CN108159046A

CN108159046A - The new application of dictamine

Info

Publication number: CN108159046A
Application number: CN201810107467.4A
Authority: CN
Inventors: 安输; 虞姣姣; 徐天瑞; 张家静
Original assignee: Kunming University of Science and Technology
Current assignee: Kunming University of Science and Technology
Priority date: 2018-02-02
Filing date: 2018-02-02
Publication date: 2018-06-15

Abstract

The present invention discloses the new application of dictamine, particularly application of the dictamine in I type hypersensitivity drugs caused by treatment Mast cell activation are prepared, belong to field of medicaments, the external inflammatory model of P815 mast cells that the present invention is induced by building C48/80, it was found that dictamine has the function of to inhibit Mast cell activation, degranulation degree of the mast cell under sensitization can effectively be inhibited, alleviate the release of β hexosaminidases；In mRNA level in-site, the expression of inflammation gene expression IL 4, IL 6, IL 12, IL 13 can be inhibited, and the phosphorylation level of ERK can be significantly inhibited in intracellular signaling pathways.

Description

The new application of dictamine

Technical field

The invention belongs to field of medicaments, and in particular to the new application of dictamine, more particularly dictamine are preparing hypertrophy Application in I types hypersensitivity drug caused by cell activation.

Background technology

Anaphylactia (allergic disease) includes allergic rhinitis, asthma, conjunctivitis, eczema, food hypersenstivity etc., It is common disease, frequently-occurring disease clinically, is classified as three big diseases of two Pius XI circle keypoint controls by the World Health Organization (WHO) One of disease is the great hygienic problems of our times.

It is counted according to world's allergy tissue (WAO), between nearly 30 years, the incidence of anaphylactia at least increases 4-5 Times, and just increased with the speed for being more than 1% every year.Whole world total prevalence rate is up to 22% at present, it is contemplated that after 20 years, industrialization The population of country 50% will suffer from anaphylactia.Global about 300,000,000 allergic asthmas of World Health Organization's estimation in 2002 Patient, 50% minimal invasive treatment therein point out this kind of breathing in developing country, the Allergic Rhinitis also more than 400,000,000 Inflammation disease is the most common chronic disease of children.By taking asthma as an example, seasonal allergic is for example untreated, 25%-38% Asthma is would develop into, eventually becomes chronic asthma, pulmonary emphysema, pulmonary heart disease, 180,000 people is had more than every year and dies of asthma, in 2005 About 250,000 people of Nian Gengyou lead to death due to asthma.

At present, the consuming in terms for the treatment of is high, this expenditure estimation of the whole world only antiallergic action (allergy) drug More than 8,000,000,000 dollars.Nearly 4,000,000,000 dollars are up to for treating the expense of anaphylactia every year European, economic costs are much More than treatment tuberculosis and the summation of AIDS.And the incidence of China's anaphylactia is more than 20%, medical expense also accounts for institute There is the forefront of disease series.

Anaphylactia causes serious threat to the normal life of the mankind and existence, to global sound development Bring huge obstruction.At present, heteropathy is mainly clinically taken to the treatment of anaphylactia, such as uses anti-group Drug amine and hormone etc. and allergen specificity immunotherapy (Allergen-specific immunotherapy), the former It can only temporarily alleviate the symptoms, and the latter's course for the treatment of is longer, costly, effect is sometimes also not very apparent.Monomer dictamine is rue The principle active component of section's plant white line root, have promote isolated frog heart have excitement, isolated rabbit ear vessel retraction, rabbit and Cavy uterine smooth muscle has the remarkable effect that strength is shunk, and has certain antibiotic effect.Dictamine extraction process is more at present Maturation, commercially available dictamine is generally all more inexpensive and matter is high.

Invention content

The present invention is directed to disclose the new application of dictamine, i.e. dictamine is preparing treatment because of caused by Mast cell activation Application in I type hypersensitivity drugs provides a kind of nontoxic, efficient, inexpensive therapy for antiallergy.

Dictamine of the present invention can also add in one or more pharmaceutically acceptable auxiliary materials, to improve drug suction It produces effects fruit or convenient for taking, such as capsule or pill, pulvis, tablet, granula, oral liquid and parenteral solution is made.

Auxiliary material of the present invention includes the filler of pharmaceutical field routine, diluent, adhesive, excipient, absorbs rush Into agent, surfactant and stabilizer etc., flavouring agent, pigment and sweetener etc. can also be added if necessary.

The dictamine that dictamine of the present invention is extracted for conventional commercial dictamine or common process.

The external inflammatory model of P815 mast cells that the present invention is induced by building C48/80 finds that dictamine has and inhibits The effect of Mast cell activation can effectively inhibit degranulation degree of the mast cell under sensitization, alleviate beta-amino hexose The release of glycosides enzyme.In mRNA level in-site, the expression of inflammation gene expression IL-4, IL-6, IL-12 and IL-13 can be inhibited, and in born of the same parents The phosphorylation level of ERK can be significantly inhibited in interior signal path, it is confirmed with certain anti-allergic effects in cellular level, Claritin is used to prepare for dictamine, and foundation is provided.

The present invention for the treatment of anaphylactia provide a kind of worth exploitation, without obvious toxic-side effects, efficient, inexpensive Compound.

Description of the drawings

Fig. 1 is the different shaggy-fruited dittany alkali concns of embodiment 1 to P815 cytotoxic effects；

Fig. 2 is the inhibiting effect that the different shaggy-fruited dittany alkali concns of embodiment 2 discharge P815 cells β-hexosaminidase；

Fig. 3 is transcription of the different shaggy-fruited dittany alkali concns of embodiment 3 to inflammatory factor IL-4, IL-6, IL-12 and IL-13mRNA Inhibiting effect；

Fig. 4 is inhibition of the different shaggy-fruited dittany alkali concns of embodiment 4 to the phosphorylation of Mast cell activation signal path molecules ERK Effect.

Specific embodiment

In order to make the purpose , technical scheme and advantage of the present invention be clearer, with reference to the accompanying drawings and embodiments, it is right The present invention is further elaborated, and the reagent used in embodiment is commercially available conventional reagent, uses examination Agent code is well known to those skilled in the art.

Embodiment 1

Experiment purpose and method：No matter which kind of drug, if excessive concentration, all can to cell generate cytotoxic effect, Dictamine excessive concentration will generate certain toxicity to P815 cells, in order to detect dictamine to P815 cytotoxicity intensity, The cell survival rate for detecting various concentration dictamine stimulation P815 cells after 24 hours is tested with CCK-8, to determine dictamine pair Use concentration of the cell without growth inhibitory activity, is as follows：

(1) culture and passage of mast cell line P815 cells

A, the culture of P815 cells：P815 is incubated at DMEM in high glucose culture medium, containing 10% fetal calf serum, 1% mycillin, It is incubated at 37 DEG C, 5%CO₂In environment；

B, the passage of P815 cells：It when cell density reaches 70% in step A, need to be passed on, be collected in culture bottle Cell culture fluid in centrifuge tube, the rinsing of the phosphate buffered saline solution of preheating three times, adds in appropriate trypsase, treats cell It completely after digestion, adding in cell culture fluid and trypsase, and blows and beats uniform, 800rpm is centrifuged 3 minutes, removes supernatant, Fresh DMEM in high glucose culture medium piping and druming mixing cell is added in, according to 1:4 ratio is passaged in new culture bottle, is jiggled Make cell distribution uniform, 37 DEG C, 5%CO₂Continue to cultivate in environment.

(2) CCK-8 detects cytotoxicity of the dictamine to mast cell P815

A, plating cells：Cell dissociation is got off after centrifugation, gone to exponential phase by step (1) P815 cell culture Clearly, mixing cell precipitation is blown and beaten with fresh DMEM in high glucose culture medium, single cell suspension is made, adjusted with the method for cell count Cell density is to 1 × 10⁵A/mL is uniformly laid on 96 orifice plates, 37 DEG C, 5%CO with the amount of every 100 μ L of hole₂Culture 24 hours；

B, shaggy-fruited dittany alkali process cell：It prepares containing various concentration：0.1 μ g/mL, 0.5 μ g/mL, 1 μ g/mL, 5 μ g/mL, 10 μ g/ ML, the culture medium of the dictamine of 50 μ g/mL siphon away original culture medium, and addition is corresponding containing each concentration dictamine culture medium, right The culture medium without dictamine, every group of 3 multiple holes, 37 DEG C, 5%CO are changed to according to group₂It is incubated 24 hours；

C, CCK-8 solution is added in：CCK-8 solution 10 μ L, 37 DEG C, 5%CO are added in per hole₂It is incubated 1.5 hours；

D. the measure of absorbance (OD values)：The absorbance in each hole is measured in 630nm and 490nm using microplate reader, analyzes number According to and map.

Experimental result：As shown in Figure 1, with various concentration：The dictamine stimulation P815 cells 24 of 0.1 μ g/mL-50 μ g/mL Hour, CCK-8 testing results show the concentration of 0.1 μ g/mL-10 μ g/mL to P815 cells without growth inhibitory activity, i.e., without carefully Cellular toxicity, subsequent experimental are determined as 0.1 μ g/mL, 1 μ g/mL, 10 μ g/mL using concentration.

Embodiment 2

Experiment purpose and method：In order to verify that can dictamine inhibit mast cell degranulation, with the shaggy-fruited dittany of various concentration Oxygenation pretreatment mast cell, can detect it inhibit the release of mast cell degranulation marker β-hexosaminidase, specifically Step is as follows：

(1) culture and passage of mast cell line P815 cells

A, the culture of P815 cells：P815 is incubated at DMEM in high glucose culture medium, containing 10% fetal calf serum (FBS), 1% green chain Mycin is incubated at 37 DEG C, 5%CO₂In environment；

B, the passage of P815 cells：It when cell density reaches 80% in step A, need to be passed on, be collected in culture bottle Cell culture fluid in centrifuge tube, the rinsing of the phosphate buffered saline solution of preheating three times, adds in appropriate trypsase, treats cell It completely after digestion, adding in cell culture fluid and trypsase, and blows and beats uniform, 800rpm is centrifuged 3 minutes, removes supernatant, Fresh DMEM in high glucose culture medium piping and druming mixing cell is added in, according to 1:4 ratio is passaged in new culture bottle, is jiggled Make cell distribution uniform, 37 DEG C, 5%CO₂Continue to cultivate in environment.

(2) development process detection dictamine is to the inhibiting effect of P815 activation degranulations

A, plating cells：Cell dissociation is got off after centrifugation, removes supernatant to exponential phase by P815 cell culture, with new Fresh DMEM in high glucose culture medium blows and beats mixing cell precipitation, and single cell suspension is made, 24 holes are uniformly laid on the amount of every 500 μ L of hole Plate, 37 DEG C, 5%CO₂Culture 24 hours；

B, dictamine pretreatment cell：Medicine group adds in shaggy-fruited dittany alkali liquor, makes its final concentration of 0.1 μ g/mL, 1 μ g/mL, 10 μ g/mL, cloudy control wells, positive control wells and total metering-orifice do not process, 37 DEG C, 5%CO₂It is incubated 30 minutes；

C, C48/80 stimulates cell：In addition to cloudy control wells, remaining each hole adds in C48/80 mother liquors, makes its final concentration of 10 μ g/mL are stimulated 1.5 hours；

D, determination of color β-hexosaminidase：It is handled with 1% Triton X-100 (Tritox-100) Total amount group makes cell rupture of membranes discharge intracellular whole β-hexosaminidase, is collected by centrifugation each group cell conditioned medium, draw 50 μ L in 96 orifice plates set 3 multiple holes, per the empty 1 × p-nitrophenyl-N- acetyl-β-D- glucosaminides (PNP- for adding in 50 μ L NAG), 37 DEG C be incubated 1 hour, 1 hour after, per hole add in 100 μ L sodium carbonate buffers terminate reaction, existed using microplate reader The absorbance in each hole is measured at 405nm, analyze data and is mapped.

Experimental result：As shown in Fig. 2, with various concentration：0.1 μ g/mL, 1 μ g/mL, the shaggy-fruited dittany oxygenation pretreatment of 10 μ g/mL After P815 cells 30 minutes, the external inflammatory model of P815 cell construction mast cells is stimulated with C48/80 (10 μ g/mL), is promoted Cell activation and degranulation discharge intracellular β-hexosaminidase, extracellular β-hexosaminidase are detected by development process, tie Fruit shows that the dictamine of above-mentioned three kinds of concentration can effectively inhibit P815 to discharge β-hexosaminidase, i.e., to the work of the cell Changing degranulation has certain inhibiting effect, and in dose-dependence, with control group significant difference.

Embodiment 3

Experiment purpose and method：Dictamine is detected to IL-4, IL-6, IL-12 and IL-13 after P815 activation in mRNA level in-site The influence of upper expression, is as follows：

(1) culture and passage of mast cell line P815 cells

(2) IL-4, IL-6, IL-12 and IL-13 exist after Real-Time Fluorescent Quantitative PCR Technique detection dictamine activates P815 The inhibiting effect of mRNA level in-site expression

A, plating cells：Cell dissociation is got off after centrifugation, removes supernatant to exponential phase by P815 cell culture, with new Fresh DMEM in high glucose culture medium blows and beats mixing cell precipitation, and single cell suspension is made, 6 orifice plates are uniformly laid on the amount of every hole 2mL, The cloudy control group of setting, positive control group and experimental group, 37 DEG C, 5%CO₂Culture 24 hours；

B, dictamine pretreatment cell：Experimental group adds in shaggy-fruited dittany alkali liquor, makes its final concentration of 0.1 μ g/mL, 1 μ g/mL, 10 μ g/mL, 37 DEG C, 5%CO₂It is incubated 1 hour；

C, C48/80 stimulates cell：In addition to control wells, C48/80 mother liquors are added in each hole, make its final concentration of 10 μ G/mL is stimulated 24 hours；

D, RNA is extracted：Light and slow piping and druming cell makes the cell of adherent part suspend, and collects whole cell suspensions in 2.0mL EP Supernatant is removed in Guan Zhong, centrifugation, and 1mL Trizol are added in per hole, overturns under mixing 10, is stored at room temperature after five minutes, per 1mL Trizol Middle addition 0.2mL chloroforms (Trizol：Chloroform=5:1) sample tube cover, is covered tightly, turning upside down makes its abundant mixing, then room under 10 Temperature stands 5 minutes, and under 4 DEG C of cryogenic conditions, 12000rpm is centrifuged 15 minutes, and lamination occurs in liquid in pipe at this time, will be upper Layer water phase is transferred in new 1.5mLEP pipes (about 400 μ L), is added in equivalent (400 μ L) isopropanol, is stored at room temperature after reverse mixing 10 minutes, then under 4 DEG C of cryogenic conditions, 12000rpm was centrifuged 10 minutes, is carefully removed supernatant, is left and taken precipitation, and often pipe adds It is primary to enter 75% ethyl alcohol (precooling) the oscillation washing RNA precipitate that 1mL now matches, rear 7500rpm low-temperature centrifugations 5 minutes, carefully Fall supernatant, precipitation is taken to put superclean bench and is blown in machine drying (about 30 minutes), then the pyrocarbonic acid diethyl ester in 20 μ L of Guan Zhongjia DEPC water dissolutions precipitate；

E, reverse transcription：

1. following system (operating on ice) is prepared in PCR tubules：

2. reaction condition：It 65 DEG C × 5 minutes, is immediately placed on ice water, at least 1 minute；

3. taking out PCR tubules, next step system (operating on ice) is prepared：

D. reaction condition：25 DEG C × 5 minutes, 42 DEG C × 60 minutes, 70 DEG C × 5 minutes；

F, real-time fluorescence PCR reacts：

1. primer：

2. reaction system:

3. reaction condition：Pre-degeneration 95 DEG C × 10 minutes, cycling condition are 95 DEG C × 10 seconds, 60 DEG C × 10 seconds, 72 DEG C × 15 seconds, totally 40 recycled.

Experimental result：With various concentration：0.1 μ g/mL, the shaggy-fruited dittany oxygenation pretreatment P815 cells 1 of 1 μ g/mL, 10 μ g/mL are small Shi Hou stimulates P815 cells 24 hours with C48/80 (10 μ g/mL), promotes relevant inflammatory factors IL-4, IL-6, IL- in cell 12nd, the transcription of IL-13mRNA detects the expression quantity of above-mentioned mRNA by Real-Time Fluorescent Quantitative PCR Technique, as shown in figure 3, on The dictamine for stating three kinds of concentration is respectively provided with inhibition inflammation gene expression IL-4, IL-6, IL-12 and IL-13 in mRNA horizontal expressions Effect.

Embodiment 4

Experiment purpose and method：Detection dictamine is specific to walk to causing the influences of the ERK signal paths of Mast cell activation It is rapid as follows：

(1) culture and passage of mast cell line P815 cells

(2) intracellular signaling pathways ERK phosphorus after immunoblotting (Western Blot) detection dictamine activates P815 The inhibiting effect of acidification

A, plating cells：Cell dissociation is got off after centrifugation, removes supernatant to exponential phase by P815 cell culture, with new Fresh DMEM in high glucose culture medium blows and beats mixing cell precipitation, and single cell suspension is made, 6 orifice plates are uniformly laid on the amount of every hole 2mL, 37 DEG C, 5%CO₂After culture 24 hours, 24 hours, DMEM in high glucose culture mediums of the 1.5mL without fetal calf serum is added in per hole to thin Born of the same parents do Nature enemy, and processing time is 12 hours；

B, dictamine pretreatment cell：Medicine group adds in shaggy-fruited dittany alkali liquor, makes its final concentration of 0.1 μ g/mL, 1 μ g/mL, 10 μ g/mL, cloudy control group and positive control group do not process, 37 DEG C, 5%CO₂It is incubated 30 minutes；

C, C48/80 stimulates cell and collects protein sample：In addition to cloudy control group, C48/80 mother liquors are added in each hole, Make its final concentration of 10 μ g/mL, stimulated 5 minutes under the conditions of 37 DEG C, be immediately placed on ice water after five minutes, blow and beat each hole cell system Into cell suspension, 1.5mL centrifuge tubes are collected in, supernatant is removed after low-temperature centrifugation, add in the phosphate buffered saline solution drift of 1mL precoolings Cell is washed, low-temperature centrifugation removes supernatant, and often pipe adds in 50 μ L Ripa lysates, on ice cracking 30 minutes, during which vortex oscillation 3- 5 times, after the completion of cracking, 12500rpm low-temperature centrifugations 15 minutes simultaneously absorb nucleic acid；

D, immunoblotting：The protein sample of collection is quantified, and be configured to using BCA protein quantifications kit The protein sample of 30 μ g, with Ripa lysates polishing to 10 μ L, 2.5 μ L of often pipe addition sample-loading buffer, 98 DEG C are heated 7 minutes, After the completion for the treatment of prepared by PAGE gel, it is put into electrophoresis tank, pours into suitable 1 × electrophoretic buffer, take out comb, it will be accurate The protein sample and albumen Marker got ready are added in each swimming lane of PAGE gel, and glue is run 30 minutes with 80V constant pressures, Glue is run with 120V constant pressures 100 minutes, after electrophoresis, the albumen on glue is gone into poly- inclined difluoro in the condition of 200mA constant currents again In vinyl film (pvdf membrane), transferring film carries out room temperature after 2 hours, with 5% skim milk (1 × TBST preparations) and closes 1 hour, The film for closing completion is cut by strip according to albumen size, under the conditions of 4 DEG C, primary antibody (occurs special with ERK on film, p-ERK Property combination) be incubated overnight, next day, the film for having incubated primary antibody is placed in 1 × TBST, is placed on shaking table, wash film 3 times, 5 points every time Clock, is then incubated at room temperature secondary antibody (with the anti-binding being incorporated on film) 1 hour, after the completion of secondary antibody is incubated, is washed with 1 × TBST Film 5 minutes every time, is finally soaked in prepared luminous substrate, using Chemiluminescence Apparatus, mixes up the time for exposure by film 3 times Develop.

Experimental result：As shown in figure 4, with various concentration：0.1 μ g/mL, 1 μ g/mL, the shaggy-fruited dittany oxygenation pretreatment of 10 μ g/mL After P815 cells 30 minutes, P815 cells are stimulated 5 minutes with C48/80 (10 μ g/mL), activation and the close phase of Mast cell activation The ERK signal paths of pass, collect protein sample, and the phosphoric acid of immunoblotting detection ERK is horizontal；The result shows that above-mentioned three kinds The dictamine of concentration has significant inhibiting effect to the phosphorylation of ERK, it is believed that dictamine makees the inhibition of P815 cell activations With may be by what regulation and control ERK signal transduction pathways were realized.

The foregoing is only a preferred embodiment of the present invention, but protection scope of the present invention be not limited thereto, Any one skilled in the art in the technical scope disclosed by the present invention, the change or replacement that can be readily occurred in, It should be covered by the protection scope of the present invention.

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Claims

1. application of the dictamine in I type hypersensitivity drugs caused by treatment Mast cell activation are prepared.