CN101632746B - Medicament for treating ulcerative colitis and preparation method thereof - Google Patents
Medicament for treating ulcerative colitis and preparation method thereof Download PDFInfo
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Abstract
The invention belongs to the medicine technical field, in particular a medicament for treating ulcerative colitis and a preparation method thereof. The medicament for treating the ulcerative colitis is prepared from the following raw materials by weight: 5-30 portions of Chinese bulbul, 4-24 portions of golden cypress, 2-12 portions of coptis chinensis, 4-24 portions of ash bark, 3-18 portions of raw astragalus root, 3-18 portions of deep-fried atractylodes, 3-18 portions of flos sophorae, 3-18 portions of sanguisorba officinalis and 2-10 portions of aconite albertdeng. In the invention, calcium pectate is also used as a coating material and an effective medicament prime tablet to prepare a colitis tablet so as to realize colonic targeting drug administration, therefore, the curative effect of the medicament for treating ulcerative colitis is obviously superior to the prior medicament.
Description
Technical field
The invention belongs to medical technical field, be specifically related to a kind of medicine for the treatment of ulcerative colitis and preparation method thereof.
Background technology
One, ulcerative colitis is one of digestive system refractory disease, and modern medicine treatment curative effect is undesirable, and side effect is big
(ulcerative colitis UC) belongs to inflammatory bowel to ulcerative colitis, is one of digestive system refractory disease, clinically with stomachache, diarrhoea and pus and blood mucus just, serves as main performance with the colon mucosa ulcer erosion under the scope.Its sickness rate is higher in western countries, in China the trend that increases is arranged also at present.But the cause of disease and pathogeny are not exclusively clear, modern medicine adopts sulfasalazine (SASP) usually to treat in conjunction with cortical hormone to primary disease more, it mainly is the control acute attack, mitigate the disease reduces recurrence, prevents and treats complication etc., because of the oral back of SASP at colonic by the azo reductase cracking of antibacterial, enter the back that is acetylation in the liver and discharged by urine, bioavailability is low, can not form effective drug level at the colitis place; Other has corticosteroids such as prednisone, succinic acid hydrocortisone etc., can make the rapid inducer remission of the ulcerative colitis that is in acute stage, but prolonged application can cause eight big side effect of hormone medicines such as moon face, electrolyte disturbance, osteoporosis etc., and also has the corticosteroid opposing among the patient; Immunosuppressant such as Ismipur and azathioprine etc., it is usually used in SASP and invalid patient and corticosteroid toxic reaction or the long-term patient who continues to rely on the use corticosteroid of corticosteroid treatment, it can block lymphopoiesis and activation, suppress the chemotactic neutrophilic granulocyte, but onset is slow, toxicity such as bone marrow depression, acute pancreatitis, feel sick, heating, hepatitis and allergy etc. are quite serious; Antibacterial agent regulator such as infliximab, daclizumab etc., its be used for through the cortex steroid hormone and (or) the severe refractory ulcerative colitis patient of immunosuppressant treatment poor effect, feeling sick is again the frequent side effect that takes place.
Two, the traditional Chinese medical herbal treatment primary disease experienced but only decoction be main, dosage form is single
The efficacious prescriptions medicine that has that is seen in report in recent years has Radix Pulsatillae Decoction, Radix Puerariae Siberian cocklebur to connect soup, wumei pills, SHENLING BAISHU SAN, and lizhong decoction closes SISHEN WAN, Zhenren Yangzang Tang, peacekeeping ball and closes more infections soup, adds flavor Sini San, Radix Scutellariae Decoction effective ingredient prescription, Radix Salviae Miltiorrhizae etc.The treatment that is used for ulcerative colitis has considerable clinical report, observes but only be confined to clinical effectiveness mostly, and decoction is main, and dosage form is single.
Three, colon targeting drug administration system
The ulcerative colitis inflammation is mainly invaded rectum, sigmoid colon, descending colon, also has minority to involve transverse colon and total colectomy, sees with the left side colon more.After conventional formulation was oral, medicine will be absorbed before arriving colon and enter the body circulation, can't act on diseased region specifically.Colon targeting drug administration system medicine does not discharge at upper digestive tract, to ileocecus or colon just begins disintegrate or corrosion discharges, and performance part or whole body therapeutic effect.
(oral colon specifi c drugdelivery system OCDDS) can be divided into multiple systems such as pH dependent form, time lag release type, pressure control type, antibacterial flip-over type according to release mechanism in the oral colon positioning feed system.Early stage research is based upon on pH dependent form and the time lag type theoretical basis mostly, but because the influence of factors such as individual variation, gastrointestinal content and patient's digestive tract pathology physiological situation often causes medicine to discharge in advance or not release at the small intestinal position.The release of pressure control type system depends on the human colon internal pressure, even but under normal circadian rhythm, colonic pressure is influenced by various physiologic factors to alter a great deal, so that the drug release individual variation is also bigger, can not guarantee that the medicine expection discharges.Antibacterial flip-over type system distributes according to the flora of human colon uniqueness, utilize bacteriogenic azo reductase, glycosidase (glycosidase) and glycuronide enzyme plurality of enzymes such as (glucuronidase), make the carrier degraded and release, thereby avoid the uncertainty of above-mentioned medicine-releasing system, become a focus of current research.
Pectin is one of natural component of food, and having to be become a kind of good carrier of antibacterial flip-over type colon medicine-releasing system, so obtain broad research by the characteristics of the distinctive bacterial enzyme degraded of colon.
Pectin is a kind of macromolecular compound of different polysaccharide, pass through 1 by D-galactopyranose aldehydic acid, the 4-glycosidic bond connects into main chain, and neutral sugar (comprising D-galactose, L-arabinose, L-rhamnose etc.) is formed side chain, and wherein the part carboxyl of galacturonic acid exists with the esterification state.Usually the pectin that esterification degree is higher than 50% (being equivalent to methoxyl content 7%～16.3%) is called hyper-methoxy pectin (high methoxylpectin, HMP), esterification degree be lower than 50% (being equivalent to methoxyl content) less than 7% then be called hypo-methoxy pectin (low methoxylpectin, LMP).Pectin is water-soluble can to form viscous solution, and its viscosity is relevant with extent of polymerization and esterification degree with dissolubility, and with the increase of methoxyl content, the dissolubility of pectin reduces, and solution viscosity increases.
Human body self can not produce the enzyme of decompose pectin, so pectin can not digested by pipe intestinal digesting liquid, but bacteroid, Fusobacterium, Eubacterium and bacillus bifidus etc. parasitic in the colon can produce polygalacturonase, pectinesterase depolymerase and pectin lyase, pectin can be decomposed into oligosaccharide, even be decomposed into galacturonic acid fully.Because these antibacterials exist only in colon, in the crowd, have the concordance of height again, so pectin as the carrier of colon targeting preparation, high specificity, the credibility of result of study and better repeatable.
Pectin is water-soluble, can not protect medicine effectively, can utilize free carboxyl and calcium ion prepared in reaction calcium pectinate in the pectin.Calcium pectinate is stable in the lower solution of pH, then swelling can take place in the solution of alkalescence.Because colon has higher pH, this specific character of calcium pectinate just can guarantee that preparation of Chinese medicine discharges at the specificity of colon.When the degree of calcification of pectin is enough high, water insoluble, sour, alkali and other solvent, but still can be degraded by intracolic pectase, produce a series of soluble oligomeric sugar.On structure, the methoxyl content in the pectin is low more, and free carboxy is many more, easy more and Ca
2+Reaction generates calcium pectinate, so LMP commonly used.
Summary of the invention
The invention discloses a kind of medicine for the treatment of ulcerative colitis and preparation method thereof.
The traditional Chinese medical science to ulcerative colitis understanding head see Difficult Classic, name of disease is " big abdominal mass is rushed down ", the pathogenesis core is based on deficiency in origin and excess in superficiality, simulataneous insufficiency and excessive, empty with spleen suffer from a deficiency of the kidney, the deficiency of vital energy, yang deficiency be the basic of morbidity, the reality duty in stagnation of damp-heat, depression of liver-QI or qi depression to blood stasis.Damp and hotly run through primary disease all the time.The insufficiency of the spleen characteristics that are primary disease with the gluing epidemic disease caused by damp-heat pathogen poison; For the treatment of primary disease, traditional medicine is many based on spleen invigorating, the kidney warming, clearing away heat-damp and promoting diuresis, promoting diuresis with drugs of tasteless flavour.Long at the primary disease course of disease, simulataneous insufficiency and excessive is similar to " chronic dysentery with frequent relapse ", and Chinese medicine compound disclosed by the invention is the unconventional and unrestrained right clinical efficacious prescriptions that have of Yangzhou, the Jiangsu five generations traditional Chinese medical science handed down from one's ancestors.This side with Radix Pulsatillae Decoction (Radix Pulsatillae, Cortex Phellodendri, Rhizoma Coptidis, Cortex Fraxini) add Flos Sophorae, Radix Sanguisorbae is taken stopgap measures, Radix Astragali, Radix Aconiti Lateralis Preparata, the Rhizoma Atractylodis Macrocephalae preparata composition that effects a permanent cure, the prescription clearing away heat-damp and promoting diuresis is main, is aided with the invigorating the spleen and benefiting QI the kidney warming, treating both the principal and secondary aspects of a disease, determined curative effect.
A kind of medicine for the treatment of ulcerative colitis, it is to be made by following bulk drugs: 5～30 parts of the Radix Pulsatillaes, 4～24 parts of Cortex Phellodendris, 2～12 parts of Rhizoma Coptidis, 4～24 parts of Cortex Fraxinis, 3～18 parts of Radix Astragali, 3～18 parts of Rhizoma Atractylodis Macrocephalae preparatas, 3～18 parts of Flos Sophorae Immaturus, 3～18 parts of Radix Sanguisorbaes, 2～10 parts of Radix Aconiti Lateralis Preparatas.The preferred intermediate quantity of each Chinese medicine weight portion.
This side is taken stopgap measures with the Radix Pulsatillae (the main pharmacodynamics composition is a protoanemonin), Cortex Phellodendri and Rhizoma Coptidis (the main pharmacodynamics composition is a berberine), Cortex Fraxini (the main pharmacodynamics composition is an esculiw etc.), Flos Sophorae Immaturus (the main pharmacodynamics composition is flavone compounds such as rutin), Radix Sanguisorbae (the main pharmacodynamics composition is tannin and saponins); (the main pharmacodynamics composition is alkaloid compound, Rhizoma Atractylodis Macrocephalae preparata (the main pharmacodynamics composition is volatile oils such as the atractylodes lactone) composition that effects a permanent cure, our treating both the principal and secondary aspects of a disease, determined curative effect for Radix Astragali (the main pharmacodynamics composition is an astragaloside etc.), Radix Aconiti Lateralis Preparata.
The medicine of above-mentioned said treatment ulcerative colitis, preparation method is as follows:
1) take by weighing each crude drug Radix Pulsatillae, Cortex Phellodendri, Rhizoma Coptidis, Cortex Fraxini, Radix Astragali, Rhizoma Atractylodis Macrocephalae preparata, Flos Sophorae Immaturus, Radix Sanguisorbae and Radix Aconiti Lateralis Preparata, standby;
2) the full recipe quantity of the Radix Pulsatillae, the full recipe quantity of Rhizoma Atractylodis Macrocephalae preparata and all the other flavour of a drug half recipe quantity with above-mentioned weight proportion mixes, and micronizing to particle diameter reaches and gets the former medicine of nano powder below the 100nm, and is standby;
3) Cortex Phellodendri, Rhizoma Coptidis, Cortex Fraxini, Radix Astragali, Flos Sophorae Immaturus, Radix Sanguisorbae and Radix Aconiti Lateralis Preparata 7 flavour of a drug half recipe quantity are decocted with water twice, merge decocting liquid twice, be concentrated into the extraction concentrated solution of proportion 1.2～1.8, standby;
4) will extract concentrated solution and be sprayed on the former medicine of nano powder and make soft material, sieving makes wet granular, 50 ℃～60 ℃ dryings; Just be prepared into the active component of medicine of the present invention.
Further, in order to reach better therapeutic, the present invention preferably utilizes calcium pectinate as pharmaceutical carrier, realizes colon targeting drug administration, for example, can be made into the Chinese medicine colon tablet, and concrete method for making is as follows:
In above-mentioned steps 4) back following steps in addition:
5) will hang down methyl ester pectin adding distil water swelling, heat is to 50 ℃～60 ℃ dissolvings, and concentration reaches 5%～15%; Impouring calcium chloride saturated solution generates to there being white flocculent deposit, and the limit edged stirs, and gets the suspendible glue of calcium content more than 10%;
6) the step 4) active component is pressed into plain sheet, the calcium pectinate suspendible glue coating that the reuse step 5) makes makes the Chinese medicine colon tablet to plain sheet weightening finish 8%～20%.
Medicine of the present invention also comprises other dosage forms except that tablet.
Our experiments show that colon calcium pectinate granule of the present invention and colon not coated granule, colon full of enteric coated granules are compared, the three all has MDA, the COX-2 of removing, increased SOD, IL-4, the reaction that reduces inflammation, the effect of promotion ulcer healing.The particulate effect of colon calcium pectinate of the present invention significantly is better than colon not coated granule, colon full of enteric coated granules.
Description of drawings
Fig. 1 is model control group effect experiment pathological section figure;
Fig. 2 is enteric coated particles group effect experiment pathological section figure;
Fig. 3 is Sasp group effect experiment pathological section figure;
Fig. 4 is calcium pectinate coated granule group effect experiment pathological section figure;
Fig. 5 is Dexamethasone group effect experiment pathological section figure;
Fig. 6 is blank group effect experiment pathological section figure;
Fig. 7 is coated granule group effect experiment pathological section figure not
The specific embodiment
The preparation tablets of embodiment 1 medicine of the present invention
The full recipe quantity of the Radix Pulsatillae, the full recipe quantity of Rhizoma Atractylodis Macrocephalae preparata and all the other flavour of a drug half recipe quantity in the prescription medical material are mixed, and micronizing to particle diameter reaches and gets the former medicine of nano powder below the 100nm.
All the other flavour of a drug half recipe quantity decocting 1h that will be except that the Radix Pulsatillae, Rhizoma Atractylodis Macrocephalae preparata leach medicinal residues and add water two and fry in shallow oil 1h, merge decocting liquid, are concentrated into proportion 1.2～1.8 and make adhesive and use.
To extract concentrated solution and be sprayed on the former medicine of nano powder and make soft material, and cross 14 mesh sieve system wet granulars, 50 ℃～60 ℃ dry 1.5h are pressed into plain sheet behind the mistake 14 mesh sieve granulate.
To hang down methyl ester pectin (esterification degree＜50%) adding distil water swelling, heat is to 50 ℃～60 ℃ dissolvings, and concentration reaches 5%～15%; Impouring calcium chloride saturated solution does not generate to there being white flocculent deposit, and the limit edged stirs, and gained suspendible glue calcium content should be at (atomic absorption spectrophotometer) more than 10%.
Plain sheet had both been got with calcium pectinate suspendible glue coating to the plain sheet weightening finish 8%～20% that makes.
The external dissolution test of embodiment 2 Chinese medicine colon tablets of the present invention
The assay method of 1 index components astragaloside
1.1 the mensuration of astragaloside maximum absorption wavelength
Precision takes by weighing astragaloside reference substance 4.895mg, be settled in the 10ml measuring bottle with the phosphate buffer of pH=8, concentration be the astragaloside reference substance solution of 0.4895mg/ml, carry out long (190～900nm) scannings of ultraviolet-visible all-wave.Determine that maximum absorption wavelength is 538nm.Other drug and Calcium Pectate 538nm place are noiseless in the prescription.
1.2 the foundation of astragaloside standard curve
Accurate astragaloside reference substance solution 0.1,0.2,0.3,0.5,0.7 and the 0.9ml that draws 0.4895mg/ml places the 50ml measuring bottle, the accurate phosphate buffer standardize solution that adds, and mixing gets the reference substance solution of 6 concentration.Measure trap in the 538nm place.With astragaloside concentration (x) trap (y) is done regression analysis, must standard curve be: C=0.096A-0.0014, r=0.9995, n=6.The range of linearity is 0.01～0.08mg/ml.
2 dissolution tests
Adopting changes the basket method, and water temperature is 37 ℃, and rotating speed is 80r/min, and dissolution medium is a simulated gastric fluid, and the stripping volume is 250ml.Timing sampling 5ml adds simulated gastric fluid 5ml simultaneously.The accurate 2ml that draws puts in the 10ml measuring bottle, according to method operation under the 3.1.2 item, surveys the trap value.The worker's intestinal juice of substituting again carries out dissolution test with method, and the result shows that self-control calcium pectinate colon tablet does not all have obviously leakage (the stripping percentage rate all is lower than 2% in 5 hours) in simulated gastric fluid, simulated intestinal fluid.
Stripping in rat cecal content solution (anthropomorphic dummy's colon).Get the rat of 6 weight at 200～300g, regularly irritate the pectin solution 1ml of stomach 15% every day, rat is put to death in the back for three days on end, takes out cecal content immediately, and the back of weighing is standby in the phosphate buffer of 1: 80 ratio adding pH7.0.Other gets 6 is equipped with the flask that changes basket beyond the Great Wall, and each adds the buffer of the above-mentioned cecal content of 100ml.Respectively 6 self-control calcium pectinate colon tablets that press being put into changes basket, and lid is tight, and feeds CO
2Bottle is placed 37 ℃ of waters bath with thermostatic control, constantly stir.Timing sampling 2ml adds the buffer of the pH7.0 of 2ml immediately, through the time 12 hours.Each sampling needs sample is centrifugal, draws supernatant 1ml, is diluted to 10ml with the buffer of pH7.0, and in 538nm place survey trap value, 1h stripping as a result is more than 70%; The 3h stripping is more than 80%; The 5h stripping is more than 90%.
Dissolution test in the particulate body of embodiment 3 Chinese medicine colons of the present invention
The plain sheet of the Chinese medicine of self-control treatment ulcerative colitis made to wrap behind the granule state self-control calcium pectinate clothing film; Get 6 rabbit of body weight about 2kg, irritate the above-mentioned calcium pectinate granule of 0.2g, jar 6 times every 6h.Put to death and dissect rabbit behind the last perfusion 3h.Find the complete calcium pectinate granule of average about 0.2g at the rabbit gastric, find the complete calcium pectinate granule of average 0.05g in small intestinal, all the other estimations are degraded in caecum.6 rabbit individual instances are similar substantially.
Embodiment 4 observation of curative effect
1 materials and methods
1.1 experiment material
1.1.1 laboratory animal
70 of SD rats, body weight 180 ± 20g, male, standard feed is fed.(the Experimental Animal Center SCXK of Zhejiang Province (Zhejiang) 2003-0001)
1.1.2 experimental drug
The plain sheet of Chinese medicine of taking from system treatment ulcerative colitis is made granule and is coated granule group not; With this granule enteric coated be the full of enteric coated granules group; With this granule bag self-control calcium pectinate clothing film is calcium pectinate coated granule group.
Positive control drug: SASP, the three-dimensional company in Shanghai produces, and lot number: 200712c13 faces with before being mixed with 72mg/ml; Dexamethasone, Zhejiang Province XianJu Pharmacy stock Co., Ltd, lot number: H33020822 faces with before being mixed with 0.18mg/ml.
1.1.3 experiment reagent
Modeling reagent: 5% this sulfonic acid of trinitro-: sigma company (Shanghai is just reaching the agency); SABC reagent: the anti-Mus cox-2 of rabbit polyclonal antibody (Beijing Bo Aosen Bioisystech Co., Ltd); SABC test kit (Beijing Bo Aosen Bioisystech Co., Ltd); 3,3 diaminobenzidine tetrahydrochlorides (Diaminbenzidine, DAB) colour reagent box (Beijing Bo Aosen Bioisystech Co., Ltd); Biochemical reagents malonaldehyde (MaleicDialdehyde MDA) test kit, lot number 20080410 (medical biotechnology research is built up in Nanjing); Superoxide dismutase (Superoxide Dismutsae SOD) test kit, lot number 20080410 (Nanjing is built up Institute of Medical Biology and provided); The Coomassie brilliant blue protein determination kit, lot number 20080410 (Nanjing is built up Institute of Medical Biology and provided); IL-4ELISA test kit: ADL (ADLITTERAMDIAGNOSTIC LABORATORIES) company (doctor's moral agency)
Dimethylbenzene, dehydrated alcohol, acetone, the haematoxylin after stain, hydrogen peroxide, neutral gum, reagent such as citrate are homemade analytical pure.
2 modelings
According to document: Chen Yingqun, this sulfonic acid of Dong Fu wheel trinitro-is induced experimentation Tongji University journal (medicine) 2006-12 of rat ulcer colitis; The described method modeling of the 27th volume the 6th phase 31-33 page or leaf, 5% trinitro-benzene-sulfonic acid (TNBS) is made into 25mg (TNBS)/ml (mixed liquor) with dehydrated alcohol with volume at 1: 1.
2.1 the rat adaptability is raised a week.
2.2 the rat fasting be can't help water 24 hours before the modeling; 70 pre-modeling rat 2% pentobarbital sodium intraperitoneal anesthesias (No. 5 syringes of 45mg/kg); After treating the rat holonarcosis, 60 rats of random choose, the plastic catheter 8cm of the light and slow insertion diameter of per anum 0.2cm, slowly inject the modeling agent (100mg/kg is 0.4ml/100g) of corresponding body weight ratio with the 2ml syringe, and then inject the air of the about 0.3ml of a segment length, remove the medicinal liquid that is attached on syringe and the coloclysis tube wall as far as possible.Rat random packet after the modeling, 10 every group, and label.Grouping back rat returns cage, is sidelong on bedding and padding, takes a low buttocks high position, prevents that rat from suffocating.Remain 10 anesthetized rats, the plastic catheter 8cm of the light and slow insertion diameter of per anum 0.2cm slowly injects the 2ml normal saline with the 2ml syringe, and then injects the air of the about 0.3ml of a segment length.As the blank group.70 modeling rats, clear-headed naturally, free diet.Modeling administration after 24 hours is observed rat, is weighed, detects stool blood, and (dose,equivalent is converted by people and animal body surface coefficient with adult's routine dose to observe situations such as spirit, fur, stool, activity, survival every day; Blank group and model group give with the volume normal saline every day, irritate stomach every day 1 time, continuous 1 week.All with common sterilization forage feed).
3 experimental specimen acquisition process and decision method as a result
3.1 blood sample collection and processing: each treated animal finishes after one week through irritating stomach, and fasting be can't help all killing inspection behind the water 24h.Behind the anesthetized rat, about abdominal cavity postcava blood sampling 4ml, put in the sterile test tube, room temperature leaves standstill after natural coagulation shrinks, and with 3000r/min centrifugal 10 minutes, separation of serum was stored in-80 ℃ of refrigerators, detects the content of IL-4 with the ELISA test kit.
3.2 colon gathers and handles: get ulcer under the cryogenic conditions fast and obviously locate colon, with ice-cold normal saline flushing, filter paper blots, shred as early as possible with the little shears of ophthalmology after weighing, place homogenizer with 9 times of normal saline homogenate (end of homogenizer places the cold water of putting ice cube), with the centrifugal 10min of 3500r/min, get supernatant and put-80 ℃ of refrigerators preservations, to be measured.Intestinal mucosa MDA, SOD measure and adopt colorimetry, and reference reagent box description is operated.Other gets 3.0～4.0cm far-end colon (ulcer is obviously located) and places 10% neutral formalin to fix, and is used for the cox-2 immunohistochemistry and detects.
3.3 decision method as a result
Negative control replaces one anti-with 0.01mol/LPBS, cytoplasm be yellow or pale brown color positive, non-coloring is negative; Area under every 5 high power field of random observation at least of cutting into slices is analyzed the average optical of positive material under each high power field with the Jetta image analyzer, gets its average.
Statistical procedures: adopt SPSS 10.0 statistical softwares to handle, experimental data is represented with X ± s, adopts one factor analysis of variance according to homogeneity of variance, and difference is meaningful on P＜0.05 expression statistics.
4 results
4.1DAI scoring
Disease activity index (10) (DAI) compares: model group, Dexamethasone group, SASP group and medicine group promptly occurred seizure of disease on the 2nd day in modeling, and model group inflammation in whole experiment continues, and did not have obvious spontaneous recovery tendency; Treatment is organized the 5th day and is begun to occur the DAI downward trend, all take a favorable turn during the experiment terminal point, the 7th day: calcium pectinate coating group DAI (0.85), enteric group DAI (1.92), coating group DAI (2) all is not lower than the positive drug group, difference has statistical significance (P＜0.05), sees Table 1, table 2, and this prompting colon granule can obviously improve rat ulcer colitis symptom.
Table 1.DAI computer chart
Table 2.DAI grade form
4.2 intestinal specimen histone assay:
Protein molecule has-NH
3 +Group, when henna Coomassie brilliant blue developer adds in protein standard liquid or the sample, the anion on the Coomassie brilliant blue dyestuff and albumen-NH
3 +The group combination makes solution become blueness, can calculate protein content by measuring absorbance.(Coomassie brilliant blue stock solution: face the time spent and use distilled water dilution (i.e. 5 times of dilutions) in 1: 4 in the desired amount, be made into and use liquid (matching while using).4 ℃ of preservations.Protein standard: 1 of 0.563g/L protein standard liquid.4 ℃ of preservations.)
Accurately take by weighing the weight of tissue to be measured, volume ratio adds normal saline and is prepared into 10% tissue homogenate by weight, 1000～3000 rev/mins, centrifugal 10 minutes (determining concrete rotating speed) according to the survey index, get tissue homogenate supernatant reuse normal saline then and be diluted to 1% tissue homogenate by 1: 9, operation sees Table 3.
Table 3. protein determination operation table
Press table 3 operation, each left standstill 10 minutes after managing mixing, measured OD value, result such as table 4 in the 595nm place.
Table 4 is respectively organized the specimen absorbance
Calculate protein content (g/L) according to following formula, result such as table 5:
Each histone content (g/L) of table 5
4.3 the mensuration of malonaldehyde
Body produces oxygen-derived free radicals by enzyme system and non-enzyme system, and the latter can attack the polyunsaturated fatty acid in the biomembrane, causes lipid peroxidation, and therefore forms lipid peroxide.As: aldehyde radical (malonaldehyde MDA), ketone group, hydroxyl, carbonyl, hydroperoxy or interior peroxy, and new oxygen-derived free radicals etc.Lipid peroxidation not only changes into the activity chemistry agent to active oxygen, i.e. the lipidolysis product of non-free radical, and, amplify the effect of active oxygen by chain type or chain type chain reaction.Therefore, an initial active oxygen can cause the formation of a lot of lipidolysis products, and in these catabolites, some are harmless, and other then can cause cellular metabolism and dysfunction, even dead.Oxygen-derived free radicals not only causes cell injury by the peroxidating of polyunsaturated fatty acid in the biomembrane, and catabolite that can also be by fat hydrogen peroxide cell injury together.Thereby the test MDA amount usually can react the snperoxiaized degree of body inner lipid, reflect the degree of cell injury indirectly.
The mensuration of MDA usually cooperatively interacts with the mensuration of SOD, the height indirect reaction of SOD vigor body remove the ability of oxygen-derived free radicals, and the height indirect reaction of MDA the body cell order of severity that attacked by free radical.
The MDA test philosophy: the malonaldehyde in the lipid peroxide catabolite (MDA) can with thiobarbituricacid (TBA) condensation, form red product, at the 532nm place maximum absorption band is arranged.
Test kit is formed and preparation: the by specification preparation, operation sees Table 6.
Table 6MDA measurement operation table
Whirlpool vortex mixer mixing, the test tube mouth is tightened with antistaling film, stings an aperture with syringe needle, and back flowing water cooling is taken out in 95 ℃ of water-baths 40 minutes, and 3500～4000 rev/mins then, centrifugal 10 minutes, get supernatant, absorbance, result such as table 7 are respectively managed in the survey of 532nm place.
Table 7.532nm surveys at the place and respectively manages absorbance
Calculate MDA content according to following formula, result such as table 8:
Table 8 is respectively organized MDA content MDA content according to what formula calculated
Statistical result such as table 9:
Table 9 is respectively organized the comparison (X ± s) of rat intestine mucosa MDA content
Experimental result shows, calcium pectinate coated granule group, enteric coated particles group and not three check groups of coating group and other each statistical significance (p＜0.05) is relatively arranged between organizing, wherein model group MDA content is the highest, two positive drug group sasp groups are higher than three check groups, Dexamethasone group is higher than calcium pectinate coated granule group, and blank group content is minimum.
4.4SOD mensuration
Superoxide dismutase plays crucial effects to the oxidation and the antioxidation balance of body, and this enzyme can be removed ultra-oxygen anion free radical protection cell and avoid damage.
Measuring principle: produce ultra-oxygen anion free radical by xanthine and xanthine oxidase response system, latter's oxidation azanol forms nitrite, presents aubergine under the effect of developer, surveys its absorbance with visible spectrophotometer.When containing SOD in the sample, then ultra-oxygen anion free radical there is narrow spectrum inhibitory action, the nitrite of formation is reduced, and the absorbance of measuring pipe during colorimetric is lower than the absorbance of control tube, calculates the SOD vigor that can obtain in the sample by formula.
Having only two kinds of SOD in the higher mammal cell is copper zinc-SOD and manganese-SOD, and the two addition equals total SOD.Manganese in the sample of sample pre-treatment-SOD vigor forfeiture, but copper zinc-SOD vigor is constant.
The mensuration of the total SOD vigor of table 10
Press table 10 preparation, behind the adding reagent 4,, put 37 ℃ of waters bath with thermostatic control 40 minutes, add developer then with the abundant mixing of whirlpool vortex mixer, mixing, room temperature was placed 10 minutes, measured trap in wavelength 550nm place, saw Table 11.
Table 11.SOD absorbance
Draw SOD vigor (U/mgprot) according to following formula, as table 12:
Table 12SOD vigor (U/mgprot)
Statistical result such as table 13:
Table 13 is respectively organized the active comparison of rat intestine mucosa SOD (X ± s)
Experimental result shows, calcium pectinate coated granule group, enteric coated particles group and not three check groups of coating group and other each statistical significance (p＜0.05) is relatively arranged between organizing, its empty group SOD content is the highest, two positive drug group Dexamethasone group only are higher than not coating group, the sasp group is lower than three check groups, and model group content is minimum.
4.5IL-4 measure
IL-4Elisa test kit test preparation: the 0.2ml suction pipe, high temperature sterilize, oven dry is preserved, and is stand-by, reads over description, checks reagent.-80 ℃ of refrigerator serum specimens are taken out, and it is stand-by to be placed on 4 ℃ of refrigerators.By specification takes out ELISA Plate, and the standard substance that add 100ul respectively are in blank micropore; The labelling sample number into spectrum adds the 100ul sample successively in each corresponding blank micropore respectively; The enzyme labelling solution that in standard substance and sample well, adds 50ul; 36 ± 2 ℃ of incubation reaction 60 minutes; Wash the plate machine and clean 5 times, left standstill 10～20 seconds at every turn; Every hole adds substrate A, each 50ul of B liquid; 36 ± 2 ℃ of following lucifuge incubation reaction 15 minutes; Every hole adds 50ul stop buffer, cessation reaction.On the microplate reader of wavelength 450nm, read the OD value in each hole, the results are shown in Table 14.(annotate: B=standard substance OD value B
00 OD value of=standard substance is with B/B
0The % value is a vertical coordinate, and the concentration of standard substance is abscissa, drawing standard curve on logarithmic paper.Standard substance absorbance: 1000=0.274,500=0.452,250=0.714,100=0.944,50=1.055,0=1.540)
Table 14 determinand absorbance
Trying to achieve computing formula according to description by excel software is: Y=-0.001X+1.1586
Calculating is respectively organized data and is tried to achieve IL-4 concentration such as table 15
Table 15IL-4 concentration
Statistical result such as table 16:
Table 16 is respectively organized rat blood serum IL-4 content (X ± s)
Experimental result shows, calcium pectinate coated granule group, enteric coated particles group and not three check groups of coating group and other each statistical significance (p＜0.05) is relatively arranged between organizing, its empty group IL-4 content is the highest, two positive drug group Dexamethasone group are a little more than coating group not, the sasp group is lower than three check groups, and model group content is minimum.
4.6COX-2 mensuration
Determine the polyclonal antibody experimental concentration.With the Mus cox-2 of rabbit Chinese People's Anti-Japanese Military and Political College polyclonal antibody (Beijing Bo Aosen) packing, each dactylethrae 20ul is distributed into one of them dactylethrae more in addition and carries out following operation after 1: 200,1: 300,1: 400:
Select 8 sections, put into 60 ℃ of baking boxs 30 minutes; After the conventional dewaxing, distillation washing 2 times; High pressure was repaired 2 minutes.(air relief valve gas leakage the time picks up counting, in 2 minutes then immediately pressure cooker is put into the cold water natural cooling); Drip PBS and handled twice in 3 minutes, drip 3% hydrogen peroxide and handle 30 minutes (room temperature); Distillation washing 2 times is dripped 5% serum confining liquid and was got rid of (can not wash off) in 30 minutes after (room temperature).Drip an anti-cox-2 (1: 200,1: 300,1: 400), select one else and drip PBS, put into 4 ℃ of refrigerator overnight as negative control.
3 minutes * of next day: PBS drip reagent B 15 minutes (37 ℃) 3 times;
3 minutes * of PBS drip reagent C 15 minutes (37 ℃) 3 times
4 PAB colour developings of 5 minutes * of PBS 3 minutes
Haematoxylin was redyed 2 minutes, and transparent mounting dewaters.
Read sheet: find that 1: 300 effective after cox-2 one anti-variable concentrations experiment, positive expression is obvious, and negative control does not have positive the expression, therefore, tests an anti-cox-2 desired concn to 1: 300 as this, determines concentration as experiment.
The positive optical density of table 17Cox-2
Statistical result such as table 18:
Table 18 is respectively organized rat blood serum cox-2 content (x ± s)
Experimental result shows, calcium pectinate coated granule group, enteric coated particles group and not three check groups of coating group and other each statistical significance (p＜0.05) is relatively arranged between organizing, its empty group cox-2 optical density content is minimum, two positive control medicine groups, Dexamethasone group is starkly lower than calcium pectinate coated granule group, the SASP group is higher than three check groups, and model group content is the highest.
Claims (4)
1. a medicine for the treatment of ulcerative colitis is characterized in that it is to be made by following bulk drugs: 5～30 parts of the Radix Pulsatillaes, 4～24 parts of Cortex Phellodendris, 2～12 parts of Rhizoma Coptidis, 4～24 parts of Cortex Fraxinis, 3～18 parts of Radix Astragali, 3～18 parts of Rhizoma Atractylodis Macrocephalae preparatas, 3～18 parts of Flos Sophorae Immaturus, 3～18 parts of Radix Sanguisorbaes, 2～10 parts of Radix Aconiti Lateralis Preparatas.
2. the preparation method of the medicine of the described treatment ulcerative colitis of claim 1, it comprises the following steps:
1) take by weighing each crude drug Radix Pulsatillae, Cortex Phellodendri, Rhizoma Coptidis, Cortex Fraxini, Radix Astragali, Rhizoma Atractylodis Macrocephalae preparata, Flos Sophorae Immaturus, Radix Sanguisorbae and Radix Aconiti Lateralis Preparata, standby;
2) the full recipe quantity of the Radix Pulsatillae, the full recipe quantity of Rhizoma Atractylodis Macrocephalae preparata and all the other flavour of a drug half recipe quantity with weight proportion described in the claim 1 mixes, and micronizing to particle diameter reaches and gets the former medicine of nano powder below the 100nm, and is standby;
3) Cortex Phellodendri, Rhizoma Coptidis, Cortex Fraxini, Radix Astragali, Flos Sophorae Immaturus, Radix Sanguisorbae and Radix Aconiti Lateralis Preparata 7 flavour of a drug half recipe quantity with remainder decocts with water twice, merges decocting liquid twice, is concentrated into proportion 1.2～1.8 and must extracts concentrated solution, and be standby;
4) will extract concentrated solution and be sprayed on the former medicine of nano powder and make soft material, sieving makes wet granular, 50 ℃～60 ℃ dryings; Just be prepared into the active component of medicine.
3. preparation method according to claim 2 is characterized in that also having following steps after step 4):
5) will hang down methyl ester pectin adding distil water swelling, heat is to 50 ℃～60 ℃ dissolvings, and concentration reaches 5%～15%; Impouring calcium chloride saturated solution generates to there being white flocculent deposit, and the limit edged stirs, and gets the suspendible glue of calcium content more than 10%;
6) the step 4) active component is pressed into plain sheet, the calcium pectinate suspendible glue coating that the reuse step 5) makes makes the Chinese medicine colon tablet to plain sheet weightening finish 8%～20%.
4. preparation method according to claim 3 is characterized in that low methyl ester pectin is the methyl ester pectin of esterification degree＜50% in the step 5).
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| CN113134065A (en) * | 2021-06-07 | 2021-07-20 | 河南大学淮河医院 | Traditional Chinese medicine decoction for treating Crohn's disease |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1130515A (en) * | 1995-03-03 | 1996-09-11 | 李玉魁 | Chinese drug for treating chronic colitis |
| CN1616011A (en) * | 2004-09-17 | 2005-05-18 | 中国人民解放军总医院 | Medicinal composition for treating ulcerative colitis and its preparing method |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1130515A (en) * | 1995-03-03 | 1996-09-11 | 李玉魁 | Chinese drug for treating chronic colitis |
| CN1616011A (en) * | 2004-09-17 | 2005-05-18 | 中国人民解放军总医院 | Medicinal composition for treating ulcerative colitis and its preparing method |
Non-Patent Citations (2)
| Title |
|---|
| 毛德新等.溃疡性结肠炎的中医药治疗概况.《陕西中医学院学报》.2004,第27卷(第2期),57-60. * |
| 汤瑜等.白头翁汤加减灌肠联合柳氮磺胺吡啶治疗非特异性溃疡性结肠炎.《临床和实验医学杂志》.2007,第6卷(第12期),107. * |
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