concentration, the present composition can
The obvious generation for suppressing melanin, when present composition concentration is 150 μ g/ml, its suppression to melanin generation reaches
It is maximum；Between 150-1600 μ g/ml, the suppression to melanin tends to be steady.
Wherein, fixed concentration is 150 μ g/ml, and the Chinese medicine composition and comparative example 1~8 that comparing embodiment 1 provides provide
Chinese medicine composition it is as shown in table 6 to the inhibitory action of tyrosinase activity：
The present composition of table 6 (the difference side of tearing open group) suppresses the influence (n-4, x ± S) of melanin generation
Note：The selection that table 6 tests each drug concentration is carried out on the basis of table 5 is preferably rear, i.e., concentration is 100 μ g/
ml；
A, b represents significant difference (P between different groups with a line be present<0.05).
From table 6, compared with blank group, control group and the different sides of tearing open group have certain for melanin generation
Inhibitory action, and compared with control group, all sides of tearing open group inhibitory action substantially reduces, in addition, what the more different sides of tearing open organized
As a result find that lack the influence of Radix Angelicae Sinensis, eclipta, the fruit of glossy privet, rose, grape seed extract for result weakens successively, and work as
Return, eclipta, the fruit of glossy privet addition whether for result have significantly affect.
The present composition has certain promotion to melanin generation in extremely low concentration, but when its concentration is between 50 μ
During g/ml~150 μ g/ml, increase with concentration, inhibitory action enhancing；It is right when present composition concentration is more than 150 μ g/ml
Melanin generation inhibitory action tends to balance, and no longer strengthens.
It can be seen that the present composition has the function that certain functions of removing chloasma, and Radix Angelicae Sinensis, eclipta, the fruit of glossy privet are to result
With significantly affecting, the component that the present invention is provided in Chinese medicine composition is indispensable.
Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Claims (9)
- A kind of 1. Chinese medicine composition of eliminating chloasma, it is characterised in thatIt is made by the raw material of following parts by weight：1 part ~ 10 parts of Radix Angelicae Sinensis, 1 part ~ 6 parts of eclipta, 1 part ~ 6 parts of the fruit of glossy privet, 1 part of rose ~ 8 parts, 0.1 part ~ 1.0 parts of grape seed extract.
- 2. Chinese medicine composition according to claim 1, it is characterised in that be made by the raw material of following parts by weight：4 parts of Radix Angelicae Sinensis ~ 8 parts, 2 parts ~ 4 parts of eclipta, 2 parts ~ 4 parts of the fruit of glossy privet, 3 parts ~ 5 parts of rose, 0.1 part ~ 0.4 part of grape seed extract.
- 3. Chinese medicine composition according to claim 1, it is characterised in that the Procyanidin in Grape Seed Extracts contains Amount is not less than 95%.
- 4. application of the Chinese medicine composition as described in any one of claim 1 ~ 3 in the product of eliminating chloasma is prepared.
- 5. application according to claim 4, it is characterised in that the product is Orally taken product.
- 6. a kind of product of eliminating chloasma, it is characterised in that including the Chinese traditional medicine composition as described in any one of claim 1 ~ 3 Thing.
- 7. the preparation method of the Chinese medicine composition as described in any one of claim 1 ~ 3, it is characterised in that including：By Radix Angelicae Sinensis, ink After non-irrigated lotus, the fruit of glossy privet and rose decocting, filter to get filtrate, will be mixed after filtrate concentration, drying with grape seed extract, Obtain Chinese medicine composition.
- 8. preparation method according to claim 7, it is characterised in that the temperature of the decocting is 80 DEG C ~ 100 DEG C, number For 1 time ~ 3 times, each decocting 60min ~ 120min.
- 9. preparation method according to claim 7, it is characterised in that the weight of the water of the decocting is the Radix Angelicae Sinensis, ink Non-irrigated lotus, 8 times ~ 10 times of the fruit of glossy privet and rose weight sum.
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