CN110327367A - Composition, preparation method, preparation containing nitrate and vitamin and application thereof - Google Patents
Composition, preparation method, preparation containing nitrate and vitamin and application thereof Download PDFInfo
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Abstract
The present invention provides a kind of composition containing nitrate and vitamin, its preparation, preparation method, its preparing the drug for improving nitrate concentration in blood, and its purposes in oncotherapy.
Description
Technical field
The present invention relates to pharmaceutical technology fields, and in particular, to a kind of composition containing nitrate and vitamin, its preparation
(resistance to Rett), preparation method and its drug of nitrate content or treatment tumour in blood are improved in preparation and/or is inhibited swollen
Purposes in the drug of tumor cell proliferation.
Background of invention
NO3-N and NO2-N is widely present in water, soil, air and plant.Absorption of human body nitrate it is main come
Source is food, and green vegetables account for major part.
Nitrate plays physiological function, including blood pressure reduction, Platelet aggregation inhibitor and blood vessel in various system activities
Protective effect (Lundberg JO et al., Cardiovasc Res, 2011；Kapil V et al,Nitric Oxide,
2014).Skeletal Muscle can absorb nitrate from peripheral blood, then pass through xanthine oxidoreductase enzymatic pathway for nitre
Hydrochlorate is reduced to nitric oxide, to increase body blood flow, and (the Weitzberg E et that can boost metabolism
al.,Annu Rev Nutr,2013).Nitrate is by increasing angiectatic external membrane of heart blood flow, reducing vascular resistence, decrease
Coronary artery steals blood and reduces preload to prevent ischemic heart disease (Pragani MA et al., Rev Cardiovasc
Med,2017).Nitrate in diet can be by inhibiting nadph oxidase and adjusting angiotensins (Ang) II by body surface
It reaches to reduce endogenous nitric oxide and generate, so that part improves the age related hypertension and metabolic activity (Hezel of mouse
M et al.,Free Radic Biol Med,2016).In addition, inorganic nitrate is pressed down by increasing neutrophil count
Acute and chronic inflammation processed, this is likely to reduced generation (Khambata RS et al., the Proc Natl Acad of atheromatous plaque
Sci USA,2017).Show after protonitrate diet 22 months in addition, a chronic dietary nitrate lacks experiment, mouse
Will appear metabolic syndrome, endothelial dysfunction and cardiovascular death (Kina-Tanada M et al., Diabatologia,
2017).Inorganic nitrate reduces platelet aggregation by enhancing Epithelial cell alive and vasodilator, play reduce blood pressure and
Improve the effect (Machha A et al., Eur J Nutr, 2011) of myocardial ischemia.
Past, nitrate were once considered harmful because under certain conditions, as acid condition gastric environment in can
Can generate carcinogenic nitrosamine, and nitrosamine and the cancer of the esophagus, gastric cancer, colon cancer and other tumours it is related (Bedale W et al.,
Meat Sci, 2016, Park JE et al, Toxicol Res, 2015).Therefore, the World Health Organization (World Health Organization) suggests
The upper limit of daily NO3-N and NO2-N intake is respectively 3.7mg/kg and 0.06-0.07mg/kg (Weitzberg E et
al.,Annu Rev Nutr,2013).However, as the epidemiological survey in recent years to nitrate and tumour shows do not have
Tangible proof proves that the nitrate in diet can increase generation (Bryan NS et al., the Food Chem of tumour
Toxicol,2012).2012, Sialin albumen was reported as nitrate cell membrane transporter body for the first time, followed in diet nitrate
Important function has been played in ring.Nitrate is concentrated in saliva by the Sialin active transport in salivary gland, is then secreted into mouth
Chamber reenters body processes (Qin LZ ea al., Proc Natl Acad Sci USA, 2012) by gastrointestinal tract.Due to
Nitrate in diet is converted into NO by oral cavity stomach function regulating bacterium by non-enzymatic synthesis, and nitrate is considered as not in physiological activity
It can or lack.
Therefore, nitrate plays important physiological regulatory action in vivo, and diet nitrate can substantially meet health
The amount of nitrate needed for people.However, for cardiovascular function damage, the stress damage of stomach and intestine, liver retrogression pathological changes, liver
For the patients such as steatosis, total body radiation damage and tumour, the intake of diet nitrate is unable to reach therapeutic agent at all
It measures (~13mg/kg).So far, in the prior art without provide be suitble to directly as drug and generate good absorption effect and
The nitrate preparations of excellent therapeutic effect.
The applicant is by in-depth study repeatedly, it has been surprisingly discovered that by nitrate and vitamin according to specific ratio
Example is prepared by mixing into composition, especially nano liposome preparations composition, can significantly improve the content of nitrate in vivo,
And the effect for the treatment of tumour is generated, so as to complete the present invention.
Summary of the invention
It is an object of the present invention to provide a kind of composition containing nitrate, the composition includes nitrate and dimension
Raw element, wherein the ratio of the amount of the substance of nitrate and vitamin is 10:1-1:10, preferably 5:1-1:5, more preferable 3:1-1:1,
Most preferably 2:1-1:1.
Wherein, the nitrate is sodium nitrate, potassium nitrate or combinations thereof, preferably sodium nitrate or potassium nitrate, most preferably nitric acid
Sodium.The vitamin is vitamin C, vitamin B6 or combinations thereof, preferred vitamin C or vitamin B6, most preferably vitamin C.
Wherein, the ratio of the amount of the substance of the nitrate and vitamin can be 10:1,5:1,2:1,1:1,1:2,1:
3,1:5,1:10, it is highly preferred that the ratio is 2:1,1:1.
It is a further object to provide the preparation sides of the composition of the present invention including nitrate and vitamin
Method comprising nitrate and vitamin are dissolved respectively, 0.22 μm of filtering with microporous membrane degerming, according to above-mentioned nitrate and
The ratio of the amount of the substance of vitamin mixes, and mixed solution is sub-packed in sterile cillin bottle, seals, at -20~-60 DEG C
2~18h is freezed, is lyophilized with freeze dryer: -20~-20 DEG C of setting primary distillation 4~20h, 20~40 DEG C of 1~30h of secondary distillation,
It takes out.
It is a further object to provide a kind of preparations comprising of the present invention includes nitrate and vitamin
Composition and one or more pharmaceutically acceptable excipient.
Wherein, the preparation can be any pharmaceutically acceptable dosage form, such as solid form or liquid form,
Such as tablet, capsule, granule, oral solution, injection, liposome, lipid microemulsion, freeze-dried powder preparation etc..
The preparation method of the preparation can be prepared in accordance with conventional methods known in the art.For specifically being made in the present invention
Standby dosage form, it is preferable that can be prepared according to specific preparation method described in the application embodiment.
In preparation of the invention, pharmaceutically acceptable excipient can be for suitable for preparing needed for different dosage forms
Any suitable excipient, such as filler, diluent, adhesive, disintegrating agent, glidant, lubricant, corrigent, inclusion material
Material, freeze-drying stabilizer.
A kind of preferred dosage form is liposome, and nanometer particle size is 150~200nm, and formula composition is as follows: sodium nitrate
0.2~2.0g, 0.2~0.9g of vitamin C, 0.5~3.5g of lecithin, 0.1~0.5g of cholesterol, 0.05~0.4g of laruyl alcohol,
Phosphate buffer (PBS) 100~200mL.
A kind of preferred liposome includes: sodium nitrate 0.2g, vitamin C 0.2-0.83g, lecithin 0.5-2.0g, and gallbladder is solid
Alcohol 0.1-0.4g, laruyl alcohol 0.05-0.2g, phosphate buffer (PBS) are appropriate.
Of the invention a further object is provides the composition or its preparation of the present invention including nitrate and vitamin
The purposes in blood in the drug of nitrate content is improved in preparation.
Studies have shown that nitrate can with vasodilator, when body ischemic injuries, damaged part pH often drop to 7 with
Under, by quickly supplementing nitrate, the content of nitrate in raising blood can be with diastole aortic annulus, to generate the adjusting heart
The effect of vascular function.Also, it studies have shown that improving the content of nitrate in blood, can produce adjusting gastrointestinal function, improve
And/or the stress damage for the treatment of stomach and intestine, prevent liver retrogression pathological changes and hepatic steatosis, mitigates damage caused by total body radiation
Wound.
Still another purpose of the invention be to provide the composition of the present invention including nitrate and vitamin or its
Purposes of the preparation in preparation treatment tumour and/or the drug for inhibiting tumor cell proliferation.Preferably, the tumour is oral cavity
Cancer.
So far, it is suitble in the prior art without providing directly as drug and generates more preferable assimilation effect and excellent control
The nitrate preparations of therapeutic effect.In the present invention, it is (especially excellent that nitrate and vitamin according to specific ratio are prepared into composition
Choosing, nano liposome preparations composition), nitrate content in blood can be significantly improved, bioavilability is improved, can be used for adjusting
Save cardiovascular function, improvement and/or treat stomach and intestine stress damage, prevention liver retrogression pathological changes and hepatic steatosis or
Mitigate damage caused by total body radiation, and without any adverse side effect.For example, measuring through animal experiment, composition 5 exists within 2 hours
In rat body blood concentration be individually be equivalent to 2.1 times of nitrate group composition 5 dosage reduce by one times when still with list
It is solely suitable to the drug effect of nitrate；And the composition 5 of nitrate and vitamin can generate the effect of significant treatment tumour；This
The composition of invention confirms through animal experiment, without any toxicity, safely and effectively.Therefore, technical solution of the present invention produces pre-
Technical effect unexpectedly has important clinical use meaning.
The dosage of the present composition or pharmaceutical composition that are administered to patient can depend on disease for example to be treated
Type or need nitrate content in blood to be achieved horizontal, and can be according to well known by persons skilled in the art dose
Amount-selection method is selected.For example, in certain embodiments, the dosage for the composition being administered to patient is with composition
The content meter of middle nitrate about 13mg/kg or higher, than such as from about 15mg/kg-30mg/kg.
In this application, if be not particularly described, wherein the ratio of the nitrate and vitamin is substance
Amount ratio (that is, molar ratio).If be not particularly described, substance, device or the equipment, substance or composition
Preparation method etc., be all that this field is conventional or well known or those skilled in the art according to the record of this specification according to normal
Rule technological means can obtain.
Detailed description of the invention
Fig. 1 be according to test example 1, according to the weight 5 kind compositions of the invention to intragastric administration on mice and individual sodium nitrate it
Nitrate content figure in blood afterwards.Wherein combination 1,2,3,4 and 5 is respectively to be according to the mass ratio of the material example prepared by embodiment 1
The sodium nitrate of 5:1,1:5,1:1,1:2,2:1 and ascorbic composition.
Fig. 2 is influence diagram of the nitrate composition diet to carcinoma of mouth CAL27 cell transplanted tumor in nude mice volume.
Fig. 3 is the influence diagram that nitrate nano-composition transplants nude mice weight to carcinoma of mouth CAL27.Note: the result shows that nitre
The application of hydrochlorate is to nude mice weight without significant change.
Fig. 4 is influence diagram of the nitrate composition to carcinoma of mouth CAL27 cellular morphology.
Fig. 5 is influence diagram of the nitrate composition to carcinoma of mouth CAL27 cell Proliferation.
Fig. 6 is influence diagram of the nitrate composition to carcinoma of mouth CAL27 cell scratch width.
Fig. 7 is that nitrate composition has the activity of anti-migration to scheme in carcinoma of mouth CAL27 cell.
Specific embodiment
In order to which the present invention is further explained, some embodiments and test example is given below.These embodiments are entirely example
Property, they are only used to that the present invention is specifically described, and are not construed as limitation of the present invention.
The composition of embodiment 1 nitrate and vitamin
Composition 1: sodium nitrate 0.85g, vitamin C 0.176g (the mass ratio of the material example 10:1)
Composition 2: sodium nitrate 0.85g, vitamin C 0.352g (the mass ratio of the material example 5:1)
Composition 3: sodium nitrate 0.85g, vitamin C 0.88g (the mass ratio of the material example 2:1)
Composition 4: sodium nitrate 0.85g, vitamin C 1.76g (the mass ratio of the material example 1:1)
Composition 5: sodium nitrate 0.85g, vitamin C 3.52g (the mass ratio of the material example 1:2)
Composition 6: sodium nitrate 0.85g, vitamin C 5.28g (the mass ratio of the material example 1:3)
Composition 7: sodium nitrate 0.85g, vitamin C 8.8g (the mass ratio of the material example 1:5)
Composition 8: sodium nitrate 0.85g, vitamin C 17.6g (the mass ratio of the material example 1:10)
Preparation method: the sodium nitrate or vitamin C of corresponding amount are weighed according to aforementioned proportion respectively, by nitrate and vitamin
C is dissolved respectively, and 0.22 μm of filtering with microporous membrane degerming mixes respectively according to different proportion, and mixed solution is sub-packed in
In sterile cillin bottle, seal, 2~18h is freezed at -20~-60 DEG C, is lyophilized with freeze drier: -20~20 DEG C of setting primary
Distil 4~20h, 20~40 DEG C of 1~30h of secondary distillation, takes out to get composition is arrived.
Tablet of the embodiment 2 containing nitrate and vitamin
The composition (1000) of tablet:
Preparation method:
Various components are weighed by recipe quantity, vitamin C, sodium nitrate and HPMC (HT-K80000S) are sieved with 100 mesh sieve, cream
The auxiliary materials such as sugar, ethyl cellulose cross 80 meshes, are uniformly mixed.Softwood is made with 1% polyvinylpyrrolidone ethanol solution, then
With 20 meshes pelletize, in 60 DEG C of dry 1h, and be added magnesium stearate mixing, cross 20 mesh sieves, tabletting, polishing, pack to get
Tablet.
This product every containing about 1.76~176mg of vitamin C, nitrate 8.5mg, slice weight is about 250mg.
Nanometer micro-emulsion of the embodiment 3 containing nitrate and vitamin
The composition of microemulsion:
Preparation method:
Step 1: under nitrogen protection, 1000g soybean oil being added to the lecithin of 8g and the polyethylene glycol PEG2000 of 20g
In, it is heated to 30~70 DEG C of formation oil mixtures；
Step 2: by the composition of the vitamin C of 0.176g~17.6g of different proportion and the sodium nitrate of 0.85g, 200g
PLURONICS F87 and 8L water for injection are mixed at 20~70 DEG C, form aqueous mixture；
Step 3: under nitrogen protection, the mixture that aqueous mixture and step 1 that step 2 obtains obtain being mixed
Close, then under the conditions of 30~80 DEG C, 5000~50000r/min after 5~30min of high speed dispersion, with 0.l mol/L NaOH or
HCl adjusts pH value to 6.0~8.5, carries out homogeneous 5~15 times under the conditions of pressure is 90~210MPa, until mixed phase is presented
Homogeneous transparent state detects the mixed phase of obtained homogeneous transparent state, is dispensed, obtains nanometer micro-emulsion.
Liposome of the embodiment 4 containing nitrate and vitamin
Preparation method:
Step 1: lecithin, cholesterol and laruyl alcohol are weighed, is placed in eggplant-shape bottle, the imitative dissolution of chlorination.Through rotary evaporation system
It is spare at dry film；
Step 2: preparing phosphate buffer, vitamin C is dissolved in appropriate phosphate buffer (PBS) 100~200mL
In, it is subsequently added into sodium nitrate, obtains solution；
Step 3: by the solution of the step 2 slowly eggplant-shape bottle of injection step 1, slowly stir, it is sonicated to be formed
Liposome.
Its nanometer of property is characterized through laser nano particle size analyzer, measuring its partial size is 150-200nm.HPLC method is used to survey
Fixed, the encapsulation rate of the liposome composition of combination 3,4 and 5 is respectively 92.5%, 94.3% and 95.2% ".
Granule of the embodiment 5 containing nitrate and vitamin
Granule 1:
Preparation method:
Sodium nitrate 8.5g and vitamin C 0.2~0.9g and HPMC (HT-K80000S) 2.0g is uniformly mixed, is used
20% ethanol solution 20mL is made softwood, and extrusion spheronization method is made particle, it is dry to moisture be no more than 3% to get sodium nitrate and
Vitamin C composition granule.
Granule 2:
Preparation method:
Potassium nitrate 10.1g and vitamin C 35.2g and HPMC (HT-K80000S) 2.0g is uniformly mixed, with 20%
Softwood is made in ethanol solution 20mL, and particle is made in extrusion spheronization method, and drying to moisture is no more than 3% to get potassium nitrate and Wei Sheng
Plain C in combination composition granule agent.
Granule 3:
Preparation method:
By sodium nitrate 8.5g and vitamin B641.0g and HPMC (HT-K80000S) 2.0g is uniformly mixed, with 20%
Softwood is made in ethanol solution 20mL, and particle is made in extrusion spheronization method, and drying to moisture is no more than 3% to get sodium nitrate and Wei Sheng
Plain B6 composition granule.
Nitrate content detection in the blood after composition of the invention is administered in test example 1
By C57BL6 mouse (male, 30 ± 5g) (70 in total, every group 10) the dosage intraperitoneal injections penta by 50mg/kg
Barbital normal saline solution is anaesthetized.The composition of sodium nitrate and five kinds of reagents containing nitrate (is respectively pressed after anesthesia
It is prepared according to embodiment 1, the mass ratio of the material example is 5:1 (combination 1), 1:5 (combination 2), 1:1 (combination 3), 1:2 (combination 4), 2:1
The sodium nitrate and ascorbic composition of (combination 5)) (in six groups of experiments, nitrate content is identical) fill according to 0.2mmol/kg
Stomach, respectively 2 after 0h, stomach-filling, 4,6,12h tail vein take blood spare.
Specific nitrate test experience method (total nitric oxide and nitrate/nitrous nitrification kit are used,
PKGE001, R&D Systems, USA):
Preparation:
1) blood sample is placed in 4 DEG C of 2h, 14000rpm is centrifuged 10min, draws serum；
2) serum of acquisition is filtered, dilutes 10 times；
3) it prepares 1 × reaction solution: 10 × reaction solution being diluted to 1 × reaction solution using distilled water/deionized water；
4) it prepares nitrate reductase: recombinating nitrate reductase with 1.0 milliliters of nitrate reductase storing liquids, be firmly vortexed,
15 minutes are stood at room temperature, stands 15 minutes again at room temperature after vortex, being vortexed again for existing side by side uses.
Nitrate reductase is diluted with reaction diluent (1 ×), preparation concentration is the nitrate reductase of mother liquor 1/5, step
It is as follows:
A. nitrate reductase (hole x+2) × 5 μ L；
B. reaction diluent (1 ×)=step a × 4 times volume；
C. the volume in step a and b is added in clean test tube, is vortexed；
D. it is placed on ice, is used in 15 minutes.
5) 5.0 ml deionized waters of NADH reagent-or distilled water recombinate NADH, stand 3 minutes and mild using preceding
Stirring, in 15 minutes using or be put on ice for.
6) preparation of nitrate standard items:
With pipette by 900 μ L reaction diluents (1 ×) move into 200 μm of ol/L pipes in, then successively prepare 100 μm of ol/L,
The nitrate standard items of 50 μm of ol/L, 25 μm of ol/L, 12.5 μm of ol/L, 6.25 μm of ol/L and 3.12 μm of ol/L.Reaction diluent
(1 ×) it is blank (0 μm of ol/L).
Nitrate content detecting step:
1) prepare all reagents, standard items and sample etc. according to the step of aforementioned preparation；
2) reaction diluent (1 ×) of 50 μ L is added in the hole of blank group；
3) 50 μ L nitrate standard items or sample are added in remaining hole；
4) 25 μ L NADH are added in the hole Xiang Suoyou；
5) 25 μ L dilution nitrate reductase is added in the hole Xiang Suoyou, is uniformly mixed, is covered with adhesive tape；
6) it is incubated for 30 minutes at 37 DEG C；
7) 50 μ L Griess I reaction solutions are added in all holes；
8) 50 μ L Griess II reaction solutions are added in all holes, gently the side of beating plate, stirs evenly；
9) it is incubated at room temperature 10 minutes；
10) optical density (OD) (O.D.) is measured under 540nm wavelength, wavelength calibration 690nm；
11) standard curve is generated according to standard items measured value, and calculates and obtains nitrate content in every group of sample, under
Table 1 and attached drawing 1.
Nitrate content (mol/g) in mouse blood after table 1 is administered
Note: above-mentioned each group numerical value is the average value of nitrate content in each group mouse blood.
As it can be seen from table 1 individual sodium nitrate and composition of the invention (combination 1- combination is administered to intragastric administration on mice
5) after, at 2 hours, compared with individual nitrate is administered, the content of nitrate in the mouse blood of administration combination 4 and 5
It has produced and has significantly improved, especially nitrate content is that independent nitre is administered in the mouse blood of the composition of administration combination 5
Twice or more of nitrate content in the mouse blood of hydrochlorate；When being up to 6 hours~12 hours, the mouse of administration combination 4 and 5
The content of nitrate is significantly higher than the content that nitrate in the mouse blood of individual nitrate is administered in blood.
The internal anti-carcinoma of mouth activity rating of the composition of the invention of test example 2
1) foundation of carcinoma of mouth Nude Mouse Model in vivo
Select 4 week old Female nude mices for experimental animal (female, 20 ± 5g), routine culture human mouth squamous cell carcinoma system
CAL27, by 2.5 × 107/ mL concentration is only injected subcutaneous into nude mice right hind groin with 0.2mL/.Quickly increase when knurl enters
Each group compound is given when long-term:.
2) nitrate composition drinking water administration and transplanted tumor in nude mice observation
Experimental animal is divided into 2 control groups (physiological saline group and sodium nitrate group) and 2 composition group (5 solids of combination
5 liposome groups of group and combination, wherein combining 5 solids to be is 2:1 (combination 5) according to the mass ratio of the material example prepared by embodiment 1
Sodium nitrate and ascorbic composition, 5 liposomes of combination are the liposome of the combination 5 prepared according to embodiment 4), every group 10
Only.2 composition groups (5 liposome groups of 5 solid groups of combination and combination) and 1 control group (sodium nitrate group) give sodium nitrate respectively
Composition (5 liposomes of 5 solids of combination or combination) or sodium nitrate drinking-water, concentration 2.5mmo1/L, the every daily ingestion of every rat
Nitrate content is about 0.5mmo1/Kg b.w., and continuous nitrate is drunk water 1 month；1 control group (physiological saline group) give with
The isometric physiological saline of above-mentioned composition, normal water.Periodic logging transplantable tumor tumor volume and nude mice weight.As the result is shown
In figure 2 and figure 3.
As can be seen that administration present composition group can significantly inhibit the growth of tumour cell from Fig. 2 and Fig. 3, and it is right
Nude mice weight is without influence.
The external anti-carcinoma of mouth activity rating of the composition of the invention of test example 3
Select 4 week old Female nude mices for experimental animal (female, 20 ± 5g), routine culture human mouth squamous cell carcinoma system
CAL27 is that 50000/mL is transferred to 96 orifice plates with concentration, detects negative control physiological saline and the present invention with the method for CCK8
Composition (according to embodiment 4 prepare combination 5 liposome) extracorporeal anti-tumor cell Proliferation activity.
Specific experimental method is as follows: l00 μ L cell suspension is inoculated in 96 orifice plates, by culture plate in incubator preculture 24
Hour (at 37 DEG C, 5%CO2Under conditions of), test compound (physiological saline or combination of the invention are added after cell is adherent
Object), every hole administered volume is 25 μ L, is then incubated for culture plate 72 hours in incubator, the culture medium l00 μ that each hole more renews
L, and l0 μ L CCK-8 solution is added to every hole.The absorbance at 450nm is measured with microplate reader after 2 hours.
As the result is shown in Fig. 4-Fig. 5.
Fig. 4 shows the influence of composition of the invention to carcinoma of mouth CAL27 cellular morphology: under inverted microscope, control
Group cell adherent growth, cell are in shuttle shape or polygonal, and endochylema is abundant, and growth is vigorous, and flanking cell growth fusion is in blocks.It is added
After the sodium nitrate of 100mM, CAL27 cell starts to become smaller, be rounded, and refractivity enhancing, cell adherence quality reduces, and presentation is typically withered
Form is died, the morphological changes such as the agglutination of visible stain matter, karyopycnosis, karyorrhexis under mirror.
Fig. 5 shows influence of the nitrate composition of the invention of various concentration to carcinoma of mouth CAL27 cell Proliferation: nitre
Hydrochlorate (30mM concentrations above) can obviously inhibit the proliferation of tumour cell.
The anti-adhesion activity of the composition of the invention of test example 4
Scarification detects Xi Bao gan and moves:
50000/mL the cell of 1.6mL is transferred in 6 orifice plates and is cultivated, cell is grown to converge completely after using 200U
Sterilizing inhales sample pipette tips for cell scratch, and 1 × PBS is washed 1 time, be added negative control physiological saline and composition of the invention (according to
The liposome of combination 5 prepared by embodiment 4) each 0.4mL, continues to cultivate 6h, restores cell, then replace with containing 0.5% tire ox
1640 culture medium of RPMI of serum continues to cultivate, and each scratch position (0h) at once is observed after mechanical scratch under inverted microscope
And take a picture, for 24 hours after, observe mechanical scratch and take a picture under inverted microscope again.
Statistical analysis is carried out using 17.0 statistics software of SPSS, multiple groups measurement data compares to be analyzed using ANOVA, P
< 0.05 is statistically significant.
By the test example as the result is shown in Fig. 6-Fig. 7.As can be seen from Figures 6 and 7, nitrate of the invention combination
Object has the activity of significant anti-migration to carcinoma of mouth CAL27 cell.
5 toxicity pharmacological experiment of test example
To water (control group) is given in mouse (every group 30, half male and half female) interior 1 stomach-filling for 24 hours, 2 kinds of compositions (it is solid to combine 5
5 liposome groups of body group and combination, wherein combining 5 solids to be is 2:1 (combination 5) according to the mass ratio of the material example prepared by embodiment 1
Sodium nitrate and ascorbic composition, 5 liposomes of combination be the liposome of the combination 5 prepared according to embodiment 4) (it is respective,
380mg/kg, as under normal circumstances, 9 times of daily dosage) after, control group, 5 solid groups of combination and 5 liposome groups of combination exist
Animal movement situation, eyelid indication, breathing, fur, excreta and secretion etc. in 14 day observation period after the administration same day and administration
It is showed no any exception；Without animal dead during experiment.Show composition of the invention under doses for animal body without
Toxicity, safety are available.
It will be appreciated by those skilled in the art that under without departing from spirit or feature of the invention, it can be with any other
The concrete form of equivalency range implements the present invention.Therefore, in the meaning and scope of the equivalence of claims of the present invention
All changes be intended to and be included within the scope of the invention.
Claims (10)
1. composition comprising nitrate and vitamin, wherein the ratio of the amount of the substance of the nitrate and vitamin is 10:
1-1:10。
2. composition according to claim 1, wherein the nitrate is selected from sodium nitrate, potassium nitrate or combinations thereof, preferably nitric acid
Sodium.
3. composition according to claim 1 is preferably tieed up wherein the vitamin is selected from vitamin C, vitamin B6 or combinations thereof
Raw element C.
4. composition as claimed in one of claims 1-3, wherein the ratio of the amount of the substance of the nitrate and vitamin
For 5:1-1:5, more preferable 3:1-1:1, most preferably 2:1.
5. preparation comprising composition of any of claims 1-4 and one or more pharmaceutically acceptable taxes
Shape agent.
6. preparation according to claim 5 is sustained release tablets, liposome, lipid microemulsion or lyophilized preparation.
7. preparation according to claim 6 is the liposome of partial size 150-200nm, formula composition is as follows: sodium nitrate 0.2
~2.0g, 0.2~0.9g of vitamin C, 0.5~3.5g of lecithin, 0.1~0.5g of cholesterol, 0.05~0.4g of laruyl alcohol, phosphoric acid
Salt buffer (PBS) 100~200mL.
8. composition as claimed in one of claims 1-4 is mentioned according to the preparation of any one of claim 5-7 in preparation
Purposes in high blood in the drug of nitrate content.
9. purposes according to claim 8, wherein the drug is used to adjust cardiovascular function, improvement and/or treatment stomach and intestine
Stress damage, prevention liver retrogression pathological changes and hepatic steatosis mitigate damage caused by total body radiation.
10. composition as claimed in one of claims 1-4 is controlled according to the preparation of any one of claim 5-7 in preparation
It treats tumour and/or inhibits the purposes in tumor cell proliferation drug.
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| CN201910544729.8A CN110327367A (en) | 2019-06-21 | 2019-06-21 | Composition, preparation method, preparation containing nitrate and vitamin and application thereof |
| PCT/CN2020/097156 WO2020253835A1 (en) | 2019-06-21 | 2020-06-19 | Nitrate and vitamin-containing composition, preparation method therefor, formulation thereof and use thereof |
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| CN201910544729.8A CN110327367A (en) | 2019-06-21 | 2019-06-21 | Composition, preparation method, preparation containing nitrate and vitamin and application thereof |
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| CN112472714B (en) * | 2020-12-03 | 2022-09-16 | 爱希（北京）国际咨询有限公司 | Nitrate-containing composition and application thereof |
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| CN113398098A (en) * | 2021-08-19 | 2021-09-17 | 首都医科大学附属北京口腔医院 | Microcapsule, preparation method thereof and application of microcapsule in preventing and/or treating salivary gland injury caused by radiotherapy |
| CN113398098B (en) * | 2021-08-19 | 2021-11-02 | 首都医科大学附属北京口腔医院 | Microcapsule, preparation method thereof and application of microcapsule in preventing and/or treating salivary gland injury caused by radiotherapy |
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| WO2023019950A1 (en) * | 2021-08-19 | 2023-02-23 | 首都医科大学 | Microcapsule, preparation method therefor, and use thereof in preventing and/or treating damage to salivary glands caused by radiation therapy |
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