CN103505452B

CN103505452B - The application of epigallocatechin gallate (EGCG) in the anti-leucoderma medicament of preparation

Info

Publication number: CN103505452B
Application number: CN201310405395.9A
Authority: CN
Inventors: 许爱娥
Original assignee: Individual
Current assignee: Individual
Priority date: 2013-09-06
Filing date: 2013-09-06
Publication date: 2016-03-30
Anticipated expiration: 2033-09-06
Also published as: CN103505452A

Abstract

The invention provides epigallocatechin gallate (EGCG) (Epigallocatechingallate, EGCG) application in the leukodermic medicine of preparation control, EGCG effectively can suppress CD8+T cell activation to be bred thus reduce CD8+T cell to reach therapeutic effect to melanocytic specific killing.Beneficial effect of the present invention is mainly reflected in: epigallocatechin gallate (EGCG) is natural in the extract green tea, can produce in a large number, substantially have no side effect, its can specific inhibition CD8+T cell for the immunologic injury of autologous melanoma cell, treat vitiligo efficiently, for new medicament screen provides the foundation.

Description

The application of epigallocatechin gallate (EGCG) in the anti-leucoderma medicament of preparation

(1) technical field

The present invention relates to the application of epigallocatechin gallate (EGCG) (Epigallocatechingallate, EGCG) in the leukodermic medicine of preparation control.

(2) background technology

Vitiligo is a kind of pigment loss dermatoses, and the sickness rate in global population is up to 0.5 ~ 2%.Its clinical manifestation is local or general property skin pigment depigmentation.The white macula of the exposure portion such as neck surface causes disfiguring infringement to patient, the serious physical and mental health affecting patient.Current research is thought, this disease is a kind of autoimmune disease, and CD8+T Cytotoxic Lymphocytes is the immediate cause causing epidermal melanophore to lack to melanocytic specific killing.It is multifactor that its cause of disease comprises h and E etc., but up to the present definite pathogenic factor is not clear, and at present this disease is lacked to the medicine of radical cure, and treatment is quite difficult.The leukodermic external used medicine for the treatment of conventional clinically mainly comprises hormones and some photosensitive class medicines, but life-time service hormone medicine can cause atrophoderma, and photosensitizer can cause the side effect such as skin foaming as Fructus Psoraleae, and offer limited effectiveness.

In order to overcome the strong shortcoming of existing treatment leucoderma medicament side effect, improve therapeutic effect, the present invention provides a kind of according to leukodermic pathogenesis and can effectively suppress CD8+T cell activation to be bred thus the new natural epigallocatechin gallate (EGCG) reducing that CD8+T cell to reach therapeutic effect to melanocytic specific killing simultaneously.Epigallocatechin gallate (EGCG) (structural formula is shown in Fig. 1) is a kind of composition in green tea, there is antioxidation, anticancer, mutation isoreactivity, also do not have epigallocatechin gallate (EGCG) suppress CD8+T cell activation propagation and treat leukodermic report at present.

(3) summary of the invention

The present invention is to provide the novelty teabag of epigallocatechin gallate (EGCG), i.e. the application of epigallocatechin gallate (EGCG) in the leukodermic medicine of preparation control.

The technical solution used in the present invention is:

The application of epigallocatechin gallate (EGCG) in the leukodermic medicine of preparation control.

Concrete, described epigallocatechin gallate (EGCG) is for the preparation of the medicine suppressing CD8+T cell activation propagation.

Described medicine can according to a conventional method, makes one of following dosage form: liniment, lotion, spray.

Preferably, in described medicine, epigallocatechin gallate (EGCG) concentration is 2 ~ 5%.

At present, the approval that the effect of melanocyte in vitiligo morbidity has obtained academia is is killed and wounded to CD8+T cell-specific, therefore have and suppress CD8+T cell activation propagation, and suppress the medicine of CD8+T cell skin homing effectively can improve the leukodermic effect for the treatment of.Because CD8+T cell needs active oxygen (ROS) as signal media at the activation initial stage, the generation of ROS in CD8+T cell is suppressed likely to suppress the activation of CD8+T cell and follow-up biological function.The present invention starts with from the strong anti-oxidation ability of epigallocatechin gallate (EGCG), inquires into its immunosuppressive action to CD8+T cell.

First the Activated in Vitro multiplicative model of mice CD8+T cell is set up, the activation and proliferation of verification table nutgall catechin gallic acid ester to CD8+T cell has inhibitory action significantly, and inhibitory action and concentration are proportionate, the external lowest concentration of drug reaching maximum suppression effect is 10ug/ml.

Simultaneously, on mice vitiligo animal model, verification table nutgall catechin gallic acid ester effectively suppresses the gathering of CD8+T cell around this decolouring speckle of Monot, the time that the vitiligo reducing monobenzone induction occurs, ratio and area, its minimum useful effect drug level is 2%.

Beneficial effect of the present invention is mainly reflected in: epigallocatechin gallate (EGCG) is natural in the extract green tea, can produce in a large number, substantially have no side effect, its can specific inhibition CD8+T cell for the immunologic injury of autologous melanoma cell, treat vitiligo efficiently, for new medicament screen provides the foundation.

(4) accompanying drawing explanation

Fig. 1 is epigallocatechin gallate (EGCG) molecular structural formula;

Fig. 2 is the in-vitro multiplication that MTS method detects mouse spleen CD8+T cell; X-coordinate is epigallocatechin gallate (EGCG) concentration, and Y-coordinate is the relative rate of increase of CD8+T cell;

Fig. 3 is that situation appears in the mice vitiligo of monobenzone induction under different condition; A uses monobenzone processed group merely, and monobenzone coating place circle marks, and non-coating position white macula arrow marks; B is that monobenzone adds epigallocatechin gallate (EGCG) treatment group.

Fig. 4 is the mice non-agents area decolouring speckle Area comparison of monobenzone induction under different condition; III, IV, V are respectively 1%, 2% and 3% nutgall catechin gallic acid ester treatment group.

Fig. 5 is that under different condition, around mice monobenzone agents area, CD8+T cellular infiltration situation compares.

(5) detailed description of the invention

Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in this:

Embodiment 1:

Laboratory animal:

C57BL/6 mice, female, in 6 ~ 8 week age, weight (20 ± 2) g, is purchased from Shanghai Slac Experimental Animal Co., Ltd.;

Experimental drug:

Epigallocatechin gallate (EGCG): Sigma Co., USA, with water dissolution;

Hyclone: LifeTechnologies company of the U.S.;

IL-2: LifeTechnologies company of the U.S.;

PBS: LifeTechnologies company of the U.S.;

RMPI-1640 culture medium: Ji Nuo biological medicine technology company limited;

Erythrocyte cracked liquid: LifeTechnologies company of the U.S.;

CD8+T cell separation test kit: German Miltenyl company;

CD3, CD28 monoclonal antibody: eBioscience company of the U.S.;

MTS cytoactive detection kit: Promega company;

CO ₂incubator: Thermo company of the U.S.;

Spectramax190 microplate reader: MolecularDevices company of the U.S.;

Tissue Culture Plate: corning company of the U.S.;

Experimental procedure:

(1) mouse spleen single cell suspension preparation

Mice is put to death in the dislocation of neck marrow, aseptic separating mouse spleen, be placed in and fill appropriate aseptic PBS plate, with syringe, spleen is pulverized, make single cell suspension, centrifugal 5 minutes of room temperature 1600rpm, abandon the erythrocyte cracked liquid that supernatant adds 3 times of cell volumes, cracking 30 seconds after mixing, then wash 3 times with PBS, abandon supernatant.

(2) CD8+T separation of lymphocytes is cultivated

With reference to German Mei Tian Ni Bioisystech Co., Ltd mouse spleen CD8+T cell separation description operation.Every 1 × 10 ⁷the unicellular 40ul dissociating buffer of mouse spleen is (containing PBSpH7.2,0.5% hyclone and 2mMEDTA) resuspended, 10 minutes are hatched in 4 degree after adding 10ulCD8+T cell Biotin-AntibodyCocktail, add 30ul dissociating buffer and 20ulAnti-BiotinMicroBeads again, hatch 15 minutes for 4 degree, add 2ml dissociating buffer, 1600rpm10min is centrifugal, abandons supernatant.With 500ul dissociating buffer re-suspended cell, isolate CD8+T cell by magnetic separating post, cell counting count board counts.

(3) CD8+T cell proliferation and pharmaceutical intervention

384 orifice plates are spent the night with 5ug/mlCD3 antibody 4 degree bag in advance, and PBS washes three times, adds 3 × 10 in every hole ⁴cD8+T cell, 1ug/mlCD28 antibody, the epigallocatechin gallate (EGCG) of 30U/mlIL-2 and variable concentrations, cumulative volume 50ul.Often group does 3 multiple holes.Cultivate and be transferred to by cell in 96 orifice plates after 48 hours, 200ulRPMI-1640 culture medium is added in every hole, and continue cultivation after 72 hours, every hole adds 20ulMTS solution, hatches 4 hours for 37 degree, detects A value by microplate reader at 490nm wavelength place.Using the cell do not activated with CD3/CD28 antibody as negative control, the simple cell activated with CD3/CD28 antibody, as positive control, the results are shown in Figure 2.

The relative rate of increase of lymphocyte (%)=A _sample-A _{negative control})/(A _{positive control}-A _{negative control})] × 100%.

Experimental result:

As seen from the figure, epigallocatechin gallate (EGCG) occurs suppressing to the activation and proliferation of CD8+T cell to CD3/CD28 antibody from 1ug/ml, and the inhibitory action when 10ug/ml reaches 100%.

Embodiment 2:

One, reagent and material:

C57BL/6 mice, female, weight (15 ± 2) g, is purchased from Shanghai Slac Experimental Animal Co., Ltd.;

Emulsifiable paste oil-in-water base: octadecanol 50g, stearic acid 100g, white vaseline 50g, liquid paraffin 50g, sodium lauryl sulphate 2.5g, polysorbas20 g, glycerol 50g, triethanolamine 5g, distilled water adds to 1000g.

Epigallocatechin gallate (EGCG): Sigma Co., USA, is prepared into 1,2,3%(w/w by No.3 People's Hospital, Hangzhou City. Drug Manufacturing Room) concentration emulsifiable paste, substrate is above-mentioned oil-in-water base;

Monobenzone: Sigma Co., USA, is prepared into 40%(w/w by No.3 People's Hospital, Hangzhou City. Drug Manufacturing Room) concentration emulsifiable paste, substrate is above-mentioned oil-in-water base;

Rabbit against murine CD8 monoclonal antibody: ZSGB-BIO company;

FITC labelling goat-anti rabbit two resists: ZSGB-BIO company;

Skin confocal laser scanning microscope, CLSM (reflectanceconfocalmicroscopy, RCM), Lucid company of the U.S., VIVASCOPE1500 type, wavelength 830nm, maximum output 16.0mw;

Confocal immunofluorescence microscopy laser scanning microscope (confocallaserscanningmicroscopy, CLSM): Japanese OLYMPUS company, FluoViewFV1000 type.

Two, experimental procedure

(1) animal grouping and process

C57BL/6 mice 50, is divided into 5 groups at random, often organizes 10, sloughs mouse back black hair 2cm × 2cm, slightly exerts oneself coating until absorb with spatula.I negative control group smears vaseline emulsifiable paste 50mg once every afternoon; II model group smears 40% monobenzone emulsifiable paste 50mg once every afternoon; III, IV and V group be external curing group, smear 1%, 2% or 3% concentration epigallocatechin gallate (EGCG) emulsifiable paste 50mg every morning and once add and smear 40% monobenzone emulsifiable paste 50mg afternoon once; Each group of mice medication 50 days, continuous 15 days perusal mouse hair colors and decolouring area change after drug withdrawal, Fig. 3 is drug withdrawal typical hair color throw photo in model group and treatment group after 15 days.

(2) hair decolouring assessment

Perusal every day hair decolours, and measures decolouring area, drug withdrawal after 15 days the decolouring of each experimental group non-agents area the results are shown in Figure 4;

(3) histopathology

Agents area and skin histology wax stone around thereof are cut to the thin slice that thickness is 4 μm on microtome, and after lacing film is dried, the conventional dewaxing of section, aquation, distilled water flushing 2min × 3 time, PBS soaks 5min.Paraffin section is placed in the multiple 30min of microwave oven 95 DEG C of microwave antigen hot repair, and distilled water flushing 2min × 2 time, PBS rinses 2min × 2 time.Cut into slices closed with 10% Normal Goat Serum, incubated at room 30min, incline serum deprivation.Add rabbit CD8 monoclonal antibody, 4 DEG C are spent the night.PBS rinses, 5min × 3 time, drips FITC labelling goat-anti rabbit two and resists, room temperature reaction 2h.PBS rinses, 5min × 3 time, 90% glycerol mounting.Detect CD8+T cell dyeing with FluoViewFV1000 type laser confocal microscope, adopt single green fluorescence channel to observe, at the above scanning of 488nm wavelength.Observe and carry scanning analysis software by computer and measure, often open the fluorescence intensity that 5 visual field (200 ×) unit of account area CD8+T cells are chosen in section.The comparison of the CD8 Fluorescence labeling intensity that is external used medicine each experimental mice last day around agents area decolouring speckle of Fig. 5 display.

(4) statistical analysis method

Data determination result is with mean ± standard deviation represent, statistical analysis is carried out in t inspection.P<0.05 is for there being significant difference.

Three, experimental result

1. the incidence rate of decolouring and time:

II model group, III-V external curing group occur decolouring at monobenzone agents area, occur that the time average of decolouring is respectively 19,19,21.6 and 29.3 days.Some animals is had to occur decolouring speckle at the non-agents area of monobenzone in each group, this non-agents area decolouring speckle similar to people's vitiligo (see Fig. 3).The incidence rate of each group of non-agents area decolouring is respectively 50%, 50%, 40%, 20%.On non-agents area, epigallocatechin gallate (EGCG) occurs that there is important impact the time of decolouring.The non-agents area of model group occurs that decolouring speckle tests 28 days average time, and III-V external curing group is on average respectively at non-agents area and occurs decolouring in 28,31 and 36 days.Result shows: 1% epigallocatechin gallate (EGCG) does not make significant difference to the ratio that non-agents area decolouring speckle occurs, 2% and 3% epigallocatechin gallate (EGCG) has certain curative effect, and non-agents area is occurred, and the incidence rate of decolouring speckle declines.Meanwhile, at non-agents area, 2% and 3% epigallocatechin gallate (EGCG) treatment group occurs that the time of decolouring is also postponed.

2. decolouring area and stability:

The decolouring area average out to 4.91cm of the non-agents area of monobenzone model group mice ², the decolouring area average out to 4.77cm respectively of the non-agents area of III-V external curing group mice ², 4.17cm ²and 3.56cm ²(see Fig. 4).The decolouring speckle area of mice non-drug position appearance is significantly reduced in the epigallocatechin gallate (EGCG) treatment of 2% and 3%.

3.CD8 ⁺the quantity variance that T cell infiltrates:

CD8+T cellular infiltration (see Fig. 5) is there is around immunofluorescence display monobenzone agents area decolouring speckle.The section of negative control group immunofluorescence has minute quantity CD8 positive cell, its quantitative values (60.159 ± 2.073) under the background of green.Model group CD8+T cellular infiltration is obvious, its quantitative values (291.637 ± 9.169), the highlighted CD8+T cell that III-V group is a small amount of as seen in green background, it quantitatively (276.149 ± 12.415), (198.328 ± 14.517), (157.728 ± 14.192), compare P<0.05 with model group, significant difference.Therefore epigallocatechin gallate (EGCG) is by significantly suppressing the infiltration of CD8+T cell, thus suppresses the vitiligo of monobenzone induction.

Claims

1. the application of epigallocatechin gallate (EGCG) in the leukodermic medicine of preparation control, in described medicine, epigallocatechin gallate (EGCG) concentration is 2 ~ 5%.

2. apply as claimed in claim 1, it is characterized in that described epigallocatechin gallate (EGCG) is for the preparation of the medicine suppressing CD8+T cell activation propagation.

3. apply as claimed in claim 1 or 2, it is characterized in that one of following dosage form made by described medicine: liniment, lotion, spray.