CN103518978B - Novel lysozyme formulation and preparation method thereof - Google Patents
Novel lysozyme formulation and preparation method thereof Download PDFInfo
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- CN103518978B CN103518978B CN201310497875.2A CN201310497875A CN103518978B CN 103518978 B CN103518978 B CN 103518978B CN 201310497875 A CN201310497875 A CN 201310497875A CN 103518978 B CN103518978 B CN 103518978B
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Abstract
The present invention relates to a kind of preparation of novel lysozyme oral formulations, namely by synthesis KLP-602 albumen prepare KLP-602 sodium alginate micro ball with this, mix with rice sprout or its decoction or its adsorbent and obtain.Said preparation obviously promotes the intestinal absorption efficiency of antalzyme protein, as novel fodder additive, can replace production performance and non-specific immune function that antibiotic strengthens the mammality cultivated animals such as pig.
Description
Technical field
The present invention relates to a kind of preparation of novel lysozyme oral formulations, the present KLP-602 sodium alginate micro ball of its mark sheet and rice sprout or its decoction or its adsorbent mixing composition, Neither of the two can be dispensed; Said preparation obviously promotes the intestinal absorption efficiency of antalzyme protein, as novel fodder additive, can replace production performance and non-specific immune function that antibiotic strengthens the mammality cultivated animals such as pig.
Background technology
Lysozyme (Lysozyme) is a kind of protein-based biology enzyme, has antibacterial, antiviral, the function such as immunological regulation and hemostasis, detumescence, quickening tissue repair, is a kind of important nospecific immunity factor, has a wide range of applications in field of medicaments.KLP-602 is the dimeric forms of lysozyme after synthesis, in recent years find that all advantages except retaining monomer are (antibacterial, antiviral, have no side effect) (WO89/11294) outward, also has stronger immunomodulation effect, two-way function immunity moderation system balancing, be embodied in the synthesis of cytokine network level being modulated TNF IL-2 IL-6 INF etc., activating phagocytic cells and related immune reaction, be particularly conducive to the disease (WO96/21463) that treatment and the disease (WO94/01127) of preventing high-level TNF to cause and immune deficiency or immunosupress cause.The acute bacterial that causes of pneumonia caused as: treatment and prevention pyemia and septic shock (particularly infected wound), acute alimentary canal and breathing problem, influenza or viral infection, aspermia or oligospermia, garget, virus and bacterial dermatosis and genital organ disease; Strengthen sperm motility, promote that bovine oocyte is ripe and be fertilized; The breeding of virus below obvious suppression: cowpox, HSV-1, canine parvovirus, mumps virus, measles virus, and influenza virus etc.KLP-602 treatment swine disease is the most effective.Almost 100% effective to following disease: in agalasisa in postpartum (MMA syndrome), dysentery, pyometritis, influenza, colibacillosis, particularly reaches 94 ~ 98% (US88/01785) to the cure rate of diarrhea of pigs.
KLP-602 has a basic premise to the treatment of above-mentioned disease and prevention effect, and namely it plays physiological function with the form of blood concentration in vivo.Existing oral lysozyme medicine or feed addictive mainly work at intestines and stomach, and its medicine target is mainly and directly acts on gastrointestinal tract pathogen, or as external medication, the efficiency that body absorption enters blood is very low, and pharmacological use is very limited.In body, injection directly can improve the blood concentration of lysozyme, but for developing the preventing preparation of non-therapeutic, the novel fodder additive for exploitation prevent disease character does not have actual application value.Therefore, the research and development oral formulations of KLP-602 or the feed addictive of oral administration, for cultivated animals prevent disease, improve non-specific immune function, vigor of keeping fit, special today of giving prominence to very much in problems such as antibiotic abuse, resistances, especially have important practical significance and huge potential age deduction.
Protein is large biological molecule, poor stability, and the oral administration harsh physicochemical environment that must stand whole digestive system, body absorption inefficiency, bioavilability is extremely low.The large molecule of protide wants smooth absorbed into serum must overcome the two road natural cover for defenses: one is decomposition and the degradation of acid, alkali and numerous digestive ferment in intestines and stomach; Two is absorption barriers of enteron aisle, and the protein macromolecule of polarity can not enter blood by absorbed intact usually.Have for this two roads barrier the absorbed intact that multiple technologies realize protein and enter blood.First development protide macromolecular carrier; wherein sodium alginate micro ball is because of the feature of its natural macromolecule amylose; there is good biocompatibility and biodegradable; the good load of the large molecule of protide, protection, controlled release can be reached and locate the targets such as release at ieoocolon, being particularly useful as the delivery system of protein.Its two be development strengthen enteron aisle to the macromolecular sorbefacient of protide.Intestinal permeation enhancers in research comprises micromolecular compound and polymer etc., as Small molecular class sorbefacients such as Medium chain fatty acid sodium-salt, cholic acid salt, nitric oxide donors, polymer class sorbefacient has crosslinked polyacrylate etc., the wherein effect of most intestinal permeation enhancers and its cytotoxicity height correlation, so find nontoxic and effective intestinal permeation enhancers is the focus that researcher pays close attention to.For reach protide large molecule absorbed intact target more than two links indispensable.
Rice sprout is a kind of food, is also a herb, cures mainly promoting digestion and removing indigestion, spleen benefiting and stimulating the appetite.First we find that rice sprout also has the function promoting the large molecule absorbed intact of protide at work, because its safety, effectively, natural, the feature such as inexpensive, we are developed as a kind of effective intestinal permeation enhancers, again by the preparation of KLP-602 sodium alginate micro ball, the two combines formation protide and divides piggyback system greatly, available protecting KLP-602 albumen also successfully promotes its intestinal absorption in the gastrointestinal tract, increase substantially bioavilability, its actual application value of practical imparting, not only can be developed as the novel fodder additive of the mammality cultivated animals such as pig, also there is the potential application of development people oral protein medicament.
Summary of the invention
The object of this invention is to provide a kind of novel lysozyme oral formulations, it to be mixed with certain proportion by KLP-602 sodium alginate micro ball and rice sprout or its decoction or its adsorbent and forms, obviously can promote the intestinal absorption efficiency of antalzyme protein, as novel fodder additive, production performance and non-specific immune function that antibiotic strengthens the mammality cultivated animals such as pig can be replaced.
Target of the present invention is achieved through the following technical solutions, and this technical scheme and embodiment are only further illustrate of the present invention, does not impose any restrictions the scope that the present invention covers:
The preparation of rice sprout or its decoction or its adsorbent:
Rice sprout dry powder: commodity are fried rice sprout and pulverized.
Rice sprout decoction: it is fried that commodity stir-fry rice sprout presses Chinese medicine conventional method, 1.0Kg is fried obtains 1.0L liquid decoction.
Rice sprout decoction cornstarch adsorbent: above-mentioned decoction with etc. heavy cornstarch absorption dry.
Rice sprout hots plate xeraphium: commodity fry rice sprout add water (1:1) hot plate drying and crushing after 0.5hr.
(all retrodicting to its dry weight during above-mentioned rice sprout decoction metering)
The synthesis of KLP-602:
Synthesis is undertaken and purifying by AlorichChemisry company description substantially, and purity is 30% ~ 95%.
The preparation of KLP-602 sodium alginate micro ball:
According to a conventional method as the preparation such as drip, spray-on process, be shaped in calcium chloride solution and fix, heat drying.Preparing major parameter is: sodium alginate concentration 1% ~ 5%(W/V), KLP-602 concentration 0.1% ~ 1.0%(W/V), calcium chloride concentration 3% ~ 10%.Prepare gained drug bearing microsphere and have good dispersiveness and stability, particle size range 1 ~ 1000 μm (moisture about 10%), drugloading rate is 0.5 ~ 5.0%.
Mouse animal experiment:
Mouse divides six groups, work, wherein four groups of experimental group (1 ~ 4), one group of control group (5), one group of blank group (6).
5/group, combination feed free choice feeding or gavage (1 times/day).
Mouse serum lysozyme enzyme activity determination:
Undertaken by GBT25879.Add quantitative lysozyme in normal mouse serum and make mouse serum lysozyme enzyme activity determination calibration curve (Figure 1A).Experimental mouse serum lysozyme sample enzyme activity determination result (Figure 1B).
Mouse serum lysozyme concentration determination (ELISA):
Adopt conventional indirect competitive ELISA method quantitative assay.Lysozyme bag quilt, lysozyme gradient titer (mouse serum lysozyme sample) and rabbit anti-lysozyme antiserum competition binding, HRP-goat anti-rabbit igg reacts, substrate colour developing also A
490measure.
Mouse serum lysozyme concentration determination calibration curve (Fig. 2 A).Mouse serum lysozyme sample concentration measurement result (Fig. 2 B).
Weanling pig is tested:
DLY weanling pig (28 age in days) establishes test group, control group and antibiotic group.KLP-602 feed addictive is added in test group daily ration.Free choice feeding, investigates its impact on Production Performance of Weaning Pigs and immunity function.
Experimental result:
Previous work proves that lysozyme is executed hello basic detection with former powder form and do not gone out blood concentration, and intestinal absorption is extremely low.Existing mouse animal experiment result (figure A B) shows, rice sprout or its decoction or its adsorbent mix with KLP-602 sodium alginate micro ball to execute feeds, significantly can improve the blood concentration of lysozyme, experiment proves that the combination of this patent rice sprout and KLP-602 sodium alginate micro ball is effective for strengthening that lysozyme intestinal-tract absorbs, improves blood concentration.Meanwhile, the lysozyme of absorbed into serum still retains the enzyme activity, shows that lysozyme can complete form absorbed into serum, is conducive to it and plays biological function in vivo.
Weanling pig experiment shows, add KLP-602 preparation in diet and can improve weanling pig average daily gain, the activate the phagocytic capacity of remarkable enhancing peritoneal macrophage and phagocytic index thereof, improve the phagocytic rate of neutrophil leucocyte in blood, thus improve weanling pig immunologic function.All animal used as test body conditions are good, do not produce any bad reaction and impact.
Conclusion:
The combination of the rice sprout that this patent is advocated and KLP-602 sodium alginate micro ball absorbs, improves the production performance of the mammality cultivated animals such as pig and immunity function is effective and safety for strengthening lysozyme intestinal-tract, and adding this preparation in weanling pig daily ration can substitute antibiotics.
Accompanying drawing explanation
Figure 1A: mouse serum lysozyme enzyme activity determination calibration curve.
Figure 1B: mouse serum lysozyme enzyme lives (successive administration 23 days).
Fig. 2 A: mouse serum lysozyme concentration determination calibration curve.
Fig. 2 B: mouse serum lysozyme concentration (successive administration 23 days).
Detailed description of the invention
Further illustrate the present invention below by specific embodiment how to realize:
The synthesis of embodiment 1, KLP-602:
Experiment material is egg white lysozyme, crosslinking agent DMS(Dimethylsuberimidatedihydrochloride), synthesis is undertaken by AlorichChemisry company description substantially.20K ultra-filtration membrane device concentrates and dialyses, dimer purity average out to 42%.
The preparation of embodiment 2, KLP-602 sodium alginate micro ball (Lys.D-SA-MS):
By the preparation of high-pressure fog method routine, preparing major parameter is: sodium alginate concentration 2.0%(W/V), KLP-602 concentration 0.1%(W/V), calcium chloride concentration 3%.Prepare gained drug bearing microsphere and have good dispersiveness and stability, particle size range 150 ~ 300 μm (after drying moisture about 10%), drugloading rate is 2.5%.
Embodiment 3, rice sprout dry powder group (experimental group 1)
Make 1000g conventional feed, wherein add Lys.D-SA-MS7.6g, rice sprout dry powder 50g, the two mixed proportion is 1:6.5, continuous 23 days of mouse free choice feeding.
Embodiment 4, rice sprout hot plate xeraphium group (experimental group 2)
Make 1000g conventional feed, wherein add Lys.D-SA-MS7.6g, rice sprout hots plate xeraphium 50g, and the two mixed proportion is 1:6.5, continuous 23 days of mouse free choice feeding.
Embodiment 5, rice sprout decoction cornstarch adsorbent group (experimental group 3)
Make 1000g conventional feed, wherein add Lys.D-SA-MS7.6g, rice sprout decoction cornstarch adsorbent 50g, the two mixed proportion is 1:6.5, continuous 23 days of mouse free choice feeding.
Embodiment 6, rice sprout decoction group (experimental group 4)
Every mouse gavage every day 1 time, dosage Lys.D-SA-MS30mg, rice sprout decoction 0.2ml(0.2g), the two mixed proportion is 1:6.7, continuous gavage 23 days.
Embodiment 7, control group (control group 5)
Make 1000g conventional feed, wherein only add Lys.D-SA-MS7.6g, continuous 23 days of mouse free choice feeding.
Embodiment 8, blank group (blank group 6)
Conventional feed, continuous 23 days of mouse free choice feeding.
Embodiment 9, KLP-602 feed addictive are on the impact of Production Performance of Weaning Pigs and immunity function
Prepared by test group diet:
90gLys.D-SA-MS and 1.5kg rice sprout is hotted plate xeraphium to mix with 1 ton of conventional diet.Amplify then in this ratio.
Prepared by control group diet:
Conventional diet.
Prepared by antibiotic group diet:
Conventional diet adds antibiotic kind and the limitation that national regulation allows interpolation.
Weanling pig is tested:
Weanling pig test group, control group and antibiotic group are respectively 36, continuous 28 days of each group diet free choice feeding.Measure Production Performance of Weaning Pigs index and salivary lysozyme.
Claims (1)
1.
a kind ofkLP-602 preparation, is characterized in that being made up of the mixing of KLP-602 sodium alginate micro ball and rice sprout or its decoction or its adsorbent; With dry weight basis, mixed proportion is by weight being 1:2 ~ 1:50, and described rice sprout decoction is that rice sprout is fried by Chinese medicine conventional method, and 1.0kg rice sprout is fried obtains 1.0L liquid decoction, retrodicts to its dry weight during its metering; Described rice sprout adsorbent be described rice sprout decoction with etc. heavy cornstarch absorption dry.
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| CN111657407A (en) * | 2020-05-14 | 2020-09-15 | 上海艾魁英生物科技有限公司 | Feed additive for improving non-specific immune function of weaned piglets and preparation method and application thereof |
| CN115975993A (en) * | 2023-02-09 | 2023-04-18 | 驻马店华中正大有限公司 | Lysozyme dimer, lysozyme dimer sodium alginate microspheres and preparation method and application thereof |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1177303A (en) * | 1995-01-13 | 1998-03-25 | 尼卡健康产品有限公司 | Application of lysozyme dimer |
| CN101947315A (en) * | 2010-09-03 | 2011-01-19 | 上海沈李科工贸有限公司 | Application of Chinese medicinal bitter apricot seeds in lysozyme sodium alginate microspheres |
| CN103040727A (en) * | 2013-01-21 | 2013-04-17 | 天津工业大学 | Preparation method of drug and protein sustained-release alginate hybrid gel |
| CN103341161A (en) * | 2013-06-20 | 2013-10-09 | 湖北省潜江市华山水产食品有限公司 | Preparation method for sodium alginate-lysozyme compound microcapsule |
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- 2013-10-22 CN CN201310497875.2A patent/CN103518978B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1177303A (en) * | 1995-01-13 | 1998-03-25 | 尼卡健康产品有限公司 | Application of lysozyme dimer |
| CN101947315A (en) * | 2010-09-03 | 2011-01-19 | 上海沈李科工贸有限公司 | Application of Chinese medicinal bitter apricot seeds in lysozyme sodium alginate microspheres |
| CN103040727A (en) * | 2013-01-21 | 2013-04-17 | 天津工业大学 | Preparation method of drug and protein sustained-release alginate hybrid gel |
| CN103341161A (en) * | 2013-06-20 | 2013-10-09 | 湖北省潜江市华山水产食品有限公司 | Preparation method for sodium alginate-lysozyme compound microcapsule |
Non-Patent Citations (1)
| Title |
|---|
| 谷芽粉在特养业中的应用;林家义等;《贵州畜牧兽医》;20011231;第25卷(第4期);第41-42页 * |
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