CN101590070B - Use of baicalin in preparation of targeted organ protection medicaments - Google Patents
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Abstract
The invention belongs to the field of Chinese medicaments and relates to the novel use of Chinese medicinal baicalin monomer in preparation of targeted organ protection medicaments, in particular to the novel use of the baicalin in the preparation of medicaments for treating allergic rhinitis. Results of animal experiments of the Chinese medicinal baicalin monomer show that the baicalin can counteract nasal mucosa permeability and exudation increase in a sensitized rat, inhibit lymphocyte proliferation in a sensitized mouse, the dose-response relationship of the lymphocyte proliferation in the sensitized rat and D10.G4.1 cell line proliferation and differentiation, regulate corresponding cytokines, improve clinical symptoms and local cellular infiltration of the sensitized mouse, and regulate serum cytokines. The baicalin of the invention can be used as a raw material of a medicinal preparation to prepare medicaments for treating nasal mucosa blood vessels and preventing and curing the allergic rhinitis.
Description
Technical field
The invention belongs to the field of Chinese medicines, relate to the new purposes of Chinese medicine monomer baicalin in preparation target-organ protection medicine, especially be specifically related to baicalin and prevent and treat new purposes in the allergic rhinitis medicine in preparation.
Background technology
Along with the acceleration of modern industrialization process, the raising of people's living standard, the change of environment, the sickness rate of allergic rhinitis constantly raises, and 60% the concurrent asthma of patient's possibility is arranged.Allergic rhinitis is a kind of allergic disease, and is clinical in rhinocnesmus, sneeze repeatedly, and clear water nasal mucus and nasal obstruction are feature, extremely painful during outbreak, though itself can be not fatal, it has a strong impact on people's quality of life.
The change of nasal membrane vascular permeability is one of main pathological basis of Allergic Rhinitis generation clinical symptoms.Lymphocyte is the main effects cell of body generation allergic inflammation, clonal expansion, activation takes place, CD4 under allergenic stimulation
+The T lymphocyte number increases, and forms t helper cell 2 (Th2) skew, is the principal character of allergic rhinitis immunologic function disorder.Multinomial studies show that: the partial t helper cell number of Allergic Rhinitis increases, and wherein major part is the Th2 cell.Th2 emiocytosis IL-4, cytokines such as IL-5, IL-13, mediation B emiocytosis IgE impels mast cell degranulation, raises the eosinophilic granulocyte, finally causes a series of pathophysiological change.Th1 emiocytosis IFN-γ and IL-2 promote the Th0 cell to break up to Th1, simultaneously the secretion of antagonism Th2 cytokines.Along with the enhancing of Th2 effect and weakening of Th1 effect, its corresponding cytokine ratio also changes, especially the progress of the ratio of IL-4/IFN-γ and disease, lapse to closely relatedly, and descend with effective treatment.The interior IgE of the imbalance of the two generation and Allergic Rhinitis body raises in close relations, is to cause IgE to reach the main cause of allergic rhinitis outbreak unusually.Suppressing Th2, promote Th1, regulate Th2/Th1 cell proportion balance, is one of strategy for the treatment of at present the respiratory tract allergic disease.
Because the nasal membrane vascular lesion due to the allergic rhinitis causes by multiple independently factor, mostly present chemosynthesis medicine is to design at a certain approach wherein, therefore often can only obtain the part curative effect.
The Chinese medicine allergic disease has its unique curative effect, and China physician has carried out a large amount of explorations and obtained certain achievement the medicine of Chinese medicine allergic rhinitis for thousands of years.The baikal skullcap root bitter in the mouth, cold in nature, return lung, liver, gallbladder, large intestine, small intestine meridian.The function heat clearing and damp drying, eliminating fire and detoxication, hemostasis, antiabortive.Can clear upper, middle and lower excessive heat in SAN JIAO, the outstanding kind part of the body cavity above the diaphragm housing the heart and lungs lung-fire that rushes down is widely used in the treatment of respiratory tract infection and anaphylactic disease with its heat clearing away and antiinflammatory, antiallergic effect, and better curative effect is all arranged.Baicalin (baicalin) is a kind of flavone compound that extracts in the dry root by labiate Radix Scutellariae Scutellariabaicalensis Georgi, it is one of main effective ingredient of Radix Scutellariae, be the major quality controlling index components of Radix Scutellariae and preparation thereof, have significant biological activity.Molecular formula is C21H18011, and molecular weight is 446.35, and its water solublity is minimum, has certain fat-solublely, is easy to dissolving under alkali condition.
Modern pharmacological research shows: baicalin is shunk by guinea-pig isolated trachea anaphylaxis and the whole animal allergic asthma all has mitigation, and collaborative with the ephedrine performance.This of baicalin kind of antiallergic action is owing to suppressed the enzyme activation system (SH-enzyme) of mastocyte and the release of anaphylaxis amboceptor, thereby has antianaphylaxis.It also has direct relexation to smooth muscle itself in addition.Baicalin can reduce the permeability of mouse ear blood capillary; Prevent the white mice pneumorrhagia that low pressure causes.
Summary of the invention
The purpose of this invention is to provide the new purposes of Chinese medicine monomer baicalin in preparation target-organ protection medicine.Relating in particular to the Chinese medicine monomer baicalin protects nasal mucosa vessels, prevents and treats the purposes in the allergic rhinitis medicine in preparation.
Chinese medicine monomer baicalin of the present invention has the structure of formula (I), and molecular formula is C21H18011, and molecular weight is 446.35.
The present invention adopts above-mentioned Chinese medicine monomer baicalin to carry out zoopery, and zoopery is the result prove, has target organ protection function, especially is embodied in following protection nasal mucosa vessels, prevents and treats the effect of allergic rhinitis:
1, antagonism sensitization rat counteract nasal mucosa permeability raises, and oozes out and increases.
2, suppress the sensitized mice spleen lymphocyte proliferation.
3, the dose-effect relationship that suppresses sensitization rat spleen lymphocyte proliferation.
4, suppress the differentiation of D10.G4.1 cell line proliferation, regulate the respective fine intracellular cytokine.
5, improve sensitized mice clinical symptoms and local cellular infiltration, regulate serum cytokines.
Baicalin of the present invention can be used as the raw material of pharmaceutical preparation; Further prepare the target-organ protection medicine; Be specifically related to the medicine protecting nasal mucosa vessels, prevent and treat allergic rhinitis.
Description of drawings
Fig. 1 is baicalin oozes out total amount to sensitization rat nasal mucosa vessels influence
Wherein, * compares P＜0.05 with model group.
Fig. 2 is the influence of baicalin to sensitization rat nasal mucosa vessels seepage discharge and time relationship.
Fig. 3 is the influence of baicalin to the sensitized mice spleen lymphocyte proliferation
Wherein: * compares P＜0.05 with model group.
Fig. 4 is that baicalin is to suppressing the influence of sensitization rat spleen lymphocytic hyperplasia dose-effect relationship
Wherein, * compares P＜0.05 with model group.
Fig. 5 baicalin is to the morphologic influence of sensitized mice lung tissue.
The specific embodiment
The present invention adopts conventional method to extract baicalin, and purity is carried out the pharmacological action experiment of baicalin more than 98%.
The antagonism of embodiment 1 baicalin raises in body sensitization rat counteract nasal mucosa permeability, oozes out and increases
Experimental technique: male Brown Norway (BN) rat 250g is divided into normal control group, model control group, baicalin group, Radix Paeoniae Alba total glucosides group.Normal group, model group, baicalin group n=7; Radix Paeoniae Alba total glucosides group n=5.
Each is organized the BN rat and makes animal sensitization with the normal saline suspension 1ml lumbar injection of ovalbumin (OVA) 1mg, gel aluminum hydroxide 0.5ml, uses the same method after seven days to strengthen sensitization once.The rats in normal control group intraperitoneal injection of saline.The 8th day beginning medication group rat oral gavage given the medicine suspension after the animal sensitization, (baicalin 40mg/kg, Radix Paeoniae Alba total glucosides 70mg/kg) once a day, each 2ml, totally 12 days.Normal control group, model control group rat are irritated drinking water 2ml, totally 12 days.After the animal sensitization the 20th day, rat was executed endotracheal intubation under narcotism.Intubate is inserted to pulmonary from trachea, to keep breathing; Another intubate is inserted to nasal cavity from esophagus, is the usefulness of nasal mucosa perfusion.Postoperative tail vein injection 1%Evans Blue dye (4ml/kg) carried out the nasal cavity perfusion 15 minutes with normal saline, used 1%OA perfusate perfusion then, and flow velocity is 0.2ml/min, collected a pipe, always 105 minutes perfusion time in per 15 minutes.The perfusate of collecting left the heart 15 minutes with 3000, and supernatant is measured Evans Blue concentration with spectrophotometer 610nm, changed situation respectively to organize the rat nasal membrane because of the vascular permeability that modeling, medication cause.Experimental result shows: model group is compared with normal group, the total amount that her Wen's basket of nasal membrane blood vessel oozed out in 105 minutes obviously raise (P＜0.05); Baicalin group and model control group relatively, the total amount that her Wen's basket of nasal membrane blood vessel oozed out in 105 minutes obviously descend (P＜0.01); Radix Paeoniae Alba total glucosides group and model control group compare, and the total amount that her Wen's basket of nasal membrane blood vessel oozed out in 105 minutes does not have significant change.Each group is when the normal saline perfusion, and her Wen orchid is oozed out seldom; Along with containing entering of allergenic perfusate, her Wen's basket of model group rat nasal membrane blood vessel oozes out obvious rising, and is in a high position (comparing with normal group, from 60min P＜0.05) all the time; Her Wen's basket of baicalin group rat nasal membrane blood vessel oozes out and obviously is in low level, and is in low level (comparing with model group, from 60min P＜0.05) all the time; Her Wen's basket of Radix Paeoniae Alba total glucosides group rat nasal membrane blood vessel oozes out and obviously is in a high position, compares no significant difference with model group.
Experimental result confirms: sensitization BN rat nasal mucosa vessels permeability obviously increases; Baicalin can resist the change of the nasal mucosa vessels permeability that causes because of atopic reaction, and Radix Paeoniae Alba total glucosides does not manifest this effect.
Experimental technique: BALB/C is male, 6 age in week mice, be divided into 3 groups at random: normal group, model group and baicalin group.Normal group, baicalin group n=17; Model group n=15.
Mice makes it sensitization in Day0 lumbar injection OVA 100 μ g and gel aluminum hydroxide mixed liquor 0.3ml, and Day7, Day14 use with quadrat method and strengthen sensitization 2 times.Day21 to Day27 sky gives 2.5%OVA normal saline solution 20 μ l collunariums and excite every day.Normal group gives normal saline lumbar injection and collunarium, and mode is identical with model group.Baicalin group mice begins gastric infusion from Day0, and consumption is 40mg/Kg, finishes to Day27.Every day every mouse stomach volume 0.5ml, normal group and model group mice give drinking water 0.5ml and irritate stomach.
Mice is anaesthetized through pentobarbital sodium, and the intracardiac blood of getting is put to death, and gets spleen, shreds, with Ficoll lymphocyte separation medium separating spleen lymphocyte.Adjusting cell concentration is 5 * 10
6/ ml is inoculated in 96 orifice plates, every hole 100 μ l.Every mouse spleen cell promotes lymphopoiesis with PHA (final concentration 25 μ g/ml) respectively, is contrast with the culture fluid, all two duplications of every sample.Add after cultivating 48h
3H-TdR1 μ Ci/ hole, after continuing to cultivate 18h, usefulness bull nutsch filter on filter membrane, is surveyed cpm number by liquid scintillation counter with cell harvesting.Calculate stimulation index according to formula.
Cpm number/control wells cpm number under proliferation index (SI)=stimulant effect
Experimental result shows: model group is compared with normal group, and the lymphopoiesis index is on the rise; The baicalin group is compared with model group, lymphopoiesis index obviously descend (P＜0.05).
Experimental result confirms: baicalin can be suppressed at the propagation of body sensitization, administration mouse spleen lymphocyte.
Experimental technique: after the baicalin powder dissolved with a small amount of DMSO with aquesterilisa dissolving, other baicalin powder, adjusting pH value was 7.0, reuse RPMI-1640 standardize solution, and concentration is 10mg/ml, DMSO content is less than 1 ‰.Above-mentioned baicalin solution is after the filtration sterilization of 0.2um microstrainer, with RPMI-1640 doubling dilution to 0.0625,0.125,0.25,0.5,1.0g/L.
6 SD male rat 250g, normal saline suspension 1ml lumbar injection with ovalbumin (OVA) 1mg, gel aluminum hydroxide 0.5ml makes animal sensitization, use the same method after seven days and strengthen sensitization once, put to death animal to 28 days under the anesthesia state, get spleen, shred, with Ficoll lymphocyte separation medium separating spleen lymphocyte, adjusting cell concentration is 2 * 10
7/ ml is inoculated in 96 orifice plates, every hole 100 μ l.Add OVA (final concentration 25 μ g/ml) and promote lymphopoiesis, every hole 10 μ l.Add baicalin solution, every hole 10 μ l, each sample all do two duplications, add behind the cultivation 48h
3H-TdR1 μ Ci/ hole, after continuing to cultivate 18h, usefulness bull nutsch filter on filter membrane, is surveyed cpm number by liquid scintillation counter with cell harvesting.
Experimental result shows: along with the increasing of baicalin dosage, the spleen lymphocyte proliferation that causes because of sensitization is suppressed step by step, and presents good dose-effect relationship.The result also shows: this effect is not subjected to the influence of solvent.
Experimental result confirms: baicalin has tangible dose-effect relationship when suppressing the spleen lymphocyte proliferation that causes because of sensitization.
Experimental technique: D10.G4.1 cell line derives from bull AKR/J mouse lymph nodal tissue, is the single cell clone to the CA specific reaction that the AKR/J mice filters out after CA sensitization.Cell type is the auxiliary lymphocyte of T, expresses the IL-1 receptor, and antigenic phenotype is H-2K.Contain IL-4, IL-5 in the cells and supernatant after this cell stimulates with CA, have the Th2 characteristic.The many researchs effect of D10.G4.1 cell observation Th in the allergy cause of disease, and explore the whole bag of tricks that suppresses the Th2 reaction.D10.G4.1 cell line (ATCC NUMBER:TIB-224
TM) provide by ATCC company.The present invention establishes the basic, normal, high dosage group of baicalin (0.5ug, 5ug, 50ug/ml), Dexamethasone group (dexamethasone dosage is 1ug/ml) and blank group.
After the baicalin powder dissolved with a small amount of PBS, adjusting pH value was 7.0, reuse RPMI-1640 standardize solution, and concentration is 10mg/ml.Dexamethasone dissolves with a small amount of Polyethylene Glycol, reuse RPMI-1640 standardize solution, and concentration is 1mg/ml, polyethyleneglycol content is less than 1 ‰.Above-mentioned medicine is after the filtration sterilization of 0.2um microstrainer, and 4 ℃ of refrigerators are preserved.Use in two weeks, be diluted to desired concn during use.
A. baicalin is to the influence of D10.G4.1 cell proliferation
Separate the D10.G4.1 cell with the Ficoll lymphocyte separation medium before the experiment, remove fragment and dead cell.Adjusting the D10.G4.1 cell concentration is 2 * 10
5Cell/ml is inoculated in 96 orifice plates, every hole 100 μ l.Add APC/10 μ l to stimulate the D10.G4.1 cell, add each medicinal liquid 10 μ l then respectively, matched group adds the culture fluid with volume RPMI-1640, and each sample is all done three duplications, cultivates 72h, and in the end 18h adds
3H-TdR1 μ Ci/ hole.Then use bull nutsch filter with cell harvesting on filter membrane, survey the cpm number by liquid scintillation counter.Experiment repeats once in addition.
The result shows: baicalin does not have obvious inhibitory action in 0.5ug/ml, 5ug/ml on cell proliferation, and 50ug/ml can significantly suppress D10G4.1 cell proliferation P＜0.01; Dexamethasone can obviously suppress D10G4.1 cell proliferation P＜0.01.
Experimental result confirms: baicalin (50ug/ml) in finite concentration has inhibitory action to the D10G4.1 cell proliferation.
Table 1 is a baicalin to the influence of D10G4.1 cell proliferation (x ± s).
Table 1
Annotate: with compare * P＜0.01
B. baicalin is to the influence of D10G4.1 emiocytosis cytokine
Adjusting cell concentration is 2 * 10
5Cell/ml is inoculated in 24 orifice plates, every hole 1ml.Adding respectively and respectively organize the concentration medicine, is contrast with the culture fluid, and each sample is all done three duplications.Cultivate 72h, centrifugal collection culture supernatant is stored in-20 ℃ of refrigerators.Cytokine IFN-γ, IL-4, IL-5, TGF-β detect and use the ELISA test kit, according to description indication operation.Experiment repeats once.
The result shows: each baicalin group is compared IL-4 in the culture supernatant with matched group, the IL-5 level obviously reduces; TGF-β level obviously raises; And to IFN-γ no change.Dexamethasone group is compared IL-4 in the culture supernatant, IL-5 with matched group, IFN-γ level obviously descends, and TGF-β level obviously rises.
The result confirms: baicalin can not only suppress the D10G4.1 cell proliferation, and can change its secreted cytokine.
Table 2 is baicalins to the influence of D10G4.1 cells and supernatant cytokine (pg/ml, x ± s)
Table 2
Annotate: with compare,
*P＜0.05,
*P＜0.01
Embodiment 5 baicalins are to the influence of sensitized mice behavioristics, morphology and serum cytokines
Experimental technique: adopt the male BALB/C mice of 6 week SPF levels in age, body weight 20 ± 2g is divided into every group each 12 of normal control groups, model control group, baicalin group, Dexamethasone group at random.Model group and each medication group mice make it sensitization in Day0 lumbar injection OVA 100 μ g and gel aluminum hydroxide mixed liquor 0.3ml, and Day7, Day14 use with quadrat method and strengthen sensitization 2 times.Day21 to Day27 sky gives 2.5%OVA normal saline solution 20 μ l collunariums and excite every day.Normal group gives normal saline lumbar injection and collunarium, and mode is respectively organized identical with other.Baicalin and dexamethasone powder are dissolved with sterilized water, be made into suspension (baicalin 40mg/Kg, Dexamethasone group is 1mg/Kg), each medication group mice begins to gavage from Day0, finish to Day27, every day every mouse stomach volume 0.5ml, normal group and model group mice give drinking water 0.5ml and irritate stomach.
A. baicalin is to the ethological influence of sensitized mice
Finish back (being Day27) in modeling and administration, each group mice collunarium is finished the sneeze in the 5min of back and grab the nose number of times and count by the special messenger.The result shows that the model group mice is the sneeze number or grabs all significantly increases of nose number.Baicalin group mice sneeze number is starkly lower than model group.Dexamethasone group sneeze number, grab the nose number average and be starkly lower than obvious model control group.The result shows: through baicalin, dexamethasone treatment, the clinical symptoms of sensitized mice is improved.
Table 3 are baicalins to sensitized mice sneeze and grab the nose number influence (inferior/5mi x ± S)
Table 3
Annotate: compare * P＜0.05 with model control group; * P＜0.01
B. baicalin is to the morphologic influence of sensitized mice lung tissue
After the modeling 28 days, mice is anaesthetized through pentobarbital sodium, gets lung tissue, through formalin fixed, makes paraffin section, and conventional H E dyeing is read sheet by Medical Center of Fudan University's pathology specialist.Light microscopic is observed lung tissue endolymph cellular infiltration, bronchial smooth muscle and epithelial proliferation, the endosmosis artificial situation of bronchial lumen down, and to 5 visuals field of each tissue specimen picked at random, carries out eosinophil count, gets its average.Photo adopts German ZEISS microscope photographing.Fig. 1: the rats in normal control group lung tissue shows no obvious abnormalities; The model control group lung tissue of rats is seen a large amount of oxyphil cells' infiltration, the moderate lymphocytic infiltration, and the bronchioles smooth muscle thickens, and the part constriction of bronchioles, comes off at epithelial proliferation; Baicalin group lung tissue of rats eosinophilic granulocyte infiltration degree reduces, and lymphocytic infiltration does not have obvious improvement; The indivedual lymphocytic infiltrations of Dexamethasone group lung tissue of rats do not see that obvious bronchioles smooth muscle thickens.The result shows: through baicalin, dexamethasone treatment, sensitized mice pulmonary alterative inflammation has improvement.
Table 4 is baicalins to the influence of sensitized mice lung tissue eosinophil count (x ± s).
Table 4
Annotate: compare * P＜0.01 with model group
C. baicalin is to the influence of sensitized mice serum cytokines
After the modeling 28 days, mice is got blood through pentobarbital sodium anesthesia, and 3000 rev/mins, centrifugal 15 minutes, separation of serum ,-20 ℃ of retentions, cytokine to be measured.Mice serum IL-4, IL-5, IFN-γ, TGF-β adopts the Biosource mice EIA of company test kit, adopt double-antibody sandwich ABC-ELISA method, with anti-mice monoclonal antibody bag by on ELISA Plate, IL-4 in standard substance and the sample, IL-5, IFN-γ, TGF-β combines with monoclonal antibody, add biotinylated anti-mice IL-4, IL-5, IFN-γ, TGF-β forms immune complex and connects onboard, the Streptavidin of horseradish peroxidase-labeled combines with biotin, add zymolyte OPD, occur yellow, add stop buffer sulphuric acid, darken, survey the OD value at the 492nm place, IL-4 concentration is directly proportional with the OD value, obtains IL-4 in the specimen by the standard curve of drawing, IL-5, IFN-γ, TGF-β concentration.
Experimental result shows: compare with the normal control group, model control group mice serum IFN-γ significantly reduces, and IL-4, IL-5 significantly raise, and TGF-β content significantly descends.Compare with model control group, baicalin group IL-5 significantly reduces, and TGF-β obviously raises.Dexamethasone group and model control group compare, and IL-4, IL-5 significantly reduce, and TGF-β obviously raises.The result confirms: baicalin can be regulated the sensitized mice serum cytokines, and changes consistent with local alterative inflammation of sensitized mice and behavioristics; Dexamethasone also has this effect.
Table 5 is baicalins to the influence of sensitized mice serum cytokines content (pg/ml x ± s).
Table 5
Annotate: compare with model group,
*P＜0.05,
*P＜0.01
Claims (6)
1. the baicalin of formula I is protected under the sensitization state in the nasal membrane of body or the purposes in the nasal membrane blood vessel drug eluting in preparation as unique active component; Described baicalin molecular formula is C21H18011, and molecular weight is 446.35,
2. by the purposes of claim 1, wherein said protection is by suppressing the propagation of splenocyte because of causing in body sensitization that exsomatizes.
3. by the purposes of claim 1, wherein said protection is the spleen lymphocyte proliferation that suppresses sensitization SD rat, and described inhibition has dose-effect relationship.
4. by the purposes of claim 1, wherein said protection is to break up by suppressing the D10.G4.1 cell line proliferation, and regulates the respective fine intracellular cytokine, and described respective fine intracellular cytokine is meant IL-4, IL-5, IFN-γ or TGF-β.
5. by the purposes of claim 1, wherein said protection is by improving sensitization clinical symptoms and local cellular infiltration, and regulates corresponding serum cytokines, and described corresponding serum cytokines is meant IL-4, IL-5, IFN-γ or TGF-β.
6. baicalin is prevented and treated purposes in the allergic rhinitis medicine as unique active component in preparation.
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| 顾一峰等.中药别敏对大鼠鼻黏膜血管通透性的影响.《中国中西医结合杂志》.2006,第26卷(第10期),918-921. * |
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