CN107375474A - Leucoderma dimension medicine compound and preparation method thereof - Google Patents

Abstract

The present invention relates to a kind of leucoderma dimension medicine compound and preparation method thereof, the compound is made up of medicinal materials such as fennelflower seed, madder, vernonia anthelmintica, cloves, common fig leaf juice and arna its roots, and tincture, gel is made using solvent extraction and pharmaceutic adjuvant.The present invention inherits the Medicinal Trait of Uygur medicine, theoretical to instruct with Uygur medicine, according to the Uygur nationality in long-term clinical practice among the people, obtains leucoderma dimension medicine compound using modern extracting method, ensures active principle content, improves bioavilability.Leucoderma dimension medicine compound has dysregulation lymphatic temperament, promoting blood circulation coloring, for leucoderma, leukoderma.External activity shows that different prescription leucoderma dimension medicine compounds are respectively provided with very strong cell melanin and increase activity.Pharmacological outcomes show that leucoderma dimension medicine compound of the present invention can treat leucoderma in various degree.

Description

Leucoderma dimension medicine compound and preparation method thereof

Technical field

The present invention relates to a kind of dimension medicine compound for leucoderma and preparation method thereof, belongs to uighur medicine and national doctor Medicine technical field.

Background technology

Leucoderma (vitiligo) is a kind of common skin pigment depigmentation disease, the function by skin and (or) hair follicle The reduction or forfeiture of property melanocyte cause.The sick illness rate is 0.5%-2% in the world, and cardinal symptom is black for large area The local or general hair property leukoderma that pigment loss is brought occurs, although the disease will not cause mortal injury tight to human body The outward appearance of ghost image sound patient and normal doings simultaneously bring stress to patient.Leucoderma pathogenesis is not yet complete at present Illustrate entirely, but increasing research supports leucoderma close ties to be present, in inherent cause and ring with abnormal immune response Epidermis feature melanocyte (MC) of lacking of proper care and cause may occur for normal cutaneous immunisation reaction in the presence of the stimulation of border Specific Damage, finally develop into leucoderma.

Leucoderma system depigmentation dermatoses, easily diagnosis, it is refractory treatment and without specific treatment.Its treatment is divided into one at present Line is treated and second line treatment.First-line treatment is that external application glucocorticoid, Calcineurin inhibitors, phototherapy and photochemistry are treated Method；Second line treatment mainly has vitamin D 3 analogs, oral glucocorticoid and operative treatment.Therapy of vitiligo common recognition (2009 Version) leucoderma is divided into progressive stage and stationary phase treated, two phases were respectively divided into homeliness type and segmental vitiligo treatment again.The past state The effect of inside and outside scholar treats leucoderma to above-mentioned treatment method and single method carried out comparative studies, the clinic of single treatment Research has tended to be ripe, and its curative effect and security are clear and definite.Therapeutic alliance in recent years turns into clinical the most frequently used Therapeutic mode, Such as phototherapy joint external medication, operative treatment joint phototherapy, the experiment of its relevant clinical also gradually increase, but by mesh Preceding also none of these methods can allow all patients to obtain satisfied effect.

Finding the natural drug for the treatment of leucoderma and extracting its active ingredient has also turned into modern medicine exploration and has developed new A kind of effective way of medicine.Therefore, the medicine with anti-leucoderma is developed from the Chinese herbal medicine and ethnic drug in China with important Social effect and economic value.

Uygur's medical science is as a part for traditional Chinese medicine, the theory of medicine system with uniqueness and many classics sides Agent, the especially classic prescriptions in terms of leucoderma is treated obtain the concern and accreditation of medical field with therapeutic effect.Uygur's medical science Teiology thinks that crossing for internal abnormal phlegm is contained, weak, the body natural force decline of the dominating organs function such as liver, the heart, brain, mistake Eat raw property food, inherent cause, damage, the reason such as compression causes leucoderma to skin for a long time more.Uygur's basic medical reason Think by Pathological Physiology because abnormal phlegm is in nature containing excessively wet and excessively cold, the device such as infringement stomach, small intestine, liver The normal physiological function of official, promote the generation of abnormal humour, generate vicious circle, make to necessary energy in material cyclic process Weaken with natural force, the colouring function of weak blood matter.And the internal abnormal phlegm calmness contained of crossing is in skin bottom, obstruction skin The normal blood circulation of skin, nutritional ingredient is seted to be unable to normal growth and development from being transported to skin position, melanocyte, so as to Depigmentation of skin, there is leucoderma.Body is in itself when also with the disease such as diabetes, hyperthyroidism, psoriasis, lupus erythematosus, alopecia areata Elemental weak and liver normal physiological function destruction causes the excessive generation of abnormal humour, the overall antiviral energy of body Power declines, and is not normally functioning in the 4th digestion that skin tissue cell is carried out and causes leucoderma.

Uygur medicine possesses the characteristics of Multiple components compatibility, toxic side effect is small, and can be according to the specific state of an illness of different patients Different treatments is imposed, therapeutic features are prominent, curative for effect, painful few, can cure the leucoderma of different type difference by stages.Cause And it is more beneficial for treating the state of an illness of patient, effective sharp weapon of maintaining healthy, new road is opened for the treatment of the disease.

The content of the invention

It is used to treat leucoderma dimension medicine compound and preparation method thereof it is an object of the present invention to provide one kind, the compound is It is made up of bulk drug fennelflower seed, madder, vernonia anthelmintica, cloves, common fig leaf juice and arna its root etc., using solvent extraction Along with tincture and gel is made in acceptable auxiliary material in pharmacy.The dimension medicine compound has treatment different type and different points The various leucoderma of phase, it is evident in efficacy, have no drug resistance.Through pharmacy in vitro, test result indicates that, this can effectively facilitate B16 cells The growth of melanin.

A kind of leucoderma dimension medicine compound of the present invention, the compound is by bulk drug fennelflower seed 5-100 parts, expelling parasite spot Turtledove chrysanthemum 5-100 parts, madder 5-80 parts, cloves 5-40 parts, common fig leaf juice 5-40 parts and arna its root 1-30 parts be made tincture or Gel.

The leucoderma ties up medicine compound preparation method, and concrete operations follow these steps to carry out：

A, raw material fennelflower seed 5-100 parts, vernonia anthelmintica 5-100 parts, madder 5-80 parts, cloves 5-40 parts, nothing are taken Flowers and fruits leaf 5-40 parts and arna its root 1-30 parts, it is that 10%-95% ethanol extracts to measure concentration with 5-30 times, and temperature is boiled for solvent Point, extract 1-4 time, each 1-6 hours, filter, merging filtrate, which is recovered under reduced pressure ethanol and obtains liquid extract or be concentrated under reduced pressure, dry must soak Cream powder, crush, obtain extract fine powder, it is standby；

B, add ethanol that tincture is made according to a conventional method the liquid extract obtained in step a so that concentration of the tincture containing ethanol For 30%-90%；

Or step a extract fine powder 1%-5% is prepared into gel according to a conventional method, gel auxiliary material is gel base Matter is carbomer, sodium alginate, xanthans, carboxyethyl cellulose, methylcellulose or sodium carboxymethylcellulose 1-20 parts, is permeated Accelerator is Laurocapram, borneol, menthol or natural borneol 1-10 parts, and NMF is propane diols, glycerine or Tween 80 1-15 parts, preservative are that sorbic acid, P-hydroxybenzoic acid, P-hydroxybenzoic acid sodium or sodium benzoate 0.1-10 parts, surplus are water.

A kind of leucoderma dimension medicine compound of the present invention, wherein respectively dimension medicine major function is：

Fennelflower seed：Uygur's name " this sub- denier ".For ranunculaceae plant nigella glandulifera Freyn (Nigella glandulifera Freyn dry mature seed).With diuresis, promoting blood circulation, tonifying brain and kidney, detoxify, stimulate the menstrual flow, promoting lactation, black hair, treatment bronchial astehma, Anti-aging, it is usually used in the diseases such as gas uncomfortable in chest is sufficient, breast is swollen, milk reduction, tinnitus, heat gonorrhea, urolithiasis, amenorrhoea, it can in addition contain stimulate Digestion, kidney-tonifying and brain tonic, stomach invigorating, sweating, wind dispelling analgesic, anti-worm and other effects, main pharmacological have anti-oxidant, anticancer, antibacterial, A variety of physiologically actives such as relieving cough and asthma, platelet aggregation-against, anti-oxidant, antitumor, liver protection, lipid-loweringing, hypoglycemic, immunological regulation etc..

Madder：Uygur's name " Wu Erdan ".Used as natural dye, its chemical composition is with anthraquinone and its glycoside chemical combination Based on thing, additionally containing naphthoquinones class, terpene, hexamethylene peptides, polysaccharide, trace element etc., there is cool blood, hemostasis, dissolving stasis, logical Through and other effects, its main pharmacological is hemostasis, increasing leukocyte, promotes body hematopoiesis function, be antitumor, suppresses toxohormone L (relevant with liver cancer), cough-relieving remove what is said or talked about, anti-acetylcholine effect (spasmolysis), antibacterial, anti-oxidant, immunosupress, anti HIV-1 virus Deng effect.

Vernonia anthelmintica：Uygur's name " card power cumin ".The main chemical compositions contained are based on flavone compound, tool Have and remove abnormal lymphatic temperament, expelling parasite, detumescence, eliminating cold to stop pain and other effects, be now used for treating raw property stomachache, hepatopathy, leucoderma Etc. illness, its pharmacological action has anticancer, activation tyrosinase activity, raising melanin content, regulation immunity of organism, improvement local micro- Circulation, directly supplement local trace element etc..

Cloves：Uygur's name " blocks blue pul ".Main chemical compositions include Phenylpropanoid Glycosides class, iridoids, driffractive ring cyclenes Ether terpene, flavonoids, Hydroxybenzenes, triterpenes, organic acid etc., there is anti-inflammatory, antibacterial, antiviral, anti-oxidant, cardiac stimulant, guarantor The pharmacological activity such as liver, hypoglycemic, decompression, booster immunization system, antipyretic, analgesia.

Common fig leaf juice：Change containing Coumarins (psoralen, Bergapten), flavones, electrolytes and minerals etc. Study point, it is tool treatment hemorrhoid, pyogenic infections, pained, while for clearing away damp-heat, solution wet poison, swelling and pain relieving effect, its pharmacological action is anti- Virus, antibacterial is antitumor, and reducing blood lipid is hypoglycemic, antioxidation activity and tranquilizing soporific, suppresses phlegm trace keloid fibroblasts, resists Osteoporosis etc..

Its root of arna：Uygur's name " Acker Kai Ha ".This product is feverfew Rome Dalmatian chrysanthemum Anacyclus Pyrethrum (L.) DC. dry root, spring and autumn excavation, is cleaned, and cut-out, is dried.With raw dry heat, Eradicates trembles with fear strengthening tendons, open-minded Retardance, excess in brain is eliminated, establishing-Yang of warming up, controlling nocturnal emission with astringent drugs, wind-expelling pain-stopping is eliminating dampness and eliminating phlegm, nourishing nerve.It is mainly used in raw property or viscous Liquid matter disease, such as paralysis, facial paralysis, of flaccid muscles, paralysis, vibration, hysteria, tongue weight, sexual hypoesthesia, seminal fluid is not solid, tooth of having a headache Bitterly, more productive coughs are coughed, neurasthenia etc., take 1g orally.

A kind of leucoderma dimension medicine compound of the present invention and preparation method thereof, with being used for leucoderma on present medical market Medicine compare, have following characteristics：

(1) every taste medicinal material in prescription, it is selected with reference to clinical reasoning and the practical experience of the local famous medical expert of dimension, It is curative for effect with long-term clinical practice and anti-leucoderma effect, quick, safety, reliably.

(2) present invention has no side effect, had no drug resistance and physical dependence compared with the Western medicine for the treatment of leucoderma.

(3) preparation technology is simple, and cost is low, has certain market competitiveness.

(4) crude drug source enriches, it is ensured that quality, is adapted to industrialized production.

Embodiment：

For the ease of the understanding of those skilled in the art, by embodiment, the invention will be further described below.

Embodiment 1

It is radix system for tincture using 1000ml：

The embodiment prescription ratio of table 1

A, in the prescription ratio gone out given in table 1 by the raw medicinal material fennelflower seed through having screened, madder, expelling parasite ringdove Its root mixing of chrysanthemum, cloves, common fig leaf juice and arna, extracted with 30 times of amount concentration for 95% ethanol, temperature is solvent boiling point, extraction 1 time, 6 hours time, filtration, ethanol is recovered under reduced pressure, obtains liquid extract, it is standby；

B, the liquid extract obtained in step a is added into ethanol, be made 70% tincture (per 100ml equivalent to former medicine materical crude slice 10g~ 20g), surplus is water, dispenses every bottle of 100mL, 200mL, tincture is made.

Embodiment 2

It is radix system for tincture using 1000ml：

A, in the prescription ratio gone out given in table 1 by the medicinal material fennelflower seed through having screened, madder, vernonia anthelmintica, Its root mixing of cloves, common fig leaf juice and arna, extracted with 20 times of amount concentration for 75% ethanol, temperature is solvent boiling point, extraction 2 Secondary, extraction 2 hours, are filtered, merging filtrate is recovered under reduced pressure ethanol, obtains liquid extract, standby every time；

B, the liquid extract obtained in step b is added into ethanol, be made 90% tincture (per 100ml equivalent to former medicine materical crude slice 10g~ 20g), surplus is water, dispenses every bottle of 100mL, 200mL, tincture is made.

Embodiment 3

It is radix system for tincture using 1000ml：

A, in the prescription ratio gone out given in table 1 by the medicinal material fennelflower seed through having screened, madder, vernonia anthelmintica, Its root mixing of cloves, common fig leaf juice and arna, to be extracted with 5 times of amount concentration for 10% ethanol, temperature is solvent boiling point, is extracted 3 times, Extraction 3 hours every time, filtration, merging filtrate is recovered under reduced pressure ethanol, obtains liquid extract, standby；

B, the liquid extract obtained in step b is added into ethanol, be made 30% tincture (per 100ml equivalent to former medicine materical crude slice 10g~ 20g), surplus is water, dispenses every bottle of 100mL, 200mL, tincture is made.

Embodiment 4

It is radix system for tincture using 1000ml：

A, in the prescription ratio gone out given in table 1 by the medicinal material fennelflower seed through having screened, madder, vernonia anthelmintica, Its root mixing of cloves, common fig leaf juice and arna, extracted with 15 times of amount concentration for 50% ethanol, temperature is solvent boiling point, extraction 4 Secondary, 1 hour every time, filtration, merging filtrate was recovered under reduced pressure ethanol, obtains liquid extract, standby；

B, the liquid extract obtained in step b is added into ethanol, be made 65% tincture (per 100ml equivalent to former medicine materical crude slice 10g~ 20g), surplus is water, dispenses every bottle of 100mL, 200mL, tincture is made.

Embodiment 5

It is radix system for gel using 1000g：

A, in the prescription ratio gone out given in table 1 by the medicinal material fennelflower seed through having screened, madder, vernonia anthelmintica, Its root mixing of cloves, common fig leaf juice and arna, to be extracted with 5 times of amount concentration for 95% ethanol, temperature is solvent boiling point, is extracted 2 times, 3 hours every time, filtration, decompression filtrate recycling ethanol, be concentrated under reduced pressure dry extract powder, pulverizes and sieves, obtains extract fine powder, It is standby；

B, weigh step a 1 part of 1 part of 1 part of extract fine powder 1%, Acritamer 940, Laurocapram, propane diols, the third three 5 parts of alcohol, 0.1 part of sodium benzoate, surplus is water, and gel is made according to a conventional method.

Embodiment 6

It is radix system for gel using 1000g：

A, in the prescription ratio gone out given in table 1 by the medicinal material fennelflower seed through having screened, madder, vernonia anthelmintica, Its root mixing of cloves, common fig leaf juice and arna, it is that 75% ethanol is extracted with 20 times of amount concentration, temperature is solvent boiling point, is carried Take 1 time, extraction time is 6 hours, and filtration, ethanol is recovered under reduced pressure in merging filtrate, and be concentrated under reduced pressure dry extract powder, crushed Sieve, obtains extract fine powder, standby；；

B, the extract fine powder 2% obtained in step a, 10 parts of sodium alginate are weighed, 10 parts of xanthans, 10 parts of borneol, is told Warm 80 15 parts, 1 part of sorbic acid, surplus is water, and gel is made according to a conventional method.

Embodiment 7

It is radix system for gel using 1000g：

A, in the prescription ratio gone out given in table 1 by the medicinal material fennelflower seed through having screened, madder, vernonia anthelmintica, Its root mixing of cloves, common fig leaf juice and arna, extracted with 15 times of amount concentration for 50% ethanol, temperature is solvent boiling point, extraction 3 Secondary, each extraction time is 3 hours, and filtration, ethanol is recovered under reduced pressure in merging filtrate, and be concentrated under reduced pressure dry extract powder, crushed Sieve, obtains extract fine powder, standby；

B, the extract fine powder 3% obtained in step a, 20 parts of sodium carboxymethylcellulose, 3 parts of menthol, natural ice are weighed 2 parts of piece, 10 parts of glycerine, 10 parts of P-hydroxybenzoic acid sodium, surplus is water, and gel is made according to a conventional method.

Embodiment 8

It is radix system for gel to prepare 1000g：

A, in the prescription ratio gone out given in table 1 by the medicinal material fennelflower seed through having screened, madder, vernonia anthelmintica, Its root mixing of cloves, common fig leaf juice and arna, extracted with 10 times of amount concentration for 30% ethanol, temperature is solvent boiling point, extraction 4 Secondary, each extraction time is 1 hour, and filtration, ethanol is recovered under reduced pressure in merging filtrate, and be concentrated under reduced pressure dry extract powder, crushes, obtains It is standby to extract fine powder；

B, the extract fine powder 4% obtained in step a, 15 parts of carboxyethyl cellulose, 5 parts of methylcellulose, bay are weighed 8 parts of Azone, 15 parts of propane diols, 8 parts of P-hydroxybenzoic acid, surplus is water, and gel is made according to a conventional method.

Embodiment 9

Dimension medicine compound of the present invention is in the evaluating drug effect for leucoderma：

1. experiment purpose：This research is intended utilizing modern medicine study theory, and mouse black is carried out to compound medicinal substances extract Plain oncocyte (B16 cells) screening active ingredients test, so as to confirm the bioactivity of the anti-leucoderma of different formulations；

2. experiment material：

2.1 experiment material：Instrument and consumptive material：Ice machine, inverted microscope, 6 porocyte culture plates, micropipettor (are inhaled Head), CO2gas incubator, electric pipettor, the pipette of different ranges, Biohazard Safety Equipment, carbon dioxide canister, cytometer Number instrument, normal temperature low speed centrifuge, refrigerated centrifuge, vortex oscillation instrument, M5 ELIASAs, cell are scraped, Tissue Culture Dish (diameter 60mm, 100mm, 150mm), 96 hole elisa Plates, 1.5mL EP pipes, 0.5mL EP pipes, disposable Thin film glove, emgloves；

2.2 reagent：High glucose medium (DMEM), hyclone, trypsase (0.25%Trypsin-EDTA), blue or green strepto- Plain dual anti-, dimethyl sulfoxide (DMSO) (DMSO), trypan blue dye liquor (Trypanblue), 8-methoxypsoralen (8-MOP), phosphoric acid delays Rush solution, 75% alcohol, sodium hydroxide (NaOH), hydrochloric acid (HCl)；

Detect medicine：Tie up medicine compound extract, concentration 50mg/mL；

2.3 solution are prepared：

2.3.1 the preparation of phosphate buffer (PBS) storing liquid：Sodium chloride (NaCl) 80g, 12 hypophosphite monohydrates are weighed respectively Disodium hydrogen (Na₂HPO₄·12H₂O) 32.5g, 2 hypophosphite monohydrate sodium dihydrogen (NaH₂PO₄·2H₂O) 4.5g is into 900mL ultra-pure waters, It is 7.4 that pH is adjusted after shaking dissolving, adds ultra-pure water to 1000ml, in 4 DEG C of preservations of temperature；

Face the used time take storing liquid 100mL add 900mL ultra-pure waters, shake up, used after sterilizing；

2.3.2 the preparation of sodium hydroxide (NaOH) lysate：4g NaOH are weighed, after adding 80mL distilled waters fully to dissolve, are added Enter 10mL dimethyl sulfoxide (DMSO)s (DMSO), ultra-pure water is settled to 100mL；

2.3.3 the preparation of 8-methoxypsoralen (8-MOP)：0.0108g 8-methoxypsoralen is weighed, is added Enter 1mL dimethyl sulfoxide (DMSO)s (DMSO) fully to dissolve, 100 μ L/ pipe lucifuges freeze；

2.3.4 cell pyrolysis liquid RIPA preparation (liquid storage)：

2.3.5 the preparation of protease inhibitors：4-NPP disodium salt hexahydrate (pNPP), ultra-pure water are prepared 1M storing liquids：0.371g 4-NPP disodium salt hexahydrate is weighed, adds 1mL ultra-pure waters, fully dissolving, temperature - 20 DEG C of degree freezes standby；

Sodium fluoride (NaF, Sodium fluoride) ultra-pure water prepares 1M storing liquids：419.9mg NaF is weighed, is added 10mL ultra-pure waters, fully dissolving, -20 DEG C of temperature freezes standby；

Phenylmethylsulfonyl fluoride (PMSF) prepares 10mM liquid storages with isopropanol：174.19mg phenylmethylsulfonyl fluoride is weighed, is added 10mL ultra-pure waters, fully dissolving, -20 DEG C of temperature freezes standby；

DL- dithiothreitol (DTT)s (DTT) prepare 100mM storing liquids with ultra-pure water；Weigh the 154.25mg sulphur threoses of DL- bis- Alcohol, 1mL ultra-pure waters, fully dissolving are added, -20 DEG C of temperature freezes standby；

Benzenecarboximidamide (Benzamidine) prepares 100mM storing liquids with ultra-pure water；120.15mg benzenecarboximidamide is weighed, is added 10mL ultra-pure waters, fully dissolving, -20 DEG C of temperature freezes standby；

Sodium orthovanadate (OV, Sodium orthavanadium) prepares 200mM storing liquids with ultra-pure water；Weigh 800.26mg Sodium orthovanadate, add 10mL ultra-pure waters, fully dissolving, -20 DEG C of temperature freezes standby；

According to the how much matching while using for collecting cell concentration；

2.3.6 the inactivation and packing of serum：Every bottle of 500mL serum places 4 DEG C of defrostings of temperature, temperature 56 after thawing completely 30min is placed in DEG C water-bath, is inactivated, and -20 DEG C of temperature freezes after the packing of 50mL/ pipes, and packaging operation is in the ultra-clean work of iuntercellular Platform is carried out；

2.3.7 dual anti-packing：4 DEG C of the dual anti-placement temperature of 100mL/ bottles is carried out after thawing completely, the packing of 5.6mL/ pipes - 20 DEG C of temperature is frozen afterwards, and packaging operation is carried out in iuntercellular superclean bench；

2.3.8 complete medium prepares (matching while using)：Contain 10% hyclone and 1% mycillin in culture medium Dual anti-, specific compound method is that the hyclone and 5.6mL green grass or young crops strepto-s of 50mL packing are added in high glucose medium (DMEM) 500mL Element is dual anti-, is used after well mixed, matching while using；

3. experimental method：

3.1 cell culture：Recovery mouse black-in tumor cell B16 (passage of this room freezes), complete medium culture to cell When degree of converging reaches more than 80%, culture medium is abandoned, phosphate buffer (1 × PBS) washs 1 time, is added according to culture area size 0.5-2mL pancreatin digests, and Microscopic observation cell edges start shrinkage and become bowlder (about 1min), and culture medium gently blows after terminating digestion Beating makes cell take off wall, and cell counter carries out cell count.Adjustment cell density is inoculated in diameter to 2.0 × 105 cell/mL In 150mm Tissue Culture Dish, 37 DEG C of temperature, 5%CO are put₂Cultivated in incubator；

3.2 passages and inoculation：Take the logarithm the B16 melanoma cells of phase, nutrient solution is discarded, with phosphate buffer (PBS) liquid Cleaning 2 times, after pancreatin digestion, 2.5 × 105/hole of cell concentration is adjusted, is accessed in 6 orifice plates, 2mL, overnight incubation are added per hole (about 12h-16h)；

3.3 drug-treated：The culture medium 2mL/ holes that next day more renews, cleaned 1 time, before dosing with phosphate buffer before changing Sample places about 30min in room temperature, fully mixes extract using vortex oscillation instrument and is administered according to 20 μ g/mL concentration, and sets Solvent control group dimethyl sulfoxide (DMSO) (DMSO) and positive controls (50 μM of 8-MOP), each concentration group set 3 multiple holes, in 37 DEG C of 5%CO of temperature₂48h (notes are cultivated in constant incubator：If cell inoculum concentration is higher, culture medium its colour changed into yellow, it should The fresh culture of drug containing is changed during processing, ensures the nutrient of abundance, is consumed for cell metabolism)；

3.4 sample collections (whole process operates on ice)：Each treatment group is put on ice for after 48h, sucks culture Liquid, cleaned 2 times with the phosphate buffer (PBS) of precooling, be sufficiently absorbed through adding the cell of 100 μ L precoolings per hole after residual liquid Lysate, rocking 6 orifice plates makes lysate fully cover attached cell.Scraped with cell and be transferred to 1.5mL after quickly cell is scraped EP manages (import), 4 DEG C of 1000rpm of temperature, and supernatant is abandoned after centrifuging 5min；The phosphate buffer (PBS) of 100 μ L precoolings is added, Vortex oscillation instrument is mixed, 1000 turns/min of temperature 4 DEG C, and supernatant is abandoned after centrifuging 5min；

3.5 melanin are collected and dissolving：After EP pipes are placed on the blending instrument of 4 DEG C of temperature fully mixing 40min, temperature 4 DEG C 12000 turns/min, 20min is centrifuged, into new EP pipes, the sodium hydroxide (NaOH) that precipitation adds 190 μ L splits transfer supernatant Liquid, temperature 60 C water-bath 2h, or 80 DEG C of water-bath 1h of temperature are solved, mixing is turned upside down during water-bath for several times, makes to be deposited in pipe The melanin granule at bottom fully dissolves；

3.6 sample protein assays：Supernatant protein content is determined, is tried using Bo Maide Bradford protein quantifications Agent box, operated according to kit operating procedure, calculate the protein concentration C in each hole；

3.7 melanin light absorption values determine：Water-bath takes 150 μ L to determine measure OD at 405nm into 96 hole elisa Plates after terminating Value；

3.8 statistical analysis：Calculate melanocyte relative amount, melanocyte relative amount=melanocyte OD405/ protein concentration C, melanocyte life Into activity ratio=experimental group melanocyte relative amount/solvent control group melanocyte relative amount × 100%；

4. experimental result：The sample solution of different prescriptions preparation various concentrations is weighed by table 1, it is black to B16 cells to detect it The influence of pigment content, the results are shown in Table 2；

Influence of the 2 different prescriptions of table to B16 cell melanin contents

Conclusion：The leucoderma dimension medicine compound of different proportion composition has under relatively low concentration promotes melanoma cells to produce The bioactivity of raw melanin, experimental result show that each group cell melanin total amount substantially increases, have good anti-leucoderma Activity.

Claims (2)

1. a kind of leucoderma ties up medicine compound, it is characterised in that the compound is by bulk drug fennelflower seed 5-100 parts, vernonia anthelmintica Tincture or gel is made in 5-100 parts, madder 5-80 parts, cloves 5-40 parts, common fig leaf juice 5-40 parts and arna its root 1-30 parts Agent.

2. leucoderma according to claim 1 ties up medicine compound preparation method, it is characterised in that concrete operations follow these steps Carry out：

A, raw material fennelflower seed 5-100 parts, vernonia anthelmintica 5-100 parts, madder 5-80 parts, cloves 5-40 parts, fig are taken Leaf 5-40 parts and arna its root 1-30 parts, it is that 10%-95% ethanol extracts to measure concentration with 5-30 times, and temperature is solvent boiling point, extraction 1-4 times, each 1-6 hours, filtration, merging filtrate is recovered under reduced pressure ethanol and obtains liquid extract or dry the extract powder that is concentrated under reduced pressure, powder It is broken, extract fine powder is obtained, it is standby；

B, add ethanol that tincture is made according to a conventional method the liquid extract obtained in step a so that concentration of the tincture containing ethanol is 30%-90%；

Or step a extract fine powder 1%-5% is prepared into gel according to a conventional method, gel auxiliary material is that gel-type vehicle is card Ripple nurse, sodium alginate, xanthans, carboxyethyl cellulose, methylcellulose or sodium carboxymethylcellulose 1-20 parts, penetration enhancer For Laurocapram, borneol, menthol or natural borneol 1-10 parts, NMF is propane diols, glycerine or Tween 80 1-15 Part, preservative is that sorbic acid, P-hydroxybenzoic acid, P-hydroxybenzoic acid sodium or sodium benzoate 0.1-10 parts, surplus are water.