CN106620662A - Application of low-iron-saturation bovine lactoferrin in preparation of medicine for treating helicobacter pylori infectious gastropathy - Google Patents
Application of low-iron-saturation bovine lactoferrin in preparation of medicine for treating helicobacter pylori infectious gastropathy Download PDFInfo
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- 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
- A61K38/40—Transferrins, e.g. lactoferrins, ovotransferrins
Abstract
The invention relates to an application of low-iron-saturation bovine lactoferrin in preparation of a medicine for treating helicobacter pylori infectious gastropathy. The low-iron-saturation bovine lactoferrin is bovine lactoferrin of which the iron saturation is less than 15%. The gastropathy includes acute or chronic gastritis, peptic ulcer, gastric mucosa fall-off and too-high gastric acidity. Low-iron-saturation lactoferrin is extracted and prepared by taking fresh and raw milk, bovine colostrum, whey powder, and transgenic milk as raw materials, can be used as an active ingredient of a medicine, a supplementary medicine and a health food for treating the acute or chronic gastritis, the peptic ulcer, the gastric mucosa fall-off and the too-high gastric acidity, is significant in curative effect and does not have any side effect. Meanwhile, the production method of the low-iron-saturation bovine lactoferrin provided by the invention has the advantage of high lactoferrin extraction and preparation purities, and can enable the iron saturation to be lowered to 6-8% at the same time to keep consistency, thereby obviously improving antibiotic activity of the lactoferrin, and achieving standardized large-scale production of a medicine while ensuring consistent quality of the medicine.
Description
Technical field
The present invention relates to gastropathy technical field of pharmaceuticals, specifically a kind of low ferrum saturation Bovine Lactoferrin is to prepare treatment deep and remote
Application in door helicobacter infection stomach medicine.
Background technology
Antibiotic being selected the current treatment to helicobacter pylori more.But life-time service antibiotic easily causes antibacterial to it
Produce drug resistance.Therefore a kind of efficiently killing drug resistance helicobacter pylorus bacteria preparation is developed, becomes the task of top priority.
Lactoferrin has antimicrobial acivity to be combined for the energy of ferrum necessary to some bacterial growths because it has
Power.Some researcheres (Bishop etc., 1977；Payne etc., research 1990) confirms that the antimicrobial acivity of lactoferrin is relied on
In its ferrum saturation.Batish et al. (1988) also has found the antibacterial activity of low ferrum saturation lactoferrin than high ferro saturation
The antibacterial activity of lactoferrin is high.Chinese patent CN200510095244.3 discloses one kind and extracts from milk or whey water
Method with high-purity lactoferrin is prepared, although wherein the lactoferrin purity for preparing is higher, due to the place of production of milk supply
The kind of difference, such as milch cow, raise ground and it is feeds utilized there is certain difference, therefore cause lactoferrin in milk
Ferrum saturation there is larger randomness, it is difficult to meet its standardization, the needs of scale application.Should to widen lactoferrin
With, in order to ensure the concordance of effect, need lactoferrin keep stable and consistent saturation；At present ferrum saturation is kept into one
By way of the high saturation of cause can be adding iron ion；But in order to ensure effect in the production process of antimicrobial DP finish
Stability, need lactoferrin keep stable and consistent low ferrum saturation, so as to realize standardization, the scale metaplasia of medicine
Produce；But the production technology that lactoferrin is realized consistent low ferrum saturation is yet there are no, causes medicine antibacterial activity to fail
Reach optimum efficiency；Therefore, the low ferrum saturation of lactoferrin how is realized, it is met as the standardized requirement of pharmaceutical raw material,
Also problem demanding prompt solution is become.
The content of the invention
For the deficiencies in the prior art, the present invention provides a kind of low ferrum saturation Bovine Lactoferrin and is preparing treatment pylorus spiral shell
Application in swing arm bacterium infected gastric disease medicine, its primary and foremost purpose is used for low saturation Bovine Lactoferrin can in stomach medicine
Effectively suppress helicobacter pylori；Simultaneously the present invention also provides a kind of low ferrum saturation Bovine Lactoferrin production technology, it is ensured that institute
The concordance of the lactoferrin iron saturation of the low ferrum saturation for obtaining, so as to realize standardization, the large-scale production of medicine.
Application of the low ferrum saturation Bovine Lactoferrin in treatment HP infected stomach medicine is prepared, it is described
Low ferrum saturation Bovine Lactoferrin is the Bovine Lactoferrin of ferrum saturation ＜ 15%.It is preferred that the Lac Bovis seu Bubali ferrum of ferrum saturation 6%～8%
Albumen.
The gastropathy includes acute or chronic gastritis, peptic ulcer, gastric mucosa comes off and gastric acidity is too high.
The preparation process of the low ferrum saturation lactoferrin is：
1st step, is adsorbed, is parsed using cation exchange resin to the lactoferrin in Lac Bovis seu Bubali；The resolving
It is that, using two grades of parsings, first order parsing is parsed using 1～2.5% saline solution, and desorbed solution is lactoperoxidase pheron,
Discard；Second level parsing is parsed using 3.5～7% saline solution, and desorbed solution is sent into the 2nd step and processed；Described saline solution is
Refer to sodium salt, potassium salt or calcium salt soln；
2nd step, is concentrated to the desorbed solution that the 1st step is obtained using seperation film；Described seperation film is molecular cut off
In the ultrafilter membrane of 1000～30K；
3rd step, the concentrated solution that the 2nd step is obtained adopts insoluble Fe3+Chelating resin is adsorbed, then using seperation film to dense
Contracting liquid is concentrated；Need to add acetic acid, acetate and citrate in concentrated solution during the adsorption operations, prepare its suction
Citrate concentration is 0.1mol/L～0.5mol/L in follower ring border, and adjusts pH to 5.0～6.7；Described seperation film is to cut
Molecular weight is stayed in the ultrafilter membrane of 1000～30K.
Further, the concentrated solution that the 2nd described step is obtained, can again through a weak-type sun before the 3rd step is sent into
The absorption of ion exchange resin, solution echo the concentration of ultrafilter membrane；Described weak-type cationic resin refers to CM resins.
Further, the 2nd step obtains concentrated solution and can add Sodium Chloride before into the 3rd step, makes Sodium Chloride
Concentration reaches 5～10g/L, then concentrated solution is filtered with the ultrafilter membrane that molecular cut off scope is 200000～400000.
Further, in the 3rd described step, described insoluble Fe3+Chelating resin refers to D403 chelating resins；Resin
Adsorption time 20～120 minutes, rotating speed of agitator speed is 20～150rpm during absorption, and adsorption temp is at 3～30 DEG C.
Further, insoluble Fe in the 3rd described step3+Chelating resin can select the chelating tree prepared by following methods
Fat, comprises the steps：
S1, by weight, by 4～6 parts of phenol, 60～80 parts of mass concentrations 20～30wt% formalin
Mixing, in 40～45 DEG C of temperature ranges 1～5h of reaction is carried out, and adds 5～8 parts of 2,2- dihydroxypropionic acids and 35～40 parts of second
Glycol, stirs, and obtains pre-polymerization liquid；
S2, adds 35～55 part by weight of liquid paraffin and 3～5 weight portion sorbitan oleates in pre-polymerization liquid,
0.5～1h is stirred after being warming up to 60～65 DEG C, hydrochloric acid 0.5～0.8 weight portion and 2～4 of the concentration in 30～35wt% is added
Weight portion hydroxyl-containing silicone, after 1～5h of polyreaction in the range of 80～85 DEG C, filtration separates solid, with washing with alcohol, drying
Afterwards, chelating resin is obtained.
Several treatment HP infected stomach medicines based on low ferrum saturation Bovine Lactoferrin presented below：
The first, its composition include 6%～8% ferrum saturation Bovine Lactoferrin 80-200mg, rabeprazole 10-20mg,
Clarithromycin 300-500mg, Tinidazole 300-500mg.
Second, its composition includes 6%～8% ferrum saturation Bovine Lactoferrin 80-200mg, omeprazole 100-
200mg, amoxicillin 200-500mg.
Second, its composition includes 6%～8% ferrum saturation Bovine Lactoferrin 10%, sodium bicarbonate 10%, sodium carbonate
30%th, citric acid 38%, Sodium Chloride 2.4%, excipient 9.6%.
The present invention is extracted as raw material with fresh milk, whey powder and prepares low ferrum saturation lactoferrin, and it can be used as controlling
Treat acute or chronic gastritis, peptic ulcer, gastric mucosa to come off the effective of the medicine too high with gastric acidity, adjuvant drug and health food
Composition, it is evident in efficacy and and without any side effects.Additionally, the present invention provide method have lactoferrin extract with prepare it is pure
The high advantage of degree, while Fe in lactoferrin can be reduced3+Ion concentration, makes ferrum saturation be reduced to 6-8%, so as to substantially carry
The high antibacterial activity of lactoferrin.In addition by adjusting Fe3+Time of chelating resin absorption, mixing speed and medium it is dense
Degree, pH, can be controlled to the ferrum saturation after lactoferrin process, full to lactoferrin iron to meet different product application
With the requirement of degree.
Description of the drawings
Fig. 1 is the curve chart of impact of the NaHCO3 concentration to bacteriostatic activity.
Specific embodiment
Embodiment 1
The mechanism of action of Bovine Lactoferrin is studied below by way of grouping experiment.
First, In vitro Bactericidal Experiments of the Bovine Lactoferrin to drug resistance helicobacter pylori (HP)
(1) test objective
This experiment refers to carrying out external overriding resistance helicobacter pylori activity rating to Bovine Lactoferrin, and with Li Zhu groups
The bismuth potassium citrate (bismuth) of Li Zhu pharmaceutical factories production is compared, with the correct antibacterial work(evaluated and develop Bovine Lactoferrin
Effect.
(2) tested medicine
1st, trial target:7% ferrum saturation Bovine Lactoferrin (LF), total protein resultant 97%, LF purity 96%.
2nd, reference substance:Bismuth (bismuth potassium citrate), is produced, every gram of medicine 110mg containing bismuth by Livzon Pharmaceutical Factory, Livzon Group.
3rd, reference substance:14% ferrum saturation Bovine Lactoferrin, LF purity 96%；20% ferrum saturation Bovine Lactoferrin, LF
Purity 96%.
(3) antibacterial:Tried antibacterial is 30 plants of helicobacter pylori clinical separation strains, is in recent years from the fresh stomach of patient
Separate the method identifications such as acquisition, and Jing urease tests, Gram's staining microscopy in mucosa sample to confirm, wherein 6 plants of pylorus spirals
Bacillus is clinical drug-resistant strain.
(4) culture medium:Using HP culture medium and brucella broth.
(5) medicine is prepared
1st, Bovine Lactoferrin is prepared
Bovine Lactoferrin is directly configured to a series of pastille plate of variable concentrations with aseptic HP culture medium.
2nd, bismuth
First dissolving bismuth with distilled water, then make a series of doubling dilutions with the culture medium of sterilizing makes to make containing variable concentrations
Drug plate.
(6) test method
1st, minimal inhibitory concentration (MIC):
The HP culture medium flat plates (the μ g/mg～0.125 μ g/mg of concentration 32) of different ferrum saturations LF.By 30 plants of pylorus spirals
Bacillus (including 6 plants of clinical resistance HP bacterial strains) culture is divided into A, B two batches (per criticizing 15 plants), is respectively matched somebody with somebody with brucella broth
Concentration is made for 1x106The bacterium solution of/ml.The ring bacterium solution streak inoculation of inoculating loop picking 1 with internal diameter as 2mm is to variable concentrations medicine
The surface of flat board, while being inoculated with the not flat board of drug containing as control, marking lengths are 1cm or so.In 37 DEG C of micro- aerobic rings
Cultivate in border and bacterial growth situation is observed after 48h.The minimum concentration of contained drug is used as minimum suppression using in without bacterial growth plate
Bacterium trip degree (MIC).
The results are shown in Table 1-1, table 1-2.
The MIC of table 1-1 difference ferrum saturation Bovine Lactoferrin and bismuth to HP bacterial strains
MIC of the ferrum saturation Bovine Lactoferrin of table 1-2 7% to clinical drug-resistant HP bacterial strain
| Strain name (bacterial strain number) | Medicine | MIC scopes |
| Helicobacter pylori (30) | 7% ferrum saturation bLF | 0.5625～3mg/ml |
2nd, killing curve
(1) it is 10 by 0.1ml concentration6The HP bacterium solutions of/ml, are separately added into ferrum saturation for 20%, 14%, 7% Lac Bovis seu Bubali ferrum
During protein concentration is for the solution of (2.5mg/ml, 5mg/ml, 10mg/ml), mix, cultivate in 37 DEG C of shaking table, respectively at
0.1ml samples are drawn when 0.5h, 1h, 1.5h and 2h and coats HP culture medium flat plates surface, be incubated after 48h in 37 DEG C of micro- oxygen environments
Viable count is counted, and draws out the time-killing curve.
(2) it is 10 by 0.1ml concentration6The HP bacterium solutions of/ml are added in bismuth solution of the 0.9ml containing 4 μ g, are equally painted by upper method
Make the controlled trial of the time-killing curve as known drug of bismuth.Different ferrum saturation bLFs and bismuth are in each time
The logarithm value for counting viable count is shown in Table 2.
The logarithm value of the different ferrum saturation bLFs of table 2 and bismuth in each time counting viable count
3rd, impact of the condition of culture to MIC
(1) impact of the pH value to MIC
The HP of the pastille culture medium of each concentration is separately adjusted to angularly into 5,7,9 three kind of state, determine the Drug plates to 2 plants of HP
The MIC changes of test strain, the results are shown in Table 3
Impact of the different PH of table 3 to MIC
(2) impact of the bacterial load to MIC
On the premise of other conditions are constant, change bacterial load (103、106、109The more different bacterium amounts of/ml are to MIC
Impact, the results are shown in Table 4.
Impact of the bacterial load of table 4 to MIC
| Quantity of microorganism inoculated | Bovine Lactoferrin MIC (mg/m1) | Bismuth MIC (μ g/m1) |
| 103/ml | 0.5625 | 1 |
| 106/ml | 1.5 | 2 |
| 109/ml | 3 | 2 |
(3) impact of the NaHCO3 concentration to bacteriostatic activity
The blf that ferrum saturation is 20%, 14%, 7% is dissolved in NaHCO3 solution, blf concentration is 2.5mg/ml, is determined
Antibacterial circle diameter to H.pylori, is as a result shown in Fig. 1.Fig. 1 show HCO3- concentration be 100mmol/L when, the inhibition zone of generation
Diameter is almost 1.7～2.2 times of matched group.This can improve blf iron-binding capacity with the concentration increase of HCO3- certain pass
System.
2nd, Experiment on therapy of the Bovine Lactoferrin to Helicobacter pylori infection animal
(1) materials and methods:
1st, laboratory animal:Mongolian gerbil, 160 ± 10g of body weight, monthly age 2～3 and male and female dual-purpose, health is without diarrhoea, and point cage is general
Logical forage feed.
2nd, Experimental agents:
(1) 8% ferrum saturation Bovine Lactoferrin；
(2) vinegar, it is permanent along board Zhenjiang mature vinegar, bottled 500ml, PH:4～5
(3) bismuth potassium citrate (bismuth), is produced, every gram of medicine 110mg containing bismuth by Livzon Pharmaceutical Factory, Livzon Group.
(4) Ampicillin, the production of Hong Kong pharmaceutical factory of federation.
3rd, experimental technique:
(1) duplication of Rats with Spleen-deficiency Helicobacter pylori infection model
Improved with reference to Peng Shi methods, the daily empty stomach gavage vinegar 2ml/100g of modeling rat, formed acidophilus monophagia, totally 10
My god, odd-numbered day fasting, even-numbered days do not limit food, and free water forms irregular diet, so that eating and drinking without temperance.11st day using constraint on an empty stomach
Water-bath (29 ± 15 DEG C of greenhouse, 0.5 DEG C of 23 scholar of water temperature) forms tired cold stimulation in 6 hours, makes rat under above-mentioned factor collective effect
Produce insufficiency of the spleen symptom.Empty stomach gavage 1x10 from 12nd day9The HP suspensions of cfu/ml, the next day dosage 1.5ml/ once, totally three times,
And fasting is raised the next day continuation.
(2) experiment packet
Rat is randomly divided into 10 groups:
1. the insufficiency of the spleen infected group of not medication, daily cold boiling water 4ml gavage.
2. positive medication control I groups are treated 10 days with bismuth potassium citrate, positive II groups Ampicillin
Treatment 3 days.
3. Lf treatments divide totally eight groups of A groups, B groups, C groups, D groups, E groups, F groups, G groups, H groups.
(3) method
Give within 17th day medicine for treatment, insufficiency of the spleen infected group feeds distilled water.Positive control I groups feed bismuth potassium citrate 0.35g/kg,
Positive II groups Ampicillin 0.53g/kg, 7% ferrum saturation Bovine Lactoferrin heavy dose A group 2.0g/kg Mus, middle dosage B group
0.45g/kg Mus, low dose of C groups 0.05g/kg, continuous use 10 days.D group 0.45g/kg Mus, medication 3 days, E groups 0.45g/kg
Mus, medication 5 days, F groups 0.45g/kg, medication 7 days, G groups 0.45g/kg, medication 10 days, H groups 0.45g/kg, 15 days positives of medication
Control II groups are put to death after treating 3 days, are taken gastric mucosa tissue and are conducted a survey.
(4) Testing index:
1st, HP cultures:45 parts of gastric mucosa biopsy specimen, is taken respectively from Antrum in Rats, and gastric mucosa tissue is put
Enter 20%GS with liquid, in 4 DEG C of 3h laboratory is delivered to, sterilizing tissue mucosa is ground into into homogenate, coat ECY culture medium,
Cultivate 3-6 days for 37 DEG C in micro- aerobic environment, observe bacterium colony after 3 days day by day.Its suspicious bacterium colony makees smear staining, microscopy, and makees fast
Fast urease test, such as has typical bacterium, thalli morphology and dyeing characteristic, and rapid urease test positive is accredited as HP.
2nd, rapid urease test.
3rd, conventional H E dyeing tissues observed morphological change.
4th, W-S silver stainings observation HP gradient of infection, HP is checked and is pressed Marshal1 standard gradings.
5th, electron microscopic observation gastric mucosa is repaired and HP infection conditions.
(2) result:
1st, detection situation of each detection method to helicobacter pylori, is shown in Table 5
Each group HP of table 5 detects situation
2nd, conventional H E dyeing tissues observed morphological change.
Main detection gastric mucosa inflammatory reaction.Inflammatory cell is distributed in stomach more based on lymphocyte and oxyphil cell,
In the bottom of mucosa and stomach submucosa connective tissue, inflammation is divided into level Four by the quantity with inflammatory cell as standard, and " 0 " is
Basic NIP cell occurs, " 1 " visible a small amount of inflammatory cell, and " 2 " are moderate inflammation cell, " 3 " visible a large amount of inflammatory cells.
The results are shown in Table 6
Table 6HE dyeing observation inflammatory conditions tables
**P<0.01, * P<0.05
3rd, Mus gastric mucosa electron microscopic observation:
Visible mucosal epithelium structural integrity under high dose group (0.5g/ml) Electronic Speculum, microvilluss are visible, and endoplasmic reticulum is clear, rich
Richness, secretory granule enrich.
Visible mucosal epithelium structure is substantially complete under middle dose group (0.11g/ml) Electronic Speculum, and micro-line hair is visible, secretory granule
It is more.
Visible most of mucosal epithelium structural integrity under low dose group (0.01g/ml) Electronic Speculum, micro-line hair is visible, secretion
Grain is less.
Visible top layer mucosal epithelial cells structural integrity under positive controls Electronic Speculum, microvilluss are visible, full of secretory granule.
Visible most of top layer mucosal epithelial cells necrosis under model group Electronic Speculum, imperfect, microvilluss are less, secretory granule
It is few.
(3), conclusion
Bovine Lactoferrin has certain inhibitory action to HP, while the gastric mucosa injury to being caused by HP has repair.
3rd, Therapy study of the Bovine Lactoferrin to infection helicobacter pylori mice
(1) materials and methods:
1st, laboratory animal:C57BL/6 mices, the scholar 20g of body weight 17,7 week old are female, and health is without diarrhoea, and point cage is common
Forage feed.
2nd, Experimental agents:
(1) 7% ferrum saturation Bovine Lactoferrin.
(2) bismuth potassium citrate (bismuth), is produced, every gram of medicine 110mg containing bismuth by Livzon Pharmaceutical Factory, Livzon Group.
(3) bacterial strain:HP bacterial strains are provided by Zhejiang University's medical college.
3rd, experimental technique:
Fasting 12 hours before all animal gavages, by with the HP bacterium gavage mices of brucella broth eluting, 0.4ml/ is only (about
109/ m1), continuous 5 times, complete within 1 week；Calculate from last time gavage HP liquid, Bovine Lactoferrin, the big agent of mice point are fed after 4 weeks
Amount group (0.1875g/kg body weight), middle dose group (0.0938g/kg body weight), low dosage (0.0469g/kg body weight), bismuth potassium citrate
Matched group (0.148g/kg body weight), model group, is administered 10 days altogether by 10 per group.Last dose one day after, puts to death mice, takes stomach
Mucous membrane tissue, HE dyeing and W-S silver stainings observe HP gradient of infection, while carry out urease test and HP culture (take Mouse Stomach stick
Film specimen, is placed in -70 DEG C of preservations in specimen preserving liquid.Specimen is taken in dismembyator, the specimen lapping liquid of Deca about 0.2ml is carried out
Grinding, lapping liquid is coated on ECY selective mediums, 5%02, 15%C02, 8.0%N2, micro- aerobic culture 5 days, observation
Colonial morphology, typical HP colonial morphologies are needle point sample size, and water white transparency, surface is smooth, moistening, glossy, picking part
Antibacterial carries out RUT experiment and gram stain microscopy, and typical HP should be RUT experiment strong positive, and microscopy is
Gram-negative, form is in flying bird shape or spiral rod)
(2) result:
1st, detection situation of each detection method to helicobacter pylori, is shown in Table 7
Detection situation of each detection method of table 7 to helicobacter pylori
(3) conclusion
Bovine Lactoferrin has certain inhibitory action to the HP of C57BL/6 mouse infections.Bacteriology's inspection therein (looks into the positive
Rate) and the result of urease-positive rate result and bismuth potassium citrate it is also low 10 percentage points.
4th, impact of the Bovine Lactoferrin to water logging stress gastric ulcer animal model
(1) test objective:
Impact of the research Bovine Lactoferrin to water logging stress in rats gastric ulcer.
(2) medicine and experimental technique
1st, 7% ferrum saturation Bovine Lactoferrin.
2nd, bismuth potassium citrate (bismuth), is produced, every gram of medicine 110mg containing bismuth by Livzon Pharmaceutical Factory, Livzon Group.
3rd, animal SD rats, body weight (200～250g), male and female half and half are provided by gradually river Provincial Medicine Research Institute Animal House.
4th, test method:
SD rats 50 are taken, body weight (200～250g), male and female half and half are randomly divided into 5 groups, 10/group, set up blank right
According to the Bovine Lactoferrin group (1.4g/kg, 0.7g/kg, 0.28g/kg) and bismuth potassium citrate positive controls (0.8g/ of three dosage
), kg after ten days, then fasting 24h, free water loads animal in special cage medicine feed, in being dipped vertically into 23 ± 0.5 DEG C of water.
Immersion depth translation thing xiphoid-process level, takes out after 24h, and dislocation of cervical vertebra is put to death.Open abdominal cavity, ligation stomach cardia and pylorus and Jing stomaches
Wall takes out stomach in immersion formalin to formalin 8m1 of stomach intracavity injection 1%, and tailing edge greater gastric curvature is cut open within 30 minutes, meter
Area of ulcer and ulcer index that number glandular stomach portion occurs.Mucosal lesion length (mm) is measured under anatomic microscope, with its summation
As ulcer index, it is compared between group.
5th, statistical method
Experimental data is expressed as mean+SDThe significance of group difference is carried out with the inspections of t mono-.
(3), result of the test
As a result show, Bovine Lactoferrin 0.7g/kg, 2.8g/kg can substantially reduce the ulcer index of rat.With blank group ratio
Compared with there were significant differences (P<O.o5 or P<o.o1).
The results are shown in Table 8
The Bovine Lactoferrin of table 8 stress cause the impact of rat acute mucosal lesion to water-bath
(X ± s, N=10)
**P<0.01,*P<0.05, compare with blank control group
(4), conclusion
This test stress cause rat acute mucosal lesion using water logging, with mucosal lesion length as index, study cattle
Lactoferrin stress cause the impact that gastric mucosa is damaged to water logging.As a result show, Bovine Lactoferrin can reduce the ulcer of rat
Index (reduce by 78%, higher than bismuth potassium citrate by 48%), suppression ratio asking in 28-78%, than bismuth potassium citrate suppression ratio more be higher by
20 percentage points.Lac Bovis seu Bubali ferrum egg self energy reduces gastric mucosa damaged length, unrestrained to water to cause rat acute mucosal lesion
There is certain protective effect.
5th, Bovine Lactoferrin causes the impact of rat gastric ulcer to acetic acid calcination.
(1) test objective
Research Bovine Lactoferrin causes the impact of rat gastric ulcer to acetic acid calcination.
(2) medicine
1st, 7% ferrum saturation Bovine Lactoferrin.
2nd, bismuth potassium citrate (bismuth), is produced, every gram of medicine 110mg containing bismuth by Livzon Pharmaceutical Factory, Livzon Group.
3rd, motilin (MTL) test kit:There is provided by medical college thrombosis research department of University Of Suzhou.
4th, prostaglandin (PEG2) test kit:There is provided by Beijing Fu Rui companies.
(3) animal:
SD rats, body weight (200～250g), male and female half and half are provided by gradually river Provincial Medicine Research Institute Animal House.
(4) test method
Take SD rats 50, body weight (200～250g), male and female half and half, fasting 24h before test, free water, in ether fiber crops
Liquor-saturated lower opening abdominal cavity, the glass tubing of the long 30mm of internal diameter 5mm is disposed vertically on body of stomach serosal surface, and ice second is added into tube chamber
Glacial acetic acid, suture operation otch are dipped in out after sour 0.2m1,1.5 minutes with cotton swab.Postoperative normal diet, is randomly divided into 5 in second day
Group, sets up blank by 10/group, the Bovine Lactoferrin group (1.4g/kg, 0.7g/kg, 0.28g/kg) of three dosage and beautiful
Pearl obtains happy positive controls (0.8g/kg), continuous medicine feed ten days.Dissect and take out stomach, and fixed with formaldehyde, measure ulcer area
(rhm2), it is compared between each group.Blood is taken simultaneously, serum is separated, and surveys motilin (MTL) and prostaglandin (PGE2).
(5), statistical method
Experimental data is expressed as mean+SDThe significance of group difference is carried out with the inspections of t mono-.
(6), result of the test
The results are shown in Table 9
Impact of the Bovine Lactoferrin of table 9 to the calcination type ulcer of acetic acid
| Group dosage (g/kg) | Number of animals (n) | Ulcer area | Suppression ratio (%) |
| Blank model control group | 10 | 41.20±10.01 | |
| Bovine Lactoferrin (1.4g/kg) | 10 | 13.93±9.78 | 66.19 |
| Bovine Lactoferrin (0.7g/kg) | 10 | 26.57±8.18 | 35.51 |
| Bovine Lactoferrin (0.28g/kg) | 10 | 35.81±9.64 | 13.08 |
| Bismuth potassium citrate (0.8g/kg) | 10 | 29.40±8.12 | 28.64 |
**P<0.01,*P<0.05, compare with blank control group
Impact of the Bovine Lactoferrin of table 10 to acetic acid calcination type rat motilin (MTL) and prostaglandin (PGE2)
*P<0.05, compare with blank control group
(7) conclusion
This test causes gastric mucosa to damage using acetic acid calcination, with ulcer area as index, studies Bovine Lactoferrin pair
Acetic acid calcination causes the impact that gastric mucosa is damaged.As a result show, Bovine Lactoferrin can reduce the ulcer index of rat (to be reduced
65%, higher than bismuth potassium citrate 52%) suppression ratio is higher by 37 percentage points between 13-66% than the suppression ratio of bismuth potassium citrate,
Causing gastric mucosa to damage acetic acid calcination has certain protective effect.
6th, Bovine Lactoferrin causes the impact of rat acute mucosal lesion to pylorus ligation
(1) test objective
Observation Bovine Lactoferrin causes the impact of acute gastric mucosal injury to pylorus ligation.
(2) medicine
1st, 7% low ferrum saturation Bovine Lactoferrin.
2nd, bismuth potassium citrate (bismuth), is produced, every gram of medicine 110mg containing bismuth by Livzon Pharmaceutical Factory, Livzon Group.
(3) animal
SD rats, body weight (200～250g), male and female half and half are provided by Inst. of Traditional Chinese Medicine, Zhejiang Prov Animal House.
(4) test method
SD rats 50 are taken, body weight (200～250g), male and female half and half are randomly divided into 5 groups, 10/group, set up blank right
According to the Bovine Lactoferrin group (1.4g/kg, 0.7g/kg, 0.28g/kg) and bismuth potassium citrate positive controls (0.8g/ of three dosage
), kg medicine feed ten days.Fasting 36 hours, free water fixes under etherization rat, and along ventrimeson an osculum is cut off, and finds out
Stomach simultaneously ligatures pylorus.Dissect stomach within postoperative 18 hours, to stomach intracavity 1% formalin 8m1 is injected.Stomach is immersed into 1% formalin
In solution, after 10min, stomach is cut off along greater gastric curvature, a situation arises for stomach ulcer before observation.The evaluation of ulcer level, Ke Yiji
The area of stomach ulcer before number, using its summation as ulcer index.Also okabe methods are commonly used, under anatomic microscope ulcer is measured
Area, is divided into 5 grades, as ulcer index by the summation of every Mus ulcer area.
Can further be calculated according to ulcer index:
Ulcer suppression percentage=(matched group ulcer index-administration group ulcer index)/matched group ulcer index * 100%
(5) statistical method
Experimental data is expressed as mean+SDThe significance of group difference is carried out with the inspections of t mono-.
(6) result of the test
The results are shown in Table 11
Impact of the Bovine Lactoferrin of table 11 to pylorus ligation ulcer
**P<0.01,*P<0.05, compare with blank control group
This test of conclusion causes rat acute mucosal lesion using pylorus ligation, with ulcer index as index, studies Lac Bovis seu Bubali
Ferritin causes the impact that gastric mucosa is damaged to pylorus ligation.As a result show, Bovine Lactoferrin can reduce the ulcer of rat and refer to
Number, suppression ratio asking in 31-51%, than bismuth potassium citrate suppression ratio more be higher by 12 percentage points. to pylorus ligation cause rat urgency
Property mucosal lesion has certain protective effect.
7th, Bovine Lactoferrin causes the impact that gastric mucosa is damaged to ethanol
(1) experiment purpose
Research Bovine Lactoferrin causes the impact of rat acute mucosal lesion to ethanol.
(2) medicine
1st, 7% ferrum saturation Bovine Lactoferrin, is made into 0.14g/ml, 0.07g/ by Bovine Lactoferrin distilled water before use
The concentration of m1,0.028g/m1, the above-mentioned solution 1ml/100g of rat ig (gavage).Equivalent to people clinical application 15g, 7.5g, 3g.
2nd, bismuth potassium citrate:Produced by Livzon Pharmaceutical Factory, Livzon Group, prepared with distilled water before use, 2 wrap the 25ml that adds water, often
100g rat oral gavage 1ml.
(3) animal:SD rats, body weight (200 ± 20) g, male and female dual-purpose is carried by Inst. of Traditional Chinese Medicine, Zhejiang Prov Animal House
For.
(4) experimental technique
SD rats 50 are taken, body weight (200 ± 20) g, male and female dual-purpose is randomly divided into 5 groups, 10/group, sets up blank
Group, the Bovine Lactoferrin group (1.4g, 0.7g, 0.28g) and bismuth potassium citrate group positive controls (0.8g/kg) of three dosage is continuous
Medicine feed 10 days, after last dose, fasting 48 hours, free water gavages dehydrated alcohol (1ml/ is only) during experiment, solve after one hour
Stomach is taken, is fixed with 1% formalin.Degree of injury is represented with ulcer index.The length of streak damage is surveyed more than 1mm person
Its length is measured, 1 point is counted per mm, if its width doubles its point more than 1mm person, score sum is the animal:Ulcer index, respectively
It is compared between group.
(5) statistical method
Experimental data is expressed as mean+SDThe significance of group difference is carried out with the inspections of t mono-.
(6) result of the test
The results are shown in Table 12
Impact of the Bovine Lactoferrin of table 12 to ethanol-type ulcer
**P<0.01,*P<0.05, compare from matched group with sky
(7) conclusion
This test causes rat acute mucosal lesion with ethanol, and with ulcer index as index, research Bovine Lactoferrin is to second
Alcohol causes the impact that gastric mucosa is damaged.As a result show, Bovine Lactoferrin can reduce the ulcer index of rat, than bismuth potassium citrate drop
15% more than the ulcer index of low rat, than bismuth potassium citrate suppression ratio more be higher by 10 percentage points, to ethanol cause rat acute
Mucosal lesion has certain protective effect.
8th, impact of the Bovine Lactoferrin to rat gastric secretion
(-) experiment purpose:Impact of the observation Bovine Lactoferrin to rat stomach liquid measure, gastric acidity and pepsin.
(2) medicine
7% ferrum saturation Bovine Lactoferrin.
Positive control drug:Bismuth potassium citrate (bismuth) is produced by Livzon Pharmaceutical Factory, Livzon Group, every gram of medicine 110mg containing bismuth.
(3) animal
SD rats, body weight (200 scholar 20) g, male and female dual-purpose is provided by Inst. of Traditional Chinese Medicine, Zhejiang Prov Animal House.
(4) test method:
SD rats are randomly divided into 5 groups, 10 per group, set up respectively high dose group (LF 0.28g/200g rats), middle dose
Amount group (LF is 0.134g/200g rats), low dosage (LF is 0.056g/200g rats), (LF is 0.053g/ to blank control group
200g rats), positive control (equivalent people's dosage, daily 4 bag), blank control group (feeding distilled water), continuous medicine feed 10 days, last
Equal fasting 24 hours, can't help water after administration.Under etherization, osculum is cut off along abdomen median line, gently finds out stomach, ligation is deep and remote
Door, suturing them otch is taken out stitches after 4～6 hours and changes abdominal cavity ligation cardia, wins full stomach, bloodstain is cleaned with filter paper, along big curved
Gastral cavity is cut off in side, pours out gastric content and is collected in graduated centrifuge tube, records stomach liquid measure, then is centrifuged about 5 with 1,000-2,000g
Minute, remove the gastric contents such as food debriss (with the acidity that acidity measures gastric juice) and survey total acidity gastric juice with acid-base titrations.Profit
Decompose hemoglobin production L-Tyrosine with pepsin, then the amount of L-Tyrosine determined with the chromogenic reaction of phenol red reagent, so as to
Connect the pepsic activity of calculating.
The measure of total acidity gastric juice:Supernatant gastric juice 1ml is taken, plus phenolic red indicator 1 drips, and is titrated with the NaoH of 0.0lmol/L,
Until gastric juice is first in switching to after yellow not disappeared in red 2 seconds for terminal, record spends NaoH amount of solution.
The NaoH amount of solution that total acidity mmo1/L=is exhausted.
(2) determination of peptic activity
Supernatant gastric juice 0.1ml is taken, adds 1% bovine serum albumin (0.1N HCL preparations) 1ml37 DEG C of temperature to incubate 20 minutes, plus
Enter 2% trichloroacetic acid, and heat 5 minutes in boiling water, cooled and filtered..Filter liquor 0.2ml is taken, is added
0.8m10.125NNa2C03, solution, while Folin-Ciocalte reagents (5ml solution As and 0.5ml second liquid) are added, it is quiet after mixing
Colorimetric (700mn) after putting 30 minutes.Pepsin activity is calculated with L-Tyrosine standard curve.Stomach egg is represented with mmol/h L-Tyrosine
White enzyme output.
(5), result
The results are shown in Table 13
As a result show, Bovine Lactoferrin can suppress rat gastric secretion, suppress gastric acidity.
Impact of the Bovine Lactoferrin of table 13 to gastric juice
(6), conclusion
As a result show, 1.4g/kg Bovine Lactoferrin can suppress rat gastric secretion, reduce gastric acidity.Wherein low dosage
Group (0.23g/kg) can reduce gastric juice 36% compared with blank control group, reduce stomach total acidity 12%.
One kind presented below is extracted from Lac Bovis seu Bubali and prepares the low ferrum saturation lactoferrin of high-purity (i.e. institute in embodiment 1
Low ferrum saturation lactoferrin) method, it is respectively with placing the agitator tank of cation exchange resin adsorbing, desorb cattle
Lactoferrin in breast, and be aided with membrane separation technique and be isolated, concentrate, it is possible to achieve to the highly purified of lactoferrin；Connect down
Come, gained lactoferrin concentrated solution is placed into insoluble Fe3+The agitator tank of chelating resin (such as D403 chelates series plastics) comes
Absorption iron ion makes it lose iron ion to be changed into undersaturated condition, and is aided with membrance separation and is concentrated and purified.
When being adsorbed to lactoferrin using cation exchange resin, being parsed, rotating speed of agitator speed that agitator tank is adjustable
Rate is 20～100rpm；There are increasing vortex baffle plate, resin bed of the tank bottoms setting with screen plate, the resin on resin bed in tank medial wall
8～12cm of layer top highly locates to arrange fast guiding suction filtering tube.Various sensor pH meters for automatically controlling, weight are set in tank
Meter, conductivity meter and thermometer, pot liquid holding temperature is 3～60 DEG C.Lactoferrin adsorbent resin is cation exchange tree
Fat, the number of times and condition that resin absorption is desorbed can set according to the requirement of different product and product purity；During the absorption of resin
Between be 20～120 minutes, adsorption temp is at 3～30 DEG C.Desorbing for resin desorbs respectively lactoperoxidase using two-stage salinity
Pheron, lactoferrin.First order salinity is 1～2.5%, desorbs lactoperoxidase pheron, desorbs the time for 20～120
Minute；Second level salinity is 3.5～7%, desorbs lactoferrin, desorbs the time for 20～120 minutes；The stir speed (S.S.) for desorbing
For 20～150rpm.The time that resin is desorbed is 20～120 minutes, and it is sodium salt, potassium salt, calcium salt that resin desorbs salt used；Breast
Ferritin solution attached liquid is degerming by lactoferrin solution attached liquid by 0.05～1.4 micron of micro-filtration membrane, go remnants fat and other
Little particle；Carry out desalination and concentration with the ultrafilter membrane of 1K～50K molecular retention amounts again.
In one embodiment, in order to improve the purity of lactoferrin, desalination can be desorbed to the above-mentioned technique absorption of Jing
The absorption again through a weak-type cationic resin (such as CM resins) of the lactoferrin dope of concentration, solution echo the dense of ultrafilter membrane
What contracting was obtained.
In one embodiment, the 2nd step obtains concentrated solution and added Sodium Chloride before into the 3rd step, makes chlorination
The concentration of sodium reaches 5～10g/L, then concentrated solution was carried out with the ultrafilter membrane that molecular cut off scope is 200000～400000
Filter.
Concentrated solution obtained above is positioned over again insoluble Fe3+Chelating resin such as D403 chelating resins, addition acetum,
, used as medium, it is 0.05mol/L～1.0mol/L to prepare its absorption ambient concentration, adjusts pH4.0～6.7 for acetate, citrate,
Iron ion in stirring and adsorbing lactoferrin in addition, resin absorption mixing time, medium concentration, rotating speed, pH can be according to products to ferrum
The requirement setting (Jing measurings, adsorption time is maximum to ferrum effect of saturation degree, and plan takes 10～150 minutes) of saturation；Resin
Adsorption time 10～150 minutes, rotating speed of agitator speed is 10～150rpm, and adsorption temp is at 3～30 DEG C.Clear liquid after absorption
Desalination and concentration are carried out with the ultrafilter membrane of 1K-50K molecular retention amounts.Lactoferrin dope is carried out into vacuum lyophilization and makes jelly
Dry powder.
Resin Jing after absorbing process process, the iron ion for being adsorbed with by 1mol/L～3mol/L soak with hydrochloric acid by resin
Regeneration is desorbed, then is available for next batch absorption to be used with the flushing of RO water.
The preparation of the cation exchange resin of embodiment 2
Cation exchange resin used in the present invention can be prepared by following method：
1st step, takes 10g chloracetylated polystyrenes-divinylbenzene microspheres carrier (PS-acyl-Cl), adds 60ml tetra-
The swelling 12h of hydrogen furan, adds 40ml methanol, then according to weight ratio (PS-acyl-Cl)：Ethylenediamine (EDA)：Sodium bicarbonate
For 1：8：0.7, ethylenediamine and sodium bicarbonate are sequentially added, it is stirring reaction 24h at 80 DEG C, after reaction terminates, product is fallen
In entering sand core funnel, neutrality is washed to distillation, methanol filter wash is dried under vacuum to constant weight 3 times, obtains crosslinking EDA microsphere supported；
2nd step, claims 60g to take crosslinking EDA microsphere supported, in being placed in three-neck flask, adds the swelling 12h of dimethylformamide
Afterwards, 2- acrylamido -2- methyl propane sulfonic acids (AMPS) 16g, N- butoxy methyl acrylamide (NBMA) 3g, propylene are added
Sour double cyclopentenyl ester (DCPA), K2CO35g, tetrabutyl ammonium bromide (TBAB) 12g, return under stirring in the oil bath of uniform temperature
Stream reaction, reaction is transferred to product in sintered filter funnel after terminating, and distilled water diafiltration is used after cleaning 4 times with the HC1 of 5wt%
To neutral, finally with draining after methanol filter wash, constant weight is dried under vacuum to, obtains cation exchange resin.
The preparation of the cation exchange resin of embodiment 3
It is not add NBMA in the 2nd step with the difference of embodiment 3.
Cation exchange resin used in the present invention can be prepared by following method：
1st step, takes 10g chloracetylated polystyrenes-divinylbenzene microspheres carrier (PS-acyl-Cl), adds 60ml tetra-
The swelling 12h of hydrogen furan, adds 40ml methanol, then according to weight ratio (PS-acyl-Cl)：Ethylenediamine (EDA)：Sodium bicarbonate
For 1：8：0.7, ethylenediamine and sodium bicarbonate are sequentially added, it is stirring reaction 24h at 80 DEG C, after reaction terminates, product is fallen
In entering sand core funnel, neutrality is washed to distillation, methanol filter wash is dried under vacuum to constant weight 3 times, obtains crosslinking EDA microsphere supported；
2nd step, claims 60g to take crosslinking EDA microsphere supported, in being placed in three-neck flask, adds the swelling 12h of dimethylformamide
Afterwards, 2- acrylamido -2- methyl propane sulfonic acids (AMPS) 16g, acrylic acid double cyclopentenyl ester (DCPA), K are added2CO3 5g、
Tetrabutyl ammonium bromide (TBAB) 12g, under stirring in the oil bath of uniform temperature back flow reaction, reaction terminate after by product turn
In moving to sintered filter funnel, with distilled water diafiltration to neutrality after cleaning 4 times with the HC1 of 5wt%, finally with draining after methanol filter wash,
Constant weight is dried under vacuum to, cation exchange resin is obtained.
The absorption of the lactoferrin of embodiment 4, solution adhesion test
3 parts of 100ml 1.6wt% lactoferrin solutions of outfit take 20 μ l in 250ml beakers, respectively and enter high performance liquid chromatography
Instrument detects that peak area is respectively 1240.0034 (ten thousand), 1241.6533 (ten thousand) and 1240.0535 (ten thousand), respectively by 100gSPC70
Respectively 100g is placed in and above-mentioned fills 250ml lactoferrin solutions cation exchange resin in resin, embodiment 1 and embodiment 2
Beaker in, absorption stir speed (S.S.) be 60rpm, per 10min sampling once detected, experimental data such as following table finds out embodiment
4 and 5 cationic resins improve the production response rate, improve absorption and desorb the consumed time.In addition, drenching through saline solution again
Wash, solve attached calculating protein content.
Drip washing, the operation for desorbing are：Lactoperoxidase pheron, lactoferrin are desorbed respectively using two-stage salinity.The
One-level salinity is 1.5%, uses NaCl aqueous solutions, desorbs lactoperoxidase pheron, desorbs the time for 70 minutes；The
Two grades of salinity are 5.0%, use NaCl aqueous solutions, desorb lactoferrin, desorb the time for 80 minutes；The stirring for desorbing
Speed is 100rpm.Lactoferrin solution attached liquid is degerming by lactoferrin solution attached liquid by 0.2 micron of micro-filtration membrane, removes the fat of remnants
Fat and other little particles；Carry out desalination and concentration with the ultrafilter membrane of 2000 molecular retention amounts again.
As can be seen from the above table, can using the cation exchange resin in SPC70 resins, embodiment 2 and embodiment 3
Preferably obtain the absorption of lactoferrin, desorb effect, but it is in the response rate or otherwise varied.
The extraction of the lactoferrin of embodiment 5 is tested with preparing
Semi-skimmed milk 1890L, pH6.7, total protein is 3.38g/L, and lactoferrin concentration is 126mg/L, by SPC70 trees
Fat adds agitator tank, adsorbs 80min, and stir speed (S.S.) is 60rpm, collects the milk after adsorbing and uses RO water rinse resins, plus
Enter first order 100L 2.5%NaCl aqueous solutions and desorb 40min, speed of agitator 40rpm, collect and received with RO water wash after solution attached liquid
Collection；Add second level 100L 5.5%NaCl aqueous solutions to desorb 40min, speed of agitator 40rpm, collect and drenched with RO water after solution attached liquid
Wash collection resin 3 times.The stripping liquid of the second level and second RO leacheate merge collects common 180L, is transported to 0.8 μm micro-
Degerming, remove impurity is carried out in filter membrane equipment；Desalination and concentration is carried out with the device for ultrafiltration membrane of 30K molecular retention amounts, sampling detects its ferrum
Saturation is 20%；Lactoferrin dope is input to equipped with, except foreigh protein removing, collection has been adsorbed after foreign protein in CM resin containers
Lactoferrin solution continue carry out desalination and concentration with the device for ultrafiltration membrane of 10K molecular retention amounts, a small amount of dope therein is entered
Row lyophilization gained pale pink dry powder.Powder moisture 3.0%, ash 1.1%, total protein is 96.3%, ferrum saturation
20%；Remaining lactoferrin dope is input to again carry out deferrization equipped with placement D403 chelating resin tanks, adds 0.1mol/
L citrates, and Acetic acid-sodium acetate buffer adjusts pH to 6.0, in addition stirring and adsorbing iron ion, 120 points of resin adsorption time
Clock, rotating speed of agitator speed 60rpm.Lactoferrin solution continuation 10K molecular retention amounts after collecting to adsorb iron ion
Device for ultrafiltration membrane carries out desalination and concentration, and collecting dope carries out lyophilization gained pale pink dry powder.Powder moisture 3.0%, ash
Divide 1.0%, total protein is 96.3%, ferrum saturation 7%.
The extraction of the lactoferrin of embodiment 6 is tested with preparing
Semi-skimmed milk 1890L, pH6.7, total protein is 3.38g/L, and lactoferrin concentration is 126mg/L, using enforcement
The operation of the step of described in example 2 production cation exchange resin, by resin agitator tank is added, and adsorbs 80min, and stir speed (S.S.) is
60rpm, collects the milk after adsorbing and uses RO water rinse resins, adds first order 100L 2.5%NaCl aqueous solutions to desorb
40min, speed of agitator 40rpm, collect and collected with RO water wash after solution attached liquid；Add second level 100L 5.5%NaCl aqueous solutions
Desorb 40min, speed of agitator 40rpm, to collect and collect resin 3 times with RO water wash after solution attached liquid.The stripping liquid of the second level and
Second RO leacheate merges collects common 180L, and being transported in 0.8 μm of microfiltration film device carries out degerming, remove impurity；Use 30K molecules
The device for ultrafiltration membrane of interception carries out desalination and concentration, and sampling detects that its ferrum saturation is 20%；Lactoferrin dope is input to
Equipped with foreigh protein removing is removed in CM resin containers, the lactoferrin solution continuation 10K molecular retention amounts after foreign protein have been adsorbed in collection
Device for ultrafiltration membrane carry out desalination and concentration, by a small amount of dope therein carry out lyophilization gained pale pink dry powder.Powder is aqueous
Amount 3.2%, ash 1.1%, total protein is 96.7%, ferrum saturation 20%；Again remaining lactoferrin dope is input to into dress
Having in placement D403 chelating resin tanks carries out deferrization, adds 0.1mol/L citrates, and adjusts pH to 6.0, in addition stirring and adsorbing
Iron ion, resin adsorption time 120 minutes, rotating speed of agitator speed 60rpm.Lactoferrin after collecting to adsorb iron ion is molten
Liquid continues to carry out desalination and concentration with the device for ultrafiltration membrane of 10K molecular retention amounts, and collecting dope carries out lyophilization gained pale pink
Dry powder.Powder moisture 3.1%, ash 1.0%, total protein is 96.7%, ferrum saturation 7%.
The extraction of the lactoferrin of embodiment 7 is tested with preparing
It is with the difference of embodiment 5：Remaining lactoferrin dope is input to equipped with placement D403 chelating resin tanks
In carry out deferrization, add 1.0mol/L citrates, and Acetic acid-sodium acetate buffer adjusts pH to 4.0, in addition stirring and adsorbing ferrum from
Son, resin adsorption time 20 minutes, rotating speed of agitator speed 150rpm.Collect to adsorb iron ion after lactoferrin solution after
Continuous that desalination and concentration is carried out with the device for ultrafiltration membrane of 10K molecular retention amounts, collecting dope carries out lyophilization gained pale pink dry powder.
Powder moisture 3.0%, ash 1.0%, total protein is 96.4%, ferrum saturation 7%.
The extraction of the lactoferrin of embodiment 8 is tested with preparing
It is with the difference of embodiment 6：Remaining lactoferrin dope is input to equipped with placement D403 chelating resin tanks
In carry out deferrization, add 0.3mol/L citrates, and Acetic acid-sodium acetate buffer adjusts pH to 6.7, in addition stirring and adsorbing ferrum from
Son, resin adsorption time 70 minutes, rotating speed of agitator speed 80rpm.Collect to adsorb iron ion after lactoferrin solution after
Continuous that desalination and concentration is carried out with the device for ultrafiltration membrane of 10K molecular retention amounts, collecting dope carries out lyophilization gained pale pink dry powder.
Powder moisture 3.0%, ash 1.0%, total protein is 96.5%, ferrum saturation 8%.
The extraction of the lactoferrin of embodiment 9 is tested with preparing
It is with the difference of embodiment 6：Insoluble Fe3+Chelating resin adopts the resin for preparing with the following method, including as follows
Step：
S1, by weight, 5 parts of phenol, 70 parts of mass concentrations is mixed in the formalin of 25wt%, at 42 DEG C
Reaction 3h is carried out in temperature range, 6 parts of 2,2- dihydroxypropionic acids and 37 parts of ethylene glycol are added, is stirred, obtain pre-polymerization liquid；
S2, adds 45 part by weight of liquid paraffin and 4 weight portion sorbitan oleates in pre-polymerization liquid, is warming up to 62
0.6h is stirred after DEG C, hydrochloric acid 0.6 weight portion and 3 weight portion hydroxyl-containing silicones of the concentration in 30wt% is added, in 82 DEG C of scopes
After interior polyreaction 2h, filtration separates solid, with washing with alcohol, after drying, obtains chelating resin.
The extraction of the lactoferrin of embodiment 10 is tested with preparing
It is with the difference of embodiment 9：Insoluble Fe3+To obtain adding Sodium Chloride in concentrated solution before chelating resin absorption,
The concentration for making Sodium Chloride reaches 5g/L, then concentrated solution is filtered with the ultrafilter membrane that molecular cut off is 200000, Ran Houjin
Row chelating resin deferrization.
Reference examples 1
It is with the difference of embodiment 10：Sodium Chloride is not added in concentrated solution, that is, obtains concentrated solution directly with retention point
Son amount is that 300000 ultrafilter membrane is filtered to concentrated solution；Then chelating resin deferrization is carried out.
Reference examples 2
It is with the difference of embodiment 10：Ultrafilter membrane is provided without in 3rd step to be filtered.Obtain adding chlorine in concentrated solution
Change sodium, the concentration for making Sodium Chloride reaches 5g/L；Then chelating resin deferrization is carried out.
Reference examples 3
It is with the difference of embodiment 10：In the preparation of chelating resin, hydroxyl-containing silicone modifying agent is not added.It is i.e. insoluble
Fe3+The preparation method of chelating resin, comprises the steps：
S1, by weight, 5 parts of phenol, 70 parts of mass concentrations is mixed in the formalin of 25wt%, at 42 DEG C
Reaction 3h is carried out in temperature range, 6 parts of 2,2- dihydroxypropionic acids and 37 parts of ethylene glycol are added, is stirred, obtain pre-polymerization liquid；
S2, adds 45 part by weight of liquid paraffin and 4 weight portion sorbitan oleates in pre-polymerization liquid, is warming up to 62
0.6h is stirred after DEG C, hydrochloric acid 0.6 weight portion of the concentration in more than 30wt% is added, after polyreaction 2h in the range of 82 DEG C,
Filtration separates solid, with washing with alcohol, after drying, obtains chelating resin.
The Testing index of the finally obtained powder of various embodiments above is as follows：
As can be seen from the above table, the method that embodiment 10 is provided has the higher purity for adsorbing yield and albumen, leads to
Embodiment 10 and the contrast of reference examples 1 are crossed as can be seen that by after salt adding degeneration, can effectively go to make a part have influence on suction
There is degeneration in the albumen of attached process, and then removed by ultrafiltration；Embodiment 9 and the contrast of reference examples 2 are as can be seen that by after salt adding
Being provided without ultra-filtration filters still cannot remove the impact of this Partial Protein, and the response rate still resulted in adsorption step is inclined
It is low；Embodiment 9 and the contrast of reference examples 3 can be seen that by adding hydroxyl-containing silicone modifying agent in the preparation of chelating resin, can
To improve the repulsive force to albumen, it is to avoid lactoferrin is adsorbed.
The method that the present invention is provided has the advantages that lactoferrin extracts high with preparation purity, while newborn ferrum egg can be reduced
Fe in white3+Ion concentration, makes ferrum saturation be reduced to 6-8%, so as to significantly improve the antibacterial activity of lactoferrin.In addition lead to
Cross adjustment Fe3+Time, the concentration of mixing speed and medium, the pH of chelating resin absorption, after can processing lactoferrin
Ferrum saturation is controlled, to meet requirement of the different product application to lactoferrin iron saturation.
Embodiment 11
One kind treats HP infected stomach medicine, and its composition includes 6%～8% ferrum saturation Lac Bovis seu Bubali ferrum egg
White 140mg, rabeprazole 15mg, clarithromycin 400mg, Tinidazole 400mg.
Embodiment 12
One kind treats HP infected stomach medicine, and its composition includes 6%～8% ferrum saturation Lac Bovis seu Bubali ferrum egg
White 140mg, omeprazole 150mg, amoxicillin 350mg.
Embodiment 13
One kind treats HP infected stomach medicine, and its composition includes 6%～8% ferrum saturation Lac Bovis seu Bubali ferrum egg
White 10%, sodium bicarbonate 10%, sodium carbonate 30%, citric acid 38%, Sodium Chloride 2.4%, excipient 9.6%.
Embodiment 11-13 these three medicines 3 days, 5 days, 7 days, the treatment wi star rat effect datas of 15 days are the following is,
To prove curative effect of the above-mentioned three kinds of medicines in treatment gastropathy.
Claims (10)
1. application of the low ferrum saturation Bovine Lactoferrin in treatment HP infected stomach medicine is prepared, described low
Ferrum saturation Bovine Lactoferrin is the Bovine Lactoferrin of ferrum saturation ＜ 15%.
2. low ferrum saturation Bovine Lactoferrin according to claim 1 is preparing the gastric disease for the treatment of Helicobacter pylori infection
Medicine in application, it is characterised in that：The low ferrum saturation Bovine Lactoferrin is the Bovine Lactoferrin of ferrum saturation 6 ~ 8%.
3. low ferrum saturation Bovine Lactoferrin according to claim 1 is preparing the gastric disease for the treatment of Helicobacter pylori infection
Medicine in application, it is characterised in that：The gastropathy includes that acute or chronic gastritis, peptic ulcer, gastric mucosa come off and stomach
Acidity is too high.
4. low ferrum saturation Bovine Lactoferrin according to claim 1 is preparing treatment HP infected gastropathy
Application in medicine, it is characterised in that the preparation process of the low ferrum saturation lactoferrin is：
1st step, is adsorbed, is parsed using cation exchange resin to the lactoferrin in Lac Bovis seu Bubali；The resolving is to adopt
With two grades of parsings, first order parsing is parsed using 1～2.5% saline solution, and desorbed solution is molten containing lactoperoxidase pheron
Liquid, it is another to use；Second level parsing is parsed using 3.5～7% saline solution, and desorbed solution is sent into the 2nd step and processed；Described saline solution
Refer to sodium salt, potassium salt or calcium salt soln；
2nd step, concentrating and desalinating is carried out to the desorbed solution that the 1st step is obtained using seperation film；Described seperation film is molecular cut off
In the ultrafilter membrane of 1000～50K；
3rd step, the concentrated solution that the 2nd step is obtained adopts insoluble Fe3+Chelating resin is adsorbed, then using seperation film to concentrated solution
Concentrated；Need to add acetic acid, acetate and citrate in concentrated solution during the adsorption operations, prepare its absorption ring
Citrate concentration is 0.1mol/L ~ 1.0mol/L in border, and adjusts pH to 3.0～6.7；Described seperation film is retention molecule
Measure the ultrafilter membrane in 1000～50K.
5. low ferrum saturation Bovine Lactoferrin according to claim 3 is preparing treatment HP infected gastropathy
Application in medicine, it is characterised in that in the 3rd described step, described chelating resin refers to D403 chelating resins；The suction of resin
20～240 minutes attached time, rotating speed of agitator speed is 10～150rpm during absorption, and adsorption temp is at 3～37 DEG C.
6. low ferrum saturation Bovine Lactoferrin according to claim 3 is preparing treatment HP infected gastropathy
Application in medicine, it is characterised in that insoluble Fe in the 3rd described step3+The preparation method of chelating resin, including following step
Suddenly：
S1, by weight, 4～6 parts of phenol, 60～80 parts of mass concentrations is mixed in the formalin of 20～30wt%,
1～5h of reaction is carried out in 40～45 DEG C of temperature ranges, 5～8 parts of 2,2- dihydroxypropionic acids and 35～40 parts of ethylene glycol are added,
Stir, obtain pre-polymerization liquid；
S2, adds 35～55 part by weight of liquid paraffin and 3～5 weight portion sorbitan oleates in pre-polymerization liquid, heats up
0.5～1h is stirred to after 60～65 DEG C, hydrochloric acid 0.5～0.8 weight portion and 2～4 weight portions of the concentration in 30～35wt% is added
Hydroxyl-containing silicone, after 1～5h of polyreaction in the range of 80～85 DEG C, filtration separates solid, with washing with alcohol, after drying,
Obtain chelating resin.
7. low ferrum saturation Bovine Lactoferrin according to claim 4 is preparing treatment HP infected gastropathy
Application in medicine, it is characterised in that the 2nd step obtains concentrated solution and added Sodium Chloride before into the 3rd step, makes Sodium Chloride
Concentration reach 5～10g/L, then concentrated solution was carried out with the ultrafilter membrane that molecular cut off scope is 200000～400000
Filter.
8. treatment Helicobacter pylori infection is being prepared according to the arbitrary described low ferrum saturation Bovine Lactoferrin of claim 1-7
Property stomach medicine in application, it is characterised in that the composition of the treatment HP infected stomach medicine include 6% ~
8% ferrum saturation Bovine Lactoferrin 80-200mg, rabeprazole 10-20mg, clarithromycin 300-500mg, Tinidazole 300-
500mg。
9. treatment Helicobacter pylori infection is being prepared according to the arbitrary described low ferrum saturation Bovine Lactoferrin of claim 1-7
Property stomach medicine in application, it is characterised in that the composition of the treatment HP infected stomach medicine include 6% ~
8% ferrum saturation Bovine Lactoferrin 80-200mg, omeprazole 100-200mg, amoxicillin 200-500mg.
10. treatment helicobacter pylori sense is being prepared according to the arbitrary described low ferrum saturation Bovine Lactoferrin of claim 1-7
Application in metachromia stomach medicine, it is characterised in that the composition of the treatment HP infected stomach medicine includes
6% ~ 8% ferrum saturation Bovine Lactoferrin 10%, sodium bicarbonate 10%, sodium carbonate 30%, citric acid 38%, Sodium Chloride 2.4%, excipient
9.6%。
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111184858A (en) * | 2020-03-16 | 2020-05-22 | 南京康容健康科技有限公司 | A preparation for preventing and treating helicobacter pylori |
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| JPH11292788A (en) * | 1998-04-01 | 1999-10-26 | Snow Brand Milk Prod Co Ltd | Infection preventive against helicobacter pylori |
| CN1840179A (en) * | 2006-01-19 | 2006-10-04 | 方雅悯 | Application of bovine lactoferricin in preparation of medicine for treating HP infected gastric disease |
| CN103550760A (en) * | 2013-11-07 | 2014-02-05 | 无锡科捷诺生物科技有限责任公司 | Application of lactoferrin in preparing medicine for treating anemia of adipose people with high inflammatory factors |
| CN106008703A (en) * | 2016-07-06 | 2016-10-12 | 方雅悯 | Method for extracting and preparing high-purity low-iron-saturation lactoferrin from cow milk |
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2016
- 2016-11-16 CN CN201611006348.7A patent/CN106620662A/en active Pending
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|---|---|---|---|---|
| JPH11292788A (en) * | 1998-04-01 | 1999-10-26 | Snow Brand Milk Prod Co Ltd | Infection preventive against helicobacter pylori |
| CN1840179A (en) * | 2006-01-19 | 2006-10-04 | 方雅悯 | Application of bovine lactoferricin in preparation of medicine for treating HP infected gastric disease |
| CN103550760A (en) * | 2013-11-07 | 2014-02-05 | 无锡科捷诺生物科技有限责任公司 | Application of lactoferrin in preparing medicine for treating anemia of adipose people with high inflammatory factors |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111184858A (en) * | 2020-03-16 | 2020-05-22 | 南京康容健康科技有限公司 | A preparation for preventing and treating helicobacter pylori |
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