CN103948915A - Nattokinase composition capable of improving stability and oral curative effect and used for preventing thrombogenesis and thrombolysis - Google Patents
Nattokinase composition capable of improving stability and oral curative effect and used for preventing thrombogenesis and thrombolysis Download PDFInfo
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Abstract
The invention provides a nattokinase composition capable of improving stability and oral curative effect and used for preventing thrombogenesis and thrombolysis. The nattokinase composition is characterized by comprising nattokinase and phosphatidylserine, wherein the weight ratio of the nattokinase to the phosphatidylserine is (100:1) to (100:1,000), preferably (100:1) to (100:100), and further preferably (100:3) to (100:30).
Description
Technical field: the present invention relates to a kind of nattokinase goods of efficient absorption, relate in particular to a kind of natto kinase composition for antithrombotic and thrombolytic and manufacture method thereof.
Background technology:
Nattokinase is a kind of Proteinkinase, it is a kind of serine protease being produced by Bacillus subtilis natto from Traditional Japanese Food (Bacil lus subtilisl natto) in soybean isoflavone by natto strain, there is thrombus, reduce blood viscosity, improve blood circulation, soften and increase the effects such as blood vessel elasticity.
The eighties in 20th century, the Hiroyuki Sumi (Xu Jian foreign firm) that is engaged in thrombosis research in the U.S. finds find antithrombotic novel substance in various natural products time, the extract of natto stickum has the artificial thrombosis ability of extremely strong dissolving, and therefrom to isolate this kind of material be serine protease, be nattokinase (Nattokinase, NK) (Sumi, H., et al., A novel fibrinolytic enzyme (nattokinase) in the vegetable cheese Natto; A typical and popular soybean food in the Japanese diet.Cellular and Molecular Life Sciences, 1987.43 (10): p.1110-1111.).Nineteen ninety-five, Fujita etc. have reported that NK is to the thrombosed dissolution of rat carotid artery vascular endothelial cell damage, make artery blood flow recovery rate up to 62.0%, and fibrinolysin only has 15.8%, elastoser is without any recovering (Fujita, M., et al., Thrombolyt ic effect of nattokinase on a chemically indueed thrombosis model in rat.Biological & pharmaceutical bulletin, 1995.18 (10): p.1387.).The discoveries such as section intelligence change, in carotid artery thrombus model rabbit, injection NK high dose (4500IUkg in duodenum
-1), low dosage (2500IUkg
-1) and normal saline, after administration in 1-1.5h, than normal saline group, the equal significant prolongation of the partial thromboplastin time of the clotting time of NK high dose group, prothrombin time, thrombin time and activation, and low dose group only has thrombin time significant prolongation; 2h after administration, euglobulin lysis time and the fibrinogen content of NK high dose group significantly reduce, fibrin degradation product (FDP) content obviously increases (section intelligence change, Jianghan Lake, Deng. Studies On Fibrinolytic Function of Nattokinase and Mechanism Study thereof. Journal of Nutrition, 2003.25 (001): p.46-51.).In the oral NK test of human body, find, the fibrinolytic of user euglobulin improves, reached peak at the 4th day, and be maintained to the 8th day (Kumada, K., T.Onga, and H.Hoshino, The effect of natto possessing a high fibrinolytic activity in human plasma.Igaku ta Seibutsugaku, 1994.128 (3): p.117-119.).Recently, Kim etc. report 20-80 year oral NK capsule of hyperpietic (2000FU/ days) is after 8 weeks, diastolic pressure and systolic pressure all obviously reduce (Kim compared with matched group (taking placebo), J.Y., et al., Effects of nattokinase on blood pressure:a randomized, controlled trial.Hypertension research, 2008.31 (8): 1583-1588.).
Find through the researchs of more than 20 years, NK has multiple pharmacological effect, can set it as a kind of prevention and treatment coronary heart disease, angina pectoris, apoplexy, the disease such as senile dementia and arthritis, therefore many patents have been produced, as prevent patent (NATTOK INASE FOR DEGRADING AND REDUCING AMYLOID FIBRILS-ASSOCIATED WITH ALZHEIMER ' the S DISEASE of alzheimer disease, PRION DISEASES AND OTHER AMYLOIDOSES US8, 137, 666B2), reduce patent (the Nattokinase for reducing whole blood viscosity of patients blood viscosity, WO2007002572A3), anticoagulant patent (JP2003357848, US20040043015Process for inhibiting platelet aggregation), arthritis etc. (Anti-inflammatory agent US8, 017, 162), US7, 951, 844Tranquilizer tranquillizer and functional food, there is patent application (nattokinase is at the purposes CN101601859B preparing in cerebral protective agent type drugs), by NK and other enzyme material combined therapy oculopathy (senile degeneration of macula, ischemic appreciation retinopathy) (treatment CN101505790A of oculopathy, WO2006025276) reduce in addition purposes (JP2004115434) and central nervous system's protective agent (CN101601859B) of the aspect such as blood glucose and blood total cholesterol and triglyceride
Generally speaking, protein/polypeptide class medicine is directly oral, is easily lost biologic activity by gastric acid/gastrointestinal tract protease destruction.Protein/polypeptide class Oral drug absorption need to overcome three large barriers: the absorption barrier of the degraded of enzyme/acid, the diffusion barrier of slime layer and mucosa.There is same problem in nattokinase oral administration, the in the situation that of pH < 5, activity is lost gradually, is exposed to 1h, almost complete deactivation under the environment of gastric acid (pH1.23).Though have article report by polyglutamic acid microcapsule technology parcel nattokinase, but the problem (Hsieh of unresolved gastric acid inactivation, CW, Lu WC, Hsieh, WC, Huang YP, Lai CH, Ko WC.Improvement of the stability of nattokinase using[gamma]-polyglutamic acid as a coating material for microencapsulation.LWT-Food Science and Technology, 2009,42:144-149).There is bibliographical information, prepare nattokinase enteric coated tablet by methacrylic acid methyl methacrylate polymer packaging technique, solve problem (the Law D of nattokinase tablet gastric acid inactivation, Zhang Z.Stabilization and target delivery of nattokinase using compression coating.Drug Dev Ind Pharm, 2007,33:495-503).Also there is patent that NK is prepared into microcapsule (natto kinase purifying process and microcapsule formulation technique CN1309826C; Natto kinase purifying process and microcapsule formulation technique CN100500839C), or add enteric material protection NK (a kind of natto kinase composition and preparation method thereof CN101862447A such as cellulosic acid esters, crylic acid resin, polyvinyl alcohol phthalate ester, methacrylic acid copolymer, Lac and zein; A kind of nattokinase enteric coated capsule and preparation method thereof CN103238829A); Or prepare cyclodextrin clathrate (Zhang Yun peak, the Preparation and identification of nattokinase hydroxypropyl-beta-cyclodextrin inclusion. Guangdong chemical industry, 2011.38 (001): p.10-10. Zhang Yun peak, prepare the method CN102406942A of nattokinase cyclodextrin clathrate etc. mono-kind of. patent .CN201010287914.2); Prepare liposome (Zhang Xia, Yin Zongning, Wang Yixin, the preparation of nattokinase liposome and release in vitro thereof. Chinese Pharmaceutical Journal, 2007.42 (4): p.279-282.); Preparation has been chosen respectively two kinds of enteric systems (calcium alginate gel and the enteric coated micropill system taking CAP as coating) and has been prepared nattokinase enteric coated preparation, does not all reach ideal effect.
Summary of the invention:
For above-mentioned technical problem, the present inventor is by the discovery of concentrating on studies, if when Phosphatidylserine (PS) is used in combination with the nattokinase NK (different microorganisms, recombiant protein) of separate sources, can solves nattokinase and easily be destroyed by gastric acid/gastrointestinal tract protease the technical problem that loses biologic activity because of oral.In addition, to solve the ubiquitous problem of NK (different microorganisms, recombiant protein) of separate sources be that the stability of oral absorption, heat/pH acid/enzyme is low to the property of the present invention is directed to.
The applicant is discovery first in surprise, although Phosphatidylserine itself does not have antithrombotic effect, Phosphatidylserine can increase the stability of nattokinase in gastric acid.Solve nattokinase and easily destroyed and lose the technical problem of biologic activity by gastric acid/gastrointestinal tract protease because of oral, thereby increased the antithrombotic effect of nattokinase.
In addition, also be surprisingly found out that, after further combining with metal ion, dispersant, antioxidant, polycation material, succinate (TPGS) class, oligosaccharide etc., oral absorption effect further improves, and acidproof, heat-resisting.As pharmaceutical dosage form can realize that spraying is dry, powder coating and supercritical coating etc.
The object of the present invention is to provide a kind of natto kinase composition for antithrombotic and thrombolytic that can increase oral drug effect, it is characterized in that, contain nattokinase and Phosphatidylserine.Can also further contain at least one being selected from antioxidant, metal ion compound, dispersant, polycation compound and polyhydric alcohol as adding ingredient.
In addition, the present invention also aims to provide a kind of preparation method of natto kinase composition, it is characterized in that, it at least comprises following operation:
Make nattokinase be dissolved or dispersed in the operation of preparing nattokinase solution in solvent;
Make Phosphatidylserine be dissolved or dispersed in the operation of preparing Phosphatidylserine solution in organic solvent; And the operation that described nattokinase solution is mixed with Phosphatidylserine solution.
Finally, the present invention also aims to provide a kind of natto kinase composition for antithrombotic and thrombolytic, it is characterized in that, comprise Nattokinase Preparation and formulations of phosphatidylserine, in described Nattokinase Preparation and/or formulations of phosphatidylserine, can also further contain at least one being selected from antioxidant, metal ion compound, dispersant, polycation compound and polyhydric alcohol.
By technique scheme of the present invention, compared with the Nattokinase Preparation of prior art, can significantly improve oral absorption and the stability of nattokinase, can improve the oral drug effect of nattokinase simultaneously.The present invention relates to a kind of natto kinase composition for antithrombotic and thrombolytic, it is characterized in that, contain nattokinase and nattokinase absorption enhancer, described nattokinase absorption enhancer is Phosphatidylserine.
The invention provides following technical scheme:
1. for a natto kinase composition for antithrombotic and thrombolytic, it is characterized in that, contain nattokinase and nattokinase absorption enhancer, described nattokinase absorption enhancer is Phosphatidylserine.
2. natto kinase composition according to claim 1, is characterized in that, also further contains antioxidant and/or metal ion compound.
3. natto kinase composition according to claim 1 and 2, is characterized in that, also further contains and is selected from least one in dispersant, polycation compound and polyhydric alcohol.
4. according to the natto kinase composition described in any one in claim 1, it is characterized in that, the weight ratio of described nattokinase and Phosphatidylserine is 100: 1-100: 1000, preferably 100: 1-100: 100. further preferably 100: 3-100: 30.
5. natto kinase composition according to claim 2, it is characterized in that, described antioxidant is selected from sulphite, bisulfites, pyrosulfite, dithiocar-bamate, cystine, cysteine, glutathion, thioctic acid, ascorbic acid, ascorbyl palmitate, hydrogen basic note legumin, tocopherol, propyl gallate, butylated hydroxyarisol, ditertbutylparacresol, nordihydroguaiaretic acid, nitrogen, helium; Described metal ion compound is selected from monovalence to sexavalence ionic compound.
6. natto kinase composition according to claim 6, it is characterized in that, described metal ion compound is selected from monovalence, the metal cation of bivalence and trivalent, more preferably silver ion, calcium ion, zinc ion, magnesium ion, plasma selenium, iron ion compound, most preferred compound comprises calcium chloride, calcium carbonate, calcium phosphate, calcium hydrogen phosphate, calcium citrate, calcium glutamate, calcium amino acid chelate, calcium gluconate, calcium levulinate, calcium ascorbate, zinc chloride, zinc carbonate, zinc phosphate, phosphoric acid hydrogen zinc, zinc citrate, zinc glutamate, zinc-amino acid chelate, zinc gluconate, levulinic acid zinc, Zine ascorbic acid, magnesium chloride, magnesium sulfate, organic selenium salt, inorganic selenium salt, copper chloride, copper sulfate, iron chloride, ferrous sulfate, ferrous fumarate, ferrous succinate, ferric ammonium citrate, iron sucrose, sodium selenite.
7. natto kinase composition according to claim 2, the mass ratio of described Phosphatidylserine and metal ion compound is 100: 0-100: 1000.
8. according to the natto kinase composition described in any one in claim 3-8, it is characterized in that, described dispersant is selected from vitamin E polyethylene glycol succinic acid ester (TPGS), poloxamer class, lactalbumin; Described polycation compound is selected from polylysine, poly arginine, and described polyhydric alcohol is selected from mannitol, sorbitol, dextran, xylitol, sucrose, glucose, oligosaccharide, trehalose, oligosaccharide, chitin.
9. natto kinase composition according to claim 3, the mass ratio of described Phosphatidylserine and dispersant is 100: 0-100: 100.
10. according to the natto kinase composition described in claim 2, it is characterized in that, the consumption of described antioxidant accounts for the 0.0-0.1w/w% of whole compositions.
11. 1 kinds of preparations, it contains the natto kinase composition described in claim 1-11 any one, and described preparation is pharmaceutical preparation, health product, functional food, drinks or food additive.
The preparation method of the natto kinase composition in 12. claim 1-11 described in any one, is characterized in that, it at least comprises following operation:
Make nattokinase be dissolved or dispersed in the operation of preparing nattokinase solution or dispersion liquid in solvent;
Make Phosphatidylserine be dissolved or dispersed in the operation of preparing Phosphatidylserine solution or dispersion liquid in organic solvent; Operation and cryodesiccated operation that described nattokinase solution is mixed with Phosphatidylserine solution.
13. 1 kinds of natto kinase compositions for the nattokinase of antithrombotic and thrombolytic, is characterized in that, comprise Nattokinase Preparation and nattokinase absorption enhancer, and described nattokinase absorption enhancer is formulations of phosphatidylserine.
14. natto kinase compositions according to claim 14, it is characterized in that, in described Nattokinase Preparation and/or formulations of phosphatidylserine, also further contain and be selected from least one in antioxidant, metal ion compound, dispersant, polycation compound and polyhydric alcohol.
15. 1 kinds of nattokinase absorption enhancers are in the application of preparing in nattokinase antithrombotic synergist, and described nattokinase absorption enhancer is Phosphatidylserine.
16. Phosphatidylserine are as the application of nattokinase absorption enhancer.
By technique scheme of the present invention, compared with the Nattokinase Preparation of prior art, can significantly improve oral absorption and the stability of nattokinase, can improve the oral drug effect of nattokinase simultaneously.
Detailed description of the invention
Below the specific embodiment of the present invention is elaborated.
(1) Phosphatidylserine (PS)
Different according to source as the Phosphatidylserine using in the present invention (PS), Phosphatidylserine is actually the general name of a compounds, comprises the PS of various different aliphatic carboxylic acid chains.Specifically can be divided into natural PS, semi-synthetic PS, complete synthesis PS.Natural PS for example can be enumerated Semen sojae atricolor PS and brain PS.The PS that carries out " hydrogenation " on the PS basis that the PS in semi-synthetic source refers at natural origin and obtain.Complete synthesis PS obtains by synthetic means.Described synthetic phospholipid acyl serine can be such as dioleoyl Phosphatidylserine (DOPS) and salt thereof, two palmityl Phosphatidylserine, distearyl Phosphatidylserine etc.
The use level of Phosphatidylserine of the present invention (PS) and nattokinase, according to weight ratio meter, preferably the weight ratio of nattokinase/Phosphatidylserine is 100: 1-100: 1000, preferably 100: 1-100: 100. further preferably 100: 3-100: 30.
Described nattokinase and the weight ratio of Phosphatidylserine are to calculate and obtain with the actual content of Phosphatidylserine.In actual application, for commercialization is used, can adopt commercially available Phosphatidylserine product, it can be the commercially produced product with the Phosphatidylserine of certain content.
(2) antioxidant
The present inventor finds by add antioxidant in compositions of the present invention, and can further improve its stability, thereby improve oral drug effect, deduction may be the cause that has improved the physical and chemical stability of said composition due to antioxidant.
As the antioxidant using in the present invention, can use the antioxidant being generally used in pharmaceutical preparation.But consider preferably sulphite, bisulfites, pyrosulfite, dithiocar-bamate, cystine, cysteine, glutathion, thioctic acid, ascorbic acid, ascorbyl palmitate, hydrogen basic note legumin, tocopherol, propyl gallate, butylated hydroxyarisol, ditertbutylparacresol, nordihydroguaiaretic acid, nitrogen, helium etc. from improving oral drug effect of the present invention and stability.
The use amount of described antioxidant, preferably accounts for the 0.0-0.1w/w% of whole compositions, further preferred 0.01-0.08w/w%, further preferably 0.03-0.06w/w%, most preferably 0.04-0.06w/w%.
(3) metal ion compound
If the present inventor also finds further to add metal ion compound in natto kinase composition of the present invention, can further improve stability and the curative effect of medication of oral drugs.
As for metal ion compound of the present invention, can enumerate monovalence, the metal cation of bivalence and trivalent, from improving stability and curative effect of medication, preferably silver ion, calcium ion, zinc ion, magnesium ion, plasma selenium, iron ion compound, most preferred compound comprises calcium chloride, calcium carbonate, calcium phosphate, calcium hydrogen phosphate, calcium citrate, calcium glutamate, calcium amino acid chelate, calcium gluconate, calcium levulinate, calcium ascorbate, zinc chloride, zinc carbonate, zinc phosphate, phosphoric acid hydrogen zinc, zinc citrate, zinc glutamate, zinc-amino acid chelate, zinc gluconate, levulinic acid zinc, Zine ascorbic acid, magnesium chloride, magnesium sulfate, organic selenium salt, inorganic selenium salt, copper chloride, copper sulfate, iron chloride, ferrous sulfate, ferrous fumarate, ferrous succinate, ferric ammonium citrate, iron sucrose, sodium selenite etc.
Phosphatidylserine is 100 with the quality of metal ion compound than scope: 0-100: 1000, preferably 100: 0.1-100: 100, more select 100: 0.5-100: and 50, more select 100: 0.5-100: 10.
(4) dispersant
If add dispersant in natto kinase composition of the present invention, can further improve stability and the curative effect of medication of oral drugs.
Described dispersant can use normally used dispersant in pharmaceutical preparation, but from improving stability of the present invention and curative effect, preferred vitamin E polyethanediol succinate (TPGS), poloxamer class; Described polycation compound is selected from polylysine, poly arginine, and described polyhydric alcohol is selected from mannitol, sorbitol, dextran, xylitol, sucrose, glucose, oligosaccharide, trehalose, oligosaccharide, chitin, lactalbumin etc.
Phosphatidylserine is 100 with the quality of dispersant than scope: 0-100: 100, preferably 100: 1-100: 50.
(5) other additives
As the additive can be used in natto kinase composition of the present invention, except additive described above, do not affecting under the prerequisite of pharmaceutically active of the present invention and stability, can also use other additive of this area routine.
Consider from improving the oral drug effect of natto kinase composition of the present invention and the viewpoint of stability, can enumerate polycation compound, polyhydric alcohol, GABA, Monas cuspurpureus Went etc.
As polycation compound, preferably polylysine, poly arginine, its use amount is that nattokinase is 100 with the quality of polycation compound than scope: 0-100: 1000
As polyhydric alcohol, being preferably mannitol, sorbitol, dextran, xylitol, sucrose, glucose, oligosaccharide, trehalose, oligosaccharide, chitin, lactalbumin, its use amount nattokinase is 100 with the quality of polyhydric alcohol than scope: 0-100: 10000.
In order to improve the bioavailability of NK, particularly reduce NK affected factor in gastrointestinal tract, reduce individual variation, guarantee curative effect.
(6) preparation method of natto kinase composition of the present invention
By PS and organic solvent solution for fat-soluble antioxidant/or be dispersed in water, add NK aqueous solution (water soluble antioxidant), be uniformly mixed, add slaine, dispersant (containing if desired mannitol, xylitol, sorbitol, sucrose), mix homogeneously, homogenizer disperses, and lyophilization/spraying is dry.
Then, obtained material can directly be made to preparation (incapsulate, compressed tablets, enteric coated tablet etc.), also can pass through powder coating, prepare various pH response coated granules (as stomach dissolution type, small intestinal lysotype, colon lysotype), then be prepared into granule, capsule, tablet etc.
Described organic solvent can be the tert-butyl alcohol, ethanol, chloroform, ether and diisopropyl ether etc.
In addition, as mentioned above, the object of the invention is also to provide a kind of natto kinase composition for antithrombotic and thrombolytic, wherein said Nattokinase Preparation and formulations of phosphatidylserine, can prepare respectively according to above-mentioned preparation method, and then mix while taking.
It should be noted that, ratio of the present invention or content except specified otherwise all taking quality as benchmark.
Compared with prior art, according to prescription of the present invention and technique, nattokinase and Phosphatidylserine, slaine, antioxidant and dispersant are combined and (according to prescription of the present invention and technique, nattokinase and Phosphatidylserine combined, or nattokinase and Phosphatidylserine, slaine, antioxidant, dispersant and polycation etc. are combined), can obviously improve oral absorption and the stability of NK, improve the oral drug effect of nattokinase simultaneously.
Embodiment
Below in conjunction with embodiment, the present invention is described in further detail, but these embodiment can not be understood to limit scope of the present invention.
In embodiment, the abbreviation of each composition used is as follows:
Nattokinase NK
Commercially available Phosphatidylserine PS
Commercially available DOPS DOPS
Commercially available DOPC DOPC
Polyethylene glycol 1000 vitamin E succinic acid ester TPGS
In the present invention, all equipment or raw materials etc. all can obtain from market or the industry is conventional, and the consumption adopting in embodiment is to obtain according to the actual sign content conversion of commercially available prod.
The comparison of embodiment 1NK and DOPS/DOPC compositions
Proportionally (NK-PS; NK-PC ratio 100: 10, w/w) NK aqueous solution is added in PS or PC tert-butyl alcohol aqueous dispersion to stirring and evenly mixing, lyophilization.Pack gained lyophilization product into rat special enteric Caplet, obtain test preparation.
30 wistar rats are divided into 5 groups at random, and 6 every group, each group is respectively:
1, blank group (Control), gavages and the same number of capsulae vacuus of administration group.
2, NK-PS capsule low dose group (NK-PS-L), dosage is 10kIUkg
-1.
3, NK-PS capsule in high dose group (NK-PS-H), dosage is 20kIUkg
-1.
4, NK-PC capsule low dose group (NK-PC-L), dosage is 10kIUkg
-1.
5, NK-PC high dose group (NK-PC-H), dosage is 20kIUkg
-1.
6, independent PS group, dosage is equivalent to the PS amount in NK-PS group.
7, independent NK group, dosage is equivalent to the NK amount in NK-PS high dose group.
Administration every day 1 time, continuous 7 days.After administration the 8th day, rat is carried out to intraperitoneal anesthesia (1ml/100g) with 3.5% chloral hydrate, operation separates bilateral common carotid arteries.The plastic tab of a 1.2cm × 3.0cm of each pad under bilateral common carotid arteries, with filter paper bar (1.0cm × 1.5cm) the covering bilateral common carotid arteries that is impregnated with 50 μ l FeCl3 solution (10%, w/w).After 20min, take off filter paper bar, cut color change interval and clean with normal saline, after filter paper suck dry moisture, measure its length, and use analytical balance precise weighing.
The weight of unit length thrombosis is designated as relative bolt heavy (mg/cm), and calculates thromboembolism preventing rate (%) with following formula.
Thromboembolism preventing rate %=(W
contrast-W
administration)/W
contrast× 100%
W
contrast-matched group average relative bolt weight; W
administration-administration group average relative bolt weight.
Press above formula and calculate thromboembolism preventing rate, and result is carried out to statistical analysis, the results are shown in Table 1.
Table 1 different dosing group thromboembolism preventing rate result
Result shows, compared with matched group, PS group, NK group, various NK preparations all have significant thromboembolism preventing effect (P < 0.05).The thromboembolism preventing effect of NK-PS group is all significantly better than NK-PC (P < 0.05.
Hence one can see that, and improving the material absorbing is PS but not PC, and PS can significance increases the absorbtivity of NK.
The comparison of the commercially available PS different purity of embodiment 2
Due to DOPS expensive (price of 10mg is 52 dollars), seriously hinder product marketization, therefore the PS of more commercially available 20%, 40%, 60% content increases the effect that NK absorbs.Proportionally NK aqueous solution is added to PS or PC tert-butyl alcohol aqueous dispersion, stirring and evenly mixing, lyophilization by (NK-PS or NK-PC ratio are 100: 10, W/w).Pack gained lyophilization product into rat special enteric Caplet, obtain test preparation.
30 wistar rats are divided into 5 groups at random, and 6 every group, each group is respectively:
1, blank group (Control), gavages and the same number of capsulae vacuus of administration group.
2, NK-PC Capsules group (NK-PC), dosage is 10kIUkg
-1.
3, NK-PS (20%) Capsules group (NK-PS-20), dosage is 10kIUkg
-1.
4, NK-PS (40%) Capsules group (NK-PC-40), dosage is 10kIUkg
-1.
5, NK-PS (60%) Capsules group (NK-PC-60), dosage is 10kIUkg
-1.
Administration every day 1 time, continuous 7 days.After administration the 8th day, rat is carried out to intraperitoneal anesthesia (1ml/100g) with 3.5% chloral hydrate, operation separates bilateral common carotid arteries.The plastic tab of a 1.2cm × 3.0cm of each pad under bilateral common carotid arteries, with filter paper bar (1.0cm × 1.5cm) the covering bilateral common carotid arteries that is impregnated with 50 μ l FeCl3 solution (10%, w/w).After 20min, take off filter paper bar, cut color change interval and clean with normal saline, after filter paper suck dry moisture, measure its length, and use analytical balance precise weighing.
The weight of unit length thrombosis is designated as relative bolt heavy (mg/cm), and calculates thromboembolism preventing rate (%) with following formula.
Thromboembolism preventing rate %=(W
contrast-W
administration)/W
contrast× 100%
W
contrast-matched group average relative bolt weight; W
administration-administration group average relative bolt weight.
Press above formula and calculate thromboembolism preventing rate, and result is carried out to statistical analysis, the results are shown in Table 2.
Table 2 different dosing group thromboembolism preventing rate result
Result shows, compared with blank group, various NK preparations all have significant thromboembolism preventing effect (P < 0.05), and the thromboembolism preventing effect of NK-PS group is all significantly better than NK-PC (P < 0.05)
Embodiment 3 different proportion scopes
Adopt commercially available 60%PS, investigate different proportion, proportionally 100: 1-100: 1000 (NK-PS; NK-PC ratio 100: 10, w/w) NK aqueous solution is added in PS or PC tert-butyl alcohol aqueous dispersion to stirring and evenly mixing, lyophilization.Pack gained lyophilization product into rat special enteric Caplet, obtain test preparation.
60 wistar rats are divided into 12 groups at random, 5 every group.
Administration every day 1 time, continuous 7 days.After administration the 8th day, rat is carried out to intraperitoneal anesthesia (1ml/100g) with 3.5% chloral hydrate, operation separates bilateral common carotid arteries.The plastic tab of a 1.2cm × 3.0cm of each pad under bilateral common carotid arteries, with filter paper bar (1.0cm × 1.5cm) the covering bilateral common carotid arteries that is impregnated with 50 μ l FeCl3 solution (10%, w/w).After 20min, take off filter paper bar, cut color change interval and clean with normal saline, after filter paper suck dry moisture, measure its length, and use analytical balance precise weighing.
The weight of unit length thrombosis is designated as relative bolt heavy (mg/cm), and calculates thromboembolism preventing rate (%) with following formula.
Thromboembolism preventing rate %=(W
contrast-W
administration)/W
contrast× 100%
W
contrast-matched group average relative bolt weight; W
administration-administration group average relative bolt weight.
Press above formula and calculate thromboembolism preventing rate, and result is carried out to statistical analysis, the results are shown in Table 3.
Remarks: NK-PS (100/0) group is NK-PC (100/10)
Table 3 different dosing group thromboembolism preventing rate result
Result shows, compared with NK-PS (100/0), various NK preparations all have the thromboembolism preventing effect of significant thromboembolism preventing effect NK-PS group to be all significantly better than (P < 0.05).And, when nattokinase: when Phosphatidylserine ratio is 100: 3, thromboembolism preventing rate is elevated to 51.71% by 29.52%, significantly increases the absorption of NK; In the time that the ratio of NK and PS reaches 100: 30, thromboembolism preventing rate is 60.41%, approaches peak.In the time that NK and PS ratio are 100: 1000, thromboembolism preventing rate reduces to 51.02%.
Embodiment 4 metal ions are on the impact absorbing
Get DOPS chloroformic solution and be placed in flask, chloroform is removed in rotation, preparation DOPS immobilized artificial membrane.Getting NK aqueous solution, is to add NK solution at 100: 10 according to NK: DOPS, stirs 10 minutes, and calculates NK/Ca++ mass ratio (100: 1) calcium chloride, calcium sulfate (Gypsum Fibrosum), calcium carbonate, calcium lactate respectively according to calcium ion.Lyophilization.Add after mix homogeneously, pour into the special enteric Caplet of mice, obtain NK Sprinkle Caps.
30 wistar rats are divided into 6 groups at random, and 5 every group, each group is respectively:
1, blank group (Control), gavages and the same number of capsulae vacuus of administration group.
2, calcium chloride group (NK-PS-CL), dosage is 5kIUkg
-1.
3, calcium sulfate group (NK-PS-S), dosage is 5kIUkg
-1.
4, calcium carbonate group (NK-PS-C), dosage is 5kIUkg
-1.
5, calcium lactate group (NK-PS-L), dosage is 5kIUkg
-1.
6, do not add calcium salt group (NK-PS), dosage is 5kIUkg
-1.
Administration every day 1 time, continuous 7 days.After administration the 8th day, rat is carried out to intraperitoneal anesthesia (1ml/100g) with 3.5% chloral hydrate, operation separates bilateral common carotid arteries.The plastic tab of a 1.2cm × 3.0cm of each pad under bilateral common carotid arteries, with filter paper bar (1.0cm × 1.5cm) the covering bilateral common carotid arteries that is impregnated with 50 μ l FeCl3 solution (10%, w/w).After 20min, take off filter paper bar, cut color change interval and clean with normal saline, after filter paper suck dry moisture, measure its length, and use analytical balance precise weighing, calculate thromboembolism preventing rate, the results are shown in Table 4.
The different calcium salt thromboembolism preventing of table 4 rate result
Result shows, compared with not adding calcium salt group, the various NK preparations that add calcium salt all further improve thromboembolism preventing effect.
Embodiment 5TPGS is on the impact absorbing
Get oral level PS (60%), dissolve, and be placed in flask with chloroform, chloroform is removed in rotation, preparation PS immobilized artificial membrane.Get NK aqueous solution, according to NK-PS100: 10 add NK solution, stir 10 minutes, and calculate NK/Zn according to calcium ion
++mass ratio (100: 1) is zinc gluconate respectively.Lyophilization.According to NK-TPGS different quality than (100: 10; 100: 30; 100: 50, w/w) add after TPGS (being designated as respectively NK-PS-Z-T1, NK-PS-Z-T3, NK-PS-Z-T5) mix homogeneously, pour into the special enteric Caplet of rat, obtain NK Sprinkle Caps.
30 wistar rats are divided into 5 groups at random, and 6 every group, each group is respectively:
1, blank group (Control), gavages and the same number of capsulae vacuus of administration group.
2, do not add TPGS group (NK-PS-Z), dosage is 5kIUkg
-1.
3, add TPGS group (NK-PS-Z-T1), dosage is 5kIUkg
-1.
4, add TPGS group (NK-PS-Z-T3), dosage is 5kIUkg
-1.
5, add TPGS group (NK-PS-Z-T5), dosage is 5kIUkg
-1.
Administration every day 1 time, continuous 7 days.After administration the 8th day, rat is carried out to intraperitoneal anesthesia (1ml/100g) with 3.5% chloral hydrate, operation separates bilateral common carotid arteries.The plastic tab of a 1.2cm × 3.0cm of each pad under bilateral common carotid arteries, with filter paper bar (1.0cm × 1.5cm) the covering bilateral common carotid arteries that is impregnated with 50 μ l FeCl3 solution (10%, w/w).After 20min, take off filter paper bar, cut color change interval and clean with normal saline, after filter paper suck dry moisture, measure its length, and use analytical balance precise weighing, calculate thromboembolism preventing rate, the results are shown in Table 5.
The different calcium salt thromboembolism preventing of table 5 rate result
Result shows, compared with not adding TPGS group, the NK preparation of the various TPGS of adding all further improves thromboembolism preventing effect.
Embodiment 6 polycation materials increase NK and absorb
NK is mixed according to 100: 100 (w/w) with the Phosphatidylserine of commercially available 40%PS, and use ethanol water dissolution, and the mass ratio that is 100: 100, NK and poly-D-lysine (or poly arginine) according to NK and milk calcium mass ratio is that 100: 20 and NK and xylitol mass ratio are to feed intake at 100: 1000, add milk calcium, poly-D-lysine/or poly arginine and xylitol, adopt spray drying process to prepare powder body.Pack the gained desciccate of spraying into rat special enteric Caplet, obtain test preparation.
20 wistar rats are divided into 4 groups at random, and 5 every group, each group is respectively:
1, blank group (Control), gavages and the same number of capsulae vacuus of administration group.
2, NK-PS poly-D-lysine group (NK-PS-PL), dosage is 10kIUkg
-1.
3, NK-PS poly arginine group (NK-PS-PA), dosage is 10kIUkg
-1.
4, NK-PS Capsules group (NK-PS), dosage is 10kTUkg
-1.
Administration every day 1 time, continuous 7 days.After administration the 8th day, rat is carried out to intraperitoneal anesthesia (1ml/100g) with 3.5% chloral hydrate, operation separates bilateral common carotid arteries.The plastic tab of a 1.2cm × 3.0cm of each pad under bilateral common carotid arteries, with filter paper bar (1.0cm × 1.5cm) the covering bilateral common carotid arteries that is impregnated with 50 μ l FeCl3 solution (10%, w/w).After 20min, take off filter paper bar, cut color change interval and clean with normal saline, after filter paper suck dry moisture, measure its length, and use analytical balance precise weighing.
The different polycation compound of table 6 thromboembolism preventing rate result
Result shows, compared with not adding polycationic compounds group, add poly-D-lysine/or the arginic NK preparation of poly all further improve thromboembolism preventing effect.
Embodiment 7 improves stability
The preparation of NK-PS compositions: with ethanol aqueous dispersion PS (60%), NK aqueous solution is added wherein to (NK/PS mass ratio is 100/1000), be uniformly mixed, add 10mM calcium ascorbate and 10mM solution of zinc sulfate, after homogenizer disperses, add TPGS (or poloxamer F68), cysteine, oligosaccharide (or oligochitosan and oligo-chitosan), mannitol and/or dextran solution, after mix homogeneously, spraying is dry again.Gained NK-PS compositions is tested as follows.
Temperature stability: former solution NK powder and NK-PS compositions powder body are positioned over respectively in 37 DEG C of environment, after 30 days, use fibrin plate method to measure the enzymatic activity of each sample, calculate residual enzyme activity.
Simulated gastric fluid stability: with concentrated hydrochloric acid/or solution of dilute hydrochloric acid preparation 0.1mol/L, solution and complex are placed therein respectively (37 DEG C), after 2 hours, use fibrin plate method to measure the enzymatic activity of each sample, calculate residual enzyme activity.
Result: NK solution and NK-PS compositions powder body are placed after 30 days in 37 DEG C of environment, and residual enzyme activity is respectively 31.6% and 76.0%, illustrates that compositions has improved the temperature stability of NK.
In simulated gastric fluid, after 2 hours, the residual enzyme activity of NK and NK-PS compositions powder body is respectively 6.3% and 53.5%, and the compositions utmost point has improved the heat stability of NK significantly.
In addition, above embodiment just provides as example embodiment of the present invention, is only that simple example can not be interpreted as limiting.Within apparent form of distortion of the present invention is also contained in protection scope of the present invention to those skilled in the art.
Utilizability in industry
Natto kinase composition of the present invention is suitable for using as pharmaceutical preparation, health product, functional food, drinks or food additive.
Claims (10)
1. for a natto kinase composition for antithrombotic and thrombolytic, it is characterized in that, contain nattokinase and nattokinase absorption enhancer, described nattokinase absorption enhancer is Phosphatidylserine.
2. natto kinase composition according to claim 1, is characterized in that, also further contains antioxidant and/or metal ion compound.
3. natto kinase composition according to claim 1 and 2, is characterized in that, also further contains and is selected from least one in dispersant, polycation compound and polyhydric alcohol.
4. according to the natto kinase composition described in any one in claim 1, it is characterized in that, described Phosphatidylserine is selected from natural phospholipid acyl serine, semi-synthetic Phosphatidylserine, complete synthesis Phosphatidylserine.
5. a preparation, it contains the natto kinase composition described in claim 1-4 any one, and described preparation is pharmaceutical preparation, health product, functional food, drinks or food additive.
6. the preparation method of the natto kinase composition described in any one in claim 1-4, is characterized in that, it at least comprises following operation:
Make nattokinase be dissolved or dispersed in the operation of preparing nattokinase solution or dispersion liquid in solvent;
Make Phosphatidylserine be dissolved or dispersed in the operation of preparing Phosphatidylserine solution or dispersion liquid in organic solvent; Operation and cryodesiccated operation that described nattokinase solution is mixed with Phosphatidylserine solution.
7. for a natto kinase composition for the nattokinase of antithrombotic and thrombolytic, it is characterized in that, comprise Nattokinase Preparation and nattokinase absorption enhancer, described nattokinase absorption enhancer is formulations of phosphatidylserine.
8. natto kinase composition according to claim 7, it is characterized in that, in described Nattokinase Preparation and/or formulations of phosphatidylserine, also further contain and be selected from least one in antioxidant, metal ion compound, dispersant, polycation compound and polyhydric alcohol.
9. nattokinase absorption enhancer is in an application of preparing in nattokinase antithrombotic synergist, and described nattokinase absorption enhancer is Phosphatidylserine.
10. Phosphatidylserine is as the application of nattokinase absorption enhancer.
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| CN106794252A (en) * | 2014-10-07 | 2017-05-31 | 塞浦路迈德有限责任公司 | For the pharmaceutical preparation of oral delivery peptide or protein matter medicine |
| CN106928085A (en) * | 2017-01-22 | 2017-07-07 | 陕西师范大学 | A kind of L theanine chelates of zinc and preparation method and tea beverage and preparation method containing the chelate |
| CN107354141A (en) * | 2017-09-11 | 2017-11-17 | 南京御匾国健生物科技有限公司 | A kind of modified Nattokinase and its application |
| CN107375913A (en) * | 2017-09-11 | 2017-11-24 | 南京御匾国健生物科技有限公司 | A kind of thrombus dredging collateral medicine for loading Nattokinase and preparation method thereof |
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| CN105054076A (en) * | 2015-08-25 | 2015-11-18 | 广州施健生物科技有限公司 | Alzheimer's disease improving nutritional composition and application thereof |
| US11065310B2 (en) | 2016-05-27 | 2021-07-20 | Nattocat, LLC | Compositions and methods for thromboembolism dissolution |
| CN106928085A (en) * | 2017-01-22 | 2017-07-07 | 陕西师范大学 | A kind of L theanine chelates of zinc and preparation method and tea beverage and preparation method containing the chelate |
| CN107412751B (en) * | 2017-04-10 | 2021-02-19 | 乐研新生物科技（成都）有限公司 | Nattokinase composition and preparation method thereof |
| CN107412751A (en) * | 2017-04-10 | 2017-12-01 | 乐研新生物科技（成都）有限公司 | Natto kinase composition and preparation method thereof |
| CN107354141A (en) * | 2017-09-11 | 2017-11-17 | 南京御匾国健生物科技有限公司 | A kind of modified Nattokinase and its application |
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| EP4151230A4 (en) * | 2020-05-15 | 2023-11-08 | JNC Corporation | Antiviral agent |
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