CN105327338B - A kind of drug and preparation method thereof for treating ulcerative colitis - Google Patents
A kind of drug and preparation method thereof for treating ulcerative colitis Download PDFInfo
- Publication number
- CN105327338B CN105327338B CN201510810779.8A CN201510810779A CN105327338B CN 105327338 B CN105327338 B CN 105327338B CN 201510810779 A CN201510810779 A CN 201510810779A CN 105327338 B CN105327338 B CN 105327338B
- Authority
- CN
- China
- Prior art keywords
- lactobacillus
- drug
- ulcerative colitis
- colostrum
- bacterium
- 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.)
- Active
Links
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/18—Growth factors; Growth regulators
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/20—Milk; Whey; Colostrum
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/66—Microorganisms or materials therefrom
- A61K35/74—Bacteria
- A61K35/741—Probiotics
- A61K35/744—Lactic acid bacteria, e.g. enterococci, pediococci, lactococci, streptococci or leuconostocs
- A61K35/747—Lactobacilli, e.g. L. acidophilus or L. brevis
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Mycology (AREA)
- Chemical & Material Sciences (AREA)
- Microbiology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- General Health & Medical Sciences (AREA)
- Epidemiology (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Gastroenterology & Hepatology (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Cell Biology (AREA)
- Developmental Biology & Embryology (AREA)
- Virology (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of drugs and preparation method thereof for treating ulcerative colitis, wherein the drug i.e. colostrum growth factor and probiotic combination for treating ulcerative colitis use.The present invention establishes Ulcerative Colitis Model using Balb/c mouse as subjects, from the apparent state of test mice, changes of weight, colon lengths, pathological conditions angularly, studies the anti-ulcerative colitis effect of drug of the present invention；Test result, which shows drug of the present invention not only, has good anti-ulcerative colitis effect, but also is in a short time not in the symptom of recurrence, can be good at repairing ulcer colitis intestinal mucosa injury, can be widely applied to preclinical medicine field.
Description
Technical field
The present invention relates to a kind of drugs and preparation method thereof for treating ulcerative colitis, i.e. three probiotics and colostrum
The drug that growth factor is used in combination, belongs to pharmaceutical technology field.
Background technique
Ulcerative colitis (ulcerative colitis, UC) is inflammatory bowel disease (inflammatory bowel
Disease, IBD) a main Types, be a kind of chronic nonspecific intestinal inflammatory that reason is unknown.Data shows, every year
U.S.'s UC illness rate is 2,00/,100,000 people, and that treats UC spends in 1 to 1.5 hundred million dollar or so, and by the end of 2012, UC patient was total
Number is 2.5 times in 2000, and recurrence rate is up to 72%；The number of patients of China UC is in obvious ascendant trend in recent years.
Ulcerative colitis lesion is predominantly located at mucous membrane of colon and submucosa；Clinical manifestation is diarrhea, mucus pus and blood stool,
Abdominal pain, it is tenesmus etc., it is in the chronic course of disease of recurrent exerbation more.The generation of ulcerative colitis has seriously affected patient's
Health and quality of life are classified as one of modern difficult treatment by the World Health Organization.The cause and onset of disease of ulcerative colitis
Mechanism is not still fully aware of.Think that the cause of disease of UC may be related with environmental factor, inherent cause, immune factor etc. more；Wherein, it loses
It passes and immune factor plays a crucial role in UC causes a disease.Though UC pathogenesis does not illustrate also completely, can affirm
Function of intestinal mucosa barrier in patient, which is lacked of proper care, takes part in the generation of UC.Gut barrier mainly includes mechanical barrier, immunization barrier and biology screen
Barrier.Mechanical barrier is made of enterocyte, intestines mucus；Immunization barrier (i.e. intestine immunity system of defense), mainly by secretory
Immunoglobulin and gut associated lymphoid tissue are constituted；Biological barrier is made of normal intestinal flora.Gut epithelium barrier breakdown sticks
Membrane permeability increases, and intestinal tissue is exposed in a large amount of antigens for a long time, leads to intestinal tract immune system overreaction and wrong identification, draw
The activation for playing macrophage and lymphocyte, discharges a series of cell factors and inflammatory mediator, activates the immune response of body, scorching
Disease reaction is amplified step by step, eventually leads to tissue damage, the pathological change of UC and clinical manifestation occurs.
The method for the treatment of ulcerative colitis mainly includes that drug therapy, nutrition treatment, psychotherapy and operation are controlled at present
It treats, drug treatment is most important method.Be used clinically for treatment UC drug mainly have minosalicylates, drugs,
Adrenal Glucocorticoid class and immunosuppressant etc..Minosalicylates, drugs can inhibit prostaglandin to synthesize well,
Scavenging activated oxygen is to mitigate inflammatory reaction；However it can not eradicate UC, and when patient's medication dose is bigger than normal, be also easy to produce many
Adverse reaction is (such as：Headache, nausea, leucocyte decline etc.).Glucocorticoid is the drug of first choice of severe or explosive UC patient；So
And such drug easily leads to the side effects such as organism metabolic disorder, water retention, can be used as emergency drug, can not be used for a long time.It is immune
Inhibit class medication medication dependence big, treatment phase is longer, thereby increases and it is possible to which intoxicating is carcinogenic, and side effect is larger, so being generally only used as auxiliary
Treatment is helped to use.On the whole, said medicine is responded well to treatment fast, good effect, can be relieved the hardship of UC patient's matter as a fire singeing one's eyebrows, but there are still very
More problems.Firstly, patient UC can not be eradicated, it is palliative；Secondly, ill-effect is big, easily causes patient's headache, nausea, make
At body digestive system, hematological abnormalities；Then, potential intoxicating carcinogenicity；Finally, antibiotics is (most such as application
Wide SASP) easily lead to body drug resistance drug resistance.
Colostrum growth factor can be by conjunction with protection of intestinal mucosal barrier cells tyrosine kinase receptor, to promote protection of intestinal mucosal barrier cells
Growth and proliferation, increase villi height, promote intestinal growth；And immunity of organism can be promoted by regulation inflammatory factor level
Power.Probiotics can be by adjusting intestinal flora balance with harmful bacteria competing binding sites, and lactobacillus plantarum can produce bacteriocin,
Inhibit the proliferation of harmful bacteria；Lactobacillus acidophilus has good prebiotic effect.Therefore we assume that colostrum growth factor is combined
Probiotics has good anti-ulcerative colitis purposes.Therefore, it is lesser safe that efficient one kind, safety and side effect are developed
The drug for treating UC, is of great practical significance.
Summary of the invention
An object of the present invention is to provide a kind of drug for treating ulcerative colitis, can effectively treat exedens
Colitis, and toxic side effect is smaller, is not easy to produce resistance.
It is a further object to provide a kind of preparation methods of drug for treating ulcerative colitis.
To achieve the goals above, the present invention provides the following technical solutions：
A kind of drug for treating ulcerative colitis, including 132~198 parts by volume probiotics and 432~684 mass parts oxen
Colostrum growth factor, wherein parts by volume/mass parts=ml/mg.
Preferably, at the beginning of the drug of above-mentioned treatment ulcerative colitis, including 165 parts by volume probiotics and 540 mass parts oxen
Newborn growth factor.The colostrum growth factor is the growth factor crude extract extracted from colostrum.The probiotics
For lactobacillus acidophilus, lactobacillus plantarum and Lactobacillus helveticus；It and is viable bacteria thallus form.The probiotics viable bacteria thallus is
The bacterium mud being collected by centrifugation respectively by the lactobacillus liquid culture for being in stable initial stage.
Preferably, the quantity ratio of the lactobacillus acidophilus, lactobacillus plantarum and Lactobacillus helveticus is 1: 1: 1.
In the drug of above-mentioned treatment ulcerative colitis, the probiotics dosage is 1.5 × 1010CFU/kg, the ox
Colostrum growth factor dosage is 216~342mg/kg.Lactobacillus acidophilus i.e. in drug used in every kilogram of animal, human body etc.,
The total quantity of lactobacillus plantarum and Lactobacillus helveticus is 1.5 × 1010CFU, colostrum growth factor dosage are 216~342mg, tool
Body, can be calculated according to the actual situation, for example, the rat that a weight is 20g, contain acidophilus cream bar in medication
The total quantity of bacterium, lactobacillus plantarum and Lactobacillus helveticus is 3 × 108CFU, colostrum growth factor dosage be 4.32~
6.84mg；One weight is the people of 50kg, and the total quantity of lactobacillus acidophilus, lactobacillus plantarum and Lactobacillus helveticus is in medication
7.5×1011CFU, colostrum growth factor dosage are 10.8~17.1g.
The preparation method of the drug of aforementioned therapies ulcerative colitis, includes the following steps：
(1) respectively by lactobacillus acidophilus, lactobacillus plantarum and Lactobacillus helveticus in MRS culture medium culture；
(2) respectively choose culture 14~18h Lactobacillus helveticus, cultivate 12~16h lactobacillus acidophilus and culture 12~
The lactobacillus plantarum of 16h；
(3) 77~115 parts by volume of Lactobacillus helveticus bacterium solution for taking step (2) to obtain respectively, lactobacillus acidophilus bacterium solution 40~
Then it is sterile to be dissolved in 10 parts by volume respectively by 60 parts by volume, 15~23 parts by volume of lactobacillus plantarum bacterium solution, centrifuging and taking bacterium mud for they
In PBS, mixing bacterium mud is obtained；
(4) colostrum growth is made to colostrum centrifugal degreasing, the heavy casein of acid, second ultrafiltration, dialysis and freeze-drying
Factor crude extract；
(5) 432~684 mass parts of colostrum growth factor crude extract are added in above-mentioned mixing bacterium mud, are stirred evenly,
Then suitable auxiliary material is added, clinically-acceptable dosage form is made.
Preferably, the preparation method for treating the drug of ulcerative colitis, includes the following steps：
(1) respectively by lactobacillus acidophilus, lactobacillus plantarum and Lactobacillus helveticus in MRS culture medium culture；
(2) probiotics at the beginning of stationary phase in the logarithm end of term is chosen, i.e., chooses Lactobacillus helveticus, the culture 14h of culture 16h respectively
Lactobacillus acidophilus and culture 14h lactobacillus plantarum；
(3) 96 parts by volume of Lactobacillus helveticus bacterium solution for taking step (2) to obtain respectively, 50 parts by volume of lactobacillus acidophilus bacterium solution,
Then they are dissolved in the sterile PBS of 10 parts by volume respectively, must mix by 19 parts by volume of lactobacillus plantarum bacterium solution, centrifuging and taking bacterium mud
Bacterium mud；
(4) colostrum growth is made to colostrum centrifugal degreasing, the heavy casein of acid, second ultrafiltration, dialysis and freeze-drying
Factor crude extract；
(5) 540 mass parts of colostrum growth factor crude extract are added in above-mentioned mixing bacterium mud, are stirred evenly, then
Suitable auxiliary material is added, clinically-acceptable dosage form is made.
Beneficial effects of the present invention：
The present invention has preferable anti-ulcerative knot with drug made of probiotics and colostrum growth factor crude extract
Enteritis effect.It is in particular in the apparent state for significantly improving ulcerative colitis mouse, increases ulcerative colitis mouse
Weight, colon lengths, and significantly improve the integrality of mouse Colon tissue.
Detailed description of the invention
Fig. 1 is lactobacillus acidophilus microscopy picture (100 times)；
Fig. 2 is lactobacillus acidophilus bacterium colony figure；
Fig. 3 is Lactobacillus helveticus microscopy picture (100 times)；
Fig. 4 is Lactobacillus helveticus bacterium colony figure；
Fig. 5 is lactobacillus plantarum microscopy picture (100 times)；
Fig. 6 is lactobacillus plantarum bacterium colony figure；
Fig. 7 is three plants of lactobacillus growth curves；
Fig. 8 is the apparent state of enteritis group mouse；
Fig. 9 is the apparent state feature of enteritis group mouse；
Figure 10 is enteritis group mouse internal anatomy；
Figure 11 is enteritis group mouse Colon length representative figure；
Figure 12 is that complete naive mice colon lengths represent figure；
Figure 13 is enteritis group mouse HE colored graph；
Figure 14 is plan design figure；
Figure 15 is the apparent state diagram of joint group mouse；
Figure 16 is joint group mouse internal anatomy；
Figure 17 is the apparent state of later observation joint group mouse；
Figure 18 is the apparent state of antibiotic group mouse；
Figure 19 is antibiotic group mouse internal anatomy；
Figure 20 is the apparent state of later observation antibiotic group mouse；
Figure 21 is the apparent state of complete naive mice；
Figure 22 is naive mice internal anatomy；
Figure 23 is the apparent state of later observation naive mice；
Figure 24 is joint group mouse Colon length representative figure；
Figure 25 is antibiotic group mouse Colon length representative figure；
Figure 26 is that complete naive mice colon lengths represent figure；
Figure 27 is joint group mouse HE colored graph；
Figure 28 is antibiotic group mouse HE colored graph；
Figure 29 is complete naive mice HE colored graph；
Figure 30 is joint group mouse later observation HE colored graph；
Figure 31 is antibiotic group mouse later observation HE colored graph；
Figure 32 is complete naive mice later observation HE colored graph.
Specific embodiment
In the case that the present inventor feels a delicacy about to the treatment of ulcerative colitis for current global field of medicaments, according to
Immunity of organisms can be enhanced according to probiotics and growth factor, have good therapeutic effect to human body intestinal canal sick (such as diarrhea),
Therefore the preferable living preparation of lactobacillus thallus of three plants of probiotic effects is combined with colostrum growth factor crude extract, drug is made, for moving
Object experimental study is detailed description of the present invention below.
Examples are merely exemplary, and it is not intended to limit the scope of the present invention in any way.Those skilled in the art should
Understand, without departing from the spirit and scope of the invention can details to technical solution of the present invention and form modify
Or replacement, but these modifications and replacement are fallen within the protection scope of the present invention.A variety of materials of the present invention, reagent or
Apparatus unless otherwise specified, can be obtained from market.
In order to prove effectiveness of the invention, applicant carried out following tests.
(1) material and reagent
1, culture medium：MRS culture medium.
MRS culture medium is prepared：Peptone 5g/L, beef extract 5g/L, tryptone 10g/L, yeast powder 5g/L, glucose
20g/L, anhydrous sodium acetate 5g/L, citric acid hydrogen diamine 2g/L, dipotassium hydrogen phosphate 2g/L, magnesium sulfate 0.58g/L, manganese sulfate
0.25g/L, 1ml/L, pH6.3+0.1,121 DEG C of Tween-80 sterilize for 15 minutes.
2, main agents：Colostrum comes from the pasture Wanda Mountain, Heilongjiang Song Bei；Dextran sulfate sodium salt comes from Beijing bit
Rich company of biotechnology Co., Ltd；Sulfasalazine comes from Shanghai Xinyi Tianping Pharmaceutical Co., Ltd.；Formaldehyde is molten
Liquid comes from the chemical inc Tianjin Bo Di；Potassium dihydrogen phosphate comes from the extremely big chemical apparatuses factory in Tianjin Dongli District；Phosphoric acid hydrogen
Disodium comes from Tianjin BASF Chemical Co., Ltd..
3, key instrument：Electron microscope comes from Motic company；Ultraviolet-uisible spectrophotometer comes from the U.S. DU800
BECKMAN company；Ophthalmology anatomy box comes from Shenyang City Jiu Ming aluminum products factory；Syringe controls the limited public affairs of space medical instrument from Jiangsu
Department.
(2) preparation of stomach-filling probiotics
1, three probiotics colony morphological observation
The lactobacillus acidophilus, Lactobacillus helveticus and lactobacillus plantarum of preservation are taken out respectively, activated for 2 generations repeatedly in solid training
It supports and carries out the setting-out of 3rd area on base, left and right, progress Gram's staining and oil mirror are observed for 24 hours for 37 DEG C of cultures.
The microscopy picture of lactobacillus acidophilus, Lactobacillus helveticus and lactobacillus plantarum sees Fig. 1, Fig. 3 and Fig. 5, bacterium respectively
It falls form and sees Fig. 2, Fig. 4 and Fig. 6 respectively, on three probiotics thalli morphologies and microscopic examination result and primary Jie Shi Bacteria Identification handbook
Describe it is almost the same, be typical lactobacillus acidophilus, Lactobacillus helveticus and lactobacillus plantarum.
2, the selection of probiotics stomach-filling time
From half of bacterium colony of picking on the solid medium of culture, after activating a generation, come into 0h, 1h, 2h, 3h, 4h, 6h, 8h,
10h, 12h, 14h, 16h, 18h, 20h, 22h, for 24 hours, 26h, 28h, 30h, the fluid nutrient medium of totally 18 periods reaches above-mentioned
It when the time, takes out and uses ultraviolet specrophotometer, measure OD600, make growth curve；Using incubation time as abscissa, OD600 value is
Ordinate draws the growth curve of 3 plants of Bacillus acidi lacticis, sees Fig. 7.According to the growth curve of three plants of lactobacillus, it is known that Switzerland's cream bar
The time that bacterium, lactobacillus acidophilus and lactobacillus plantarum enter stationary phase is respectively 16h, 14h and 14h.
Due to the activity of probiotic highest of logarithmic phase, and the probiotics bacterium number of stationary phase reaches maximum, so choosing logarithm
Probiotics at the beginning of stationary phase in the end of term treats ulcerative colitis mouse, i.e., cultivates Switzerland's cream of 16h, 14h and 14h respectively
Bacillus, lactobacillus acidophilus and lactobacillus plantarum.
3, the measurement of stomach-filling bacterium number
Lactobacillus helveticus, lactobacillus acidophilus and lactobacillus plantarum are cultivated respectively to 16h, 14h and 14h, gradient has been carried out
Plate count, the results showed that, the bacterium number at the beginning of Lactobacillus helveticus, lactobacillus acidophilus and lactobacillus plantarum reach stationary phase is respectively
1.25×109CFU/mL、2.42×109CFU/mL、6.3×109CFU/mL。
Research obtains, and the stomach-filling amount of mouse is 3 × 108When CFU/20g (bacterium number/mouse weight), has and repair well
Intestinal mucosa injury effect；This experiment mice average weight is calculated with 20g, according to 1: 1: 1 ratio, Lactobacillus helveticus, acidophilus cream bar
Bacterium, lactobacillus plantarum should be respectively 1 × 108CFU, therefore, the Lactobacillus helveticus bacterium solution 0.96mL for taking equal stationary phase to go out, acidophilus cream
Then bacillus bacterium solution 0.5mL, lactobacillus plantarum bacterium solution 0.19mL, centrifuging and taking bacterium mud are refitted in 0.1mLPBS respectively, for every
The probiotics given low of mouse.
(3) preparation of colostrum growth factor crude extract
As raw to colostrum made from colostrum centrifugal degreasing, the heavy casein of acid, second ultrafiltration, dialysis and freeze-drying
Long factor crude extract；The processing methods such as signified centrifugation, ultrafiltration, dialysis, drying of the invention, can be used ordinary skill people
Any method well known to member, as long as it is able to maintain growth factor effect, preferably, centrifugation is dry using cold by 4 DEG C of centrifugations
It is lyophilized dry.
(4) preparation of the drug of ulcerative colitis is treated
Colostrum growth factor crude extract, given low are 5.4mg/20g (colostrum growth factor crude extract quality/small
Mouse weight)；The stomach-filling form of colostrum growth factor crude extract is to take out the acidophilus cream bar being dissolved in the sterile PBS solution of 0.1mL
Bacterium, lactobacillus plantarum, Lactobacillus helveticus bacterium mud tubule, mixing, are then added the colostrum growth factor crude extract of the dosage.
(5) antibiotic group intragastric administration on mice drug：The given low of antibiotic is 9.2mg/20g (willow nitrogen thiamines pyridine silty
Amount/mice weights)；The stomach-filling form of willow nitrogen thiamines pyridine is that taking-up is dissolved in the sterile PBS solution of 0.3mL, is mixed, then 4 DEG C
It saves, in case stomach-filling.
It is studied below with reference to zoopery is further to drug of the invention, to illustrate effectiveness of the invention.
(1) production and experimental design of ulcerative colitis mouse model
Dextran sulfate sodium (DSS) is dissolved in water bottle, Balb/c mouse is allowed freely to drink 8 days, is burst with provocative test
Ulcer colitis mouse model is verified small then with mouse weight variation, apparent situation, colon lengths, pathology etc. for index
Whether mouse Ulcerative Colitis Model is successfully established.
Mouse weight rate of descent is 13.76% during modeling；And enteritis group mouse is apparently not in good state, and is in particular in small
Mouse is One's eyesight is restrained, the obscure curling of hair, has Qiang's hair phenomenon, sees Fig. 8；Also, there is blood cake at enteritis group mouse root of the tail, illustrate mouse
There is dominant hematochezia phenomenon, sees Fig. 9；There is phenomenon of festering at enteritis group mouse Colon after dissection, intestinal tissue has hair purulence, turbidity existing
As seeing Figure 10；Moreover, enteritis group mouse Colon length is about 8.64cm, compared with naive mice colon during modeling
12.82cm, it is significant to shorten, see Figure 11, Figure 12；Also, by HE colored graph (see Figure 13) it is found that model group mouse Colon tissue,
Intestinal mucosa atrophy, mucous membrane surface passivation, crypts destroy.
In conclusion acute ulcer Colitis Model is successfully established, next step drug therapy test, this experiment can be carried out
Experimental design scheme is shown in Figure 14.
(2) experiment conclusion
(1) explanation is tested
During treatment, PBS stomach-filling only is carried out to enteritis naive mice, without treatment, as a result enteritis is empty during stomach-filling
White group dead mouse 11；On the one hand illustrate that Ulcerative Colitis Model is successfully established, still further aspect illustrates ulcerative colitis
Scorching mouse cannot be fully recovered by self-regeneration；So subsequent comparison, is across comparison, i.e. the comparison with group front and back is small
Mouse treatment after with the comparison before treatment.
(2) drug of the invention is obviously improved the apparent state of mouse
After the stomach-filling of two courses for the treatment of, the poly- mind of medicine group mouse sight of the present invention, action are active, and mouse hair is suitable
Sliding, brilliant white, is shown in Figure 15, compared with apparent state when enteritis, difference is extremely obvious；And with the apparent state of complete naive mice
(Figure 21) and the apparent state of antibiotic group mouse (Figure 18) are compared, and difference is not significant.From the point of view of mouse internal anatomy, medicine of the invention
Object significantly improves the festering of the colonic tissue of enteritis mouse, blood cake phenomenon, sees Figure 16；And medicine group mouse of the present invention and completely
Naive mice (Figure 22), antibiotic group mouse Colon tissue are compared to (Figure 19) without significant difference.From the point of view of later observation figure, with
The apparent state of antibiotic group mouse (see Figure 20) and the apparent state of naive mice (23) completely are compared, the apparent shape of joint group mouse
State is normal, has no adverse reaction, and sees Figure 17, illustrates that drug of the invention has good effect to treatment ulcerative colitis, and short
Without recurrence sign in phase.
(3) drug of the invention (joint group) increases mouse Colon length
Mouse Colon length changes table during table 1 is treated
The mouse Colon length of joint group, antibiotic group and complete blank group, is shown in Figure 24, Figure 25, Figure 26 respectively, and by
It is found that after two courses for the treatment of, drug of the invention (joint group) can significantly increase the colon group of mouse by table 1, Figure 24, Figure 25, Figure 26
It knits, by the 8.64cm of mouse Colon tissue after modeling, rises to 12.93 or so, and difference is not shown compared with complete blank group
It writes, and is significantly better than antibiotic group, illustrate that the drug therapy of the present invention of two courses for the treatment of will be because of knot caused by ulcerative colitis
Intestinal tissue atrophy restores to normal level, and therefore, from the point of view of mouse Colon length angle, drug of the invention can rise appreciably small
Mouse colonic tissue length, enhancing gut barrier effect.
(4) drug of the invention increases mouse weight
Mouse weight changes table during table 2 is treated
As shown in Table 2, after one course for the treatment of of stomach-filling, drug effect of the invention is significant, shows that medicine group mouse of the present invention is non-
But without because of weight loss and dead, body weight increase 11.02% instead the reason of enteritis, after two courses for the treatment of, this hair
Bright medicine group mouse is relative to modeling initial stage body weight increase 25.42%, and compared with antibiotherapy, difference is not significant, explanation
Utilize the method for drug of the invention and Medication in Ulcerative Colitis traditional on the market, no significant difference；Later observation 2
Week the result shows that, there is not the sign recurred in medicine group mouse weight sustainable growth of the present invention.And complete naive mice
Body weight growth rate is 11.01% after two courses for the treatment of, it may be possible to which, since this group of mouse does not carry out establishing model treatment, weight is not
It reduces, is at a normal level, then mouse weight growth space is smaller；It is larger additionally, due to this group of mouse weight radix, then mouse
Growth rate is also relatively small.Therefore, from the point of view of weight angle, drug of the invention is used with preferable anti-ulcerative colitis
On the way, and in a short time do not have the sign of recurrence.
(5) drug of the invention repairs mouse intestinal mucosa injury
After the treatment for carrying out two courses for the treatment of to mouse using drug of the invention, mouse is dissected, its colonic tissue is taken, is carried out
Hematoxylin-eosin dyes (HE dyeing), the pathology of enteritis group, medicine group of the present invention (joint group), antibiotic group, complete blank group
Figure is shown in Figure 13, Figure 27, Figure 28, Figure 29 respectively；After two weeks later observations, the same processing is repeated, medicine group of the present invention, completely
Blank group, antibiotic group pathology figure see Figure 30, Figure 31, Figure 32 respectively.
As seen from the figure, enteritis group mouse, intestinal mucosa atrophy, mucous membrane surface passivation, crypts destroy, and are substantially at intestinal mucosa screen
Hinder imbalance state；After the treatment for carrying out two courses for the treatment of to mouse using drug of the invention, protection of intestinal mucosal barrier cells arrangement is substantially neat,
Intestinal mucosa structural integrity, inorganization necrosis phenomena illustrate that drug of the invention can be very good repairing ulcer colitis mice
Intestinal mucosa injury；And compared with antibiotic group, complete naive mice, no significant difference；The pathology chart of later observation
Bright, drug of the invention all has good anti-ulcerative colitis effect in short-term and long-term.
A kind of preparation method for the drug for treating ulcerative colitis of embodiment 1, includes the following steps：
(1) respectively by lactobacillus acidophilus, lactobacillus plantarum and Lactobacillus helveticus in MRS culture medium culture；
(2) Lactobacillus helveticus, culture 12h lactobacillus acidophilus and the plant cream bar for cultivating 12h of culture 14h are chosen respectively
Bacterium；
(3) 77 parts by volume of Lactobacillus helveticus bacterium solution for taking step (2) to obtain respectively, 40 parts by volume of lactobacillus acidophilus bacterium solution,
Then they are dissolved in the sterile PBS of 10 parts by volume respectively, must mix by 15 parts by volume of lactobacillus plantarum bacterium solution, centrifuging and taking bacterium mud
Bacterium mud；
(4) colostrum growth is made to colostrum centrifugal degreasing, the heavy casein of acid, second ultrafiltration, dialysis and freeze-drying
Factor crude extract；
(5) 432 mass parts of colostrum growth factor crude extract are added in above-mentioned mixing bacterium mud, are stirred evenly, then
Suitable auxiliary material is added, clinically-acceptable dosage form is made.
A kind of preparation method for the drug for treating ulcerative colitis of embodiment 2, includes the following steps：
(1) respectively by lactobacillus acidophilus, lactobacillus plantarum and Lactobacillus helveticus in MRS culture medium culture；
(2) Lactobacillus helveticus, culture 16h lactobacillus acidophilus and the plant cream bar for cultivating 16h of culture 18h are chosen respectively
Bacterium；
(3) 115 parts by volume of Lactobacillus helveticus bacterium solution for taking step (2) to obtain respectively, 60 parts by volume of lactobacillus acidophilus bacterium solution,
Then they are dissolved in the sterile PBS of 10 parts by volume respectively, must mix by 23 parts by volume of lactobacillus plantarum bacterium solution, centrifuging and taking bacterium mud
Bacterium mud；
(4) colostrum growth is made to colostrum centrifugal degreasing, the heavy casein of acid, second ultrafiltration, dialysis and freeze-drying
Factor crude extract；
(5) 684 mass parts of colostrum growth factor crude extract are added in above-mentioned mixing bacterium mud, are stirred evenly, then
Suitable auxiliary material is added, clinically-acceptable dosage form is made.
A kind of preparation method for the drug for treating ulcerative colitis of embodiment 3, includes the following steps：
(1) respectively by lactobacillus acidophilus, lactobacillus plantarum and Lactobacillus helveticus in MRS culture medium culture；
(2) probiotics at the beginning of stationary phase in the logarithm end of term is chosen, i.e., chooses Lactobacillus helveticus, the culture 14h of culture 16h respectively
The lactobacillus plantarum of lactobacillus acidophilus and culture 14h；
(3) 96 parts by volume of Lactobacillus helveticus bacterium solution for taking step (2) to obtain respectively, 50 parts by volume of lactobacillus acidophilus bacterium solution,
Then they are dissolved in the sterile PBS of 10 parts by volume respectively, must mix by 19 parts by volume of lactobacillus plantarum bacterium solution, centrifuging and taking bacterium mud
Bacterium mud；
(4) colostrum growth is made to colostrum centrifugal degreasing, the heavy casein of acid, second ultrafiltration, dialysis and freeze-drying
Factor crude extract；
(5) 540 mass parts of colostrum growth factor crude extract are added in above-mentioned mixing bacterium mud, are stirred evenly, then
Conjunction auxiliary material living is added, clinically-acceptable dosage form is made.
Claims (7)
1. a kind of drug for treating ulcerative colitis, which is characterized in that including 1.32 ~ 1.98ml probiotics and 4.32 ~
6.84mg colostrum growth factor；The probiotics be lactobacillus acidophilus KLDS20010, lactobacillus plantarum KLDS00170 and
Lactobacillus helveticus KLDS10010；It and is viable bacteria thallus form.
2. the drug for the treatment of ulcerative colitis according to claim 1, which is characterized in that including 1.65ml probiotics and
5.4mg colostrum growth factor.
3. the drug for the treatment of ulcerative colitis according to claim 1 or 2, which is characterized in that the colostrum is raw
The long factor is the growth factor crude extract extracted from colostrum.
4. the drug for the treatment of ulcerative colitis according to claim 1, which is characterized in that the lactobacillus acidophilus
The quantity ratio of KLDS20010, lactobacillus plantarum KLDS00170 and Lactobacillus helveticus KLDS10010 are 1：1：1.
5. it is according to claim 1 treatment ulcerative colitis drug, which is characterized in that the viable bacteria thallus be by
It is in the bacterium mud that the lactobacillus liquid culture at stable initial stage is collected by centrifugation respectively.
6. the preparation method of the drug for the treatment of ulcerative colitis described in claim 1,4-5 any one, which is characterized in that
Include the following steps：
（1）Lactobacillus acidophilus KLDS20010, lactobacillus plantarum KLDS00170 and Lactobacillus helveticus KLDS10010 are existed respectively
MRS culture medium culture；
（2）The Lactobacillus helveticus KLDS10010 of 14 ~ 18h of culture is chosen respectively, cultivates the lactobacillus acidophilus of 12 ~ 16h
The lactobacillus plantarum KLDS00170 of 12 ~ 16h of KLDS20010 and culture；
（3）Step is taken respectively（2）Obtained Lactobacillus helveticus KLDS10010 0.77 ~ 1.15ml of bacterium solution, lactobacillus acidophilus
Then KLDS20010 0.4 ~ 0.6ml of bacterium solution, 0.15 ~ 0.23ml parts of lactobacillus plantarum KLDS00170 bacterium solution, centrifuging and taking bacterium mud are divided
They are not dissolved in the sterile PBS of 0.1ml, obtains mixing bacterium mud；
（4）To colostrum centrifugal degreasing, the heavy casein of acid, second ultrafiltration, dialysis and it is freeze-dried obtained colostrum growth factor
Crude extract；
（5）4.32 ~ 6.84mg of colostrum growth factor crude extract is added in above-mentioned mixing bacterium mud, is stirred evenly, then plus
Enter suitable auxiliary material and clinically-acceptable dosage form is made.
7. the preparation method of the drug for the treatment of ulcerative colitis according to claim 6, which is characterized in that including as follows
Step：
（1）Lactobacillus acidophilus KLDS20010, lactobacillus plantarum KLDS00170 and Lactobacillus helveticus KLDS10010 are existed respectively
MRS culture medium culture；
（2）The probiotics at the beginning of stationary phase in the logarithm end of term is chosen, i.e., chooses Lactobacillus helveticus KLDS10010, the training of culture 16h respectively
Support the lactobacillus plantarum KLDS00170 of the lactobacillus acidophilus KLDS20010 and culture 14h of 14h；
（3）Step is taken respectively（2）Obtained Lactobacillus helveticus KLDS10010 bacterium solution 0.96ml, lactobacillus acidophilus KLDS20010 bacterium
Liquid 0.5ml, 0.19ml parts of lactobacillus plantarum KLDS00170 bacterium solution, centrifuging and taking bacterium mud, then respectively by they be dissolved in 0.1ml without
In bacterium PBS, mixing bacterium mud is obtained；
（4）To colostrum centrifugal degreasing, the heavy casein of acid, second ultrafiltration, dialysis and it is freeze-dried obtained colostrum growth factor
Crude extract；
（5）Colostrum growth factor crude extract 5.4mg is added in above-mentioned mixing bacterium mud, is stirred evenly, it is suitable to be then added
Auxiliary material clinically-acceptable dosage form is made.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510810779.8A CN105327338B (en) | 2015-11-23 | 2015-11-23 | A kind of drug and preparation method thereof for treating ulcerative colitis |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510810779.8A CN105327338B (en) | 2015-11-23 | 2015-11-23 | A kind of drug and preparation method thereof for treating ulcerative colitis |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105327338A CN105327338A (en) | 2016-02-17 |
| CN105327338B true CN105327338B (en) | 2018-11-23 |
Family
ID=55278243
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510810779.8A Active CN105327338B (en) | 2015-11-23 | 2015-11-23 | A kind of drug and preparation method thereof for treating ulcerative colitis |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105327338B (en) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112218646B (en) * | 2018-05-31 | 2024-06-11 | 深圳华大生命科学研究院 | Composition and application thereof |
| AU2021229110B2 (en) * | 2020-02-26 | 2024-05-23 | Gi Biome | Lactobacillus Helveticus strain and composition containing same for prevention or treatment of inflammatory diseases |
| CN114231470B (en) * | 2022-01-26 | 2023-04-28 | 江南大学 | Lactobacillus acidophilus capable of relieving ulcerative colitis and application thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103146738A (en) * | 2013-01-31 | 2013-06-12 | 武汉工业学院 | Construction method and purpose of recombinant lactobacillus acidophilus expressing pig epidermal growth factors |
-
2015
- 2015-11-23 CN CN201510810779.8A patent/CN105327338B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103146738A (en) * | 2013-01-31 | 2013-06-12 | 武汉工业学院 | Construction method and purpose of recombinant lactobacillus acidophilus expressing pig epidermal growth factors |
Non-Patent Citations (2)
| Title |
|---|
| 牛初乳中生长因子的研究进展;王珺等;《农产品加工》;20110215(第3期);第71页-73页 * |
| 牛初乳生长因子粗提物修复溃疡性结肠炎的研究;刘宾等;《中国乳品工业》;20150731;第43卷(第7期);第21页引言第2段及第22页1.2.3节 牛初乳生长因子粗提物的提取 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105327338A (en) | 2016-02-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105343133B (en) | A kind of compound probiotic that treating ulcerative colitis, drug and preparation method thereof | |
| KR102085787B1 (en) | Nanovesicles derived from Bacillus bacteria and Use thereof | |
| US11944652B2 (en) | Nano-vesicles derived from genus Sphingomonas bacteria and use thereof | |
| JP2007518693A (en) | Stable liquid probiotic composition, its preparation and application | |
| TWI607758B (en) | Lactobacillus paracasei strain gmnl-653 and composition having the same for improving psoriasis symptoms | |
| CN105012350B (en) | Probiotics clostridium butyricum bacterial strain | |
| CN105327338B (en) | A kind of drug and preparation method thereof for treating ulcerative colitis | |
| JP7264531B2 (en) | Morganella-derived nanovesicles and uses thereof | |
| JP2022058656A (en) | Nanovesicles derived from micrococcus bacteria and use thereof | |
| CN103446552B (en) | For the fermentation composition of prevention and therapy digestive system disease | |
| KR20160140565A (en) | Composition comprising extracellular vesicles derived from Akkermansia muciniphila and Bacteroides acidifaciens as an active ingredient for treating or preventing inflammatory disease | |
| JP4540376B2 (en) | Lactic acid bacteria production substances | |
| CN105343132B (en) | Composition, the drug and preparation method thereof for treating colitis | |
| CN112121068A (en) | Use of lactobacillus johnsonii for the prevention and/or treatment of inflammatory bowel disease | |
| CN110464756A (en) | It is a kind of for adjusting the gegen qinlian decoction water extract of fat body | |
| CN104120093B (en) | Bifidobacterium longum and application thereof, and functional food composition and preparation method thereof | |
| RU2546253C2 (en) | Method of obtaining personified autoprobiotic product and method of treating syndrome of irritable bowl with thereof application | |
| CN112043723B (en) | Application of bacillus amyloliquefaciens exopolysaccharide | |
| CN107400635A (en) | Import rhodotorula glutinis recombinant bacterial strain of α MSH polypeptides and its preparation method and application | |
| US20220090171A1 (en) | Nanovesicles derived from bacteria of genus deinococcus, and use thereof | |
| RU2505304C2 (en) | Method for preparing autoprobiotic containing living bifidus bacteria and lactic acid bacilli | |
| Chen et al. | Lactobacillus gasseri relieves diarrhea caused by enterotoxin-producing Escherichia coli through inflammation modulation and gut microbiota regulation | |
| RU2176668C1 (en) | Strain of bacterium lactobacillus acidophilus nv ep 317/402 "narine" tnci used for preparing curative-prophylactic preparations for intestine microflora normalization | |
| CN112933116B (en) | Application of lactobacillus plantarum in preparation of vascular protecting agent | |
| TWI791918B (en) | Use of mycelium of deinococcus spp. for manufacturing pharmaceutical composition for angiogenesis inhibition |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |