CN110433188A - Application of the tuftybell extract in the drug that preparation treats or prevents inflammatory bowel disease - Google Patents
Application of the tuftybell extract in the drug that preparation treats or prevents inflammatory bowel disease Download PDFInfo
- Publication number
- CN110433188A CN110433188A CN201910779431.5A CN201910779431A CN110433188A CN 110433188 A CN110433188 A CN 110433188A CN 201910779431 A CN201910779431 A CN 201910779431A CN 110433188 A CN110433188 A CN 110433188A
- Authority
- CN
- China
- Prior art keywords
- tuftybell
- extract
- bowel disease
- inflammatory bowel
- drug
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/34—Campanulaceae (Bellflower family)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
- A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
Landscapes
- Health & Medical Sciences (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Life Sciences & Earth Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Mycology (AREA)
- Epidemiology (AREA)
- Microbiology (AREA)
- Medical Informatics (AREA)
- Botany (AREA)
- Biotechnology (AREA)
- Alternative & Traditional Medicine (AREA)
- Medicines Containing Plant Substances (AREA)
Abstract
The present invention relates to field of medicaments, and in particular to application of the tuftybell extract in the drug of preparation treatment inflammatory bowel disease.The present invention is raw material with natural plants tuftybell, by soxhlet extraction, extracts effective active composition-tuftybell extract of tuftybell, the control and application for inflammatory bowel disease.Tuftybell plant extracts of the invention can be used as a kind of therapeutic agent of safe and effective inflammatory bowel disease.
Description
Technical field
The present invention relates to field of medicaments, treat or prevent inflammatory bowel disease in preparation more particularly to tuftybell extract
Application in drug.
Background technique
Inflammatory bowel disease (Inflammatory Bowel Disease, IBD) is a kind of to involve ileum, colon, rectum
Chronic, idiopathic bowl inflammatory diseases mainly include Crohn disease (Crohn Disease, CD) and ulcerative colitis
(Ulcerative Colitis,UC).Pathological characters are mainly that continuously or discontinuously property inflammation is anti-for mucous membrane of colon and submucosa
It answers, clinical manifestation is abdominal pain, diarrhea, hematochezia, out of strength, trophic disturbance, weight loss etc..In recent years, the morbidity of inflammatory bowel disease
Rate and illness rate increase year by year, and clinical treatment expense constantly increases, and some patientss may occur in which enterorrhagia and enterobrosis, or even knot
Intestinal cancer is the big disease for seriously threatening human life.It mainly include 5- aminosalicyclic currently used for the drug of inflammatory bowel disease
Acids, glucocorticoid, immunosuppressor such as imuran and biological agent TNF-α antibody macromolecular.Although these drug energy
Alleviate the clinical symptoms and Signs of patients with inflammatory bowel disease, but Most patients are still easy to recur, protracted course of disease, and these drugs
There is also serious adverse reactions, and the risk for causing infection and malignant tumour to occur, and therefore, find novel effectively safety
Drug just seem especially urgent.
All the time, traditional Chinese medicine has its unique curative effect for clinical chronic refractory disease.It is some to be planted from natural
The Chinese medicine of object for example Herba Andrographitis, Michelia floribunda, aloe, wheat straw, acanthaceous indigo extract had been reported for inflammatory bowel disease.Tuftybell
(Wahlenbergia marginata) is a kind of Campanulaceae tuftybell of growth distribution on the south Southwestern China and the Yangtze river basin
The plant of category.Its root or herb are widely applied to cold in children, cough due to deficiency of the lung, broncho-pulmonary as a kind of ethnic minority traditional medicine
The respiratory diseases such as inflammation, infantile malnutrition, toothache, malaria, the treatment of hypertension etc., but controlling for inflammatory bowel disease is not reported
It treats.
Summary of the invention
In view of the foregoing deficiencies of prior art, the purpose of the present invention is to provide tuftybell extracts treats in preparation
Or the application in the drug of prevention of inflammation enteropathy.
In order to achieve the above objects and other related objects, the present invention provide a kind of tuftybell extract preparation treatment and/
Or the application in the drug of prevention of inflammation enteropathy or its related disease.
Optionally, the tuftybell extract is to extract to obtain by soxhlet extraction.
Optionally, the extracting method of the tuftybell extract includes: that tuftybell is ground into powder, and Soxhlet extraction is added
In the casing of device, solvent is added into cucurbit, is heated to reflux, obtains extracting solution, the extracting solution is dry, obtain solid powder
Last shape tuftybell extract.
Optionally, when extracting the tuftybell extract, the organic solvent of use be selected from solvent described in second mystery be selected from ether,
At least one of methylene chloride, chloroform, ethyl acetate, acetonitrile, ethyl alcohol, methanol, water.
Optionally, the drying mode of the extracting solution is vacuum drying or air drying.
Optionally, the inflammatory bowel disease includes but is not limited to ulcerative colitis, Crohn disease.
Optionally, the clinical manifestation of the inflammatory bowel disease includes weight loss, diarrhea, hematochezia.
Optionally, the tuftybell extract inhibits the generation of inflammatory cytokine in inflammatory bowel disease colon.
Optionally, the inflammatory cytokine includes TNF-α, INF- γ, IL-1 β, IL-6, IL-17A.
Optionally, the tuftybell extract inhibits the infiltration of inflammatory cell in inflammatory bowel disease colon.
Optionally, the inflammatory cell includes leucocyte, macrophage, neutrophil leucocyte, CD4+T lymphocyte, Th1 leaching
Bar cell, Th17 lymphocyte.
Optionally, the tuftybell extract reduces the level of oxidized molecules in inflammatory bowel disease colonic tissue, increases anti-
The level of oxidized molecules.
Optionally, the oxidized molecules include oxygen radical (ROS), malonaldehyde (MDA).
Optionally, the antioxidant molecule includes glutathione (GSH), superoxide dismutase (SOD), catalase
(CAT)。
Optionally, the tuftybell extract mitigates inflammatory bowel disease mucous membrane of colon epithelial barrier dysfunction.
The present invention also provides a kind of preparation methods of tuftybell extract, comprising: using soxhlet extraction to tuftybell into
Row extracts and obtains the tuftybell extract.
Optionally, the tuftybell is made in the casing that the Soxhlet extractor is added after powder, institute is added in solvent
It states in the cucurbit of Soxhlet extractor, flows back repeatedly under heating condition, obtain extracting solution.
Optionally, the solvent is selected from least one of organic solvent, inorganic solvent.
Optionally, the organic solvent is in ether, methylene chloride, chloroform, ethyl acetate, acetonitrile, ethyl alcohol, methanol
It is at least one.
Optionally, at least one of the inorganic solvent in distilled water, deionized water, ultrapure water.
Optionally, the mass ratio of the tuftybell powder and the solvent is 1:(4-20).
Optionally, the time of reflux is 4-8h.
Optionally, gained extracting solution is dry, the tuftybell extract is obtained, which is powdered.
Optionally, the drying mode is vacuum drying.
Optionally, the drying temperature of the extracting solution is 10-30 DEG C.
The present invention also provides tuftybell extracts made from above-mentioned preparation method.
The present invention also provides above-mentioned tuftybell extract containing effective dose for prevent and treat inflammatory bowel disease drug or
Health care product.
The present invention also provides the above-mentioned tuftybell extracts containing effective dose for alleviating the medicine of diarrhea or hematochezia symptom
Product or health care product.
As described above, the beneficial effects of the present invention are: the present invention provides a kind of preparation method of tuftybell extract,
It is extracted from tuftybell by soxhlet extraction and obtains effective active composition-tuftybell extract, and by largely testing
Tuftybell extract is applied in inflammatory bowel disease and its related disease, can improve and mitigate inflammatory by card research, discovery
The problems such as weight loss caused by enteritis, diarrhea, intestinal bleeding, colon shorten, colonic tissue pathologic damage inhibits inflammatory
The exudation of inflammatory cell (macrophage, neutrophil leucocyte, T lymphocyte and bone-marrow-derived lymphocyte) in enteritis colonic tissue, enhancing are scorching
The generation of cytokine TNF-α, INF- γ, IL-1 β, IL-6, IL-17A in disease property enteritis colonic tissue enhance inflammatory enteritis
Polyphenoils glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT) are horizontal and living in colonic tissue
Property, reducing oxygen radical ROS and malonaldehyde (MDA) in colonic tissue generates, improve inflammatory enteritis gut epithelium barrier function,
And safely and effectively, adverse reaction is few.The present invention develops a kind of new application of tuftybell extract, can be made into drug
Or health care product is the preventing/treating inflammatory bowel disease of clinical inflammatory enteropathy for preventing and treating inflammatory bowel disease and its related disease
Provide a new selection.
Detailed description of the invention
Fig. 1 is shown as in the embodiment of the present invention 4, and tuftybell extract (EWM) induces inflammatory bowel disease mouse weight to DSS
Variation diagram.
Fig. 2 is shown as in the embodiment of the present invention 4, and tuftybell extract (EWM) induces inflammatory bowel disease mouse disease to DSS
The influence result figure of activity index (DAI).
Fig. 3 is shown as in the embodiment of the present invention 5, and tuftybell extract (EWM) is to DSS inducing mouse inflammatory bowel disease colon
Substantial length result of variations figure.
Fig. 4 is shown as in the embodiment of the present invention 5, and tuftybell extract (EWM) is to DSS inducing mouse inflammatory bowel disease colon
The statistical results chart of length variation.
Fig. 5 is shown as in the embodiment of the present invention 5, and tuftybell extract (EWM) is to DSS inducing mouse inflammatory bowel disease colon
Organize HE coloration result figure.
Fig. 6 is shown as in the embodiment of the present invention 6, and tuftybell extract (EWM) is to DSS inducing mouse inflammatory bowel disease colon
Organize the experimental result picture of leukocyte infiltration (red-label is CD45 positive leukocytes in figure).
Fig. 7 is shown as in the embodiment of the present invention 6, and tuftybell extract (EWM) is to DSS inducing mouse inflammatory bowel disease colon
Organize the experimental result picture of different type leukocyte infiltration.
Fig. 8 is shown as in the embodiment of the present invention 6, and tuftybell extract (EWM) is to DSS inducing mouse inflammatory bowel disease colon
The experimental result picture of inflammatory cytokine in tissue (wherein ND is to be not detected).
Fig. 9 is shown as in the embodiment of the present invention 7, and tuftybell extract (EWM) is to DSS inducing mouse inflammatory bowel disease colon
The experimental result picture of tissue oxygen and antioxidant molecule.
Figure 10 is shown as in experimental example 9 of the present invention, and tuftybell extract (EWM) is to DSS inducing mouse weight loss, disease
The experimental result picture of activity index and colon lengths (wherein ND is to be not detected).
Figure 11 is shown as in experimental example 10 of the present invention, and tuftybell extract (EWM) is to DSS inducing mouse weight loss, disease
The experimental result picture of sick activity index and colon lengths (wherein ND is to be not detected).
Specific embodiment
Illustrate embodiments of the present invention below by way of specific specific example, those skilled in the art can be by this specification
Other advantages and efficacy of the present invention can be easily understood for disclosed content.The present invention can also pass through in addition different specific realities
The mode of applying is embodied or practiced, the various details in this specification can also based on different viewpoints and application, without departing from
Various modifications or alterations are carried out under spirit of the invention.
The present invention intends with natural plants tuftybell being raw material, by the method for chemical extraction, extracts effective work of tuftybell
Property ingredient-tuftybell extract, the control and application for inflammatory bowel disease.
The present invention chooses tuftybell plant extracts, the therapeutic agent as a kind of safe and effective inflammatory bowel disease.
One of technical solution of the present invention is to provide the preparation method of the tuftybell extract for preventing and treating inflammatory bowel disease, tool
Body comprising the following three steps: be mechanically pulverized dry without mouldy tuftybell herb into powder, obtain tuftybell powder；With matter
It measures the ratio than 1:4-20 and is added to Soxhlet extractor, In respectively by the organic solvent of tuftybell powder and water or opposed polarity
It is impregnated under reflux temperature 4-8 hours, extracts 2-5 times repeatedly, obtain tuftybell extract liquor；It is then that tuftybell extract liquor room temperature is true
Sky is dry, obtains solid powdery tuftybell extract.Required organic solvent include but is not limited to ether, methylene chloride, chloroform,
At least one of ethyl acetate, acetonitrile, ethyl alcohol, methanol.
Technical solution of the present invention second is that above-mentioned tuftybell extract or above-mentioned tuftybell extract containing effective dose can
For preventing and treating the drug or health care product of inflammatory bowel disease.
Technical solution of the present invention third is that above-mentioned tuftybell extract or above-mentioned tuftybell extract containing effective dose can
For alleviating the drug or health care product of diarrhea or hematochezia symptom.
Technical solution of the present invention fourth is that above-mentioned tuftybell extract can in dosage (100-500mg/kg) rely on ground improve
The inflammatory bowel disease of dextran sulfate sodium (DSS) induction, including alleviate mouse weight and mitigate, Disease Activity Index is reduced, is reversed
Colon lengths shorten, and mitigate colonic tissue inflammatory reaction and pathologic damage.
Technical solution of the present invention fifth is that above-mentioned tuftybell extract can in dosage (100-500mg/kg) rely on ground mitigate
The inflammatory bowel disease colon local inflammation reaction of dextran sulfate sodium (DSS) induction, including reduce cell factor in colonic tissue
The generation of TNF-α, INF- γ, IL-1 β, IL-6, IL-17A inhibits macrophage, neutrophil leucocyte, T lymph in colonic tissue
The infiltration of the inflammatory cells such as cell, bone-marrow-derived lymphocyte.
Technical solution of the present invention fifth is that above-mentioned tuftybell extract can in dosage (100-500mg/kg) rely on ground mitigate
The inflammatory bowel disease colon part Oxygen stress of dextran sulfate sodium (DSS) induction, including reduce oxygen freedom in colonic tissue
Base ROS and malonaldehyde (MDA) generate, and increase glutathione (GSH), superoxide dismutase (SOD) and catalase
(CAT) horizontal and activity.
Technical solution of the present invention sixth is that above-mentioned tuftybell extract can in dosage (100-500mg/kg) rely on ground mitigate
The inflammatory bowel disease gut epithelium barrier dysfunction of dextran sulfate sodium (DSS) induction, including reduce DSS and induce FITC-
Dextran leaks into the concentration in blood from intestinal mucosal epithelial.
Embodiment 1
The preparation method of tuftybell extract:
Dry tuftybell is mechanically pulverized into powder, sieve looks over so as to check to obtain average diameter about 0.8mm powder, weighs 10 grams
Tuftybell powder is put into Soxhlet extractor, and round-bottomed flask is added in 100 ml methanols, then heats round-bottomed flask, solvent evaporation,
Condensation is leached, reflux, is extracted 6 hours in reflux temperature, is collected tuftybell extract liquor in round-bottomed flask, is dried in vacuo under room temperature,
2.18 grams of methanolic extract of celadon solid sheet tuftybell are obtained, yield 21.8%.
Embodiment 2
Dry tuftybell is mechanically pulverized into powder, sieve looks over so as to check to obtain average diameter about 0.8mm powder, weighs 10 grams
Tuftybell powder is put into Soxhlet extractor, and 100 milliliters of methylene chloride are added round-bottomed flask, then heat round-bottomed flask, solvent
Evaporation condensation, is leached, reflux, is extracted 5 hours in reflux temperature, and tuftybell extract liquor in round-bottomed flask, vacuum under room temperature are collected
It is dry, 2.24 grams of dichloromethane extract of celadon solid sheet tuftybell are obtained, yield 22.4%.
Embodiment 3
The preparation method of tuftybell extract:
Dry tuftybell is mechanically pulverized into powder, sieve looks over so as to check to obtain average diameter about 0.8mm powder, weighs 10 grams
Tuftybell powder is put into Soxhlet extractor, and 100 milliliters of methylene chloride are added round-bottomed flask, then heat round-bottomed flask, solvent
Evaporation condensation, is leached, reflux, is extracted 5 hours in reflux temperature, and tuftybell extract liquor in round-bottomed flask, vacuum under room temperature are collected
It is dry, obtain tuftybell dichloromethane extract.By tuftybell remaining in above-mentioned Soxhlet extractor, add 100 by rechanging
Ml methanol round-bottomed flask is again heated to solvent evaporation, condensation, leaches, reflux 5 hours, it is blue to collect methanol in round-bottomed flask
Flower ginseng extract liquor, normal-temperature vacuum is dry, obtains methanolic extract after tuftybell methylene chloride extracts.
Tuftybell extract used by embodiment 4-8 (Extract of Wahlenbergia marginata, EWM)
It is all from embodiment 1.
Embodiment 4
Tuftybell extract (Extract of Wahlenbergia marginata, EWM) improves dextran sulfate sodium
(DSS) the mouse inflammatory bowel disease weight loss and Disease Activity Index induced:
Experimental material: dextran sulfate sodium is purchased from MP Biomedicals company, and experimental animal is 4-8 weeks, and weight 18~
22 grams of SPF grades of male C57/BL6J mouse, derive from Medical University Of Chongqing's Experimental Animal Center, and animal is using credit number
SYXK (Chongqing) 2015-0001 is raised in temperature (24 ± 2) DEG C, 12/12 hour illumination/dark (i.e. 12h illumination, 12h dark)
Interior, conventional feed are fed, free water feed, and mouse adapts to begin setting up model and pharmaceutical intervention experiment after a week.
Experimental group and intervention: mouse is randomly divided into 6 groups, respectively normal group of blank, drug control group (500mg/kg),
DSS inflammatory enteritis model group, low dose of blue peanut extract intervention group (100mg/kg), middle dosage indigo plant peanut extract intervention
The blue peanut extract intervention group (500mg/kg) of group (300mg/kg), large dosage, totally 6 groups, every group 7.DSS inflammatory enteritis mould
Type group and various dose tuftybell extract intervention group, mouse are freely drunk containing the water-soluble of 2.5% (quality percent by volume) DSS
It liquid 7 days, then changes no DSS water into and drinks 3 days；Normal group of blank and drug control group only drink no DSS water, drug control group
With various dose tuftybell intervention group, corresponding dosage tuftybell extract is given in daily stomach-filling, wherein drug control group and big agent
Tuftybell extract intervention group is measured, stomach-filling drug dose is 500mg/kg, low dose of, middle dosage tuftybell extract intervention group
Stomach-filling drug dose is respectively 100mg/kg or 300mg/kg.
Animal observation evaluation: since drinking DSS, mouse active state, mental condition are observed daily, stool has
No diarrhea and hematochezia occur, and weighing record mouse weight daily.
Animal observes result: blank is normally organized with the activity of drug control group mouse freely, and the state of mind is good, DSS enteritis mould
Type group mouse, since third day, dysphoria, the state of mind is deteriorated, until slow in reacting, the state of mind is very poor at the tenth day,
Various dose tuftybell extract intervention group, animal activity and the state of mind have and improve to a certain degree；Different experiments group mouse
As shown in Figure 1, from figure 1 it appears that since third day, DSS colitis model group mouse weight is gradually reduced changes of weight
The mouse weight that tuftybell extract can reverse DSS to induce in dose-dependant is lost；Further referred to according to weight loss, diarrhea
Several and hematochezia degree calculates mouse disease activity index (Disease activity index, DAI) scoring and (refers to Wirtz, S
Et al.Nature protocols 2017,12,1295), as a result as shown in Fig. 2, from figure 2 it can be seen that containing drinking
The water of 2.5%DSS the tenth day, relative to the mouse for drinking no DSS water, DAI scoring is significantly increased, and in tuftybell extract
(EWM) intervention group, DAI scoring are reduced in dose-dependant.
Embodiment 5
Tuftybell extract mitigates the mouse inflammatory bowel disease colonic pathological change of dextran sulfate sodium (DSS) induction:
Experimental material: hematoxylin, Yihong, paraformaldehyde
Experimental group and intervention: animal packet and intervention are with embodiment 4, and after mouse drinks DSS the 10th, anesthesia is put to death,
Collect mouse Colon tissue.
Analysis and detection method: observation colon general structure variation measures colon lengths, then takes lower distal colon tissue more
Polyformaldehyde is fixed, and is sliced after dehydration, paraffin embedding, row hematoxylin eosin staining (HE), its pathological change of microscopically observation,
And it scores.
Colonic pathological change result: Fig. 3 and Fig. 4 is respectively that each group DSS inducing mouse inflammatory bowel disease colon is substantially in this implementation
Length result of variations figure and statistical results chart, from Fig. 3 and Fig. 4 as can be seen that relative to normal group of blank and drug control group
Mouse Colon length, DSS colitis model group mouse Colon obviously shorten, various dose tuftybell extract (EWM) intervention group, then
Increase to colon lengths dose-dependant；Fig. 5 is each group DSS inducing mouse inflammatory bowel disease colonic tissue leucocyte in the present embodiment
The experimental result picture (red-label is CD45 positive leukocytes in figure) of infiltration, from figure 5 it can be seen that further colonic tissue
HE dyes pathological analysis discovery, and DSS inflammatory bowel disease model group, mucous membrane and the exudation of submucosa inflammatory cell are obvious, on mucous membrane
Chrotoplast is lost, and mucous membrane structure is destroyed, and various dose tuftybell extract intervention group, has and different degrees of alleviation is presented.It is real
Apply example 6
Tuftybell extract mitigates the mouse inflammatory bowel disease colitis cellular infiltration of dextran sulfate sodium (DSS) induction
With the generation of inflammatory cytokine:
Experimental material: antibody CD45-PE (article No. 103106), CD45-FITC (article No. 103108), CD11b-APC (article No.
101212) and F4/80-FITC (article No. 123108) is purchased from Biolegend company, antibody Ly6G-PE (article No. 551461), CD3-
FITC (article No. 553061), CD3-PE (article No. 553063), CD4-PE-Cy5 (article No. 553654), CD4-APC-Cy7 (article No.
552051), CD8-PE (article No. 555635), NK1.1-PE (article No. 557391), Foxp3-PE (article No. 560408), TNF-α (goods
Number 561063), INF- γ-PE (article No. 554412), IL-17A-PE (article No. 559502), IL-4-PE (article No. 554435), thin
Intracellular cytokine TNF-α (article No., IL-1 β (article No., INF- γ (article No., IL-6 (article No. ELISA kit is purchased from U.S. Becton,
Dickinson and Company company, IL-17A ELISA kit (article No. M1700) are purchased from R&D Systems company.
Experimental group and intervention: animal packet and interference method are with embodiment 4, after mouse drinks DSS the 10th, at anesthesia
Extremely, mouse Colon is collected, is divided into three parts, wherein first part's paraformaldehyde fixes 6 hours, and OCT is embedded after sucrose dehydration, row ice
Freeze slice, is used subsequently to histogenic immunity fluorescence analysis；Second part takes 200mg colonic tissue to shred, and collagenase digesting is added afterwards
15 minutes, inflammatory cell in colonic tissue, flow cytometry analysis were separated and collected after centrifugation；100mg colonic tissue addition group
After knitting lysate homogenate, 12000 turns of centrifugations take supernatant, are ELISA.
Detection and analysis method: the colon cryopreserved tissue for taking OCT to embed is cut at 10 μm of uplink of Lycra CM1950 slicer
After the closing of 5% fetal calf serum, the CD45 antibody of PE label is added in piece, and after TBST rinsing, DAPI dyeing is added in 4 DEG C of overnight incubations
5 minutes, with rear defence fluorescent quenching mountant mounting, fluorescence microscopy microscopic observation was simultaneously taken a picture, as a result as shown in Figure 6；Different experiments
The nonparenchymal cell that colonic tissue is extracted is organized, total number of cells in the mentioned tissue of cytometric analysis are then separately added into difference
The cell marker molecules streaming antibody of fluorescent marker is protected from light dyeing 30 minutes, and after PBS washing, flow cytometer detection is different
Leucocyte and its hypotype；Coating different cytokines are added in 50 μ l of supernatant after taking different experiments group colonic tissue Tissue Lysis
96 orifice plates of antibody, 4 DEG C of overnight incubations, wash away unbonded tissue supernatant, and the secondary antibody incubation at room temperature 1 that biotin labeling is added is small
When, after washing away unbonded secondary antibody, the Avidin that HRP crosslinking is added is combined 30 minutes, and color developing agent TMB, microplate reader is then added
450nm detects absorbance, and the content of cell factor is calculated according to standard curve.
Colitis reaction result: Fig. 6 is that the inflammatory bowel disease colonic tissue of each group DSS inducing mouse in the present embodiment is white
The experimental result picture (red-label is CD45 positive leukocytes in figure) of cellular infiltration, can from the immunofluorescence analysis result of Fig. 6
See, relative to blank and tuftybell extract control group, leucocyte (CD45 in DSS model group colonic tissue+) dramatically increase,
Various dose tuftybell extract then dose-dependently reduces the exudation of leucocyte in DSS induction colonic tissue；Further stream
The result of formula analysis exudation leukocyte cell types is as shown in fig. 7, macrophage (CD45 in DSS processing group colonic tissue+CD11b+)、
Neutrophil leucocyte (CD45+Ly6G+)、CD4+T lymphocyte (CD3+CD4+), Th1 lymphocyte (CD3+CD4+INFγ+)、Th17
Lymphocyte (CD3+CD4+IL17+) dramatically increase, Treg lymphocyte slightly increases (CD3+CD4+Foxp3+), and tuftybell
Intervention group then mitigates to dose-dependant the exudation of these types of leukocytic cells；(wherein ND is not detect to ELISA result as shown in Figure 8
To), relative to blank and tuftybell extract control group, cytokine TNF-α in DSS model group colonic tissue, INF- γ,
IL-1 β, IL-6 and IL-17A significantly increase, and the colonic tissue that tuftybell intervention group then inhibits to dose-dependant DSS to induce
In above-mentioned cell factor increase.
Embodiment 7
Tuftybell extract influences the mouse inflammatory bowel disease colon oxidative and anti-oxidative of dextran sulfate sodium (DSS) induction
Molecule generates:
Experimental material: oxygen radical (ROS), malonaldehyde (MDA), glutathione (GSH), superoxide dismutase (SOD),
The green skies Bioisystech Co., Ltd in Nanjing is purchased from catalase (CAT) detection kit.
Experimental group and intervention: animal packet and intervention are with embodiment 4, and after mouse drinks DSS the 10th, anesthesia is put to death,
Collect mouse Colon tissue.
Detection and analysis method: 100mg colonic tissue is taken, after Tissue lysates cracking, 4 degree of centrifuging and taking supernatants are pressed respectively
Corresponding reagent box explanation, is analyzed with microplate reader.
Colon oxidative stress result: Fig. 9 is oxygen in the inflammatory bowel disease colonic tissue of each group DSS inducing mouse in the present embodiment
Change the experimental result with antioxidant molecule, it can be seen in figure 9 that, in blank group and tuftybell control group colonic tissue, ROS
It is lower with MDA content, and the content of ROS and MDA significantly increases in DSS model group, colonic tissue, further tuftybell is dry
The ROS and MDA enhancing that pre- group then inhibits DSS to induce in dose-dependant；It is worth noting that, antioxidant molecule result then shows
For relative to blank group, Lan Huasheng control group glutathione (GSH) content, superoxide dismutase (SOD) and catalase
(CAT) activity is slight increases, and DSS model group colonic tissue GSH-PX activity (GSH) content, superoxide dismutase (SOD)
It is then remarkably decreased with catalase (CAT) activity, various dose tuftybell intervention group then increases gluathione in dose-dependant
Peptide (GSH) content, superoxide dismutase (SOD) and catalase (CAT) activity.
Embodiment 8
Tuftybell extract mitigates the damage of the mouse inflammatory bowel disease gut epithelium barrier of dextran sulfate sodium (DSS) induction
Wound:
Experimental material: FITC-Dextran (4KD) is purchased from Sigma-aldrich company.
Experimental group and intervention: animal packet and interference method are with embodiment 4, after mouse drinks DSS the 10th, stomach-filling
400mg/kg FITC-Dextran (0.5ml), mouse is put to death in anesthesia after 4 hours, and serum is collected, is protected from light.
Detection and analysis method: FITC-Dextran detects (excitation with sepectrophotofluorometer in different experiments group serum
Wavelength 488nm, launch wavelength 520nm).
Gut epithelium barrier result: it can be evaluated on intestinal mucosa by the FITC-Dextran concentration leak into detection serum
The degree of injury of skin barrier, the results show that FITC-Dextran in normal and tuftybell control group (500mg/kg EWM) serum
Concentration is respectively 0.91 ± 0.05 μ g/ml and 0.87 ± 0.09 μ g/ml, and DSS model group is 10.47 ± 1.32 μ g/ml, low,
Middle and high dosage tuftybell intervention group, FITC-Dextran concentration is respectively 9.12 ± 1.17 μ g/ml, 7.02 ± 0.84 μ in serum
g/ml,4.39±0.53μg/ml.The prompt of this result, blue peanut improve gut epithelium barrier function in dose-dependant.
Embodiment 9
It using tuftybell extract made from embodiment 2, is tested referring to the method for embodiment 4 and 5, evaluates tuftybell
Influence of the extract to mouse weight, disease activity index (DAI) and colon lengths, experimental result (wherein ND as shown in Figure 10
To be not detected), it can be seen from fig. 10 that indigo plant made from the effect of tuftybell extract made from embodiment 2 and embodiment 1
The effect of flower conopsea extraction is almost the same.
Embodiment 10
It using tuftybell extract made from embodiment 3, is tested referring to the method for embodiment 4 and 5, evaluates tuftybell
Influence of the extract to mouse weight, disease activity index (DAI) and colon lengths, experimental result (wherein ND as shown in figure 11
To be not detected), it can be seen from fig. 11 that indigo plant made from the effect of tuftybell extract made from embodiment 2 and embodiment 1
The effect of flower conopsea extraction is almost the same.
In conclusion it is a large amount of by applicant experimental studies have found that, the effective component-indigo plant extracted from tuftybell
Flower conopsea extraction, can be used for preventing and treating inflammatory bowel disease and its related disease, improves and mitigate weight caused by inflammatory enteritis
The problems such as mitigation, diarrhea, intestinal bleeding, colon shorten, colonic tissue pathologic damage, inhibits in inflammatory enteritis colonic tissue
The exudation of inflammatory cell (macrophage, neutrophil leucocyte, T lymphocyte and bone-marrow-derived lymphocyte) enhances inflammatory enteritis colon group
The generation of middle cytokine TNF-α, INF- γ, IL-1 β, IL-6, IL-17A are knitted, antioxygen in inflammatory enteritis colonic tissue is enhanced
Compound glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT) are horizontal and active, reduce colonic tissue
Middle oxygen radical ROS and malonaldehyde (MDA) generate, and improve inflammatory enteritis gut epithelium barrier function, and safely and effectively, bad
Reaction is few.The present invention develops a kind of new application of tuftybell extract, drug or health care product can be made into, for preventing
Control inflammatory bowel disease and its related disease, for clinical inflammatory enteropathy preventing/treating inflammatory bowel disease provide one it is new
Selection.
The above-described embodiments merely illustrate the principles and effects of the present invention, and is not intended to limit the present invention.It is any ripe
The personage for knowing this technology all without departing from the spirit and scope of the present invention, carries out modifications and changes to above-described embodiment.Cause
This, institute is complete without departing from the spirit and technical ideas disclosed in the present invention by those of ordinary skill in the art such as
At all equivalent modifications or change, should be covered by the claims of the present invention.
Claims (10)
1. application of the tuftybell extract in the drug of preparation treatment and/or prevention of inflammation enteropathy or its related disease.
2. application according to claim 1, it is characterised in that: the tuftybell extract is extracted by soxhlet extraction
It obtains.
3. application according to claim 2, which is characterized in that the extracting method of the tuftybell extract includes: will be blue
Flower ginseng is ground into powder, is added in the casing of Soxhlet extractor, solvent is added into cucurbit, be heated to reflux, obtain extracting solution,
The extracting solution is dry, obtain solid powdery tuftybell extract.
4. application according to claim 3, it is characterised in that: the solvent is selected from ether, methylene chloride, chloroform, acetic acid
At least one of ethyl ester, acetonitrile, ethyl alcohol, methanol, water；
And/or the drying mode of the extracting solution is vacuum drying or air drying.
5. application according to claim 1, it is characterised in that: the inflammatory bowel disease includes ulcerative colitis, Crow
Grace disease；And/or the clinical manifestation of the inflammatory bowel disease includes weight loss, diarrhea, hematochezia.
6. application according to claim 1, it is characterised in that: the tuftybell extract inhibits in inflammatory bowel disease colon
The generation of inflammatory cytokine, it is preferable that the inflammatory cytokine includes TNF-α, INF- γ, IL-1 β, IL-6, IL-17A；
And/or the tuftybell extract inhibits the infiltration of inflammatory cell in inflammatory bowel disease colon, it is preferable that the inflammation
Cell includes total leucocyte, macrophage, neutrophil leucocyte, CD4+T lymphocyte, Th1 lymphocyte, Th17 lymph are thin
Born of the same parents；
And/or the tuftybell extract reduces the level of oxidized molecules in inflammatory bowel disease colonic tissue, increases anti-oxidant point
The level of son, it is preferable that the oxidized molecules include oxygen radical (ROS), malonaldehyde (MDA), and the antioxidant molecule includes
Glutathione (GSH), superoxide dismutase (SOD), catalase (CAT)；
And/or the tuftybell extract mitigates inflammatory bowel disease mucous membrane of colon epithelial barrier dysfunction.
7. a kind of preparation method of tuftybell extract, comprising: extracted, obtained described to tuftybell using soxhlet extraction
Tuftybell extract.
8. preparation method according to claim 7, it is characterised in that: tuftybell is made after powder and the Soxhlet is added mentions
It takes in the casing of device, solvent is added in the cucurbit of the Soxhlet extractor, is flowed back repeatedly under heating condition, extracted
Liquid；
And/or the solvent is selected from least one of organic solvent, inorganic solvent, it is preferable that the organic solvent is selected from second
At least one of ether, methylene chloride, chloroform, ethyl acetate, acetonitrile, ethyl alcohol, methanol, the inorganic solvent select distilled water, go
At least one of ionized water, ultrapure water；
And/or the mass ratio of the tuftybell powder and the solvent is 1:(4-20)；
And/or the time of reflux is 4-8h；
And/or gained extracting solution is dry, obtain the tuftybell extract；
And/or the drying mode is vacuum drying or air drying；
And/or the drying temperature of the extracting solution is 10-30 DEG C.
9. according to tuftybell extract made from preparation method described in claim 7-8 any one.
10. the drug or health care product of tuftybell extract described in the claim 9 containing effective dose, it is characterised in that: described
Drug or health care product are for treating or preventing inflammatory bowel disease；
And/or the drug or health care product are for alleviating diarrhea or hematochezia symptom.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910779431.5A CN110433188A (en) | 2019-08-22 | 2019-08-22 | Application of the tuftybell extract in the drug that preparation treats or prevents inflammatory bowel disease |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910779431.5A CN110433188A (en) | 2019-08-22 | 2019-08-22 | Application of the tuftybell extract in the drug that preparation treats or prevents inflammatory bowel disease |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110433188A true CN110433188A (en) | 2019-11-12 |
Family
ID=68437180
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910779431.5A Pending CN110433188A (en) | 2019-08-22 | 2019-08-22 | Application of the tuftybell extract in the drug that preparation treats or prevents inflammatory bowel disease |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110433188A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113310963A (en) * | 2021-05-31 | 2021-08-27 | 四川大学华西医院 | Improved immunofluorescence detection method for neutrophil NETs |
| CN114381488A (en) * | 2022-03-02 | 2022-04-22 | 淮安唯尔美化妆品有限公司 | Active peptide, anti-skin aging application and commercialized application in cosmetics |
| CN115137708A (en) * | 2022-06-24 | 2022-10-04 | 重庆医科大学 | Liver fibrosis targeting nanoparticle loaded with YAP1 nucleic acid sequence and preparation method and application thereof |
-
2019
- 2019-08-22 CN CN201910779431.5A patent/CN110433188A/en active Pending
Non-Patent Citations (5)
| Title |
|---|
| 何志成: "《全国高等医药院校药学类规划教材 制剂单元操作与制剂工程设计 （供药学类专业用）》", 31 January 2006 * |
| 张宽等: ""HPLC法同时测定蓝花参中芦丁、橙皮苷及槲皮素的含量"", 《中国民族民间医药》 * |
| 朱坤福: "《中医外治疗法》", 31 May 2019 * |
| 杨彬等: "蓝花参对硫酸钠致小鼠腹泻的作用及机制", 《中药材》 * |
| 药渡: "炎症性肠病-处于临床前的小分子药物研究进展", 《百度》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113310963A (en) * | 2021-05-31 | 2021-08-27 | 四川大学华西医院 | Improved immunofluorescence detection method for neutrophil NETs |
| CN113310963B (en) * | 2021-05-31 | 2023-12-01 | 四川大学华西医院 | Improved neutrophil NETs immunofluorescence detection method |
| CN114381488A (en) * | 2022-03-02 | 2022-04-22 | 淮安唯尔美化妆品有限公司 | Active peptide, anti-skin aging application and commercialized application in cosmetics |
| CN115137708A (en) * | 2022-06-24 | 2022-10-04 | 重庆医科大学 | Liver fibrosis targeting nanoparticle loaded with YAP1 nucleic acid sequence and preparation method and application thereof |
| CN115137708B (en) * | 2022-06-24 | 2023-05-16 | 重庆医科大学 | Liver fibrosis targeting nanoparticle loaded with YAP1 nucleic acid sequence as well as preparation method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110433188A (en) | Application of the tuftybell extract in the drug that preparation treats or prevents inflammatory bowel disease | |
| Aher et al. | Immunomodulatory effect of alcoholic extract of Terminalia chebula ripe fruits | |
| CN103800390B (en) | Health-care product with immunity-reinforcing and liver-protecting functions, preparation method and application thereof | |
| CN105542024B (en) | Red algae asparagus polysaccharide and its preparation, anti-tumor activity detection method and application | |
| CN102242069A (en) | Paecilomycescicadae (Miq.)Samson and application thereof | |
| CN101862351A (en) | Application of active parts of gallnut in preparing anti-ulcerative colitis medicine | |
| CN101724025A (en) | Anti-tumor toad polypeptide and preparation method and application thereof | |
| CN112386665B (en) | Fermentation liquor of natural raw materials and application of fermentation liquor in preparation of composition for inhibiting anaphylactic reaction or improving immunity | |
| CN102212581A (en) | Preparation method and application of cordyceps polysaccharide germanium | |
| CN107669565A (en) | A kind of natural antibacterial bath foam containing radix glycyrrhizae | |
| KR20120059258A (en) | Oriental medicine extract having biological activity and manufacturing method thereof | |
| Gupta et al. | Immunosuppressive activity of crude terpenoids from extracts of Emblica officinalis, Ficus racemosa and Strychnos nux-vomica | |
| CN108685951B (en) | A kind of stingless bee Mel extract and its extracting method and application | |
| CN107213145A (en) | Application of the Rabdocetsin B in the product for suppressing esophageal squamous cell cancer cell multiplication is prepared | |
| Khatkar et al. | Biological and medicinal properties of Azadirachta indica: a review | |
| CN105853406B (en) | Application of procyanidine in preparation of medicine for preventing and treating porcine reproductive and respiratory syndrome | |
| CN103316062A (en) | Novel application of mulgedium tataricum | |
| Nagadesi et al. | Taxonomy and Bioactive chemicals from Ganoderma and Phellinus of India | |
| CN101596268B (en) | Application of garlic total saponin in preparing medicaments and foods for resisting oxidative stress damage | |
| CN106177035B (en) | Preparation method and application of effective rosa chinensis flower extract with blood sugar reducing and anticancer functions | |
| CN109498644A (en) | Application of the Chinese medicine sulphation Codonopsis pilosula polysaccharide as antioxidant | |
| CN106266473A (en) | A kind of green Bulbus Allii extract with antihepatocarcinoma effect and Preparation method and use | |
| Kumar et al. | Phytochemical analysis of Boerhaavia diffusa, Emblica officinalis, Terminilia chebula, Terminilia bellirica, Withania somnifera | |
| Otorkpa | Comparative Analysis of the in-vitro Susceptibility of Salmonella Typhi to Chloramphenicol and Leaf Extracts of Moringa Oleifera | |
| CN106913600A (en) | A kind of maidenhair extract, preparation method and its applied in cancer therapy drug is prepared |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20191112 |