CN106215174A - A kind of formulation preparation method of human lactoferrin - Google Patents
A kind of formulation preparation method of human lactoferrin Download PDFInfo
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- CN106215174A CN106215174A CN201610630888.6A CN201610630888A CN106215174A CN 106215174 A CN106215174 A CN 106215174A CN 201610630888 A CN201610630888 A CN 201610630888A CN 106215174 A CN106215174 A CN 106215174A
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- human lactoferrin
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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
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Abstract
The formulation preparation method of a kind of human lactoferrin, belongs to pharmaceutical technology field.The present invention, using porous-starch as carrier, carries out absorption embedding to human lactoferrin.Use porous starch embedding restructuring lactoferrin, develop a kind of new dosage form human lactoferrin microcapsule, oral human lactoferrin can be made the most digested destruction of gastric, arrive the release of intestinal position and play its biologic activity, on the one hand solve oral preparation drug administration amount and greatly and be administered shortcoming frequently, also remain the biologic activity of protide product simultaneously, improve its bioavailability.Simultaneously can overcome drug stability problems, delay controlled release put the effect of medicine additionally, the particulate delivery system of this encapsulating reaches intestinal.
Description
Technical field
The formulation preparation method of a kind of human lactoferrin, belongs to pharmaceutical technology field.
Background technology
Human lactoferrin (rhLF) is a kind of multifunctional protein, belongs to transferrins family, has promotion ferrum and absorbs, resists
The physiological functions such as bacterium, antiviral, immunomodulating, antitumor, antioxidation.Therefore baby milk powder, nutrition it are widely used in
Health food, cosmetics, feed additive, food preservative, the aspect such as ancillary drug treatment.
Wuxi Kejienuo Biotechnology Co., Ltd. utilizes somatic cell clone technique acquisition to turn human lactoferrin first
Gene clened cows, wherein human lactoferrin transgene clone cattle expression is up to 3g/L, it is thus achieved that maximum, heredity in the world
Stable transgenic dairy breeding populations.With transgenic milk as raw material, through pretreatment, chromatography, inactivation of virus and go
Remove, be concentrated by ultrafiltration and the processing step such as buffer exchange, aseptic filtration, lyophilization or spray drying, prepare purity and be more than
The restructuring lactoferrin of 90%.
After recent studies have found that human lactoferrin oral administration, major part lactoferrin is disappeared by the pepsin of gastric juice
Change, cause these active substance low absorption and instability, especially lower with Oral administration bioavailability, because it is at intestinal
Instability in pH environment, easily by enzyme hydrolysis, biological half-life is short, and oral rear active component is easily degraded, it is impossible to complete arrival
The effective absorption site of intestinal plays its physiological active functions.Poor stability, be easily degraded, be difficult to absorb and bioavailability low,
This is also the general character shortcoming of albumen, many peptides oral administration.The safest and the preparation as substitute Bovine Lactoferrin in the future one
Simply, the multifunctional protein of low cost------restructuring lactoferrin, how to improve its stability and bioavailability urgently
Need to solve, therefore build a kind of effective lactoferrin carrier imperative.
Utilize natural polymer carrier-porous-starch also known as micropore starch, be a kind of New Modified Starch, refer at enzymolysis
Manage and make granule present cavernous starch, there is compared with native starch bigger specific surface area, specific pore volume and good suction
Water, oil absorbency, and low cost, nonhazardous, can natural degradation.Porous-starch mainly has following spy compared with native starch
Point: (1) bigger specific pore volume；(2) large specific surface area；(3) bulk density, grain density are relatively low: (4) well water suction, oil suction energy
Power: (5) have good mechanical strength in the dry state；(6) it is dispersed in water and other solvent and can keep obvious structural intergrity:
(7) course of processing does not use chemical reagent, and safe and nontoxic, using dosage is unrestricted.Porous-starch produces the biggest because of porous
Specific surface area, therefore porous-starch is mainly used as the carrier of adsorption function material.
Use porous starch embedding restructuring lactoferrin, develop a kind of new dosage form---human lactoferrin microcapsule, it is possible to
Make oral human lactoferrin the most digested destruction of gastric, arrive the release of intestinal position and play its biologic activity, on the one hand solve
Oral preparation drug administration of having determined amount is big and is administered shortcoming frequently, also remains the biologic activity of protide product simultaneously, improves
Its bioavailability.Drug stability problems can be overcome, additionally, the particulate delivery system of this encapsulating reaches the slow control of intestinal simultaneously
Release medicine.
Summary of the invention
It is an object of the invention to provide the formulation preparation method of a kind of human lactoferrin, using porous-starch as load
Body, carries out absorption embedding to human lactoferrin.
Technical scheme: the formulation preparation method of a kind of human lactoferrin, it is characterised in that compound concentration is
The human lactoferrin solution of 40-65 mg/mL, is added in the ratio that w v is 1 10 of porous-starch human lactoferrin solution
0.4-0.8g porous-starch, is stirred at room temperature 10 h with magnetic stirring apparatus, stands 5h afterwards, centrifugal 10-under 5000rpm
20min, precipitation carries out lyophilization, is i.e. able to the formulation human lactoferrin that porous-starch is carrier.
Preferably preparation method is: compound concentration is the human lactoferrin solution of 60 mg/mL, by porous-starch human milk ferrum
The ratio that w v is 1 10 of protein solution adds 0.4g porous-starch, is stirred at room temperature 10 h with magnetic stirring apparatus, afterwards
Standing 5h, 5000rpm and be centrifuged 10min, precipitation carries out lyophilization, is i.e. able to the formulation human milk ferrum egg that porous-starch is carrier
In vain.
Beneficial effects of the present invention: develop a kind of orally available lactoferrin novel formulation, it is to avoid oral protein class medicine
In gastric juice, it is hydrolyzed destruction, makes arrival intestinal play a role.
Accompanying drawing explanation
The carrying capacity of porous-starch during Fig. 1 different people lactoferrin initial concentration.
The human lactoferrin that Fig. 2 porous-starch bag carries release profiles in intestinal juice.
Detailed description of the invention
The preparation of embodiment 1 porous-starch
The preparation technology flow process of porous-starch is: with corn starch as raw material, through sizing mixing, enzyme-added enzymolysis, is centrifuged, washs, precipitates
Constant pressure and dry, pulverize and sieve, obtain porous-starch.Precise 15 g corn starch joins in the triangular flask of 100 mL, adds
The 0.2 mol/L Na of 50 mL pH 5.82HPO4-0.1 mol/L citrate buffer solution, adds two toluene and (prevents starch from gluing
Wall), preheat 10 min at 45 DEG C, adding α-amylase saccharifying enzyme volume ratio is the mixed enzyme of 13, and enzyme dosage is amount of starch
1.0%, 45 DEG C of constant temperature uniform stirring reaction 15h, enzymolysis solution is centrifuged and washs at 4000 rpm 3 times, and abandoning supernatant is deposited in
Constant pressure and dry at 40 DEG C, pulverizes and sieves and get final product.The porous-starch oil absorbency of preparation reaches 79.3%, and yield is 51.2%, and porous is formed sediment
Powder oil absorbency is 2.9 times of ative starch, and starch granules keeps complete, and punching is uniformly.
The saturated adsorption curve of the manned lactoferrin of embodiment 2 porous-starch bag
The concentration of human lactoferrin solution is the key factor affecting porous-starch adsorbance.Human lactoferrin to be obtained the highest
Carrying capacity, needs to calculate its saturated adsorption concentration in porous-starch.
Table 1 porous-starch bag manned lactoferrin initial optimization
Human lactoferrin during concentration is the human lactoferrin liquid of 20,40,60 mg/mL as can be seen from the above table is by porous-starch
Absorption, and along with the liter high adsorption capacity of concentration gradually increases.From the beginning of 80mg/mL, adsorbance is a negative, illustrates,
From the beginning of 80mg/mL, human lactoferrin causes the winding between macromole, gathering to make molecular dimension increase, relatively due to high concentration
In the cellular hole of porous-starch difficult to get access, the ability of porous-starch water suction is better than far away the energy of starch adsorption human lactoferrin
Power, causes the human lactoferrin concentration in supernatant to increase on the contrary.Therefore, the concentration selecting human lactoferrin solution is 45,55,
65,70 mg/mL optimize further, filter out the human lactoferrin initial adsorption concentration that carrying capacity is maximum.
The manned lactoferrin of table 2 porous-starch bag optimizes further
According to above-mentioned data, make the graph of a relation (Fig. 1) of human lactoferrin initial concentration and porous-starch carrying capacity, it is thus achieved that human milk ferrum
The saturated adsorption concentration of albumen is 60mg/mL.With this understanding, porous-starch is 227.3 to the de-luxe compartment carrying capacity of human lactoferrin
mg/g。
The digestion characteristics research of the formulation human lactoferrin of embodiment 3
The preparation of simulation digestion buffer
SGF(gastric juice): the dense HCl of 10mL 2g/L NaCl, 7mL；
SIF(intestinal fluid): (needing now with the current) CaCl2Solution: take 3.663g solid CaCl2It is made into 200mL solution.
Gastric juice simulation digestion experiment: take 10mL gastric juice simulation digestion buffer (SGF), after adding 10mg pepsin
Add in digestion system, regulate pH to 2.0, add the digested sample of 1g (human lactoferrin of porous starch embedding), react 1h.
Sample respectively at 0,10,20,30,40,60min time, measure wherein human lactoferrin content, as shown in table 3.
Intestinal juice simulation digestion experiment: regulation pH to 7, adds the trypsin solution of 13.2mL 6mg/mL, adds the shallow lake of 20 μ L
Powder saccharifying enzyme (AMG) and the CaCl of 1mL2Solution (SIF) regulation pH to 7, the addition digested sample of 1g be (porous starch embedding
Human lactoferrin), react 2h, respectively at 0,10,30,50,80,120min samplings, measure wherein human lactoferrin content, such as table
Shown in 4.
Porous-starch bag carry human lactoferrin simulation gastro-intestinal Fluid in release and digestion characteristics
As can be seen from Table 3, in simulated gastric fluid, substantially do not had in 0-50min by the human lactoferrin of porous starch embedding
Release, discharges less in 1 hour.And the human lactoferrin not being embedded is in simulated gastric fluid, the most discharge greatly at front 10min
Part, and it is hydrolyzed (unlisted result).Illustrate that porous-starch can protect human lactoferrin not to be degraded in gastric juice.
In table 3 simulated gastric fluid, human lactoferrin is at the release concentration of different time
By table 4 and Fig. 2, in simulated intestinal fluid, can be released by the human lactoferrin of porous starch embedding, and in 120min
Basic release is completely.In simulated gastric fluid, the porous-starch as wall material will not be hydrolyzed by pepsin, therefore human milk ferrum egg
Can preferably be protected and not degraded by protease in vain.Arrive in simulated intestinal fluid, under the effect of pancreatin and saccharifying enzyme, porous
Starch is progressively hydrolyzed, and human lactoferrin is released, and is hydrolyzed by protease the most again.Illustrate that porous-starch can be at intestinal juice
In complete effective release of human lactoferrin.
In table 4 simulated intestinal fluid, human lactoferrin is at the release concentration of different time
。
Claims (2)
1. the formulation preparation method of a human lactoferrin, it is characterised in that compound concentration is the human milk ferrum of 40-65 mg/mL
Protein solution, adds 0.4-0.8g porous-starch in the ratio that w v is 1 10 of porous-starch human lactoferrin solution, uses magnetic
Power agitator is stirred at room temperature 10 h, stands 5h, 5000rpm afterwards and is centrifuged 10-20min, and precipitation carries out lyophilization, i.e.
It is able to the formulation human lactoferrin that porous-starch is carrier.
The most formulation preparation method of human lactoferrin, it is characterised in that compound concentration is 60 mg/
The human lactoferrin solution of mL, adds 0.4g porous in the ratio that w v is 1 10 of porous-starch human lactoferrin solution and forms sediment
Powder, is stirred at room temperature 10 h with magnetic stirring apparatus, stands 5h, 5000rpm afterwards and is centrifuged 10min, and precipitation carries out freezing dry
Dry, i.e. it is able to the formulation human lactoferrin that porous-starch is carrier.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109276560A (en) * | 2018-09-25 | 2019-01-29 | 华南理工大学 | A kind of pH response type micro-capsule and its preparation method and application containing lactoferrin |
| CN115349638A (en) * | 2022-09-16 | 2022-11-18 | 齐鲁工业大学 | High-probiotic resistant starch and preparation method thereof |
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| CN101117351A (en) * | 2007-04-30 | 2008-02-06 | 北京济普霖生物技术有限公司 | Method for purifying restructuring lactoferrin from transgenic cow's milk |
| CN101601862A (en) * | 2009-07-03 | 2009-12-16 | 中国人民解放军第三军医大学 | Porous-starch and derivant thereof are as the application of pharmaceutical carrier |
| CN102766206A (en) * | 2012-07-02 | 2012-11-07 | 北京济福霖生物技术有限公司 | Product for improving iron deficiency anemia and preparation method thereof |
| 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 |
| CN104768971A (en) * | 2012-10-08 | 2015-07-08 | 墨累古尔本合作有限公司 | Improved process for purifying lactoferrin from milk and products thereof |
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2016
- 2016-08-04 CN CN201610630888.6A patent/CN106215174A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101117351A (en) * | 2007-04-30 | 2008-02-06 | 北京济普霖生物技术有限公司 | Method for purifying restructuring lactoferrin from transgenic cow's milk |
| CN101601862A (en) * | 2009-07-03 | 2009-12-16 | 中国人民解放军第三军医大学 | Porous-starch and derivant thereof are as the application of pharmaceutical carrier |
| CN102766206A (en) * | 2012-07-02 | 2012-11-07 | 北京济福霖生物技术有限公司 | Product for improving iron deficiency anemia and preparation method thereof |
| CN104768971A (en) * | 2012-10-08 | 2015-07-08 | 墨累古尔本合作有限公司 | Improved process for purifying lactoferrin from milk and products thereof |
| 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 |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109276560A (en) * | 2018-09-25 | 2019-01-29 | 华南理工大学 | A kind of pH response type micro-capsule and its preparation method and application containing lactoferrin |
| CN109276560B (en) * | 2018-09-25 | 2021-07-20 | 华南理工大学 | Lactoferrin-containing pH response type microcapsule and preparation method and application thereof |
| CN115349638A (en) * | 2022-09-16 | 2022-11-18 | 齐鲁工业大学 | High-probiotic resistant starch and preparation method thereof |
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