CN100448470C - Medicinal composition for immunological disease and its preparing method - Google Patents
Medicinal composition for immunological disease and its preparing method Download PDFInfo
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- CN100448470C CN100448470C CNB2003101029437A CN200310102943A CN100448470C CN 100448470 C CN100448470 C CN 100448470C CN B2003101029437 A CNB2003101029437 A CN B2003101029437A CN 200310102943 A CN200310102943 A CN 200310102943A CN 100448470 C CN100448470 C CN 100448470C
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Abstract
The present invention relates to a medicinal composition for treating immunological diseases and a preparation method thereof. The medicinal composition for treating immunological diseases comprises the ethanolic extracts of Chinese yam, oriental waterplantain rhizome, tuckahoe and scutellaria root. The preparation method comprises the steps of carrying out heat extraction with ethanol of a specific concentration range, filtering, regulating the pH-value, purifying, concentrating, moderately mixing, freeze drying and preparing granular powder. Experiments in vitro and in vivo are carried out in order to verify the curative effect of a specific medicinal plant extract composition. The medicinal herb extract composition can enhance the anti-inflammation reaction and regulate the secretion of cytohormones, such as eotaxin, interferon-4(1L-4), etc. The medicinal composition for treating immunological diseases prevents asthma by means of the functions of relieving cough, enhancing lung functions and reducing specific allergen IgE in blood when asthma occurs.
Description
Technical field
The invention relates to a kind of medical composition and manufacture method thereof that is used for the treatment of anaphylactic disease, particularly a kind of Chinese herbal medicine medical composition and manufacture method thereof that is used for the treatment of allergic asthma.
Background technology
The disease that anaphylactogen caused, for example asthma remains one of serious in the world health problem.Recently these irritated relevant immunological diseases also are inclined to young group.In other words, the irritated immune disease symptom that causes takes place in more child and teenager.Numerous doctors and scientist believe, the irritated correlation immunity disease that these are early sent out is because environmental pollution causes.
Although the seriousness of affected group ratio and symptom thereof constantly increases, cause certain reason of this phenomenon also fuzzy.Therefore, present antasthmatic method still mainly rely on empirical law with unexpected seek method be main, but not utilize scientific method.The Drug therapy of anti-now asthma mainly focuses on now therapeutic modality of improvement (as: Hangzhoupro short of money leukotriene (antl-leukotriene), or slow down and eliminate inflammatory response (as: suction steroid) by suppressing the relevant cytohormone of inflammation.Yet, the threat that has the child of eczema and pollinosis medical history also to be panted especially easily, and the problem that is produced is, often the chronic asthma sufferer of outbreak needs every day and takes the medicine of anti-asthma, and takes all the year round.In order to respond the problem of this class, the curative effect of testing traditional Chinese herbal medicine is necessary that especially many documents point out that all these medical herbs have good effect for anaphylactic disease such as allergic dermatitis.
Traditional Chinese herbal medicine has been used for treating the allergic dermatitis and the asthma of heredity.The result shows, uses traditional Chinese herbal medicine after, most of patient's general symptom all is improved, but the small part patient still because the reason of failing to understand can't cure.Some medical herbs can manifest effect in a short time, and has only the side effect of minority, but for the then still more scientific research of prophylactic long-term efficacy.Therefore, need the further research of traditional Chinese herbal medicine.Following document points out that medical herbs has the immunoreactive function of adjusting, but wherein major part still has the tangible uncertain factor, and needs careful research.
With United States Patent (USP) the 09/949th, No. 610 application cases are example, a kind of medical composition that is used for rhinitis of this disclosure of the Invention, it comprises: the aqueous solution extraction thing of Radix Ophiopogonis (Tuber Ophlopogon), the Rhizoma Pinelliae (Tuber Pinelliae), Radix Glycyrrhizae (Radix Glycyrrhizae) and Radix Panacis Quinquefolii (Radix Pancis Quinquefoli), and 50% ethanolic extract of Herba Tridacls procumbentis.This prescription comprises 5 kinds of medical herbs, and has the good result of antiinflammatory.Yet,, so be not easy to control its long-term efficacy because this prescription only has the releive effect of symptom of short-term.
WO 02/78723A1 discloses a kind of medicinal herb components for preparing tool the kidney invigorating and anti-asthma effect, and this Chinese herbal medicine comprises: Folium Perillae (Perilla Frutescens), Semen Armeniacae Amarum (Prunus Armeniaca), Radix Glycyrrhizae (Glycyrrhiza Uralensis), Radix Scutellariae (Scutellaria Baicalensis), Rhizoma Coptidis (Coptis Chinesis), coltsfoot (Tusilago Farfara), Radix Stemonae (Stemona Sessllifolia), Bulbus Fritillariae Uninbracteatae (FritllariaCirrhosa), ginseng is hair earthworm (Pheretima Aspergillum) also, Fructus Psoraleae (Psoralea Corylifolia), Radix Codonopsis (Codonopsis Pilosula), six row Fructus Hordei Vulgaris (Hordeum Vulgara), six songs (MassaFermentata Medicalis), Fructus Schisandrae Chinensis (Schisandra Chinensis) and Gypsum Fibrosum (Gypsum).Because this prescription comprises 15 kinds of different medical herbs, so the quality of medical herbs extract is difficult to grasp.And be not easy to observe each composition pharmacological reaction separately in the prescription.
WO 02/32440 discloses a kind of antasthmatic Folium Et Cacumen Murrayae (Murraya koenigii) extract.
GB 2274059 discloses a kind of medical composition of the asthma of releiving, and comprises Radix Paeoniae in its composition.The single plant component acute asthma symptom for the treatment of or releive is used in these inventions, but all can't keep secular curative effect.
Therefore, providing a kind of simpler composition and method of making the same, effective ingredient can not changed before the treatment disease, and can improve the long-term efficacy of treatment immunological diseases, is to be worth research with the method for getting rid of foregoing problems.
Some has the Chinese herbal medicine of anti-inflammatory reaction effect, may produce effect to the treatment immune dysfunction diseases.For example, before twoth century of Christian era, just there is the document record with Radix Scutellariae (Scutellarla Baicalensis) anti-inflammatory in China, especially as treatment damp-heat syndrome (hot﹠amp; Damp) one of class disease such as dysentery and diarrheal main component.Yet document there is no record and how to be made into homogenizing powder with long preservation period, and should how to make and be used for treating allergic asthma.Therefore, the present invention discloses a processing procedure, prepares the effective ingredient that comprises Radix Scutellariae (Scutellaria Baicalensls) and other three kinds of medical herbs extracts with minimum step, and by in vitro reaching in vivo experiment, shows the effect that it is good.In other words, Radix Scutellariae (Scutellaria Baicalensis) can provide the advantage that prolongs several active component stability with the combination of other three kinds of medical herbs extracts, and demonstrates the effect for antianaphylaxis asthma, and reduces the toxicity of medicine to human body.
Summary of the invention
The object of the present invention is to provide a kind of medical composition of immune disease, can relax allergic asthma, and can treat the allergic asthma of sufferer effectively.
For achieving the above object, medical composition provided by the invention comprises:
One Rhizoma Dioscoreae (Radix Dioscorea) extract;
One Rhizoma Alismatis (Rhizoma Alismatis) extract;
One Poria (Poria cocos (schw) Wolf) extract; And
One Radix Scutellariae (Scutellaria Baicalensis) extract.
Rhizoma Dioscoreae wherein, Rhizoma Alismatis and tuckahoe extract are the alcohol extractions with 40-95%.
Wherein the Radix Scutellariae extract is the alcohol extraction with 40-99%.
Wherein this Rhizoma Dioscoreae extract, Rhizoma Alismatis extract, tuckahoe extract and Radix Scutellariae extract extract respectively from Rhizoma Dioscoreae, Rhizoma Alismatis, Poria and Radix Scutellariae.
Wherein this Rhizoma Dioscoreae extract, Rhizoma Alismatis extract, tuckahoe extract and Radix Scutellariae extract are the mixture that extracts simultaneously from Rhizoma Dioscoreae, Rhizoma Alismatis, Poria and Radix Scutellariae.
Wherein also comprise an excipient.
Wherein the weight ratio of this Rhizoma Dioscoreae, Rhizoma Alismatis, Poria and Radix Scutellariae is 1: 1-2: 0.5-2: 1-3.
Wherein this medical composition is used for the treatment of the allergic asthma sufferer.
The present invention also provides the method for preparing above-mentioned medical composition, comprises the following step:
Respectively with alcohol extraction Rhizoma Dioscoreae, Rhizoma Alismatis, Poria and Radix Scutellariae, to produce the extract of Rhizoma Dioscoreae, Rhizoma Alismatis, Poria and Radix Scutellariae;
Filter and concentrate this extract respectively, to produce the concentrate of Rhizoma Dioscoreae, Rhizoma Alismatis, Poria and Radix Scutellariae; And
The concentrate of this Rhizoma Dioscoreae, Rhizoma Alismatis, Poria and Radix Scutellariae is mixed;
Wherein, this concentration of alcohol is 40-95%.
This method also comprises respectively or concentrates this each concentrate jointly, granulates with the combination drying that carries out Rhizoma Dioscoreae, Rhizoma Alismatis, Poria and Radix Scutellariae.
This method is added an excipient to this Rhizoma Dioscoreae, Rhizoma Alismatis, Poria and Radix Scutellariae concentrate before also being included in and forming granule.
Wherein this granule is to coat with capsule.
Wherein this concentration of alcohol is 40-90%.
Wherein the weight ratio of this Rhizoma Dioscoreae, Rhizoma Alismatis, Poria and Radix Scutellariae is respectively 1: 1-2: 0.5-2: 1-3.
Preparation method of the present invention can also be the following step:
Mix Rhizoma Dioscoreae, Rhizoma Alismatis, Poria and Radix Scutellariae to produce a mixture;
With this mixture of alcohol extraction, to produce the ethanolic extract of Rhizoma Dioscoreae, Rhizoma Alismatis, Poria and Radix Scutellariae; And
Filter and concentrate this extract, with the concentrate of generation Rhizoma Dioscoreae, Rhizoma Alismatis, Poria and Radix Scutellariae,
Wherein, should be 40-95% in order to the concentration of alcohol of extraction.
Also comprise this Rhizoma Dioscoreae, Rhizoma Alismatis, Poria and Radix Scutellariae concentrate, make granule with spraying-seasoning.
Also be included in and form a preceding excipient, starch and the calcium phosphate (Ca of adding of granule
3(PO
4)
2) to the concentrate of this Rhizoma Dioscoreae, Rhizoma Alismatis, Poria and Radix Scutellariae.
Wherein this granule is to coat with capsule.
Wherein the concentration of alcohol of this extraction usefulness is 40-50%.
Wherein the weight ratio of this Rhizoma Dioscoreae, Rhizoma Alismatis, Poria and Radix Scutellariae concentrate is respectively 1: 1-2: 0.5-2: 1-3.
Description of drawings
Fig. 1 is a kind of sketch map (drawing from ' Nature ' 402 12-17 (1999), P.G.Holt, C.Macabas, PA.Stumbles and P.D.Sly) that causes the possibility mechanism of allergic asthma; Wherein IL-4 is the main cytohormone of secretion from the Th2 cell, with important fat medium-leukotriene, plays an important role in acute and chronic irritated inflammatory response simultaneously;
Fig. 2 a shows with various compositionss and suppresses experiment in vitro that cell strain EL-4 and THP-1 secrete IL-4 and TNF-alpha figure as a result.Symbol " aq " in the literal of diagram below is represented the aqueous solution extraction thing of medical herbs, and symbol " et " then is an ethanolic extract of representing medical herbs;
Fig. 2 b show variable concentrations TCM-B suppress RBL-1 emiocytosis cysteamine acyl group leukotriene (cysteinyl-leukotrienes, c-LTs) and Cytotoxic experiment in vitro figure as a result;
Fig. 3 a detects experiment in vitro that variable concentrations TCM-B suppresses BEAS-2B emiocytosis eotaxin and cell survival rate (cytotoxicity) figure as a result with MTT experiment;
Fig. 3 b shows to comprise 36% starch and 1.9% calcium phosphate (Ca
3(PO
4)
2) the TCM-B powder, the influence of test EL-4 cell strain secretion IL-4.Abscissa shows the concentration of powder, and excipient starch and calcium phosphate were tested the phenomenon of not disturbing EL-4 cell strain secretion IL-4 in advance;
Fig. 3 c shows that use comprises the medical herbs extraction powder of TCM-B and excipient, has impartial effect for the phenomenon that suppresses EL-4 cell strain secretion IL-4;
Fig. 4 a shows that various compositionss and TCM-B mixture suppress the huge experiment in vitro result who has a liking for emiocytosis TNF-alpha at peritoneum position;
Fig. 4 b shows the huge cytophilic cytotoxicity that acts on the peritoneum position with MTT experiment various compositionss of detection and TCM-B mixture;
Fig. 5 shows when measuring pulmonary function, the signal that pressure increases, (B) Penh value from 0.9 to 3.95 among the figure, demonstrate its pulmonary function decline of irritated mouse of experiment usefulness, be that one diverse parameter (is quoted the inImmunology from E.Hamelmann and E.W.Gelfand in Current Protocols, John Wiley ﹠amp when detecting mouse; Sons, Inc., edited by J.E.Coligan, A.M.Kruisbeek, D.H.Matgulies, E.M.Shevach, and W.Strober, Vol.3, Chapter15,18,1-18.13 (1999));
Fig. 6 shows and the normal physiological saline solution, TCM-B, and the TCM-A that removes behind the Rhizoma Alismatis extract compares, TCM-B can prevent because of the dosage of the ugly methyl gallbladder of acetyl element (methacholine) from low to high, the pulmonary function decline that is caused;
Fig. 7 is in 5 groups of experiment mouse serums, the titration value of IgE;
Fig. 8 is in the serum and BAL flushing liquor of 5 groups of experiment mouse, the concentration of IL-5;
Fig. 9 is in the serum and BAI flushing liquor of 5 groups of experiment mouse, the concentration of IFN-γ;
Figure 10 is that TCM-B (1217B) promotes the figure as a result that regulates the T cell ability in the blood in 5 groups of experiment mouse.
The specific embodiment
The asthma that antigen or anaphylactogen cause is the respiratory disorder that is caused by the chronic inflammation reaction.Inflammatory response is the inflammatory factor that is discharged by cell, such as platelet activating factor (platelet activation factor), histamine (histamine), prostaglandin (prostaglandin) and leukotriene (leukotriene) regulation and control.Inflammatory response can stimulate the T lymphocyte, especially the antigen-reactive of CD4+ cell.Mainly contain two kinds of CD4+ cells.T assistant's cell 1 (Th1) secretory cell hormone such as IFN-γ in the CD4+ cell, T assistant's cell 2 (Th2) then secrete interferon (interleukin) as IL-4 and IL-5.The blood of diagnosis allergy asthma sufferer, bronchoalveolar lavage fluid and bronchial mucosa sample find that the Th2 cytoactive is stronger.Anaphylactogen stimulates the Th2 cell to produce excessive IL-4, impels bone-marrow-derived lymphocyte to make justacrine IgE, as shown in Figure 1.The secreted eotaxin of tracheal epithelial cell attracts numerous eosmophils to be gathered in this inflammation occurrence positions simultaneously.Medical composition of the present invention can prevent disengaging of inflammatory factor and IL-4.
Below explanation only is the embodiment of the invention, should be as the i.e. system of the scope of the invention.
Embodiment 1: the preparation of medical composition TCM-A
A kind of medical composition is made by following five steps:
(1) gets 400 g Rhizoma Dioscoreae (Radix Dioscorea) stem or root, 300 g Poria (Poriacocos (schw) Wolf) and 500 g Radix Scutellariae (Scutellaria Baicalensis), be chopped into small pieces respectively.
(2) Rhizoma Dioscoreae and Poria are soaked in respectively in 2000 ml waters, Radix Scutellariae was soaked in 70% ethanol 30 minutes.Every kind of solution is respectively with 85 ℃ of heating 60 minutes.Obtain the extract of Rhizoma Dioscoreae, Poria and Radix Scutellariae afterwards respectively.
(3) after the extract cooling, with it respectively with screen filtration (about 100 orders).Collect filtrate, and under vacuum condition (30torr), concentrate and obtain the medicinal herbs concentrated solution with 50 to 60 ℃.
(4) before with every kind of concentrated solution lyophilization, add capacity carboxy methyl cellulose (carboxyl methylcellulose) as excipient, then it is made particulate powder respectively.
(5) again that discrete powder mixes is even, formed hybrid particles is called TCM-A, and is made into capsule.
Embodiment 2: the preparation of medical composition TCM-B
A kind of medical composition can prepare via following five steps:
(1) gets stem or root, 400 g Rhizoma Alismatis, 300 g Poria and 500 g the Radix Scutellariae of 400 g of Rhizoma Dioscoreaes, be chopped into small pieces respectively.
(2) Rhizoma Dioscoreae and Poria are soaked in respectively in 2000 milliliters the water, and Rhizoma Alismatis is soaked in 50% ethanol, and Radix Scutellariae then was soaked in 70% ethanol each 30 minutes in addition.Every kind of solution obtains the extract of Rhizoma Dioscoreae, Rhizoma Alismatis, Poria and Radix Scutellariae respectively afterwards respectively at 85 ℃ of heating 60 minutes.
(3) etc. after the extract cooling, it is become filtrate with screen filtration (about 100 orders) respectively.Discrete filtrate under vacuum condition (30torr), concentrates with 50 to 60 ℃ after collecting, and obtains the extraction concentrated solution of each medical herbs.
(4) before each concentrated solution carries out lyophilization, add capacity carboxy methyl cellulose (as excipient), and make graininess respectively.
(5) each medical herbs granule is mixed, form blended granule and be TCM-B, and carry out capsule and make.
Embodiment 3: the preparation of medical composition TCM-C
A kind of medical composition can prepare via the following step:
(1) gets stem or root, 400 g Rhizoma Alismatis, 300 g Poria and 500 g the Radix Scutellariae of 400 g of Rhizoma Dioscoreaes, be chopped into small pieces respectively.
(2) first three is planted medical herbs and be soaked in together in 2000 milliliters 50% ethanol, and Radix Scutellariae is soaked in 70% ethanol in addition.The alcoholic solution heating obtained the ethanolic extract of Rhizoma Dioscoreae, Rhizoma Alismatis, Poria and Radix Scutellariae after 30 minutes.
(3) etc. after the extract cooling, it with screen filtration (about 100 orders), and is mixed and form a filtrate.This filtrate is (30torr) under vacuum condition, concentrates with 50 to 60 ℃.Under vacuum condition, concentrated solution is made powder with freeze-drying then.
(4) add capacity carboxy methyl cellulose or calcium phosphate (Ca
3(PO
4)
2) (as excipient) to the dried powder and mix homogeneously.Whole mixture is made graininess with freeze-drying, and called after TCM-C.
(5) granule is made capsule under dry environment, and fill the nitrogen bottling.
Embodiment 4: the preparation of medical composition TCM-D
A kind of medical composition can prepare via the following step:
(1) get 400 g Rhizoma Dioscoreae stem or root, 400 g Rhizoma Alismatis, 300 g Poria and 500 g Radix Scutellariae, be cut into fractionlet respectively, remix becomes a mixture.
(2) mixture of aforementioned four kinds of medical herbs is soaked in together 6000 milliliters 50% ethanol.This alcoholic solution heating after 60 minutes, is obtained mixing the ethanolic extract of Rhizoma Dioscoreae, Rhizoma Alismatis, Poria and Radix Scutellariae.
(3) wait the cooling of this extract after, with it with screen filtration (about 100 orders).Collect filtrate, and under vacuum condition (30torr), concentrate with 50 to 60 ℃.The concentrated solution that obtains is carried out lyophilization under vacuum.
(4) carboxy methyl cellulose or the calcium phosphate (Ca of interpolation capacity
3(PO
4)
2) (as excipient) is in dried powder, and mix homogeneously.Whole mixture is made granule with freeze-drying, and called after TCM-D.
Embodiment 5: in vitro test
Below will test the effect of several medical herbs inhibition IL-4.As Fig. 2 a, 2b and shown in Figure 3, clearly illustrate that Rhizoma Dioscoreae, Rhizoma Alismatis and Radix Scutellariae can suppress the release of IL-4, TNF-alpha and eotaxln.
(A) suppressing the cultured cells strain produces cytohormone (1L-4, TNF-alpha) in the experiment schedule, medical herbs is diluted to terminal point concentration (0.5ug/ml, 5ug/ml or look required debita spissitudo configuration) in cell culture fluid.Measure the influence that every kind of herbal composition or its mixture produce IL-4, TNF-alpha respectively, and measurement cysteamine acyl group leukotriene (cysteinyl-leukotrienes, c-LTs).
A1: assess IL-4 with the EL-4 cell strain:
Mouse thymocyte cell strain EL-4 was incubated at every kind of compositions or mixture 2 hours, and A23187 and the PMA with 100ng/ml stimulates again.The sample of tissue culture medium is collected after 24 hours at cell culture, and is stored in subzero negative 70 degree Celsius until carrying out analytical test.
The ELISA check cover group (R﹠amp that adopts commercialization to buy; D Systems, Minneapolis Minn), and according to its use indication, carries out the assessment of cytohormone.In brief, in the microtiter plates that contains the anti-cell hormone antibody that lays in advance,, add the standard/sample solution 100ul that diluted according to the experiment demand again, be incubated at room temperature respectively at adding 100ul testing liquid in each titration hole.After 2 hours, this titration dish with lotion flushing 3 times, after secondary antibodies and detecting are 1: 200 with antibody with the diluent dilution, is added 100ul in each titration gift, the titration disk seal is tight, incubated at room temperature 2 hours.Be diluted to 1: 200 streptavidin-HRP reaction with diluent and be subjected to matter respectively at adding in each titration hole afterwards, under dark room temperature, cultivated 20 minutes.Each titration hole will not be after will being incorporated into the streptavidin-HRP absorption in the hole and cleaning, and adding is subjected to qualitative response liquid (TMB), cultivates 30 minutes under dark room temperature, adds the sulfuric acid solution of 50ul 2N more respectively with cessation reaction.Detect its light reaction data with the microtiter plates reading machine down in the 450nm wavelength.
The method of the aforementioned IL-4 in step territory of assessment TNF-alpha is identical, only the cultured cells strain is replaced with THP-1 by EL-4, and the activity of being assessed is the antibody with anti-TNF-alpha.
Shown in Fig. 2 a, because the inhibitory action of the ethanolic extract of Rhizoma Alismatis and Radix Scutellariae, the performance amount of IL-4 and TNF-alpha reduces (being respectively 90% TNF-alpha, 17% IL-4 and 78% TNF-alpha, 47% IL-4) significantly.Under the condition of concentration 0.2mg/ml, the ethanolic extract of Rhizoma Dioscoreae suppresses 40% IL-4 and 20% TNF-alpha respectively.It is relevant with the dosage of its use that most of extract suppresses the effect of cytohormone, and when the concentration of cytohormone when reducing as the cell culture fluid, the i.e. increase of protein decomposition.At least with regard to certain part, have immunosuppressive effect in vivo as the medical herbs extract, in the in vitro tests, the reduction of cytohormone output also results from the minimizing of EL-4 and THP-1 cell strain growth amount.
Except changing cell strain into THP-1, and be culture fluid with RPMI, other experimental procedures are identical with aforementioned assessment IL-4 all.Supernatant in the titration hole comprises the antibody of TNF-alPha, and determines the secretory volume of TNF-alpha via the ELISA method.
A2: the cytotoxicity that detects each medical herbs in each titration hole with mtt assay:
With the ability that the EL-4 cell decomposes MTT, assess the cytotoxicity of various compositionss or mixture.MTT (3 ', 5 '-Dimethyl-thiazol-2y1-2,5-diphenyltetrazolium bromide), be a kind of yellow compound, can be decomposed into purple crystal product (being called formazan) by the Mitochondria ferment, and can provide or the compatibility or the cytotoxicity of trigger cell.
By preceding method assessment IL-4, TNF-alpha c-LTs, can learn the cytotoxicity of EL-4, THP-1 and RBL-1 cell strain.With the MTT of 5mg/ml (Sigma Chemical Co., St.Louis, MO.) be dissolved in pH7.4 phosphate buffer (phosphate buffer saline, PBS) in, with preparation MTT concentrated solution.After cultivating 21 hours under the specified condition, the MTT of 5ul is added each cultivate in titration hole.After cultivating through 4 hours again, add DMSO with stopped reaction.With the microtiter plates reading machine of MolecularDevice, the wavelength of 560nm measures the optical density (OD) of sample.Data is the optical density (OD) of measuring samples and the ratio of untreated matched group.
A3: with RBL-1 cell strain assessment cysteamine acyl group leukotriene (Cysteinyl-leukotrienes, c-LTS):
Stimulated RBL-1 cell strain (rat basophilic lymph corpuscle-1 cell) 16 hours with 0.1ug/ml formic acid (Retinoic acid), with LTC4 synzyme in the activating cell.Then the medical herbs composition mixture is added in the culture fluid, and cultivated 2 hours in 37 ℃.The A23187 that adds 10uM subsequently again makes c-LTs to stimulate.Cultivate after 15 minutes, stop experiment, and collect supernatant 100ul.(Cayman Chemical, Michigan USA), assess according to its service manual the concentration of c-LTs with ElISA check cover group in the cell conditioned medium liquid.
A4: assess TNF-alpha with the mouse peritoneal macrophages:
Peritoneal macrophages is to take from the big male Balb/c mouse of 6-8 week that carries out lumbar injection 3%Brewer thioelycolate culture fluid in advance.After 7 days, with the RPMI-1640 culture fluid and the 10%FBS lavation abdominal cavity of ice, collecting peritoneum cell, and it is incubated at 37 ℃, 2 hours.The cell of non-adherence will be rinsed, and test is used as TNF-alpha.In this test, the kinase whose inhibitor-SB203580 of one special P-38MAP will be as the positive matched group (positive control) of each test.Shown in Fig. 4 a, by the inhibitory reaction that SB203580 causes, also can be as the index of cell to the LPS reaction.
(B) suppress human BEAS-2B emiocytosis eotaxin
This test employed BEAS-2B cell be take from U.S. strain preserve the center (AmericanType Culture Collection, ATCC).The BEAS-2B epithelial cell separates from the non-human bronchial epithelial cell of dying from the corpse of cancer.
This experiment comprises two parts, the cytotoxicity test, and suppress eotaxin secretion test.Each medical composition and mixture will carry out Cytotoxic test earlier, are described as follows, and represent with the survival percentage ratio of cell.Shown in Fig. 3 a, percentage ratio is high more, and the material pair cell of representative test is avirulent.Usually, the cell survival rate of avirulent medical herbs mixture is up to more than 80%, and this mixture will be used to suppress the excretory test of eotaxin.
In eotaxin secretion test, the BEAS-2B cell is incubated in the 96 hole titration dishes with the DMEM/F12 culture fluid, cultivates one day under 37 ℃ of environment.The BEAS-2B cell culture in the culture fluid of the medical herbs mixture that adds specific concentrations, 37 ℃, 2 hours; Add the PBS 20ul that contains 100ng/ml IL-13 and 100ng/ml TNF-α again, in 37 ℃ of cultivations 72 hours, to stimulate the BEAS-2B cell.Afterwards, collect supernatant 100ul, detect the content of eotaxin with the ELISA method.(amount of its emiocytosis etotain is a basic value for pituitary adenylate cyclasepolypeptides, culture fluid PACAP) not add any medical herbs mixture or pituitary adenylate enzyme victory peptide.Add the amount of the eotaxln that PACAP suppresses of 0.001uM, then as the positive matched group of confirmatory experiment correctness.
The data of Fig. 3 a shows, comprises the TCM-B mixture 0.5ug/ml of Rhizoma Dioscoreae, Rhizoma Alismatis, Poria, Radix Scutellariae, can obviously suppress BEAS-2B secretion eotaxin, simultaneously can't the effect of pair cell toxigenicity.The data of Fig. 3 b then show the inhibition situation of TCM-B for EL-4 cell strain secretion IL-4.This inhibitory reaction is relevant with dosage, and maximum dose level 500ug/ml promptly makes a difference to the growth of EL-4 cell strain.To comprise 36% starch and 1.9% calcium phosphate (Ca
3(PO
4)
2) the TCM-B powder, the influence of test EL-4 cell strain secretion IL-4.This test is simultaneously also in order to check general excipient, and as starch and calcium phosphate, whether IL-4 makes a difference for the secretion of EL-4 cell strain.The result shows, excipient is as starch and calcium phosphate, for the not influence of phenomenon of EL-4 cell strain secretion IL-4.The powder that contains TCM-B and excipient has consistent inhibition effect for the phenomenon of EL-4 cell strain secretion IL-4, and shown in Fig. 3 c, so starch and calcium phosphate is prepared in the particulate process at herbal powder and adds, as excipient.
Fig. 4 a shows that the TCM-B of 50ug/ml concentration suppresses effect for the huge phenomenon tool of having a liking for emiocytosis TNF-alpha of peritoneum.But the inhibition effect of TCM-B is compared with the Rhizoma Alismatis extract of equivalent, and is more not obvious.Yet shown in Fig. 4 b, the Rhizoma Alismatis extract of 50ug/ml concentration but has to a certain degree cytotoxicity for peritoneal macrophages.Suppressing BEAS-2B emiocytosis eotaxln, increase in the experiment of cell survival rate, same condition also takes place.Therefore, mix the compositions TCM-B of Rhizoma Dioscoreae, Rhizoma Alismatis, Poria, Radix Scutellariae, not only can keep it and suppress the excretory effect of TNF-alpha, also can reduce TCM-B the issuable cytotoxicity of institute's cultured cells strain.
Example 6. in vivo tests
(A) laboratory animal
Male Balb/c mouse is from national Experimental Animal Center, and raises the environment in specific pathogen free.It is big that these mouse are 6-8 week.According to the processing mode difference, 6 mouse are divided into one group, and every group mouse is all separately raised.The Dermatophoides pteronyssinus group 2 (Der p 2) of reorganization is to be provided by doctor Cai, and Chinese medicine A (Traditional Chinese Medicine A, TCM-A, 1217A among Fig. 7,8,9 and 10) and Chinese medicine B (Traditional Chinese MedicineB, TCM-B, the 1217B among Fig. 7,8,9 and 10) then grinds institute and give birth to the doctor center from the worker of Hsin-chu.
(B) cause anaphylaxis trachea inflammatory response
Mouse in respectively the 0th day with the mat woven of fine bamboo strips 7 days, in the mode of lumbar injection, injection is with 4ug/0.062ml aluminium oxide (aluminum hydroxide, Al (OH)
3, Whitehall Lab Ltd, Punchbowel, Australia) emulsive 1ug/0.1ml Der p 2 makes it that immunoreation take place.Afterwards, with 3 weeks of TCM feeding mouse.The mouse of different groups, difference feeding: ground plug Wu Song (Dexamethasome) (every Mus 1ug every day), TCM-A (every Mus 20mg every day), TCM-B (every Mus 20mg every day) or the every Mus of general normal saline solution 500ul every day.In the time of the 28th and 35 day, with mouse with the sodium phenobarbital of 60mg/kg dosage (sodium pentobarbital, Sigma Chemical Co., St.Louis, MO, USA) make slight anesthesia via peritoneal injection after, from the Der p 2 of trachea inoculation 1ug/50ul.And, measure pulmonary function in inoculating back 24 hours for the second time, subsequently mouse is sacrificed.
(C) measure pulmonary function
With every mouse thus in the air pressure volume measuring instrument (Buxco electronics, Troy, NY) in.This measuring instrument includes two empty chambers: one is the main chamber, or is called animal housing's (7.5 centimeters of internal diameters, 3.5 centimeters high); Another then is reference chamber (7.5 centimeters of an internal diameter, 3.5 centimeters high).One pressure differential translator is used to detect the pressure differential of aforementioned two empty chambers.After this pressure differential is amplified via analog/digital signal conversion, signal is sent to one is loaded with BioSystem XA system (Buxco, Electronics, Troy, computer NY), and carry out the respiration parameter analysis of sample.Similar to people's (1997) such as Hamelmann report, strengthen (enhanced pause sexual refractoriness, Penh), intermittently (pause), tidal volume (tidalVolume, VT), and respiratory frequency (breathing frequency, f), the highest inspiration capacity (peak inspiratoryflow, PIF), shout most tolerance (peak expiratory flow, PEF), air-breathing not (end-inspiratory pause intermittently, EIP) and exhale not intermittently (end-expiratory pause, EEP) etc. parameter can record, the result is as shown in Figure 5.
With 5 milliliters saline solutions or the ugly methyl gallbladder of acetyl element (methacholine, 1.56 to 50.00mg/ml) solution places ultrasound atomizer (ultrasonic nebulizer, DeVilbiss, Somerset, PA) in, can produce fog, and be connected to the animal housing of air pressure volume measuring instrument via three-way pipe.The mean size of fog is about 3um, according to the specification of different manufacturers, its magnitude range from 1 to 5um.Usually in 15 to 20 seconds, fog can be full of the sky chamber.During beginning, allow mouse suck the Sal aqueous vapor 3 minutes earlier, measured its respiration parameter again 3 minutes.Then, replace the Sal aqueous vapor so that the ugly methyl gallbladder of acetyl is plain, and allow mouse suck 3 minutes.Behind the aeration, immediately that sky is indoor fog is got rid of.After sucking the plain fog of the ugly methyl gallbladder of acetyl, measured respiration parameter 3 minutes.The plain dose-response curve of the ugly methyl gallbladder of acetyl is to be represented by low dosage to high dose.As shown in Figure 5, TCM-B can improve the immune inflammatory response of pulmonary function and irritated animal with ground Sai Meisong.Between per twice aeration, 15 minutes interval is arranged.Data are meansigma methods ± SEM.Not on the same group between the difference of parameter, will analyze its parameter.If, significant difference is not arranged between on the same group, will be with the statistical difference between wantonly two groups of Newman-Keuls method analysis.The numerical value difference of aeration before or after saline solution or the ugly methyl gallbladder of acetyl element will be analyzed with paired t method.Saline solution matched group and the difference between the experimental group not will be analyzed with non-paired t method.If the p value is less than 0.05, this difference is considered as significantly.
(D) sample collection and preparation
After measuring pulmonary function, (bronchoalveolar lavage BAL) will carry out according to following method bronchoalveolar lavage.Divide 1 milliliter of sterilization of 2 injections, do not have endotoxic saline solution, be injected in the Mus lung from trachea.Reclaim about 1.8 milliliters BAL flushing liquor.This BAL flushing liquor that has gas is stored in-70 ℃ before experiment.Behind full lencocyte count, 100 milliliters of BAL flushing liquors are through cell centrifugation, and (Tonyar Diagnostic Inc, Taipei shien Taiwan) also calculate cell respectively with the dyeing of Liu stain.The results are shown in Fig. 6 and following table 1, TCM-A and TCM-B all can reduce the cell number that inflammatory response is arranged in the BAL flushing liquor.Blood sample picks up from socket of the eye guest (orbital sinus), and the serum of obtaining is stored in-70 ℃ before experiment.
Table 1
(E) measure tool Der p 2 narrow spectrum TgG1, IgG2a and IgE antibody
In experiment beginning and end front and back, (retro-orbital venous plexus) gets blood from eye socket.By the ELISA method, measure IgE, the IgG1 of anti-Der p 2 and tiring of IgG2a in the serum.(USA) surface is covered with the 100ul Der p 2 that concentration is 0.5ug/ml, is placed in 4 ℃ of refrigerators overnight for Nunc Lab, IL with microtiter plates.The titration dish washes three times with PBS-Tween-20 (PBST), and is stored in-70 ℃ up to use.Add the IgE of mouse serum with dilution in 1: 5, the IgG1 of dilution in 1: 100, and behind the IgG2a of dilution in 1: 5, will drop down price fixing and be placed in 4 ℃ overnight, adding the antibody (antibody of the anti-ageing Mus of goat of HRP-bond, the antibody of wherein anti-IgE and anti-IgG2a is dilution in 1: 800, and anti-IgG1 antibody is dilution in 1: 2000, available from Southern Biotech Assoc, Inc, Birmingham, AL, USA) preceding cleaning is 3 times.37 ℃ cultivate 1 hour after, with PBST flushing 3 times, add again the ferment reaction be subjected to matter ABTS (2,2 " Azion-bis (3-ethylbenzothiazolin-6-sulfonicacid) diammonilum salt, Bio-Rad, USA).After 15 minutes, add 50ul 4NH
2SO
4With cessation reaction, and with the spectrophotometer of multi-wavelength (Salzburg is Austria) with 450nm wavelength measurement luminosity for A-5682, SLT Lab Instruments.The result represents with ELISA unit (EU).An EU is defined as the inverse after the serum dilution, and measured shading value is 1.0.This result all drops on the linear segment of dilution curve.In order to confirm repeatability, in each experiment with a known serum as standard value.Fig. 7 is in the test serum, the titration concentration of IgE.
(F) test of the cytohormone in the mouse sample
Measure IFN-γ and IL-5 with the ELISA check cover group that commodity are buied, and use the monoclonal antibody of mouse, with the different antigen decision bit (epitopes) in decision cytohormone surface.Minimum detection range is 10pg/ml.The result is shown in Fig. 8,9.
(G) the lymphocytic cultivation of bronchoalveolar lavage gained, immunofluorescence dyeing and flow cytometry (flow cytometry analysis)
The method that proposes according to people such as Assenmacher (1994) can flow cytometry be measured the cytohormone of mouse T assistant cell activation.Two stainings are used to analyze the performance at CD4 or cd8 cell IFN-γ and IL-5.From the leukocyte of peripheral blood, be through PMA (50ng/ml), ionomycin (2uM) and GolgiStop (Cytofix/Cytoperm Plus Cat No.2076KK, Pharmingen, San Diego, CA) 5 hours stimulation is again with PBS flushing 2 times.At room temperature, cell dyeed 30 minutes with CD4-FITC or CD8-FITC, was cleaned again.Cell is fixed 30 minutes with cytofix/cytoperm under room temperature, again with the antibody staining of anti-cell hormone 30 minutes, and flushing again.Cell is suspended among the 0.5mlPBS that contains 0.1% Hydrazoic acid,sodium salt (sodium azide) again.(Becton Dichnson, CA USA) read the fluorescent value with Becton Dickinson flow cytometry.Each sample will be analyzed 2000 cells.With reference to Figure 10, show that TCM-B has the ability of T cell in the preferable adjusting blood.
(H) statistical analysis
The result shows with arithmetic mean number ± SEM.The difference of group is to analyze with the Mann-WhitineyU method.If the P value less than 0.05, then is considered as statistically significant difference.
Aforesaid example focuses on the curative effect of assessment medical composition of the present invention.This prescription can suppress the immunity inflammatory response of respiratory tract, improves pulmonary function, reduces IgE content in the blood, and prevents that asthma from taking place.
Though the present invention by the most preferred embodiment description, is not deviating under spirit of the present invention and the category, all can all changes and modification be arranged to the present invention, to be applicable to variety of applications and situation.In other words, to the omission of the form of filling a prescription among the present invention and details, modification, impairment, with change, do not deviating under spirit of the present invention and the category, all can be undertaken by haveing the knack of this technology.
Must not be described in further detail again, believe that above-mentioned explanation can give full expression to the present invention, other people can have all changes to the present invention under the situation that the feature of forming necessity of the present invention is not ignored.
Claims (13)
1, a kind of medical composition that relaxes with the treatment allergic asthma, the consisting of of crude drug:
One Rhizoma Dioscoreae (Radix Dioscorea) 40-95% ethanolic extract;
One Rhizoma Alismatis (Rhizoma Alismatis) 40-95% ethanolic extract;
One Poria (Poria cocos (schw) Wolf) 40-95% ethanolic extract;
One Radix Scutellariae (Scutellaria Baicalensis) 40-95% ethanolic extract; And
Wherein the weight ratio of this Rhizoma Dioscoreae, Rhizoma Alismatis, Poria and Radix Scutellariae is 1: 1-2: 0.5-2: 1-3.
2, medical composition as claimed in claim 1 is characterized in that, wherein this Rhizoma Dioscoreae extract, Rhizoma Alismatis extract, tuckahoe extract and Radix Scutellariae extract extract respectively from Rhizoma Dioscoreae, Rhizoma Alismatis, Poria and Radix Scutellariae.
3, medical composition as claimed in claim 1 is characterized in that, wherein this Rhizoma Dioscoreae extract, Rhizoma Alismatis extract, tuckahoe extract and Radix Scutellariae extract are the mixture that extracts simultaneously from Rhizoma Dioscoreae, Rhizoma Alismatis, Poria and Radix Scutellariae.
4, medical composition as claimed in claim 1 is characterized in that, wherein also comprises an excipient.
5, a kind of method for preparing the described medical composition of claim 1 comprises the following step:
Respectively with alcohol extraction Rhizoma Dioscoreae, Rhizoma Alismatis, Poria and Radix Scutellariae, to produce the extract of Rhizoma Dioscoreae, Rhizoma Alismatis, Poria and Radix Scutellariae;
Filter and concentrate this extract respectively, to produce the concentrate of Rhizoma Dioscoreae, Rhizoma Alismatis, Poria and Radix Scutellariae; And
The concentrate of this Rhizoma Dioscoreae, Rhizoma Alismatis, Poria and Radix Scutellariae is mixed;
Wherein, this concentration of alcohol is 40-95%, and the weight ratio of this Rhizoma Dioscoreae, Rhizoma Alismatis, Poria and Radix Scutellariae is 1: 1-2: 0.5-2: 1-3.
6, method as claimed in claim 5 is characterized in that, also comprises respectively or concentrated jointly this each concentrate, granulates with the combination drying that carries out Rhizoma Dioscoreae, Rhizoma Alismatis, Poria and Radix Scutellariae.
7, method as claimed in claim 6 is characterized in that, also is included in before combination drying granulates, and adds an excipient to this Rhizoma Dioscoreae, Rhizoma Alismatis, Poria and Radix Scutellariae concentrate.
8, method as claimed in claim 5 is characterized in that, this concentration of alcohol is 40-90%.
9, a kind of method for preparing the described medical composition of claim 1 comprises the following step:
Mix Rhizoma Dioscoreae, Rhizoma Alismatis, Poria and Radix Scutellariae to produce a mixture;
With this mixture of alcohol extraction, to produce the ethanolic extract of Rhizoma Dioscoreae, Rhizoma Alismatis, Poria and Radix Scutellariae; And
Filter and concentrate this extract, with the concentrate of generation Rhizoma Dioscoreae, Rhizoma Alismatis, Poria and Radix Scutellariae,
Wherein, should be 40-95% in order to the concentration of alcohol of extraction, and the weight ratio of this Rhizoma Dioscoreae, Rhizoma Alismatis, Poria and Radix Scutellariae be 1: 1-2: 0.5-2: 1-3.
10, method as claimed in claim 9 is characterized in that, also comprises this Rhizoma Dioscoreae, Rhizoma Alismatis, Poria and Radix Scutellariae concentrate, makes granule with spraying-seasoning.
11, method as claimed in claim 10 is characterized in that, also is included in to make the preceding concentrate that adds an excipient to this Rhizoma Dioscoreae, Rhizoma Alismatis, Poria and Radix Scutellariae of granule.
12, method as claimed in claim 9 is characterized in that, wherein the concentration of alcohol of this extraction usefulness is 40-50%.
13, method as claimed in claim 11 is characterized in that, wherein excipient is starch and calcium phosphate.
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| CN1298724A (en) * | 2000-11-21 | 2001-06-13 | 李佳阳 | Medicine for treating immunodefiiciency disease |
Non-Patent Citations (4)
| Title |
|---|
| 治疗乙肝47种常用中药临床药理统计分析. 戴俊达.中药材，第4期. 1985 |
| 治疗乙肝47种常用中药临床药理统计分析. 戴俊达.中药材，第4期. 1985 * |
| 芩桔汤治疗慢性鼻窦炎IgG变化和疗效的关系. 苏崇周等.中西医结合杂志，第4卷第12期. 1984 |
| 芩桔汤治疗慢性鼻窦炎IgG变化和疗效的关系. 苏崇周等.中西医结合杂志，第4卷第12期. 1984 * |
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