CN107582574A - Land for building dish alcohol extract is preparing the application in treating inflammatory bowel medicine - Google Patents
Land for building dish alcohol extract is preparing the application in treating inflammatory bowel medicine Download PDFInfo
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Abstract
The invention discloses a kind of land for building dish alcohol extract to prepare the application in treating inflammatory bowel medicine, wherein the preparation method of the land for building dish alcohol extract includes：Land for building dish is taken, using low-carbon alcohols as solvent, is extracted under heating condition, extract solution is collected, recycling design, produces.Applicants experimentally found that the UC model mices body weight that land for building dish alcohol extract is obviously improved DSS inductions reduces, suppress DAI scoring rises, mitigate UC induced mices colon lengths and shorten, while reduce inflammatory cell infiltration and tissue damage.In addition, land for building dish alcohol extract substantially reduces the content of colon cytokine TNF α and IL 6, and with it to suppress the key transcription factor NF kB activations of inflammatory signal path closely related for this effect.The experimental result of applicant shows that land for building dish alcohol extract can be used for treating IBD, mitigates the UC modeled inflammations reaction of DSS inductions, improves the UC orders of severity.
Description
Technical field
The present invention relates to the application of plant extracts, and in particular to land for building dish alcohol extract is preparing treatment IBD medicine
Application in thing.
Background technology
IBD (inflammatory bowel disease, IBD) is that one kind mainly involves ileum, rectum, knot
A kind of idiopathic bowl inflammatory diseases of intestines, clinical manifestation is diarrhoea, stomachache, or even can have bloody stool.The disease includes exedens
Colitis (ulcerative colitis, UC) and Crohn disease (crohn ' s disease, CD).Ulcerative colitis is knot
Intestinal mucosa layer and submucosa continuity inflammation, disease generally first involve rectum, are gradually spread to total colectomy；Crohn disease can tire out
And all-digestive tract, it is noncontinuity holostrome inflammation, it is terminal ileum, colon and crissum most often to involve position.UC is as a kind of allusion quotation
The IBD of type, 30-40 year between twenty and fifty, and asexuality difference is apt to occur in, shows as suffering from diarrhoea, suffer from abdominal pain, mucus pus and blood stool etc.
Clinical symptoms, more concurrent enterobrosis, polyp, ulcer etc., have a strong impact on patients ' life quality and existence.Epidemiological study table
Bright, the UC incidences of disease of European Region are up to 505/100,000, and the incidence of disease in Canada area is 248/100,000, the hair in the U.S.
Sick rate is 214/100,000.With the change of life style, dietary, Asia and the Middle East etc. area the UC incidences of disease be in by
The trend that year increases.
At present.Drug therapy and surgery excision are the main methods for controlling UC, and conventional medicine has following 5 class：(1) ammonia
Base salicylic acid, such as salicylazosulfapyridine and Mei Shala piperazines；(2) glucocorticoids, such as dexamethasone and dipropionic acid times chlorine rice
Pine；(3) immunodepressant, such as Ismipur and cyclosporine；(4) biological agent, such as TNF-α inhibitor infliximab；
(5) anti-infectives, such as antibiotic metronidazole and Ciprofloxacin.These medicines are mainly the immune response by suppressing exception,
Mitigate colon local inflammation, prevent disease progression, but exist curative effect is unstable, adverse reaction is more, poor resistance and be not suitable for grow
Phase medication or it is expensive the deficiencies of.Therefore, find that curative for effect, adverse reaction is few from Chinese medicine, and quality controllable anti-UC
Medicine has important value.
Increasing research shows that excessive cell factor, chemotactic factor (CF) and growth factor and active oxygen metabolism produce
Thing (such as ROS and NO) inducible cascade of response of inflammation, causes Traumatic Colon.In these media, IL-1 β, TNF-α and IL-6
It is considered as playing an important role in UC inflammatory reactions.In UC patient's colonic mucosa, blood and excrement, IL-1 β, TNF-α
With IL-6 expression showed increased.Treatment UC important means, Duo Zhongkang are turned into for TNF-α and IL-6 targeted therapy
TNF-α preparation (such as infliximab and adalimumab) has been used for treating UC patient, has for IL-6 monoclonal antibody
It is desirably used for treating UC.But because such drug price is high, route of administration is single, and effect of missing the target occur in some patientss, because
This such medicine is difficult to clinically widely use.
NF- κ B are the important transcription factors for participating in inflammatory reaction gene, it is considered to be the target spot of inflammation disease.It is early
The research of phase shows, in the colon of IBD patient and colitis model animal, NF- κ B activation substantially increases.NF- κ B lead to
The heterodimer being often made up of p50 and p65 Liang Ge subunits, compound is formed with repressible protein I κ B during tranquillization state and is existed
In cytoplasm.A variety of stimulations can activate NF- κ B-I κ B compounds, cause I kB proteins phosphorylation, ubiquitination and degraded.Then NF-
The rapid transposition of κ B heterodimers enters core, is combined with the DNA binding sites of target gene, regulates and controls a variety of target genes such as TNF-α and IL-6
Transcription.Although can turn into treatment UC alternative medicine for single inflammatory factor, therapeutic effect is generally bad.In fact,
Such as the gene of the monofactor such as cell factor, chemotactic factor (CF) or adhesion molecule only represents one of downstream target gene, and
NF- κ B are the final common pathway or rate-limiting step of cascade of response of inflammation.
Land for building dish, scientific name Nostoc Commune (Nostoc commune Vauch.), alias Nostoc commune, land for building dish, it is soft,
Dish etc. is stepped on ground, is the sheet algae of division cyanophyta Nostoc, is liked being grown in wetland surface, or be mixed in weeds base portion
In the base portion set between cauline leaf, greatly and bryophyte group, almost all it is distributed throughout the country.Land for building dish is cool in nature, sweet enter liver
Through；The effect of with heat-clearing improving eyesight, convergence QI invigorating, it is rich in the mineral matter such as protein, multivitamin and phosphorus, zinc, calcium, with color
The scientist for arranging the graceful research of Wei thatch studies discovery, and a kind of composition contained by the dish of land for building can suppress the acetylcholine ester in people's brain
The activity of enzyme, so as to produce curative effect to senile dementia.In addition, Publication No. CN103059112A patent of invention, open
Land for building dish can be extracted under cryogenic with PBS and obtain molecular weight through PAGE gel electrophoresis
For the 35-40KDa albumen with anti-intestinal cancer activity.But have not yet to see and land for building dish alcohol extract is applied to treatment inflammation
Relevant report in disease property enteropathy.
The content of the invention
The technical problem to be solved in the present invention is to provide land for building dish alcohol extract in treatment inflammatory bowel medicine is prepared
Using.
The technical scheme is that：Land for building dish alcohol extract answering in treatment inflammatory bowel medicine or health products is prepared
With.
In technical scheme of the present invention, the preparation method of the land for building dish alcohol extract preferably includes：Land for building dish is taken, with low
Carbon alcohol is solvent, is extracted under heating condition, collects extract solution, recycling design, that is, obtains land for building dish alcohol extract.Thus method obtains
The land for building dish alcohol extract obtained may be directly applied to prepare treatment inflammatory bowel medicine or health products.In order to improve gained land for building dish
The purity of alcohol extract, the preparation method of above-mentioned land for building dish alcohol extract further comprise purification step, specifically by gained land for building dish
Alcohol extract is recrystallized with low-carbon alcohols, that is, obtains land for building dish alcohol extract after purification.
In the preparation method of above-mentioned land for building dish alcohol extract, after the land for building dish can be fresh or dry
, preferably dried land for building dish.
In the preparation method of above-mentioned land for building dish alcohol extract, described low-carbon alcohols can be in the alcohol containing 1-6 carbon atom
One or more kinds of combinations, preferably methanol, ethanol or propyl alcohol, most preferably ethanol.The concentration of the low-carbon alcohols can
To be 10-100v/v%, preferably 50-100v/v%, 70-100v/v%.
In the preparation method of above-mentioned land for building dish alcohol extract, extraction is carried out in a heated condition, is on the one hand made contained therein
Protein ingredient cracking, denaturation, on the other hand with preferably obtain inflammatory bowel medicine can be worked it is various effectively into
Point.The extraction can be carried out in 50 DEG C to solvent of boiling point temperature range, preferably in 60 DEG C to solvent of boiling point temperature
Carry out in the range of degree, more preferably carried out in 70 DEG C to solvent of boiling point temperature range.The mode of extraction is preferably that backflow carries
Take, the number of extraction can be carried out as needed, usually 1-3 times；The amount of solvent for use and existing traditional extraction during extraction every time
Operate identical, can be specifically 3-12 times of raw material weight；The time extracted every time can be 0.5-3h or longer
Time.
Present invention additionally comprises a kind of medicine or health products for treating IBD, the land for building containing the upper effective dose for the treatment of
Dish alcohol extract.Land for building dish alcohol extract described here is prepared by preceding method.The formulation of the medicine or health products can
To be pharmaceutically acceptable formulation, such as capsule, tablet or granule regular dosage form.
Applicant has found that land for building dish alcohol extract can substantially mitigate dextran sulfate sodium (dextran by many experiments
Sulphate sodium, DSS) induction colitis model mouse weight reduce, suppress colitis model mice disease activity index
The rise of (disease activity index, DAI) scoring, improve colitis model mice colon lengths and shorten, mitigate knot
Colitis model colon inflammatory cell infiltration.In addition, land for building dish alcohol extract can substantially reduce model mice colon rush
Scorching factor TNF-α and IL-6 contents, it is close that a kind of this effect with it suppresses the key transcription factor NF- kB activations of inflammatory signal path
Cut is closed.It follows that land for building dish alcohol extract can mitigate the IBD model mice inflammatory reaction of DSS inductions, improve
IBD disease severity, available for the medicine or health products for preparing treatment IBD.
Brief description of the drawings
Fig. 1 is the influence curve that the UC mouse weights that land for building dish alcohol extract is induced DSS reduce, wherein, normal is represented
Normal group, DSS represent model group, and AENC (20mg/kg) represents land for building dish alcohol extract low dosage (20mg/kg) group, AENC
(100mg/kg) represents land for building dish alcohol extract high dose (100mg/kg) group, and Mesaiazine (200mg/kg) represents mesalazine
(200mg/kg) group；
Fig. 2 is the influence curve for the UC mouse DAI scorings that land for building dish alcohol extract is induced DSS, wherein, normal is represented just
Normal group, DSS represents model group, and AENC (20mg/kg) represents land for building dish alcohol extract low dosage (20mg/kg), AENC (100mg/
Kg land for building dish alcohol extract high dose (100mg/kg) group) is represented, Mesaiazine (200mg/kg) represents mesalazine (200mg/
Kg) group；
Fig. 3 is the influence relation for the UC mouse Colon length that land for building dish alcohol extract is induced DSS, wherein, DSS represents model
Group, AENC (mg/kg) represent land for building dish alcohol extract group, and Mesaiazine (mg/kg) represents mesalazine group；
Fig. 4 is the block diagram of the influence for the UC mouse MPO vigor that land for building dish alcohol extract is induced DSS, wherein, DSS is represented
Model group, AENC (mg/kg) represent land for building dish alcohol extract group, and Mesaiazine (mg/kg) represents mesalazine group；
Fig. 5 is the HE Color figures of colon, wherein, (a) is normal group, and (b) is model group, and (c) is land for building dish
Alcohol extract low dosage (20mg/kg) group, (d) are land for building dish alcohol extract high dose (100mg/kg) group, and (e) is mesalazine
(200mg/kg) group；
Fig. 6 is the block diagram of the influence for the UC mouse Pathomorphology that land for building dish alcohol extract is induced DSS, wherein, DSS tables
Representation model group, AENC (mg/kg) represent land for building dish alcohol extract group, and Mesaiazine (mg/kg) represents mesalazine group；
Fig. 7 is the block diagram of the influence for the UC mouse proinflammatory TNF-α contents that land for building dish alcohol extract is induced DSS, its
In, DSS represents model group, and AENC (mg/kg) represents land for building dish alcohol extract group, and Mesaiazine (mg/kg) represents mesalazine
Group；
Fig. 8 is the block diagram of the influence for the UC mouse proinflammatory IL-6 contents that land for building dish alcohol extract is induced DSS, its
In, DSS represents model group, and AENC (mg/kg) represents land for building dish alcohol extract group, and Mesaiazine (mg/kg) represents mesalazine
Group；
Fig. 9 is the block diagram of the influence for the UC mouse proinflammatory IL-1 β contents that land for building dish alcohol extract is induced DSS, its
In, DSS represents model group, and AENC (mg/kg) represents land for building dish alcohol extract group, and Mesaiazine (mg/kg) represents mesalazine
Group；
Figure 10 is the Western blot figures for the UC mouse proinflammatory NF- kB activations that land for building dish alcohol extract is induced DSS,
Wherein, DSS represents model group, and AENC (mg/kg) represents land for building dish alcohol extract group, and Mesaiazine (mg/kg) represents U.S. salad
Piperazine group, p-p65 represent p65 phosphorylation, i.e. NF- kB activations, and GAPDH represents internal reference；
Figure 11 is the block diagram of the influence for the UC mouse proinflammatory NF- kB activations that land for building dish alcohol extract is induced DSS, its
In, DSS represents model group, and AENC (mg/kg) represents land for building dish alcohol extract group, and Mesaiazine (mg/kg) represents mesalazine
Group.
Embodiment
Embodiment 1
Land for building dish is taken, is dried 8 hours for 80 DEG C in electric heating constant-temperature blowing drying box, obtains dried land for building dish；By institute
Obtain dried land for building dish to be placed in extractor, add 80v/v% alcohol refluxs and extract 3 (additions of solvent when extracting every time
Amount respectively is dry after 10 times of land for building dish quality amounts, 8 times of amounts and 5 times of amounts, it is small that extraction time of 3 times respectively is 2
When, 1.5 hours and 1 hour)；Merge extract solution, filtering, concentrate the filtrate to no alcohol, obtain land for building dish alcohol extract.
Embodiment 2
Land for building dish is taken, is dried 5 hours for 60 DEG C in electric heating constant-temperature blowing drying box, obtains dried land for building dish；By institute
Obtain dried land for building dish to be placed in extractor, add 100v/v% alcohol refluxs and extract 1 time (after the addition of solvent is dries
12 times of amounts of land for building dish quality, extraction time are 3 hours)；Filtering, filtrate are concentrated into no alcohol, obtain land for building dish alcohol extract.
Embodiment 3
Land for building dish is taken, is dried 12 hours for 40 DEG C in electric heating constant-temperature blowing drying box, obtains dried land for building dish；By institute
Obtain dried land for building dish to be placed in extractor, add 20v/v% methanol eddies and extract 2 (additions of solvent when extracting every time
Amount respectively is dry after 10 times of land for building dish quality amounts, 10 times of amounts, extraction time of 2 times respectively be 3 hours, it is 2 small
When)；Merge extract solution, filtering, concentrate the filtrate to no alcohol, obtain land for building dish alcohol extract.
Embodiment 4
Land for building dish is taken, is dried 8 hours for 60 DEG C in electric heating constant-temperature blowing drying box, obtains dried land for building dish；By institute
Obtain dried land for building dish to be placed in extractor, the 3 (additions of solvent when extracting every time of addition 100v/v% propyl alcohol refluxing extraction
Amount respectively is dry after 6 times of land for building dish quality amounts, 5 times of amounts and 5 times of amounts, it is small that extraction time of 3 times respectively is 2
When, 1 hour and 1 hour)；Merge extract solution, filtering, concentrate the filtrate to no alcohol, obtain land for building dish alcohol extract.
Embodiment 5
Land for building dish is taken, is dried 8 hours for 80 DEG C in electric heating constant-temperature blowing drying box, obtains dried land for building dish；By institute
Obtain dried land for building dish to be placed in extractor, the 3 (additions of solvent when extracting every time of addition 60v/v% butanol refluxing extraction
Amount respectively is dry after 10 times of land for building dish quality amounts, 8 times of amounts and 5 times of amounts, it is small that extraction time of 3 times respectively is 2
When, 1 hour and 1 hour)；Merge extract solution, filtering, concentrate the filtrate to no alcohol, obtain land for building dish alcohol extract.
With reference to specific experiment with illustrate land for building dish alcohol extract to treat IBD application.
Zoopery is using classical 2.5%DSS solution induction C57/BL6 mouse structure UC models, while gavage is given
Model mice land for building dish alcohol extract low dosage (20mg/kg), high dose (100mg/kg) and positive drug mesalazine (200mg/
Kg), dish alcohol extract in comparative analysis land for building further investigates it to proinflammatory TNF-α, IL-6 and IL- β to UC improvement result
The influence of content and NF- kB activations, inquire into the land for building anti-UC of dish alcohol extract mechanism.
1. material and method
1.1 experimental animal
SPF level C57/BL6 mouse, female, 6-8 week old, 20 ± 2g of body weight, be purchased from Hunan Si Laike scapes has up to experimental animal
Limit company, credit number：SCXK (Hunan) 2016-0002, quality certification number：SYXK (osmanthus) 2013-0001.Raise in temperature 25 ± 2
DEG C, in the environment of humidity 55 ± 10%, freely ingest and drink water.Used after adapting to raising one week.
1.2 experiment reagent
Land for building dish alcohol extract, prepared by the methods described of the embodiment of the present invention 1；Mesalazine, the hair pharmacy of Shanghai love are limited
Company, article No.：151007；Dextran sulfate sodium (dextran sulfate sodium, DSS), U.S. MP Biomedicals
Company, article No.：160110；Myeloperoxidase (myeloperoxidase, MPO) kit, bio-engineering research is built up in Nanjing
Institute, lot number：20170717；NF- κ B p65 polyclonal antibodies, Proteintech companies, article No.：10745-1-AP；P-p65 is more
Clonal antibody, Bioworld companies, product batch number：CA36131；Mouse IL-1 β Enzyme-linked Immunosorbent Assays (ELISA) detection reagent
Box, Elabscience companies, article No.:AK0016MAR09007；Mouse IL-6 Enzyme-linked Immunosorbent Assays (ELISA) detection kit,
CUSABIO companies, article No.:Z01018255；Mouse TNF-α Enzyme-linked Immunosorbent Assay (ELISA) detection kit, Elabscience
Company, article No.：AK0016AUG30005；Polyvinylidene fluoride (PVDF) film, Millipore companies of the U.S., goods
Number：K5NA8022H；Bicinchoninic acid (bicinchoninic acid, BCA) protein quantification kit, green skies biotechnology
Research institute, article No.：P0010S；TRIzol reagents, Invitrogen companies of the U.S., article No.：152104；bovine serum
Albumin (BSA), solarbio companies, lot number：316S052, article No.：A8020；Glycine (glycine), solarbio are public
Department, lot number：1203P0633；Trishydroxymethylaminomethane (Tris-base), solarbio companies, lot number：1217S074；Tell
- 20 (tween-20) of temperature, Chemical Reagent Co., Ltd., Sinopharm Group, lot number：F20100722；Lauryl sodium sulfate (sodium
Dodecyl sulfate, SDS), solarbio companies, lot number：1207G034；Phenylmethylsulfonyl fluoride
(phenylmethanesulfonyl fluoride, PMSF), solarbio companies, article No.：P0100, lot number：20160826；
Tetramethylethylenediamine (tetramethylethylenediamine, TEMED), company：Green skies biotechnology research institute, product
Numbering：ST728；ECL chemical luminescence for liquid, Gu Tai biotechnologys Co., Ltd of Wuhan City, article No.：G2020-2；Other reagents
It is pure, the Chemical Reagent Co., Ltd., Sinopharm Group of commercially available analysis.
1.3 laboratory apparatus
2. experimental method
The foundation of 2.1 mouse colitis models
Female C57BL/6 mouse, 6~8 week old, 20 ± 2g of body weight.In addition to normal group, remaining group freely drinks 2.5%
DSS 7 days, then changes drinking purpose of tap water into 3 days.The beginning modeling same day is designated as d1, and now mouse weight is used as just initial body to record
Weight.
To investigate land for building dish alcohol extract to the inhibitory action of DSS inducing mouse colitis, mouse is randomly divided into following 5
Group：Normal group (normal), model group (DSS), land for building dish alcohol extract low dosage (20mg/kg) group (AENC (20mg/kg)) are high
Dosage (100mg/kg) group (AENC (100mg/kg)) and mesalazine (200mg/kg) group (Mesaiazine (200mg/kg)),
Every group of 8 animals.Start gastric infusion (0.1mL/10g), once a day, continuous 10 days on the day of modeling.Normal group and model group
Gavage gives the mixed solution of respective volume (by ethanol and PBS by 5：95 volume ratio composition).
2.2 disease activity index are evaluated
The observation mouse state of mind, hair color, stool daily, active state and situations such as having blood in stool, record mouse weight,
Scours index and fecal occult blood situation, disease activity index (DAI) is calculated, (body weight reduction+Scours index+excrement is hidden by DAI=
Blood)/3.DAI standards of grading refer to table 1.
Table 1：DAI standards of grading
Occult blood detection：Using ortho-aminotoluene method, with a little excrement of cotton swab picking, ortho-aminotoluene glacial acetic acid solution is first added dropwise
0.3mL, then it is added dropwise rapidly in 3% hydrogenperoxide steam generator 0.3mL, 2min and shows blue brown for the positive.
2.3 collection of specimens
1h after last dose, taken a blood sample from eyeground vein clump, 4 DEG C of standing 2h, (3500rpm, 4 DEG C) 15min is centrifuged, in absorption
Layer serum simultaneously dispense, -80 DEG C freeze it is standby.In taking out colon at anus 1cm, ruler measurement colon lengths are simultaneously taken pictures, PBS
Clean colon 2 times, take colon 0.4cm from close to rectum looking somebody up and down, be placed in 4% paraformaldehyde solution fixed, remaining -80 DEG C of colon
Freeze.
2.4 colon MPO assays
Colon 40mg is weighed, phosphate buffer is added and is prepared into the homogenate of 10% tissue, according to MPO kit explanations
Book, absorbance (OD) is determined at 460nm, MPO contents are calculated according to formula.Calculation formula is as follows：
MPO unit of activity/g tissues=the colon of (measure pipe OD values-control tube OD values) × 2/22.6 × sampling amount (g) 2.5
Histopathological examination
The colon for being fixed on 4% paraformaldehyde solution (being no less than 24h) is taken, with 5% metabisulfite solution and 36h,
Flowing water rinses overnight, the dehydration of gradient concentration absolute ethyl alcohol, specimens paraffin embedding slices, conventional dewaxing, is contaminated through haematoxylin Yihong (H＆E)
Color, resinene mounting are as follows after observation by light microscope intestinal wall Histopathologic changes, specific score by rules：(1) intestinal wall
The order of severity of inflammation, 1,2 and 3 point is designated as, represents slight, moderate and hyperphlogosis respectively；(2) lesion severity, it is designated as
1st, 2 and 3 points, i.e. lesion is located at mucous layer, mucous membrane and submucosa and transmural damage；(3) crypt damage degree, be designated as 1,2,
3 and 4 points.That is 1/3 crypt damage, 2/3 crypt damage, crypts missing but superficial epithelium completely all lack with crypts superficial epithelium.
2.6 cytokines measurement
40mg colons are weighed, physiological saline is added and prepares 10% tissue homogenate, centrifuge (3,000rpm, 4 DEG C)
10min, colon's supernatant is prepared, protein concentration is determined using Coomassie Brilliant Blue.Determine cytokine TNF-α and IL-6
Level, operated according to kit specification.Standard curve of the content of cell factor according to calculated by standard items calculates,
And ensure that the OD values of sample fall in standard curve range, if exceeding standard curve range, detected after diluting again.Knot
The concentration of the enterocyte factor is expressed as：Pg/ml colons albumen.
2.7Western blot are analyzed
2.7.1 the extraction of animal tissue's total protein is with quantifying
Weigh test serum 40mg, with the PBS of precooling twice after, shred.Addition protein lysate (1%RIPA,
50mM Tris-HCl pH 8.0,0.02%NaN3, 150mM NaCl, per 1mL lysates in add the μ L of 100mM PMSF 10)
400 μ L, 10min is ground, in ice bath after cell lysis 20min, 12000rpm centrifugations 10min.Supernatant is transferred to 1.5mL
Eppendorf pipes in, carry out protein quantification with reference to BCA kit specifications methods describeds.
2.7.2 gel electrophoresis and transferring film
According to the difference of molecular weight of albumen to be detected, the separation gel and 4% for preparing 8% or 10% compress glue, record SDS-
PAGE gels.After adding appropriate 1 × electrophoretic buffer, pre-dyed albumen maker (1 μ L) and detected sample are sequentially added.80V is steady
Piezoelectricity swimming about 30min, after sample enters separation gel, adjustment voltage to 120V continues electrophoresis.With reference to pre-dyed albumen maker, treat
When purpose band reaches correct position, terminate electrophoresis.Size according to destination protein cuts the gel of relevant position, is put into transferring film
Balanced in buffer solution.The pvdf membrane sheared is placed in methanol and activates 1min, is subsequently placed into transferring film buffer solution and soaks 30min,
Filter paper is put into transferring film buffer solution simultaneously to be soaked.According to " (+) clamping plate-filter paper-pvdf membrane-gel-filter paper-clamping plate (-) " order
Make " sandwich " transferring film, it is ensured that without carrying out transferring film after bubble.According to testing protein molecular size range set transferring film voltage and
Time.After the completion of transferring film, pvdf membrane is contaminated into 1min or so with Ponceau S dye liquor, observes the albumen on film, subsequent deionized water is clear
Wash 3min.
2.7.3 development
Pvdf membrane is placed in the confining liquid prepared in advance, is incubated at room temperature 2h.PBST is washed 4 times, each 5min.With
Afterwards, put it into hybridization bag, 4 DEG C of primary antibody coatings are overnight.Next day, PBST are washed 4 times, each 5min, are put it into new miscellaneous
Hand in bag, room temperature secondary antibody coating 2h.PBST is washed 4 times, each 5min.Film is carried out using Bio-Rad gel imaging systems
Take pictures, IPP software analysis optical density.
2.8 data analysis
All data represent that significant difference uses one-way in SPSS softwares between group with means ± S.E.M.
ANONA and Dunnett ' s is examined.P value, which is less than 0.05, is considered as having significant difference.
3. experimental result
3.1 land for building dish alcohol extracts induce DSS the influence that UC mouse weights reduce
Mouse drinks DSS and causes colitis symptoms, reduced with body weight, suffer from diarrhoea or loose stools and it is macroscopic have blood in stool based on
Want feature.Fig. 1 is the influence that land for building dish alcohol extract induces DSS UC mouse weights to reduce.#p<0.05,##p<0.01versus is just
Normal group；*p<0.05,**p<0.01versus DSS model groups.This result of study shows, compared with normal group, DSS model groups are small
Mouse body weight substantially reduces (p ＜ 0.01).Compared with DSS model groups, gavage give land for building dish alcohol extract low dosage (20mg/kg),
High dose (100mg/kg) and positive drug (200mg/kg), which weaken model mice body weight, to be reduced.Wherein, the high agent of land for building dish alcohol extract
The improvement result that amount (100mg/kg, p ＜ 0.01) reduces to model mice body weight and positive drug (200mg/kg, p ＜ 0.01) phase
When.
The influence for the UC mouse DAI scorings that 3.2 land for building dish alcohol extracts are induced DSS
The colitis mice Disorders Inflammation activity index DAI of DSS inductions gradually rises, and is mainly shown as body weight reduction, abdomen
Rush down and have blood in stool.Fig. 2 is the influence that land for building dish alcohol extract induces DSS UC mouse weights to reduce.##p<0.01versus normal groups；*
p<0.05,**p<0.01versus DSS model groups.This result of study shows, compared with normal group, DSS model group mouse DAI
Score significantly raised (p ＜ 0.01).Gavage give land for building dish alcohol extract low dosage (20mg/kg), high dose (100mg/kg) and
Positive drug (200mg/kg) substantially reduces model mice disease activity index DAI scorings.Wherein, land for building dish alcohol extract low dosage
Inhibitory action that (20mg/kg, p ＜ 0.05) scores DAI with positive drug (200mg/kg, p ＜ 0.01) quite, high dose
(100mg/kg) is better than positive drug (200mg/kg, p ＜ 0.01) to the model mice DAI inhibitory action to score.
The influence for the UC mouse Colon length that 3.3 land for building dish alcohol extracts are induced DSS
The colitis mice colon lengths of DSS inductions substantially shorten.Fig. 3 is that the UC that land for building dish alcohol extract is induced DSS is small
The influence of mouse colon lengths.##p<0.01versus normal groups；**p<0.01versus DSS model groups.This result of study shows,
Compared with normal group, DSS model group mouse Colon length substantially shortens (p ＜ 0.01).Gavage gives low dose of land for building dish alcohol extract
Measuring (20mg/kg, p ＜ 0.01) and high dose (100mg/kg, p ＜ 0.01), substantially protection model mice colon shortens, and it suppresses
The ability that colon shortens is suitable with positive drug (200mg/kg, p ＜ 0.01).
The influence for the UC mouse MPO vigor that 3.4 land for building dish alcohol extracts are induced DSS
The colitis mice colon neutrophil infiltration of DSS inductions is obvious, and MPO vigor is significantly raised.Fig. 4 is ground
Skin dish alcohol extract induces DSS the influence of UC mouse MPO vigor.##p<0.01versus normal groups；*p<0.05,**p<
0.01versus DSS model groups.This result of study shows, compared with normal group, DSS model group mouse Colon tissue MPO vigor
Significantly raised (p ＜ 0.01).Gavage gives land for building dish alcohol extract low dosage (20mg/kg, p ＜ 0.05) and high dose (100mg/
Kg, p ＜ 0.01) substantially suppress model mice MPO vigor, its rejection ability and positive drug (200mg/kg, p ＜ to MPO vigor
0.01) quite.
The influence for the UC mouse Pathomorphology that 3.5 land for building dish alcohol extracts are induced DSS
Normal group mouse Colon tissue mainly involves viscous without obvious pathological change, DSS model group mouse Colon lesion tissues
Film layer and submucosa, inflammatory cell type are mainly mononuclear macrophage and neutrophil leucocyte.Inflammation critical regions local mucous membrane
Layer holostrome necrosis, forms ulcer.
Fig. 5 is the colored graph of colon HE dyeing, and Fig. 6 is the UC mouse pathology shapes that land for building dish alcohol extract is induced DSS
The influence of state.##p<0.01versus normal groups；*p<0.05,**p<0.01versus DSS model groups.This result of study table
It is bright, compared with normal group, DSS model group mouse Colon tissue inflammatories cellular infiltration, crypt damage (p ＜ 0.01).Gavage is given
Land for building dish alcohol extract low dosage (20mg/kg, p ＜ 0.05) and high dose (100mg/kg, p ＜ 0.01) substantially mitigate above-mentioned tissue
Pathological change, it is suitable with positive drug (200mg/kg, p ＜ 0.01) to the action intensity for reducing lesion tissue scoring.
The influence for the UC mouse proinflammatory TNF-α contents that 3.6 land for building dish alcohol extracts are induced DSS
A variety of proinflammatories participate in UC occurrence and development, particularly important with TNF-α and IL-6.Fig. 7 is land for building dish alcohol extract
Influence to the UC mouse proinflammatory TNF-α contents of DSS inductions.##p<0.01versus normal groups；*p<0.05,**p<
0.01versus DSS model groups.This result of study shows, compared with normal group, DSS model group mouse Colon tissue T NF- α contain
The obvious increase (p ＜ 0.01) of amount.Gavage gives land for building dish alcohol extract high dose (100mg/kg, p ＜ 0.05) and positive drug
(200mg/kg, p ＜ 0.01) significantly reduces model mice colon TNF-α content.
The influence for the UC mouse proinflammatory IL-6 contents that 3.7 land for building dish alcohol extracts are induced DSS
The colitis mice colon IL-6 expression of DSS inductions is significantly raised.Fig. 8 is that land for building dish alcohol extract is lured DSS
The influence for the UC mouse proinflammatory IL-6 contents led.##p<0.01versus normal groups；*p<0.05,**p<0.01versus
DSS model groups.This result of study shows, compared with normal group, DSS model group mouse Colon tissue IL-6 contents substantially increase (p
＜ 0.01).Gavage gives land for building dish alcohol extract low dosage (20mg/kg, p ＜ 0.05) and high dose (100mg/kg, p ＜ 0.01)
Significantly reduce colon's IL-6 contents, and its reduction degree to IL-6 contents and positive drug (200mg/kg, p ＜ 0.01) phase
When.
The influence for the UC mouse proinflammatory IL-1 β contents that 3.8 land for building dish alcohol extracts are induced DSS
The mouse models of colitis mouse Colon tissue IL-1 β contents of DSS inductions are significantly raised.Fig. 9 is land for building dish alcohol extracting
The influence for the UC mouse proinflammatory IL-1 β contents that thing is induced DSS.##p<0.01versus normal groups；*p<0.05versus
DSS model groups.This result of study shows, compared with normal group, DSS model group mouse Colon tissue IL-1 β contents substantially increase
(p ＜ 0.01).Gavage, which gives land for building dish alcohol extract low dosage (20mg/kg) and high dose (100mg/kg), slightly reduces IL-1 β
Content, it is suitable that its reduction Chengdu to IL-1 β contents is weaker than positive drug (200mg/kg, p ＜ 0.05).
The influence for the UC mouse proinflammatory NF- kB activations that 3.9 land for building dish alcohol extracts are induced DSS
NF- κ B are a crucial transcription factors, and inflammation target gene is such as proinflammatory in participation regulation and control UC Development process
Expression.Figure 10 is the Western blot figures for the UC mouse proinflammatory NF- kB activations that land for building dish alcohol extract is induced DSS；Figure
11 be the block diagram that the UC mouse proinflammatory NF- kB activations that land for building dish alcohol extract is induced DSS influence.##p<0.01versus
Normal group；*p<0.05,**p<0.01versus DSS model groups.This result of study shows, compared with normal group, DSS model groups
Mouse Colon tissue NF- kB activations are remarkably reinforced.(p ＜ 0.01).Gavage gives land for building dish alcohol extract low dosage (20mg/kg, p
＜ 0.05) and high dose (100mg/kg, p ＜ 0.01) substantially suppress colon NF- kB activations, and it is to NF- kB activations
Inhibition level is suitable with positive drug (200mg/kg, p ＜ 0.01).
4. conclusion is with discussing
The study find that the UC model mices body weight that land for building dish alcohol extract is obviously improved DSS inductions reduces, suppress DAI scorings
Rise, mitigate UC induced mices colon lengths and shorten, while reduce inflammatory cell infiltration and tissue damage.In addition, land for building dish alcohol
Extract substantially reduces colon's cytokine TNF-α and IL-6 content, and this effect suppresses inflammatory signal path with it
Key transcription factor NF- kB activations it is closely related.These conclusions prompting land for building dish alcohol extract can be used for treating IBD,
Mitigate the UC modeled inflammations reaction of DSS inductions, improve the UC orders of severity, its mechanism may be with suppressing the regulation of NF- κ B signals path
The expression such as cytokine TNF-α and IL-6 it is related.
Claims (8)
1. application of the land for building dish alcohol extract in treatment inflammatory bowel medicine or health products are prepared.
2. application according to claim 1, it is characterised in that：The preparation method of the land for building dish alcohol extract includes：Take ground
Skin dish, using low-carbon alcohols as solvent, extracted under heating condition, collect extract solution, recycling design, that is, obtain land for building dish alcohol extract.
3. application according to claim 2, it is characterised in that：The preparation method of the land for building dish alcohol extract further comprises
Purification step.
4. application according to claim 3, it is characterised in that：Described purification step is：Take gained land for building dish alcohol extract
Recrystallized with low-carbon alcohols, that is, obtain land for building dish alcohol extract after purification.
5. according to the application any one of claim 2-4, it is characterised in that：Described low-carbon alcohols are former containing 1-6 carbon
Combination more than one or both of alcohol of son.
6. according to the application any one of claim 2-4, it is characterised in that：The extraction is the boiling at 50 DEG C to solvent
Carried out in point temperature range.
7. a kind of medicine or health products for treating IBD, the land for building dish alcohol extract containing the upper effective dose for the treatment of.
8. medicine according to claim 7 or health products, it is characterised in that：The formulation of the medicine or health products is pharmacy
Upper acceptable formulation.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103059112A (en) * | 2013-01-22 | 2013-04-24 | 山西大学 | Method of extracting anti-intestinal cancer active protein from nostoc commune |
| CN105769748A (en) * | 2016-04-19 | 2016-07-20 | 桂林理工大学 | Cassava starch hydrogel nostoc commune extract mask matrix and preparation method thereof |
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2017
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103059112A (en) * | 2013-01-22 | 2013-04-24 | 山西大学 | Method of extracting anti-intestinal cancer active protein from nostoc commune |
| CN105769748A (en) * | 2016-04-19 | 2016-07-20 | 桂林理工大学 | Cassava starch hydrogel nostoc commune extract mask matrix and preparation method thereof |
Non-Patent Citations (1)
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| 李海平等: "响应面法优化超声提取地皮菜多糖工艺研究", 《北方园艺》 * |
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| EP4104845A1 (en) * | 2021-06-18 | 2022-12-21 | Medizinische Universität Innsbruck | Extract with anti-inflammatory effect |
| WO2022263580A1 (en) * | 2021-06-18 | 2022-12-22 | Medizinische Universität Innsbruck | Extract with anti-inflammatory effect |
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