KR102553967B1 - Two-component type spray formulation for nasal or oral for inhibiting virus infection - Google Patents

Abstract

The present invention relates to a first solution, which is a nitrogen oxide donor solution containing natural nitrite ions (NO ₂ ^- ) produced by microbial fermentation, and nitrogen oxide acting as an accelerator to promote the generation of nitric oxide by a reduction reaction with the first solution. The present invention relates to a two-component nasal or oral spray formulation in which a second liquid, which is a reducing liquid, is independently separated, and a first liquid, which is a nitrogen oxide donor, and a second liquid, which is a nitrogen oxide reducing liquid, are simultaneously sprayed to rapidly generate nitrogen oxide. .
Two-component nasal or oral spray formulations for suppression of respiratory viral infections use natural fermented products, not synthetic chemicals, as raw materials, and use nitric oxide donating solutions that contain various antioxidants contained in these materials as raw materials, At the same time, as the nitric oxide reducing solution, the risk to the human body is minimized by using materials used as food raw materials such as erythorbic acid, ascorbic acid or citric acid.
In addition, the two-component nasal or oral spray formulation of the present invention is stored in a small spray container, so it is convenient to carry and use. Of course, it has a helpful effect on relieving symptoms, and as has already been found in several research papers, it also shows side effects on cold prevention, rhinitis, sinusitis, and other inflammatory diseases through the supply of sufficient amounts of nitric oxide in the nasal cavity.

Description

Two-component type spray formulation for nasal or oral for inhibiting virus infection}

The present invention relates to a two-component nasal or oral spray formulation independently distinguished for the suppression of respiratory viral infection, and specifically, a nitrogen oxide donating solution containing nitrite ion (NO ₂ ^- ) and a reduction reaction with the nitrous oxide donating solution It relates to a two-component nasal or oral spray formulation in which a nitric oxide reducing solution, which is a trigger for generating nitric oxide, is independently separated.

As COVID-19 shows signs of gradually turning into an endemic (endemic disease), many pharmaceutical bio companies are actively developing spray-type (spray) products as a convenient way to prevent and treat corona, while domestic biotech companies also spray on the nose. Nasal Spray Corona prevention and treatment are being developed in the form of medical devices.

Until now, research and development in response to corona infection had been approached as a treatment, but recently, amid active research that can be used for preventive purposes, the technology field that is attracting particular attention is a spray type that is sprayed into the nose in the form of a liquid nasal spray. mainly uses the result of a recent study that angiotensin converting enzyme 2 (ACE2), a viral receptor, is distributed in a relatively large amount in nasal epithelial cells (Non-Patent Document 1).

When nasal spray liquid is sprayed into the nose (intranasal cavity), gaseous nitric oxide (Nitric Oxide) generated around the nasal epithelial cells interferes with the binding of virus spike protein and ACE2 located on the epithelial cell membrane, or causes super Oxide (O ₂ ^- ) Combined with feroxynitrite (ONOO ^- ) It has been studied to inhibit the blocking and replication of corona virus by creating a strong active nitrogen species and destroying the DNA of the infectious virus (Non-Patent Document 2).

Nitric Oxide is a gaseous physiologically active substance produced in the body and is known as a neurotransmitter and physiologically active component for various metabolic activities in the human body, such as vasodilation, blood clot inhibition, anti-inflammation, antibacterial, antiviral, neuron, etc. It served as an opportunity for three Americans, Ignarro, Murad, and Perchcote, who proved its efficacy and role, to win the 1998 Nobel Prize in Physiology or Medicine.

Since then, more than 100,000 papers have been published in succession, and in 2004, it was known that nitric oxide inhibits the replication cycle of SARS corona virus by a research report by the Swedish Center for Infectious Disease Control (Non-Patent Document 3), but SARS quickly disappeared after that. and MERS incident, it was forgotten by researchers, but after the 2019 pandemic, many researchers began to verify the treatment effect through nitric oxide.

Nitric oxide gas inhalation has already been approved as a treatment for the pulmonary artery, so even without a separate clinical trial, many corona treatment hospitals obtained results of shortening the treatment period by administering nitric oxide gas to the respiratory tract for severely ill patients, but it exists as a gas. Complex and huge treatment equipment, including a storage container for storing nitric oxide and an electronic control device that finely adjusts it to suit the patient's condition, can be used only in large hospitals such as general hospitals, so the explosively increasing corona There was a lot of distance to apply to patients.

However, as the antiviral efficacy of nitric oxide has already been proven, on the other hand, studies for the purpose of treatment and prevention using nitric oxide have begun to be attempted. After developing a spray-type treatment NONS (Nitric Oxide Nasal Spray) and obtaining emergency use approval in Israel with only the FDA 2 clinical results, sales were subsequently obtained in India, Bahrain, Thailand, Indonesia, and Singapore (Non-Patent Document 4 ).

Although it is late compared to overseas, companies currently developing spray prevention and treatment agents in Korea include GeneOne Life Science, Salvation, and SK Bioscience. It is currently undergoing phase 2 clinical trials in the United States for the purpose of preventing corona infection by developing a drug re-creation method using the principle that quinine, an ingredient, stimulates the nasal mucosa to release nitric oxide.

Regarding the prior art, for example, in relation to a composition for nasal or oral spray for suppression of respiratory virus infection, Patent Document 1 includes Manuka honey, propolis, xylitol, frankincense, myrrh, aesthetica, Taoin, Samneung, bongchul and vitamin C as active ingredients. Patent Document 2 discloses an anti-influenza virus agent comprising 0.01 to 0.3 w/v% of povidone-iodine, 1 to 5 w/v% of one or more osmotic taste agents selected from sodium chloride or glycerol, and 0.0001 to 0.1w of flavoring agent. Disclosed is an antiviral and antibacterial composition for ophthalmology, oral cavity, nasal cavity or inhalation, characterized in that it is an aqueous solution containing /v%.

In addition, Patent Document 3 discloses (i) a nitrite compound at a concentration of about 0.667 mg NO ₂ VmL to about 100 mg NO ₂ VmL; (ii) taste masking agents; and (iii) is formulated with a pH buffer. A pharmaceutically acceptable nitrite compound preparation composition for pulmonary delivery comprising an aqueous solution of a nitrite compound having a final pH of greater than 7.0 and less than 9.0 is disclosed, and in Patent Document 4, a liquid carrier introduced into the upper airway through the nasal cavity and when introduced into the liquid carrier Nasal formulations with a pH ranging from 4 to 10 comprising low molecular weight arginine-rich peptides derived from protamine which are present in liquid carriers to increase nitric oxide (NO) levels in the upper respiratory tract are disclosed.

KR 1627065 B KR 10-1935250 B PCT/US2008/088340 PCT/US2015/057154

Ji Hoon Ahn, JungMo Kim,Seon Pyo Hong et al., Nasal ciliated cells are primary targets for SARS-CoV-2 replication in the early stage of COVID-19, The Journal of Clinical Investigation, Jul. 2021, 131(13):e148517. Ji HoonAhn, JungMoKim, et al., Nasal ciliated cells are primary targets for SARS-CoV-2 replication in the early stage of COVID-19, The Journal of Clinical Investigation,2021, 131(13). Sara Akerstrom et al., Nitric Oxide Inhibits the Replication Cycle of Severe Acute Respiratory Syndrome Coronavirus, Journal of Virology, Feb. 2005, 1966-1969. Stephen Winchester, Sarah John, et al., Clinical efficacy of nitric oxide nasal spray (NONS) for the treatment of mild COVID-19 infection, Journal of Infection, May. 2021, 21:49.

The problem to be solved by the present invention is a first solution that is a nitric oxide donating solution and a nitric oxide reducing solution that acts as a trigger to promote the generation of nitric oxide by a reduction reaction with the first solution to suppress respiratory virus infection. It relates to the provision of a two-component nasal or oral spray formulation in which the second liquid is independently distinguished.

More specifically, the first liquid, which is a nitrogen oxide-donating liquid containing natural nitrite ions (NO ₂ ^- ) produced by microbial fermentation, and the oxidation acting as an accelerator to promote the generation of nitric oxide by a reduction reaction with the first liquid. Provision of a two-component nasal or oral spray formulation in which the second liquid, which is a nitrogen reducing liquid, is independently separated, and the first liquid, which is a nitrogen oxide donor and the second liquid, which is a nitrogen oxide reducing liquid, are simultaneously sprayed to rapidly generate nitrogen oxide. is intended for

As a solution to the problem according to the present invention, a two-component nasal or oral spray formulation for suppressing respiratory viral infections contains nitric oxide (NO) containing natural nitrite ions (NO ₂ ^- ) produced by microbial fermentation of nitrate-rich plants. ) It consists of a two-component type in which the first liquid, which is a donor liquid, and the second liquid, which is a nitrogen oxide reducing liquid composed of an acidity regulator of pH 2 or higher that rapidly generates nitrogen oxide by a reduction reaction, are separately stored. .

One embodiment according to the two-component nasal or oral spray formulation for inhibiting respiratory viral infection of the present invention contains nitric oxide (NO) containing natural nitrite ion (NO ₂ ^- ) produced by microbial fermentation of nitrate-rich plants. The first liquid, which is a donor liquid, and the second liquid, which is a nitrogen oxide reducing liquid composed of an acidity regulator with a pH of 2 or higher that rapidly generates nitrogen oxide by a reduction reaction with the first liquid, are independently stored and separated, and nitrogen oxide is generated. It consists of a two-component type in which the first liquid and the second liquid are injected at the same time.

Another embodiment according to the two-component nasal or oral spray formulation for inhibiting respiratory viral infections of the present invention is produced by microbial fermentation of nitrate-rich plants selected from lettuce, beets, spinach, chicory, wasabi, and garlic. The first liquid, which is a nitrogen oxide (NO) donating solution containing natural nitrite ions (NO ₂ ^- ), and ascorbic acid and erythorbic acid, which generate nitrogen oxide by a reduction reaction with the first liquid Alternatively, it is composed of a two-component type in which a second liquid, which is a nitric oxide reducing liquid composed of an acidity regulator selected from citric acid, is independently distinguished.

A more specific embodiment according to the two-component nasal or oral spray formulation for inhibiting respiratory viral infections of the present invention is produced by microbial fermentation of nitrate-rich plants selected from lettuce, beet, spinach, chicory, wasabi, and garlic The first solution, which is a nitrogen oxide (NO) donating solution containing natural nitrite ions (NO ₂ ^- ), and ascorbic acid, erythorbic acid, or A second liquid, which is a nitric oxide reducing liquid composed of an acidity regulator selected from citric acid, is separately stored and separated, and the first liquid and the second liquid are simultaneously sprayed at the same ratio to generate nitrogen oxide. It is made up of two components.

The first liquid, which is a nitric oxide (NO) donating solution selected for the two-component nasal or oral spray formulation for suppressing respiratory viral infections according to the present invention, is sodium nitrite (NaNO ₂ ) or nitrous acid, which is commonly used as a nitric oxide gas donating solution It is characterized by using only natural nitrite ions obtained by microbial fermentation of nitrate-rich natural plants as a nitric oxide (NO) donating solution instead of chemical products such as potassium (KNO ₂ ), and nitric oxide reduction composed of an acidity regulator In the second liquid, which is a liquid, one or more components are selected from ascorbic acid, erythorbic acid, or citric acid rich in hydrogen ions (H ⁺ ) used as food raw materials harmless to the human body.

The two-component nasal or oral spray formulation for suppression of respiratory viral infection of the present invention uses a natural fermented product rather than a synthetic chemical as a raw material, and uses a nitric oxide donor solution containing various antioxidant components included in these materials as a raw material. At the same time, as the nitric oxide reducing solution, ascorbic acid or its isomer, erythorbic acid or citric acid, which is used as a food raw material, is used, thereby minimizing the risk to the human body.

In addition, the two-component nasal or oral spray formulation of the present invention is stored in a small spray container and is convenient to carry and use. As well as having a beneficial effect on relieving symptoms, as has already been found in several research papers, it also shows side effects on cold prevention, rhinitis, sinusitis, and other inflammatory diseases through the supply of a sufficient amount of nitric oxide in the nasal cavity.

1 is a video of an experimental device composed of a composite gas measuring device (MultiRAE Lite model) with a built-in nitrogen oxide measuring sensor
2 is a measurement of NO production of <Example 1> sample using natural fermentation raw materials
3 is a graph of measurement of NO generation amount of <Comparative Example 1> sample
Figure 4 is a relative comparison graph of NO production measurement of [Example 2] sample and <Comparative Example 1> sample
Figure 5 is a comparison graph of measurement of NO ₂ generated by side reactions of <Example 2> and <Comparative Example 1> samples
Figure 6 is a graph of the results of the cytotoxicity test evaluation of the sample

Hereinafter, the present invention will be described in more detail through specific details and <Examples> and <Test Examples> for carrying out the present invention, but the present invention is not limited by the description below.

The two-component nasal or oral spray formulation for suppressing respiratory viral infection of the present invention is a first nitric oxide (NO) donor solution containing natural nitrite ions (NO ₂ ^- ) produced by microbial fermentation of nitrate-rich plants. It consists of a two-component type in which a liquid and a first liquid and a second liquid, which is a nitrogen oxide reducing liquid composed of an acidity regulator of pH 2 or higher that rapidly generates nitrogen oxide by a reduction reaction, are separated and stored independently.

The first liquid, which is the nitric oxide (NO) donating liquid according to the present invention, contains nitrite ion, which is a precursor of nitric oxide, and contains natural nitrite ion (NO ₂ ^- ) produced by microbial fermentation of nitrate-rich plants (vegetables). It consists of containing

The first liquid contains nitrite ions (Nitrite) at a concentration of 1,000 to 20,000 ppm, and in an alkalized state, 0.5 to 0.5 to hydroxypropyl methylcellulose (HPMC) having a moisturizing and stabilizing effect based on the weight of the first liquid It consists of containing 5% by weight and maintaining the pH at 7.0 to 10.

It is possible to add one or more components selected from various sweeteners, flavoring agents, plasticizers, and surfactants to give coolness and coolness, such as menthol, peppermint, and herbal extracts, to the first liquid (nitric oxide donating liquid).

The nitrate-rich plant consists of one or more plants (vegetables) selected from lettuce, beetroot, spinach, chicory, wasabi, and garlic, and it is preferable to select plants (vegetables) containing a nitrate concentration of 100 ppm or more.

In addition, the microorganism (bacteria) selected for microbial fermentation of the nitrate-rich plant selects a single or mixed microorganism of microorganisms exhibiting nitrate-reducing properties among GRAS listed microorganisms specified by the Ministry of Food and Drug Safety and the US FDA, specifically, Lactobacillus fermentum ( Mixed species of Lactobacillus fermentum and Weicessella cibaria (KCTC13782BP), Lactobacillus casei (KCTC13899BP), Lactobacillus plamtarum (KCTC13902BP), Lactobacillus rhamnosus (KCTC) 13903BP ), Weissella paramesenteroides (KCTC13905BP), Lactobacillus paracasei (KCTC13906BP), Lactobacillus pentosus (KCTC13897BP), Bacillus amyloliquefaciens (KCTC138) 98BP), saccharin Strains selected from Saccharomyces cerevisiae (KCTC13900BP) and Bifidobacterium longum (KCTC13901BP) can be exemplified.

The second liquid, which is the nitrogen oxide reducing liquid according to the present invention, is composed of generating nitrogen oxide by a reduction reaction with the first liquid, and contains at least one from citric acid, erythorbic acid or ascorbic acid (vitamin C) used for food. In terms of composition, citric acid or erythorbic acid is advantageous in terms of economy, but in terms of marketing, ascorbic acid (vitamin C), which is well known to the general public, can be said to be advantageous, but is not limited to these two ingredients.

The pH of the second liquid should be maintained at pH 2.0 to 6.0 so that there is a sufficient hydrogen ion concentration to react with the first liquid sprayed into the nasal cavity or oral cavity and reduce and vaporize it into a nitric oxide (NO) gas state.

Since the first liquid, which is a nitrogen oxide (NO) donating liquid, and the second liquid, which is a nitrogen oxide reducing liquid according to the present invention, are rapidly generated by a chemical reaction from the moment they are mixed together, each must be independently It consists of storing separately, and the first liquid and the second liquid are simultaneously sprayed into the oral cavity or nasal cavity for the generation of nitric oxide.

When the first liquid and the second liquid are sprayed, when the pH condition of the second liquid (nitric oxide reducing liquid) is strongly acidic at pH 2 or less, the pH of the first liquid (nitric oxide donating liquid) becomes too low for a short time The production of nitric oxide is increased in the body and the amount released into the air through respiration is increased, shortening the duration of the effect, and strong acids can cause damage to the cells of the nasal cavity.

In addition, when the pH condition of the second solution (nitrogen oxide reducing solution) is weakly acidic or alkaline at pH 6 or higher, it meets the first solution (nitrogen oxide donating solution) in the pH range of 7.0 to 10.0 to maintain a neutral or alkaline state, After all, since hydrogen ions necessary for the reduction of nitrite ions are not supplied, it is difficult to generate nitrogen oxide gas, making it difficult to expect the original efficacy.

The same amount (1:1) of the first liquid and the second liquid is injected into the oral cavity or nasal cavity, and the sprayed mixed liquid has a nitrite ion concentration of 500 to 10,000 ppm and a pH of 4.0 to 6.9. Alternatively, nitric oxide that can relieve harmful bacteria, viruses, and inflammation in the oral cavity can be generated for a certain period of time.

Here, if the nitrite ion concentration is less than 500 ppm, it is difficult to expect a therapeutic effect by nitric oxide, and if it is higher than 10,000 ppm, more than necessary nitrite ion concentration is peroxinite combined with superoxide (O ₂ ^- ), an active acid species. This is because it creates a strong active nitrogen species called light (ONOO ^- ) to destroy the DNA of the infectious virus, but it can cause an inflammatory reaction in normal cells.

As described above, a characteristic feature of the present invention is that bacteria having nitrate reducing ability use nitrate, which is abundantly stored in the vacuole of plant chlorophyll, to microbial growth and metabolism when maintaining a specific temperature and pH in an anaerobic state in which oxygen is insufficient. In order to obtain the necessary oxygen, a decomposition process such as 2NO ₃ ^- → 2NO ₂ ^- + O ₂ is created, and then through several chemical reactions as shown in [Scheme 1] below, the anion, nitrite ion, becomes the hydrogen ion of the acid solution. It is based on the research result of the inventors of the present invention that nitrogen oxide gas is released by the reduction reaction combined with and, on the contrary, becomes chemically stable in an alkali solution (OH ^- rich condition).

[Scheme 1]

NO ₂ ^- + H ⁺ → HNO ₂

2HNO ₂ → N ₂ O ₃ + H ₂ O

NO ₂ ^- + 2H ⁺ → H ₂ O + NO↑ (gasification)

N ₂ O ₃ → 1/2O ₂ + 2NO↑ (vaporization)

And, as revealed in many medical journals, the above facts are converted into nitrite ions by oral anaerobic bacteria when ingesting fresh vegetables rich in nitrate, and pass through the gastrointestinal tract, and then reduced and decomposed by stomach acid to form nitric oxide It can be clearly seen that the principle of the metabolic process in the body that is partially released is applied.

In addition, the two-component nasal or oral spray formulation for suppressing respiratory virus infection of the present invention is provided with a container (storage means) capable of completely storing the first liquid and the second liquid separately, and the first liquid and The second liquid can be sprayed using a spray means capable of spraying the same amount at the same time, and a commercially available spray means is also possible, but when using a commercially available spray means, the first and second liquids must be independently stored. It is very important to check whether the first liquid and the second liquid can be sprayed in the same amount at the same time.

In the actual productization stage, when the liquid phase is put into a double-separated container, compared to the liquid phase stored in an aluminum pouch, in the case of the liquid stored simply in the container, there is a risk of loss due to vaporization during long-term storage, and the entire liquid phase on the bottom of the container Since it cannot be pumped, it should not be overlooked that the amount of liquid stored in the container must be slightly larger than the amount injected into the aluminum pouch, and the ratio of the two liquids is not 1:1, but 1:1.05 ~ 1:1.20. While determining, it is preferable to determine a large amount of an inexpensive nitrogen oxide reducing solution, such as citric acid, in consideration of economic feasibility.

Hereinafter, the two-component nasal or oral spray formulation for suppression of respiratory virus infection of the present invention will be described in more detail by <Example> and <Test Example>.

<Example 1>

A. Preparation of Nitric Oxide Donation Solution (First Solution)

One). Preparation of natural nitrate extract

Requested from a lettuce hydroponic cultivation company located in Anseong, Gyeonggi-do, received 10Kg of romaine lettuce grown hydroponically with a specially designed culture medium, dried for 12 hours in a low-temperature hot air dryer at 45 ~ 50℃ to make about 600g of dried lettuce into powder, and purified water 198g, which is 6.6% based on the weight ratio of 3 liters, was added, hot water extraction was performed at 80 ~ 90 ° C for more than 2 hours to obtain the first extract, and new purified water was added to make 2 liters, and then extracted again under the same conditions. By adding the secondary extract obtained by the above, about 4.2 liters of nitrate extract was finally obtained.

After making a color reaction using a Gries reagent for the extract, UV analysis was performed to obtain a lettuce hot water extract having a nitrate concentration as shown in [Table 1] below.

division Yield (ml) NO ₃ ^- content (ppm) 1st extraction 2,535 4,309 2nd extraction 1,655 1,832 Sum of 1 + 2nd extract 4,190 2,873

2). Microbial inoculation and fermentation

In order to reduce nitrate to nitrite through fermentation process, Lactobacillus plamtarum (KCTC 13902BP) strain was selected and cultured in NA medium to 10*9 colonies, which is 1% of 4,190㎖ of lettuce extract. After inoculating 42㎖, fermentation was carried out at 25 ~ 30 ℃ and pH 6.5 or higher for 72 hours to obtain a fermentation broth containing nitrite ions. It was confirmed through three measurements that it was sufficiently contained, and the results of component analysis of the fermentation broth are shown in [Table 2] below.

division Nitrite (NO ₂ ^- ) Residual Nitrate (NO ₃ ^- ) NH4+ pH 1 measurement 2,689 ppm 592 ppm 56 ppm 6.7 2 measurements 2,655 ppm 702 ppm 68 ppm 6.7 3 measurements 2,674 ppm 652 ppm 64 ppm 6.6 medium 2,673 ppm 649 ppm 63 ppm 6.7

Through the component analysis in [Table 2], 4,190 ppm of nitrate extract was converted to an average of 2,673 ppm in the metabolic process of nitrate reducing bacteria. This is because the specific fermentation odor increases if it is longer than 72 hours.

3). Powdering of fermentation broth

After nitrate extracted from lettuce is reduced to nitrite ions through fermentation of Lactobacillus plantarum strains, alkalinization is required to neutralize and remove hydrogen ions (H ⁺ ) remaining in the slightly acidic fermentation broth. At the same time, the pH is adjusted to 7.5 using magnesium hydroxide (Mg(OH) ₂ ), an acidity regulator for food, and then sterilized in an autoclave at 121°C, 1.5 atmospheric pressure for 15 minutes, and then finalized using a vacuum concentrator. As a result, 680 ml of lettuce extract fermented concentrate was obtained, and the component results of the lettuce extract fermented concentrate are shown in [Table 3] below.

division Nitrite
(NO ₂ ^- ) Residual Nitrate
(NO ₃ ^- ) NH ₄ ⁺ pH Sugar content
(Brix.) Salinity (%) 1 measurement 9,582 ppm 2,201 ppm 167 ppm 8.7 1.8 0.9 2 measurements 9.603 ppm 2,817 ppm 98 ppm 8.5 1.8 1.1 3 measurements 9.661 ppm 2,394 ppm 141 ppm 8.4 1.9 1.2 medium 9,615 ppm 2,471 ppm 135 ppm 8.5 1.8 1.1

The nitrite ion in the fermentation broth was concentrated about 3.6 times through the vacuum concentrator, but it was confirmed that ammonia, which is highly volatile, volatilized and disappeared in a considerable amount under reduced pressure conditions and was concentrated about 2.1 times. Uniquely, it is the fact that the salt content of the fermented concentrate was 1.1%, which is similar to 0.9% of the body fluid. It can be found that there is a feature of using natural salt derived from plants.

For long-term storage of the nitrite ion-containing fermentation broth, it is preferable to further concentrate the concentrate to Brix.

4). Nitric oxide donor preparation

In order to finally prepare a nitric oxide donor solution, HPMC, which is commonly used for wet stability, was added to the extract in an amount of 0.5% by weight, and a nitric oxide (NO) donor solution having a pH of 8.5 was prepared.

Of course, there is no need to add refined salt here, and Benzalkonium Chloride (C ₆ H ₅ CH ₂ N (CH ₃ ) ₂ R), which is used for the purpose of a bactericidal preservative in some antiviral nasal spray treatments, is domestically available. It was used as a humidifier additive, but it was not used in the present invention because repeated inhalation was found to be the cause of lung fibrosis and caused a great social impact. Being able to do that is another difference.

B. Preparation of Nitric Oxide Reduction Solution (Second Solution)

As a component of the acidic nitric oxide reducing solution, instead of ascorbic acid (vitamin C) or erythorbic acid, which are easy to oxidize and have poor storage properties, edible citric acid, which is economical and more stable thermally and chemically, is used. A nitric oxide reducing solution was prepared.

The above-prepared nitric oxide reducing solution at pH 3.0 is pH 5.5 to 6.0 when mixed 1:1 with the previously prepared nitrogen oxide donating solution (first solution) at pH 8.5, providing an appropriate amount of nitric oxide without stimulating nasal cells. It is a value intentionally designed to release for a long time, and unlike the nitric oxide donating solution (first solution), the reason why sterilization was not performed is that purified water was used when diluting citric acid and there is no concern about spoilage due to low acidity.

C. Storage of the spray bottle

The spray means includes a storage container for storing the first liquid, and a second liquid is stored in the storage container for the first liquid, and a sealed aluminum pouch is separately provided so that the first liquid and the second liquid are stored independently, A two-component spray means capable of spraying two liquids at the same time was arbitrarily manufactured and prepared by the present inventors.

The prepared aluminum pouch of the two-component spray means was filled with 25 ml of the nitric oxide donating solution (first solution) of pH 8.5 prepared above, and the nitrogen oxide reducing solution (second solution of pH 3.0 prepared from citric acid) was filled in the storage container. Liquid) 30 ml was filled to finally complete a prototype containing a two-component nasal or oral spray formulation for suppression of respiratory virus infection.

<Example 2>

After purchasing 10 'ENOVID' products, a nasal spray treatment product manufactured only with chemical ingredients sold for the purpose of preventing corona virus only with FDA 2 clinical results overseas, and analyzing the components and concentration of the product, this study It was compared with the prototype manufactured by the invention, and the results are shown in [Table 4].

division Product of the present invention Enovid products composition 2 component 2 component 1st amount (NO donation amount) Lettuce extract fermented liquid
(Contains NO2- ions) NaNO2
(Sodium Nitrite) HPMC 0.5% HPMC - NaCl - Benzalkonoum Chloride Purified water Purified water 2nd liquid (NO reducing liquid) citric acid citric acid

As can be seen in [Table 4], the design difference between the two products is clearly different, and since the concentration of NO ₂ ^- is the most important factor in comparing the nitric oxide generating ability, which is the most important technical component, <Example In Example 2>, it was prepared in the same manner as in <Example 1>, but the concentration of NO ₂ ^- was manufactured at the same concentration as that of the commercially available Enovid product to compare the nitric oxide generating ability of the two products, and finally < [Example 1] and <Example 2>, prototypes with two different compositions were prepared as shown in [Table 5] below.

division NO2 ^- Concentration NO ₃ ^- Concentration pH note Example 1 9,615 ppm 2,471 ppm 8.5 ppm Example 2 2,106 ppm 2,817 ppm 6.7 ppm Same composition as commercially available products

<Test Example 1> High-temperature storage storage stability comparison

For <Example 1> and <Example 2>, two self-manufactured samples and three commercially available Enovid products <Comparative Example 1> were stored at 50 ° C for a certain period of time, and then the change in nitrite ion content was measured. A long-term storage stability test was conducted, and the results are shown in [Table 6].

division NO2 ^- Concentration 1 week later 2 weeks later note Example 1 9,615ppm 8,471ppm 7,909ppm Example 2 2,106ppm 2,012ppm 1,968ppm Same composition as Enovid Comparative Example 1 2,136ppm 91ppm 1ppm Enovid products

Based on the long experience of the present inventors, after storage at 50 ° C for 2 weeks, nitrite ion was measured, and if it was not scavenged by more than 30%, it was judged that the shelf life of 1 year was secured, and to guarantee a shelf life of 2 years, 50 After 4 weeks of storage at ℃, we have secured our own standard that should not be scavenged more than 30%.

As shown in [Table 6], <Example 1>, which is a high-concentration product manufactured through <Example> of the present invention, and <Example 2>, which is a product manufactured with the same content as commercially available products, show NO ₂ after 2 weeks. ^- Although the concentration value decreased by 17.7% and 6.5%, respectively, in the case of <Comparative Example 1>, the sample was scavenged very quickly and was exhausted by vaporization after 2 weeks. condition, because the acidity of the nitric oxide reducing agent was too strong, and although the storage container was divided into two parts, it was expected that there would be an exchange of vaporized gases through a fine gap separating the space. That is, in the case of <Comparative Example 1>, which is a commercially available product, it was found that it had a disadvantage that it had to be refrigerated after purchase or used within 1 to 2 months after manufacture because the product was still not complete.

On the other hand, the pH of the nitric oxide donor solution of both prototypes according to the present invention prepared in <Example 1> and <Example 2> is weakly alkaline, making it difficult to scavenge NO ₂ ⁻ , and also the nitric oxide donor solution Because it is thoroughly sealed with an aluminum pouch, it can be predicted that the exchange of gasified gas is impossible even under high temperature conditions.

<Test Example 2>

In order to prepare the new samples composed in <Test Example 1> and compare and measure the nitric oxide generating ability between the samples, 25 ml of each of the nitric oxide donating solution and the nitric oxide reducing solution were placed in three types of Erlenmeyer flasks, respectively. After mixing at a 1:1 ratio, nitrogen oxide that starts to be generated was measured in real time, but NO gas measurement records were accumulated for at least 6 hours (300 minutes) or more.

The equipment used at this time is a custom-made equipment with a specially mounted nitrogen oxide gas measuring sensor based on a portable wireless gas measuring device (MultiRAE Lite model) from Rae System, a Japanese company specializing in complex gas measuring devices (see [Figure 1]) was used and the results are shown in [Table 7] below, and [Figure 3] to [Figure 5] are attached.

division peak the lowest cumulative generation note Example 1 >250ppm 13.2 ppm 24,879 ppm Example 2 62.7 ppm 1.4 ppm 3,536 ppm Same composition as Enovid Comparative Example 1 15.4 ppm 1.7ppm 1,738 ppm Enovid products

As can be seen from [Table 7], the sample of <Example 1> exhibited a high concentration of NO 2 ⁻ due to the high concentration of NO ₂ - so that the nitrogen oxide gas peak value (more than 250 ppm) exceeded the limit of the measuring instrument, which < It was confirmed that the value was more than 16 times higher than that of Comparative Example 1>, and the cumulative amount generated for 300 minutes was 24,879 ppm, which was 14.3 times higher than that of <Comparative Example 1>, which was made by Enovid.

In addition, although the NO ₂ ^- concentration of <Example 1> was 4.5 times higher than that of <Comparative Example 1>, the reason why the NO gas peak value and cumulative generation amount were much higher at 16 and 14.3 times, respectively, has not been precisely investigated. However, it was judged that what can be estimated can only be understood by the influence of various antioxidant components contained in natural ingredients.

And in [Table 7], <Example 2> is made at the same NO ₂ ^- concentration as <Comparative Example 1> and is the result of additional verification to compare the NO gas generating ability under the same NO ₂ ^- concentration condition. Similarly, the sample of <Example 2> using natural raw materials had a peak NO gas value about 4 times higher than that of <Comparative Example 1> prepared only with synthetic chemical raw materials, and the cumulative generation amount was measured more than twice as much. As described above, various components other than NO ₂ ^- of natural fermentation materials inhibit side reactions such as shown in [Scheme 2] below, mainly inducing NO ₂ ^- + 2H ⁺ → 2NO↑ (vaporization) reaction, resulting in oxidation It was assumed that this was the cause of increasing the nitrogen production efficiency.

[Scheme 2]

NO ₂ ^- + 2H ⁺ → NO ₂ ↑ (vaporization) + H ₂ ↑ (vaporization)

2NO ₂ ^- + 4H ⁺ → NH ₄ ↑ (vaporization) + 2O ₂ ↑ (vaporization) + 2H ₂ ↑ (vaporization)

In order to confirm this, it was decided to check the samples of <Example 2> and <Comparative Example 1> having the same NO ₂ ^- content through NO ₂ gas measurement. According to the judgment that if the gasified NO ₂ level appears high, it will be close to the prediction of the present inventors, and the NO ₂ gas generated as a side reaction is shown in [Table 8] below and [FIG. 5] is attached.

division NO ₂ ^- content
(ppm) NO gas (ppm) NO2 gas (ppm) note peak value cumulative amount peak value cumulative amount Example 2 2,106 64 3,536 20 2,234 Same composition as Enovid Comparative Example 1 2,136 15.5 1,739 11.6 1,602 Enovid products

As shown in [Table 8], compared to the cumulative NO gas production amount of 3,535 ppm in <Example 2> using natural fermentation raw materials as NO ₂ ^- donors, <Comparative Example 1> using artificially synthesized chemical raw materials accumulated While the amount of NO gas produced was as low as 49.1% at 1,739ppm, the cumulative amount of NO2 gas produced was 71.7%, confirming the fact that the conversion efficiency of the sample _in <Comparative Example 1> to NO gas was lowered. It can be expected that the amount of O ₂ and H ₂ gas will also decrease.

Although the above phenomenon will require in-depth research through scientific analysis in the future, the use of natural fermentation raw materials can convert NO gas more efficiently than using pure chemical raw materials by the above experimental examples of the present invention. It can be found that there is

The equipment used at this time is a custom-made equipment with a specially mounted nitrogen oxide gas measuring sensor based on a portable wireless gas measuring device (MultiRAE Lite model) from Rae System, a Japanese company specializing in complex gas measuring devices (see [Figure 1]) was used and the results are shown in [Table 7] below, and [Figure 3] to [Figure 5] are attached.

<Test Example 3>

The samples composed in [Table 8] were prepared and the cytotoxicity evaluation (MTT ASSAY) of the two samples was conducted at the Department of Oriental Resources, Kyung Hee University, and the results were shown schematically in [Figure 6]. Despite all of the same nitrite ion concentration, the cytotoxicity of the sample designed in the present invention, which contains nitrite ion by fermentation of natural raw materials and does not contain benzalkonium chloride, a bactericidal preservative, is higher than that of a sample composed of pure chemical components. The cytotoxicity to the human-derived cultured nasal cell line (RPMI2650) was lower than that of the human-derived cultured nasal cell line (RPMI2650).

1: A sample in which the nitrogen oxide donating solution and the reducing solution are dissolved in a 1:1 ratio, 2: Thermometer, 3: Thermostat for maintaining the temperature at 37℃, 4: Magnetic stirrer for temperature maintenance and stirring, 5: Generated gas discharge Passage, 6: portable complex gas meter, 7: real-time graph based on data transferred in conjunction with gas meter

Claims (6)

Nitric oxide (NO) donor solution containing natural nitrite ion (NO ₂ ^- ) produced by microbial fermentation of plants selected from lettuce, beet, spinach, chicory, wasabi, and garlic containing 100 ppm or more of nitrate. The first liquid and the second liquid, which is a nitrogen oxide reducing liquid composed of one or more components from citric acid, ascorbic acid, or erythorbic acid as an acidity regulator of pH 2 or higher that rapidly generates nitrogen oxide by a reduction reaction with the first liquid, are independently It is composed of a two-component type that is stored separately, and the first liquid, which is a nitric oxide (NO) donating liquid, and the second liquid, which is a nitric oxide reducing liquid, are simultaneously sprayed into the nasal cavity or oral cavity in the same amount. Inhibition of respiratory viral infection A two-component nasal or oral spray formulation for
delete delete The method according to claim 1, wherein the first liquid, which is a nitrogen oxide (NO) donating liquid, contains nitrite ions (Nitrite) at a concentration of 1,000 to 20,000 ppm, and in an alkalized state, hydroxypropyl methylcellulose having moisturizing and stabilizing effects (HPMC) is contained in an amount of 0.5 to 5% by weight based on the weight of the first liquid, and the pH is maintained at 7.0 to 10, and the second liquid, which is a nitric oxide reducing liquid, reacts with the first liquid sprayed into the nasal cavity or oral cavity to form nitric oxide ( NO) A two-component nasal or oral spray formulation for suppressing respiratory viral infection, characterized in that the pH is maintained at 2.0 to 6.0 so that there is a sufficient concentration of hydrogen ions for reduction and vaporization into a gaseous state.
The method according to claim 4, wherein the mixture of the first liquid and the second liquid sprayed into the oral cavity or nasal cavity has a nitrite ion concentration of 500 to 10,000 ppm and a pH of 4.0 to 6.9. A liquid nasal or oral spray formulation. The method according to claim 1, wherein the microorganism of the microbial fermentation is a mixture of Lactobacillus fermentum and Weicessella cibaria, which are microorganisms exhibiting nitrate reducing properties (KCTC13782BP), Lactobacillus casei (KCTC13899BP) , Lactobacillus plamtarum (KCTC13902BP), Lactobacillus rhamnosus (KCTC13903BP), Weissella paramesenteroides (KCTC13905BP), Lactobacillus paracasei (KCTC13906BP) ), Lactobacillus At least one from Lactobacillus pentosus (KCTC13897BP), Bacillus amyloliquefaciens (KCTC13898BP), Saccharomyces cerevisiae (KCTC13900BP), Bifidobacterium longum (KCTC13901BP) A two-component nasal or oral spray formulation for inhibiting respiratory viral infection, characterized in that selected.

Images