WO2020253835A1

WO2020253835A1 - Nitrate and vitamin-containing composition, preparation method therefor, formulation thereof and use thereof

Info

Publication number: WO2020253835A1
Application number: PCT/CN2020/097156
Authority: WO
Inventors: 王松灵; 王玉记; 李国情; 秦力峥; 胡亮
Original assignee: 爱希（北京）国际咨询有限公司
Priority date: 2019-06-21
Filing date: 2020-06-19
Publication date: 2020-12-24
Also published as: CN110327367A

Abstract

A nitrate and vitamin-containing composition, a formulation thereof, a preparation method therefor, use thereof for preparing a medicament for improving the concentration of nitrates in blood, and use thereof in tumor treatment.

Description

Composition containing nitrate and vitamin, its preparation method, preparation and its use

Technical field

The present invention relates to the technical field of medicine, in particular, to a composition containing nitrate and vitamins, its preparation (Naruit), its preparation method, and its use in the preparation of drugs for increasing blood nitrate content or treating tumors and/ Or use in drugs that inhibit tumor cell proliferation.

Background of the invention

Nitrate and nitrite are widely found in water, soil, air and plants. The main source of nitrate absorption by the human body is food, and green vegetables account for the main part.

Nitrate plays a physiological function in various system activities, including blood pressure reduction, platelet aggregation inhibition and vascular protection (Lundberg JO et al., Cardiovasc Res, 2011; Kapil V et al, Nitric Oxide, 2014). Rat skeletal muscle cells can take up nitrate from peripheral blood, and then reduce nitrate to nitric oxide through the xanthine oxidoreductase pathway, thereby increasing the body’s blood flow and promoting metabolism (Weitzberg E et al., Annu Rev Nutr, 2013). Nitrate prevents ischemic heart disease by increasing vasodilatory epicardial blood flow, reducing vascular resistance, reducing coronary blood stealing and reducing preload (Pragani MA et al., Rev Cardiovasc Med, 2017). Nitrate in the diet can reduce endogenous nitric oxide production by inhibiting NADPH oxidase and regulating the expression of angiotensin (Ang) II receptors, thereby partially improving age-related hypertension and metabolic activity in mice (Hezel M et al., Free Radic Biol Med, 2016). In addition, inorganic nitrates inhibit acute and chronic inflammation by increasing neutrophil counts, which may reduce the occurrence of atherosclerotic plaques (Khambata RS et al., Proc Natl Acad Sci USA, 2017). In addition, a long-term dietary nitrate deficiency experiment showed that after 22 months of a low-nitrate diet, mice will experience metabolic syndrome, endothelial dysfunction, and cardiovascular death (Kina-Tanada M et al., Diabatologia, 2017) . Inorganic nitrates can reduce blood pressure and improve myocardial ischemia by enhancing the activity of epithelial cells and relaxing blood vessels, reducing platelet aggregation (Machha A et al., Eur J Nutr, 2011).

In the past, nitrates were considered harmful because under certain conditions, such as acidic gastric environment, carcinogenic nitrosamines may be produced, and nitrosamines are related to esophageal cancer, gastric cancer, colon cancer and other tumors (Bedale W et al., Meat Sci, 2016, Park JE et al, Toxicol Res, 2015). Therefore, the World Health Organization (WHO) recommends that the upper limit of daily intake of nitrate and nitrite is 3.7mg/kg and 0.06-0.07mg/kg respectively (Weitzberg E et al., Annu Rev Nutr, 2013) . However, with recent epidemiological investigations of nitrate and tumors, there is no clear evidence that dietary nitrates can increase the occurrence of tumors (Bryan NS et al., Food Chem Toxicol, 2012). In 2012, Sialin protein was first reported as a nitrate cell membrane transporter, which played an important role in the dietary nitrate cycle. Nitrate is actively transported by Sialin in the salivary glands, concentrated in saliva, then secreted into the mouth, and re-enters the body circulation through the gastrointestinal tract (Qin LZea al., Proc Natl Acad Sci USA, 2012). Since nitrate in the diet is converted into NO by oral and stomach bacteria through non-enzymatic synthesis, nitrate is considered to be indispensable in physiological activities.

Therefore, nitrate plays an important physiological regulation role in the body, and dietary nitrate can basically meet the amount of nitrate required by healthy people. However, for patients with cardiovascular function damage, gastrointestinal stress damage, liver degeneration, liver steatosis, systemic radiation damage and tumors, the intake of dietary nitrates simply cannot reach the therapeutic dose (~13mg /kg). So far, the prior art has not provided nitrate preparations that are suitable for direct use as drugs and produce good absorption effects and excellent therapeutic effects.

After repeated and in-depth research, the applicant unexpectedly found that mixing nitrate and vitamins in a specific ratio to prepare a composition, especially a nanoliposomal formulation composition, can significantly increase the content of nitrate in the body and produce The effect of treating tumors, thus completing the present invention.

Summary of the invention

An object of the present invention is to provide a composition containing nitrates, said composition comprising nitrates and vitamins, wherein the ratio of the amount of nitrates and vitamins is 10:1 to 1:10, preferably 5:1 -1:5, more preferably 3:1-1:1, most preferably 2:1-1:1.

Wherein, the nitrate is sodium nitrate, potassium nitrate or a combination thereof, preferably sodium nitrate or potassium nitrate, and most preferably sodium nitrate. The vitamin is vitamin C, vitamin B6 or a combination thereof, preferably vitamin C or vitamin B6, most preferably vitamin C.

Wherein, the ratio of the amount of the nitrate and the vitamin may be 10:1, 5:1, 2:1, 1:1, 1:2, 1:3, 1:5, 1:10, more preferably In particular, the ratio is 2:1, 1:1.

Another object of the present invention is to provide a preparation method of the composition comprising nitrate and vitamins according to the present invention, which includes dissolving nitrate and vitamins separately, filtering and sterilizing with a 0.22 μm microporous filter membrane, according to the above nitrate and vitamin The ratio of the amount of the vitamin substance is mixed, the mixed solution is divided into sterile vials, sealed, frozen at -20～-60℃ for 2～18h, freeze-dried with a lyophilizer: set -20～- Sublimation once at 20℃ for 4-20h, and sublimation twice at 20-40℃ for 1-30h, then take it out.

Another object of the present invention is to provide a preparation comprising the composition of the present invention including nitrate and vitamins and one or more pharmaceutically acceptable excipients.

Wherein, the preparation can be any pharmaceutically acceptable preparation form, such as solid form or liquid form, such as tablet, capsule, granule, oral liquid, injection, liposome, lipid microemulsion, freeze-dried Powder preparations and so on.

The preparation method of the formulation can be prepared according to conventional methods known in the art. For the specific preparation form of the present invention, preferably, it can be prepared according to the specific preparation method described in the embodiment of the present application.

In the formulations of the present invention, the pharmaceutically acceptable excipients can be any suitable excipients suitable for the preparation of different formulations, such as fillers, diluents, binders, disintegrants, glidants Agents, lubricants, flavoring agents, inclusion materials, freeze-dried stabilizers.

A preferred form of preparation is liposome, with a nanometer particle size of 150-200nm, and the formula composition is as follows: sodium nitrate 0.2-2.0g, vitamin C 0.2-0.9g, lecithin 0.5-3.5g, cholesterol 0.1-0.5g, Lauryl alcohol 0.05～0.4g, phosphate buffered saline (PBS) 100～200mL.

A preferred liposome includes 0.2 g of sodium nitrate, 0.2-0.83 g of vitamin C, 0.5-2.0 g of lecithin, 0.1-0.4 g of cholesterol, 0.05-0.2 g of lauryl alcohol, and an appropriate amount of phosphate buffered saline (PBS).

Another object of the present invention is to provide the use of the composition comprising nitrate and vitamin or its preparation in the preparation of a medicine for increasing the nitrate content in blood.

Studies have shown that nitrate can relax blood vessels. When the body is injured by ischemia, the pH of the damaged part often drops below 7. By quickly replenishing nitrate and increasing the content of nitrate in the blood, it can relax the aortic ring and produce regulation. The role of cardiovascular function. Moreover, studies have shown that increasing the nitrate content in the blood can regulate gastrointestinal function, improve and/or treat gastrointestinal stress damage, prevent liver degeneration and liver steatosis, and reduce damage caused by systemic radiation.

Still another object of the present invention is to provide the use of the composition comprising nitrate and vitamins or preparations thereof in the preparation of drugs for treating tumors and/or inhibiting the proliferation of tumor cells. Preferably, the tumor is oral cancer.

So far, the prior art has not provided nitrate preparations that are suitable for direct use as drugs and produce better absorption effects and excellent therapeutic effects. In the present invention, nitrate and vitamin are prepared into a composition (especially preferably, nanoliposome preparation composition) according to a specific ratio, which can significantly increase the nitrate content in the blood, increase the bioavailability, and can be used to regulate cardiovascular function , Improve and/or treat gastrointestinal stress damage, prevent liver degeneration and liver steatosis, or reduce damage caused by whole body radiation without any adverse side effects. For example, according to animal experiments, the blood concentration of composition 5 in rats in 2 hours is 2.1 times that of the nitrate group alone, which is equivalent to that of composition 5 when the dose is reduced by one time and the effect of nitrate alone is still equivalent And the composition 5 of nitrate and vitamins can produce a significant effect of treating tumors; the composition of the present invention is confirmed by animal experiments, without any toxicity, safe and effective. Therefore, the technical solution of the present invention produces unexpected technical effects, and has important clinical significance.

The dose of the composition of the present invention or the pharmaceutical composition administered to the patient may depend on, for example, the type of disease to be treated or the level of nitrate content in the blood that needs to be reached, and may be based on a dose-selection method known to those skilled in the art Make a selection. For example, in certain embodiments, the dosage of the composition administered to the patient is about 13 mg/kg or higher based on the nitrate content of the composition, such as about 15 mg/kg to 30 mg/kg.

In this application, if there is no special description, the ratio of the nitrate to the vitamin is the ratio of the amount of the substance (ie, the molar ratio). If not specifically stated, the materials, devices or equipment, the preparation methods of the materials or compositions, etc., are all conventional or well-known in the art, or those skilled in the art can obtain them according to the description of this specification according to conventional technical means. of.

Description of the drawings

Fig. 1 is a graph of nitrate content in blood after gavage of 5 kinds of compositions of the present invention and sodium nitrate alone according to test example 1, according to body weight. Wherein the

combinations

1, 2, 3, 4 and 5 are the amount ratios of the substances prepared according to Example 1 of 5:1, 1:5, 1:1, 1:2, 2:1 sodium nitrate and vitamin C. combination.

Figure 2 is a graph showing the effect of a nitrate composition diet on the volume of transplanted tumors of oral cancer CAL27 cells in nude mice.

Figure 3 is a graph showing the effect of nitrate nanocomposites on the body weight of nude mice transplanted with oral cancer CAL27. Note: The results show that the application of nitrate has no significant change in the body weight of nude mice.

Figure 4 is a graph showing the effect of nitrate composition on the morphology of oral cancer CAL27 cells.

Figure 5 is a graph showing the effect of nitrate composition on the proliferation of oral cancer CAL27 cells.

Figure 6 is a graph showing the effect of nitrate composition on the scratch width of oral cancer CAL27 cells.

Figure 7 is a graph showing the anti-migration activity of a nitrate composition on oral cancer CAL27 cells.

Detailed ways

To further illustrate the present invention, some examples and test examples are given below. These embodiments are completely exemplary, and they are only used to specifically describe the present invention, and should not be construed as limiting the present invention.

Example 1 Composition of nitrate and vitamin

Composition 1: Sodium nitrate 0.85g, vitamin C 0.176g (substance ratio 10:1)

Composition 2: Sodium nitrate 0.85g, vitamin C 0.352g (substance ratio 5:1)

Composition 3: Sodium nitrate 0.85g, vitamin C 0.88g (substance ratio 2:1)

Composition 4: Sodium nitrate 0.85g, vitamin C 1.76g (substance ratio 1:1)

Composition 5: 0.85g of sodium nitrate, 3.52g of vitamin C (substance ratio 1:2)

Composition 6: sodium nitrate 0.85g, vitamin C 5.28g (substance ratio 1:3)

Composition 7: 0.85g of sodium nitrate, 8.8g of vitamin C (substance ratio 1:5)

Composition 8: 0.85g of sodium nitrate, 17.6g of vitamin C (substance ratio 1:10)

Preparation method: Weigh the corresponding amount of sodium nitrate or vitamin C according to the above ratio, dissolve the nitrate and vitamin C separately, filter and sterilize with a 0.22μm microporous filter, mix separately according to different proportions, and pack the mixed solution. In a sterile vial, sealed, frozen at -20～-60℃ for 2～18h, freeze-dried with a freeze dryer: set at -20～20℃ for 4～20h once sublimation, 20～40℃ for second sublimation 1 ～30h, take it out and get the composition.

Example 2 Tablets containing nitrate and vitamins

Composition of tablets (1000 tablets):

Preparation:

Weigh various components according to the prescription, pass the vitamin C, sodium nitrate and HPMC (HT-K80000S) through a 100-mesh sieve, and pass the lactose, ethyl cellulose and other auxiliary materials through an 80-mesh sieve, and mix well. Use 1% polyvinylpyrrolidone ethanol solution to make soft material, then use 20 mesh sieve to granulate, dry at 60°C for 1 hour, add magnesium stearate and mix well, pass through 20 mesh sieve to size, compress, polish, package, Ready to get tablets.

Each tablet of this product contains about 1.76~176mg of vitamin C, 8.5mg of nitrate, and the tablet weight is about 250mg.

Example 3 Nano microemulsion containing nitrate and vitamins

Composition of microemulsion:

Preparation:

Step 1: Under the protection of nitrogen, add 1000g soybean oil to 8g lecithin and 20g polyethylene glycol PEG2000, and heat to 30～70℃ to form an oil phase mixture;

Step 2: Mix the composition of 0.176g～17.6g vitamin C and 0.85g sodium nitrate, 200g poloxamer 188 and 8L water for injection in different proportions at 20～70℃ to form an aqueous mixture;

Step 3: Under the protection of nitrogen, mix the water phase mixture obtained in step 2 with the mixture obtained in step 1, and then disperse at a high speed for 5 to 30 minutes under the conditions of 30 to 80°C and 5000 to 50000 r/min, then use 0.1 mol/L NaOH or HCl adjust the pH to 6.0-8.5, homogenize for 5-15 times under the pressure of 90-210MPa, until the mixed phase is in a uniform and transparent state, and the obtained uniform and transparent mixed phase is detected, Divide the packaging to obtain nano microemulsion.

Example 4 Liposomes containing nitrate and vitamins

Preparation:

Step 1: Weigh lecithin, cholesterol and lauryl alcohol, put them in an eggplant-shaped bottle, and dissolve them with chloroform. Make dry film by rotary evaporation for use;

Step 2: Prepare phosphate buffer solution, dissolve vitamin C in an appropriate amount of phosphate buffer solution (PBS) 100-200mL, and then add sodium nitrate to obtain a solution;

Step 3: Slowly inject the solution of step 2 into the eggplant-shaped bottle of step 1, agitate slowly, and form liposomes after ultrasonic treatment.

The nanometer properties were characterized by a laser nanoparticle sizer, and the particle size was measured to be 150-200nm. The encapsulation efficiency of the liposome composition of

combinations

3, 4, and 5 is 92.5%, 94.3%, and 95.2%, respectively, as determined by the HPLC method."

Example 5 Granules containing nitrate and vitamins

Granule 1:

Preparation:

Mix 8.5g of sodium nitrate, 0.2～0.9g of vitamin C, and 2.0g of HPMC (HT-K80000S), and use 20ml of 20% ethanol solution to make soft material, and make pellets by extrusion spheronization method, and dry until the moisture does not exceed 3% , That is, sodium nitrate and vitamin C composition granules.

Granule 2:

Preparation:

Mix 10.1g of potassium nitrate, 35.2g of vitamin C, and 2.0g of HPMC (HT-K80000S) evenly, use 20ml of 20% ethanol solution to make soft material, extrude and spheronize to make pellets, and dry until the moisture does not exceed 3%, that is Obtain potassium nitrate and vitamin C composition granules.

Granule 3:

Preparation:

Sodium nitrate and 8.5g vitamin B ₆ 41.0g, and HPMC (HT-K80000S) 2.0g mixed with 20% ethanol solution 20mL obtain a soft material, extrusion and spheronization into particles, dried to a moisture less than 3%, Sodium nitrate and vitamin B6 composition granules are obtained.

Test Example 1 Detection of nitrate content in blood after administration of the composition of the present invention

C57BL6 mice (male, 30±5 g) (70 in total, 10 in each group) were anesthetized by intraperitoneal injection of pentobarbital physiological saline solution at a dose of 50 mg/kg. After anesthesia, the sodium nitrate and five nitrate-containing reagent compositions (prepared in accordance with Example 1 respectively, and the ratio of the amount of substances is 5:1 (combination 1), 1:5 (combination 2), 1:1 ( Combination 3), 1:2 (combination 4), 2:1 (combination 5) sodium nitrate and vitamin C composition) (in the six groups of experiments, the nitrate content is the same) by intragastric administration at 0.2 mmol/kg, respectively At 0h, blood was taken from the tail vein at 2, 4, 6, 12 h after intragastric administration.

Specific nitrate testing method (using total nitric oxide and nitrate/nitrite determination kit, PKGE001, R&D Systems, USA):

Ready to work:

1) Place the blood sample at 4°C for 2h, centrifuge at 14000rpm for 10min, and absorb the serum;

2) Filter the obtained serum and dilute 10 times;

3) Prepare 1× reaction solution: use distilled water/deionized water to dilute 10× reaction solution to 1× reaction solution;

4) Preparation of nitrate reductase: Reconstitute nitrate reductase with 1.0 ml of nitrate reductase stock solution, vortex vigorously, let stand at room temperature for 15 minutes, vortex and let stand at room temperature for another 15 minutes, vortex again and immediately use.

Dilute nitrate reductase with reaction diluent (1×) to prepare nitrate reductase with a concentration of 1/5 of the mother liquor. The steps are as follows:

a. Nitrate reductase (x hole + 2) × 5μL;

b. Reaction diluent (1×)=step a×4 times the volume;

c. Add the volumes in steps a and b to a clean test tube, vortex;

d. Put on ice and use within 15 minutes.

5) NADH reagent-Use 5.0 ml deionized water or distilled water to reconstitute NADH, let it stand for 3 minutes and gently stir before use, use within 15 minutes or place it on ice.

6) Preparation of nitrate standards:

Use a pipette to transfer 900 μL of reaction diluent (1×) into a 200 μmol/L tube, and then prepare 100 μmol/L, 50 μmol/L, 25 μmol/L, 12.5 μmol/L, 6.25 μmol/L, and 3.12 μmol/L in sequence Nitrate standards. The reaction diluent (1×) is blank (0μmol/L).

Nitrate content detection steps:

1) Prepare all reagents, standards, samples, etc. according to the aforementioned preparation steps;

2) Add 50μL of reaction diluent (1×) to the wells of the blank group;

3) Add 50μL of nitrate standard or sample to the remaining wells;

4) Add 25μL NADH to all wells;

5) Add 25μL of diluted nitrate reductase to all wells, mix well, and cover with tape;

6) Incubate at 37°C for 30 minutes;

7) Add 50μL Griess I reaction solution to all wells;

8) Add 50μL Griess II reaction solution to all wells, tap the side of the plate gently, and stir evenly;

9) Incubate for 10 minutes at room temperature;

10) Measure the optical density (O.D.) at a wavelength of 540nm, with a wavelength correction of 690nm;

11) Generate a standard curve based on the measured values of the standard products, and calculate the nitrate content in each group of samples, see Table 1 below and Figure 1 below.

Table 1 Nitrate content in blood of mice after administration (mol/g)

Note: The above values in each group are the average values of nitrate content in the blood of mice in each group.

It can be seen from Table 1 that after intragastric administration of sodium nitrate alone and the composition of the present invention (combination 1-combination 5) to mice, at 2 hours, compared with administration of nitrate alone, The nitrate content in the blood of mice in

combination

4 and 5 has been significantly increased, especially the nitrate content in the blood of mice administered the composition of combination 5 is the same as that in the blood of mice administered nitrate alone The content is more than twice as long as 6 hours to 12 hours, the blood nitrate content of mice administered with

combination

4 and 5 is still significantly higher than the blood nitrate content of mice administered nitrate alone .

Test Example 2 Evaluation of in vivo anti-oral cancer activity of the composition of the present invention

1) Establishment of nude mice transplanted tumor model of oral cancer in vivo

Female nude mice aged 4 weeks were selected as experimental animals (female, 20±5g), human oral squamous cell carcinoma cell line CAL27 was routinely cultured, and the concentration was 2.5×10 ⁷ /mL, and 0.2mL/mouse was injected into the right hind limb of nude mice subcutaneously . When the tumor body enters the rapid growth phase, give each group of compounds:.

2) Drinking water administration of nitrate composition and observation of transplanted tumor in nude mice

The experimental animals were divided into 2 control groups (physiological saline group and sodium nitrate group) and 2 composition groups (combination 5 solid group and combination 5 liposome group, where the combination 5 solid was the amount of the substance prepared according to Example 1. A composition of sodium nitrate and vitamin C in a ratio of 2:1 (combination 5), and the combination 5 liposomes are the liposomes of the combination 5 prepared according to Example 4), each group has 10 mice. Two composition groups (combination 5 solid group and combination 5 liposome group) and 1 control group (sodium nitrate group) were given sodium nitrate composition (combination 5 solid or combination 5 liposome) or sodium nitrate drinking water, respectively. The concentration is 2.5mmo1/L, the daily nitrate content of each rat is about 0.5mmo1/Kgbw, and the nitrate drinking water is continuous for 1 month; a control group (physiological saline group) is given the same volume as the above composition Normal saline, drink water normally. The volume of the transplanted tumor and the weight of the nude mice were recorded regularly. The results are shown in Figure 2 and Figure 3.

It can be seen from Fig. 2 and Fig. 3 that the administration of the composition of the present invention can significantly inhibit the growth of tumor cells without affecting the body weight of nude mice.

Test Example 3 Evaluation of in vitro anti-oral cancer activity of the composition of the present invention

Female nude mice aged 4 weeks were selected as experimental animals (female, 20±5g), human oral squamous cell carcinoma cell line CAL27 was cultured routinely, and transferred to a 96-well plate at a concentration of 50,000 cells/mL, and the negative control physiological saline was detected by the CCK8 method The anti-tumor cell proliferation activity of the composition of the present invention (the liposome of combination 5 prepared according to Example 4) in vitro.

The specific experimental method is as follows: inoculate 100μL of cell suspension in a 96-well plate, pre-culture the plate in an incubator for 24 hours (at 37°C, 5% CO ₂ ), and add test compound (physiological saline) after the cells adhere to the wall. Or the composition of the present invention), each well is administered with a volume of 25 μL, and then the culture plate is incubated in an incubator for 72 hours, each well is replaced with 100 μL of new medium, and 10 μL of CCK-8 solution is added to each well. After 2 hours, the absorbance at 450nm was measured with a microplate reader.

The results are shown in Figure 4-Figure 5.

Figure 4 shows the effect of the composition of the present invention on the morphology of oral cancer CAL27 cells: under an inverted microscope, the control group cells grow adherently, the cells are fusiform or polygonal, the cytoplasm is rich, the growth is vigorous, and the adjacent cells grow and fuse into sheet. After adding 100mM sodium nitrate, CAL27 cells began to become smaller, rounded, increased refractive index, decreased cell adhesion, and showed typical apoptotic morphology. The morphology of chromatin aggregation, nuclear pyknosis, and nuclear fragmentation can be seen under the microscope. change.

Figure 5 shows the effect of different concentrations of the nitrate composition of the present invention on the proliferation of oral cancer CAL27 cells: nitrate (at a concentration of more than 30 mM) can significantly inhibit the proliferation of tumor cells.

Test Example 4 Anti-adhesion activity of the composition of the present invention

Scratch method to detect cell migration:

Transfer 1.6mL 50,000 cells/mL cells to a 6-well plate for culture. After the cells grow to complete confluence, use a 200U sterile pipette tip to scratch the cells, wash once with 1×PBS, and add negative control saline and this The composition of the invention (the liposome of combination 5 prepared according to Example 4) was 0.4 mL each, and continued to culture for 6 hours to recover the cells, and then replaced with RPMI 1640 medium containing 0.5% fetal bovine serum to continue the culture, in the inverted microscope Observe the mechanical scratch immediately (0h) after the mechanical scratch and take a photo. After 24h, observe the mechanical scratch under an inverted microscope and take a photo again.

SPSS 17.0 statistical software was used for statistical analysis. The measurement data of multiple groups were compared by ANOVA analysis, and P<0.05 was statistically significant.

The results of this test example are shown in Figs. 6-7. It can be seen from Figure 6 and Figure 7 that the nitrate composition of the present invention has significant anti-migration activity on oral cancer CAL27 cells.

Test Example 5 Toxicity Pharmacology Experiment

Mice (30 in each group, half male and female) were given water (control group) and 2 compositions (combination 5 solid group and combination 5 liposome group) once within 24 hours. The amount ratio of the substance prepared in Example 1 is a composition of sodium nitrate and vitamin C in a ratio of 2:1 (combination 5), and the combination 5 liposomes are the combination 5 liposomes prepared according to Example 4) (each, 380 mg/ kg, which is 9 times the daily dose under normal circumstances), the control group, the combination 5 solid group and the combination 5 liposome group on the day of administration and the observation period of 14 days after administration of animal movement, eyelid finger There were no abnormalities in symptoms, breathing, fur, excreta and secretions; no animal died during the experiment. It is shown that the composition of the present invention is non-toxic to animals at a certain dose and is safe and usable.

Those skilled in the art should understand that, without departing from the spirit or characteristics of the present invention, the present invention can be implemented in any other specific form within the equivalent scope. Therefore, all changes within the meaning and scope of equivalence of the claims of the present invention are intended to be included in the scope of the present invention.

Claims

The composition includes nitrate and vitamin, wherein the ratio of the amount of the nitrate and vitamin is 10:1 to 1:10.
The composition according to claim 1, wherein the nitrate is selected from sodium nitrate, potassium nitrate or a combination thereof, preferably sodium nitrate.
The composition according to claim 1, wherein the vitamin is selected from vitamin C, vitamin B6 or a combination thereof, preferably vitamin C.
The composition according to any one of claims 1 to 3, wherein the ratio of the amount of the nitrate and vitamin substances is 5:1 to 1:5, more preferably 3:1 to 1:1, most preferably 2: 1.
A formulation comprising the composition of any one of claims 1 to 4 and one or more pharmaceutically acceptable excipients.
The formulation according to claim 5, which is a sustained-release tablet, liposome, lipid microemulsion or lyophilized formulation.
The preparation according to claim 6, which is a liposome with a particle size of 150-200nm, and its formula is composed of: 0.2-2.0g sodium nitrate, 0.2-0.9g vitamin C, 0.5-3.5g lecithin, and 0.1-0.5g cholesterol , Lauryl alcohol 0.05 ~ 0.4g, phosphate buffered saline (PBS) 100 ~ 200mL.
Use of the composition according to any one of claims 1 to 4 or the preparation according to any one of claims 5-7 in the preparation of a medicament for increasing blood nitrate content.
The use according to claim 8, wherein the medicament is used for regulating cardiovascular function, improving and/or treating gastrointestinal stress damage, preventing liver degeneration and liver steatosis, or reducing damage caused by whole body radiation.
Use of the composition according to any one of claims 1 to 4 or the preparation according to any one of claims 5-7 in the preparation of a drug for treating tumors and/or inhibiting tumor cell proliferation.