LT7047B - Plant oil composition for the preventive protection against sars-cov-2 infection - Google Patents

Abstract

The invention includes a new oil composition for body protection against SARS-CoV-2 infection. All compound products are, according to scientific research, green, with an effective antiviral effect. The selected antiviral activity component of the viral spike protein can bind to the host response blocking as well as neutralizing the virus. The composition includes black seed, tea tree, medicinal thyme, bay leaf and sunflower oils and variegated phytosomes.

Description

The invention relates to a new oil composition for the protection of the body against SARS-CoV-2 infection.

STATE OF THE ART

Severe acute respiratory syndrome (SARS) is a recently recognized severe respiratory disease caused by a novel coronavirus infection (SARS CoV) [1]. It is believed that the first cases of the disease were recorded among people as early as 2002. in November in China [2]. The syndrome was clarified in 2003. in February

Today, the structure of the virus and the mechanism of how the virus attaches to the host cell have been fully elucidated. Like other coronaviruses, the SARS-CoV-2 virus is composed of an RNA genome that encodes viral replication proteins and structural proteins, including viral spike (S), membrane proteins (M), viral envelope proteins (E), and other uncharacterized viral proteins [ 3]. Infection is established when the virus binds to a host cell receptor via the spike protein (S). This connection is one of the most important in the development of drugs, preventive preparations and vaccines against this infection.

Currently, vaccination remains one of the main methods used to prevent the spread of the disease. in 2021 July 20 according to data, six vaccines are available on the market: AstraZeneca - Vaxzevria, Janssen Ad26.COV 2.5, Moderna - mRNA-1273, Pfizer BioNTech - Comirnaty, COVID-19 vaccine BIBP and Sinovac-CoronaVac.

Due to possible side effects and controversial opinion, vaccination is not an acceptable method of preventive protection against SARS-CoV-2 viral infection for all individuals. Therefore, the search for alternative and safe methods to protect oneself from the coronavirus infection has begun.

The search for active substances that would be suitable and safe for human use and have antiviral properties against the SARS-CoV-2 virus has begun.

Most of the compounds described in the literature are of synthetic origin. In order to create a product of natural origin, plant-based products that have the necessary properties for the prevention and treatment of SARS-CoV-2 viral infection have been analyzed. There is ample evidence from in vitro studies reported in the scientific literature that essential oils can be used as antiviral agents to treat human viral infections, including SARS coronaviruses. These antiviral essential oils have mostly been tested against viruses such as Herpes simplex virus type 1, Junin virus, influenza virus, dengue virus type 2, and SARS coronaviruses, as well as non-enveloped viruses such as coxsackievirus B1, poliovirus, and type 3 adenovirus. Most of these clinically useful antiviral agents are agents that act on specific steps of viral biosynthesis, specifically inhibiting viral replication. On the other hand, antiviral agents can denature the structure or glycoproteins of the virus, thus reducing or completely blocking the infectivity of the virus particles. The angiotensin-converting enzyme 2 (ACE2) receptor in host cells has been shown to play an essential role in viral entry into cells; therefore, ACE2 blockers may be a potential target for antiviral intervention.

A study by Senthil Kumar and colleagues tested the ACE2 inhibitory effects of 10 essential oils. Of these, 8 essential oils exhibited significant ACE2 inhibition. Interestingly, some essential oils strongly inhibited ACE2 activity without showing cytotoxicity. The main components of essential oils - citronellol, geraniol, limonene, linalool and nerylacetate inactivate ACE2 receptor activity in host epithelial cells [4].

Many of the components described above can also be found in Nigella sativa oil. In traditional medicine, black seed has been used for centuries to treat a variety of ailments, including asthma, colds, headaches, nasal congestion, rheumatic diseases, warts, and many others. A review of the scientific literature found at least 8 in silico studies that investigated the effects of compounds in Nigella sativa oil on the SARS-CoV-2 virus. Molecular binding of compounds from Nigella sativa and some antiviral drugs (chloroquine, hydroxychloroquine, favipiravir, and saquinavir) to molecular targets of the SARS-CoV-2 virus, such as major proteases (6LU7 and 6Y2E), major peptidase (2GTB), angiotensin-converting enzyme enzyme 2 (ACE2)) and heat shock protein A5. Binding of the compounds to the molecular targets of the virus can prevent the attachment of the coronavirus to host cells. Nigella sativa active components bound to viral active sites in some cases formed complexes with higher energy scores than antiviral drugs.

The antimicrobial activity of tea tree (Melaleuca alternifolia) essential oil against gram-positive and gram-negative bacteria, aerobic and anaerobic bacteria, yeasts and molds has been described in the scientific literature. Tea tree oil has also been reported in the scientific literature to have antiviral activity against DNA and RNA viruses such as influenza A/PR8 virus H1N1, Herpes simplex viruses (HSV-1) and (HSV-2), Echovirus 9, Poliovirus 1, Coxsackievirus B1 and Adenovirus. 2, can also help get rid of viruses that cause colds and flu [5]. The combination of monoterpenes in tea tree oil shows a synergistic antiviral effect. All these properties indicated the possibility of efficacy of tea tree oil against SARs-CoV-2 [6].

The antibacterial activity of thymol, the main ingredient of essential oil of medicinal thyme (Thymus vulgaris), against L. monocytogenes, E. coli, S. enterica, S. aureus, C. botulinum,C. perfringens, S. son nei, S. lutea, M. flavus, B. hermosphacta, L. innocua, L. monocytogenes, P. putida and S. putrefaciens bacteria in in vitro studies. Antiviral activity of thyme against H1N1 type A, HSV-1 and HSV-2 viruses has also been described [7]. The monoterpene hydrocarbons found in the essential oil of T. vulgaris have antiviral properties, and α-terpineol, α-pinene, terpinen-4-ol, 1, 8-cineole show strong potential for inhibitory effects against the COVID-19 virus [6], [8].

The antibacterial and antiviral activity of bay leaf (Laurus nobilis) essential oil has been described in the scientific literature. The antibacterial activity of the essential oil was shown against Staphylococcus aureus, Bacillus cereus, B. cereus (Gram-positive) and Escherichia coli, Pseudomonas aeruginosa (Gram-negative) bacteria. β-Ocimene, α- and β-pinenes, and 1,8-cineole, components of L. nobilis essential oil are believed to be potential components in the development of agents against COVID-19 by inhibiting the major viral protease (Mpro) [9].

Vitamin E, a powerful fat-soluble antioxidant, is present in higher concentrations in immune cells compared to other blood cells. Vitamin E deficiency has been shown to impair normal immune function in animals and humans, which can be corrected by dietary supplementation with vitamin E. Although vitamin E deficiency is rare, vitamin E supplementation has been shown to improve immune function and reduce the risk of infection, especially in the elderly [10], [11]. The mechanisms responsible for the effects of vitamin E on the immune system and inflammation have been investigated in cellular preclinical and clinical interventional studies. Vitamin E modulates T cell function by directly affecting T cell membrane integrity, signal transduction, and cell division, as well as indirectly by affecting inflammatory processes [12]. Modulation of immune function by vitamin E has clinical relevance as it affects host susceptibility to infectious diseases such as respiratory tract infections or allergic diseases such as asthma [13]. Studies examining the role of vitamin E in the immune system have mostly focused on α-tocopherol, but emerging evidence suggests that other forms of vitamin E, including other tocopherols and tocotrienols, may also have potent immunomodulatory functions [14].

Phytosomes are a new patented technology by which standardized plant extracts are attached to phospholipids, thus obtaining oil-soluble molecular complexes. Most biologically active plant constituents are polar or water-soluble molecules. However, water-soluble phytoconstituents such as flavonoids, tannins, glycosidic aglycones, etc. are poorly absorbed by the body. Due to their large molecular size, the molecules cannot be absorbed by passive diffusion or by lipid solubility. These properties severely limit their ability to penetrate lipid-rich biological membranes [15]. The increased bioavailability of phytosomes compared to simpler, uncomplicated plant extracts has been demonstrated by pharmacokinetic and activity studies [16]. Silymarin is a flavonoid complex extracted from the Silybum marianum plant. It acts as a potent antioxidant through multiple mechanisms. However, in addition to its antioxidant effects, silymarin/silybin has an immunomodulatory effect. Silymarin has been shown to have anti-inflammatory effects as well as different immunomodulatory activities in a dose- and time-dependent manner. Silymarin, being an immunomodulator, suppresses the function of T-lymphocytes in low doses, and stimulates inflammatory processes in high doses. Studies have shown that silymarin attenuated autoimmune, allergic, preeclamptic, cancer, and immune-mediated liver diseases, as well as inhibited oxidative and nitrosative immunotoxicity [17].

Since the beginning of the SARS-CoV-2 virus infection, there are already ingredients described in patent applications to fight this infection. Most of the compounds described are of synthetic origin, so they will not be suitable for a group of users who prefer products of natural origin. The mentioned applications also lack biomedical and clinical research-based efficacy and safety for the use of these compounds for the prevention and treatment of symptoms of SARS-CoV-2 virus infection.

Patent application WO2020080682A1 describes a quinolinone compound that has an excellent inhibitory effect on coronavirus infection. It can be used in pharmaceutical compositions to prevent or treat diseases caused by coronavirus infection.

Patent CN109111405B describes the potential use of an aromatic thioether compound in the production of a drug against SARS-CoV-2 virus infection, as the described compound acts as an inhibitor of the main protease of the SARS coronavirus.

Patent CN111184707B describes the anti-coronavirus therapeutic agent tolfenam salt, which is suitable for the prevention and treatment of SARS coronavirus infection, acting as an inhibitor of the 3CL hydrolase (Mpro) of the coronavirus.

A library of FDA-approved drugs was examined. Thirty-one compounds were selected and studied, and their effectiveness was evaluated. Twenty-nine compounds with antiviral properties have been identified and can be used in the development of preparations for the prevention and treatment of SARS CoV viral infection. Among the selected compounds, the compound vinpocetine described in patent application KR102185947B1, the compound meclizine described in KR102185946B1 and the compound flunarizine described in KR102185945B1. All three isolated compounds exhibited strong antiviral properties.

The closest product to the created composition is described in the patent document CN111184805B. Herein, we describe herbal compositions used in traditional Chinese medicine that are suitable for the prevention and treatment of SARS coronavirus infection, but there are no biomedical and clinical studies to prove this.

Patent document CN111184805B describes several compositions. Plant components and minerals used in the composition: Taiwanese trifolium (Tripterygium wilfordii), hydrated calcium sulfate, Dioscorea polystachya, traditional medicinal preparation from the brown barberry (Scrophularia ningpoensis), forsythia (Forsythia suspensa) fruit, cicada (Periostracum cicadae) extract, Codonopsis pilosula, Mint, Burdock, Iron(III) oxide, Chai Hu root (Radix bupleuri), Pinellia ternata, Scutellaria baicalensis, Phragmites australis, Citrus reticulata ) fruit exocarp, erect bellflower (Adenophora stricta), salvia root (Peucedanum praeruptorum), large-flowered broadleaf bellflower (Platycodon grandiflorus), almond (Prunus amygdalus), honey-roasted licorice twig (Glycyrrhiza glabra), long-fruited tuft (Trichosanthes cucumerina), wild onion (Allium macrostemon), poria cocos mushroom (Wolfiporia extensa), traditional medicinal preparation from pinellia (Pinellia ternata), dried orange peel, bitter lemon tree (Citrus aurantium), bamboo shavings, narrow-leaved ayer (Acorus gramineus).

The effectiveness and safety of similar products is based only on the scientific studies of the individual components that have already been carried out. In order to ensure that the developed product is effective and safe for use, pre-clinical and clinical studies of the formulation are carried out. Our goal is to create a purified, effective composition that has scientifically proven activity against SARS-CoV-2 virus infection. Therefore, after the analysis of the scientific literature, the herbal ingredients described in the latest articles were selected, which are more familiar to the European consumer and have a scientific basis for the fight against SARS-CoV2 virus infection.

ESSENCE OF THE INVENTION

Taking into account the spread of SARS-CoV-2 virus infection and the methods currently used to protect against this infection, a product was developed for daily preventive protection against the entry of the virus through the external respiratory tract (nose and mouth). In order to create an effective product, the properties of secondary metabolites of different plants capable of inhibiting/stopping the attachment of the virus to the host cell were analyzed and plant products were selected, which, according to scientific research, had the most effective protective properties.

The composition effective against the SARS-CoV-2 virus consists of 68-99% black seed oil (Nigella sativa), 0.1-5.0% tea tree (Melaleuca alternifolia) leaf essential oil, 0.5-5.0% medicinal thyme (Thymus vulgaris) leaf/flower essential oil, 0.1-5.0% bay leaf (Laurus nobelis) essential oil, 0.4-10.0% vitamin E dissolved in sunflower (Helianthus annuus) seed oil and 0.17, 0% Silybum marianum phytosomes.

BRIEF DESCRIPTION OF THE DRAWINGS

Preferred embodiments of the invention are described below with reference to the drawings.

Fig. Effect of undiluted test preparation 1 on cell viability of the A549-hACE2-TMPRSS2 line. Cell viability was assessed by crystal violet assay and normalized to control t. i.e. cells that were grown in untreated medium. K - inactive cells.

Fig. Effect of undiluted test preparation on cell viability of the A549-hACE2-TMPRSS2 line. Cell viability was assessed by MTT assay and normalized to control t. i.e. cells that were grown in untreated medium. K untreated cells, TK - positive control after exposure to 1 mg/ml SDS.

Fig. Effect of undiluted test preparation on infectivity of SARS-CoV-2 Delta virus isolate in A549-hACE2-TMPRSS2 cell line. The vertical axis represents the number of virus copies per microliter of medium.

Fig. Effect of undiluted test preparation on infectivity of SARS-CoV-2 Omikron virus isolate in A549-hACE2-TMPRSS2 cell line. The vertical axis represents the number of virus copies per microliter of medium.

Fig. Cytotoxicity testing by direct contact and agar penetration methods samples after incubation with MTT solution.

Fig. In vitro relative cytotoxic effect on L929 cells, after 24 h. direct contact. Test material 1 - YOU&OIL mouth and nose spray.

Fig. In vitro relative cytotoxic effect on L929 cells, after 4 h. and 24 hours by the agar penetration method. Test material 1 - YOU&OIL mouth and nose spray.

DETAILED DESCRIPTION OF THE INVENTION

The developed product is intended for daily preventive protection against the entry of the virus through the external respiratory tract (nose and mouth). All the compound raw materials of the product, based on scientific studies, have shown effective antiviral effects. The antiviral activity of the selected components is manifested by blocking the ability of the spike protein of the virus to bind to the receptor of the host cell, as well as by neutralizing the virus.

When developing the product, it was decided to concentrate only on possible plant components of natural origin. All ingredients selected for the product are of plant origin, isolated from different parts of the plant. Vegetable oil is extracted from the seeds of the plant, essential oils are extracted from the aerial parts of the plant, phytosomes of plant components are added to the composition.

The components of the developed product were selected based on the information provided in the scientific literature. Individual ingredients have been selected according to their properties suitable for the prevention and treatment of SARS coronavirus infection: black seed oil (Nigella sativa), tea tree (Melaleuca alternifolia) leaf essential oil, thyme (Thymus vulgaris) leaf/flower essential oil, bay leaf (Laurus nobilis) essential oil, vitamin E (α-tocopherol), phytosomes (modified soy lecithin with marigold (Silybum marianum) extract).

When creating an effective composition, it was decided to focus on the effect of black seed (Nigella sativa) against the SARS-CoV-2 virus described in the scientific literature. The vegetable black seed oil contains compounds that, by binding to the main molecular targets of the virus, can help prevent the virus from attaching to the cells of the human body. Due to these properties, black seed oil was selected as the main component of the product.

To enhance the effect, the product is supplemented with plant essential oils, which have antiviral and antioxidant properties, thus enhancing the antiviral effect of the product. The bioactive compounds of essential oils, due to their lipophilic nature, can penetrate viral cells and thereby stop viral replication. Also, essential oils complement the product with properties that reduce the symptoms of respiratory tract inflammation. The composition is supplemented with essential oils of tea tree, medicinal thyme, and bay leaves. Tea tree (Melaleuca alternifolia) essential oil complements the product with antiviral and anti-inflammatory effects. The compounds in its composition have shown high activity against cold and flu viruses, these properties are very important in creating an effective product.

The product is supplemented with bay leaf (Laurus nobilis) essential oil, due to its components β-ocimene, α- and β-pinene. These compounds have a strong inhibitory effect against the COVID-19 virus.

Medicinal thyme (Thymus vulgaris) essential oil is included in the product composition due to its properties to alleviate the symptoms caused by respiratory tract infections, the active compounds of the essential oil reduce inflammation of the mucous membrane caused by respiratory tract infections. Also, medicinal thyme essential oil contains compounds with a strong antiviral effect, which enhance the product's performance.

Vitamin E added to the product was selected due to several beneficial properties. Its first property is that it acts as a strong antioxidant. Its effect on the human body has been proven by clinical studies. Vitamin E taken as a dietary supplement has a proven health claim: "Helps protect cells from oxidative damage." Also, vitamin E is important for improving the immune system, thereby reducing the risk of viral infection. These properties are extremely important when creating an oil composition for protection against COVID19.

The product is also supplemented with silybum marianum extract. The flavonoid complex silymarin/silybin in its composition has many beneficial properties for the product: immunomodulatory and antioxidant effects. These properties are very important in the development of a product against viral infection. These compounds are hydrophilic, which is why the patented form of the extract - phytosomes - was selected. This technology allows the use of hydrophilic active ingredients in oily compositions.

Black seed oil was selected as the main ingredient of the created product, its concentration in the product reaches more than 50%. Essential oils are highly concentrated extracts of plant volatile components, so their concentrations in the final product are relatively low, and essential oils have established limits for internal and external use. Taking into account the safe consumption limits of essential oils and other components of the product, the created composition consists of: 68-99% black seed oil (Nigella sativa), 0.1-5.0% tea tree (Melaleuca alternifolia) leaf essential oil, 0.5 -5.0% thyme (Thymus vulgaris) leaf/flower essential oil, 0.1-5.0% laurel leaf (Laurus nobilis) essential oil, 0.410.0% vitamin E (α-tocopherol) dissolved in sunflower (Helianthus annuus) ) in seed oil, 0.1-7.0% mottled phytosome (modified soy lecithin with mottled (Silybum marianum) extract).

The product consists of two separate bottles of the same product: a nasal spray and an oral spray. It was decided to present the product in two different bottles for the convenience of the user. Having the product in different bottles with special separate dispensers for the nose and mouth avoids the inconvenience of cleaning/changing the dispensers for the user. The nasal spray is used by injecting 12 sprays into each nostril as needed. Repeated spraying is recommended every 2-3 hours. It is not recommended to spray more than 5 doses in each nostril per day. Oral spray is used by injecting 1-2 sprays into the oral cavity as needed. After injection for at least 15 min. eating or drinking is not recommended. Repeated spraying is recommended after 2-3 hours, if there is a need. It is not recommended to spray more than 5 doses per day.

The safety of the product for use has been investigated and is based on studies of pharmacological effects (cytotoxicity) and physiological effects (sensitization). Product efficacy is based on in vitro antiviral efficacy studies using formulations of product components against the SARS-CoV-2 virus. The stage of clinical trials was carried out to evaluate the effectiveness of the product.

ANTIVIRUS PROTECTION STUDY

In order to investigate the efficacy of the product and the effective concentrations of the components, a product test is performed to assess the infectivity and viability of cell cultures with the SARS-CoV-2 virus. The study examines the effect of different concentrations of the components on the viability of human lung epithelium and virus cells. The research hypothesis was raised: the infectivity of cell cultures with the SARS-CoV-2 virus decreases and the viability increases after exposure to the product. VRBTEK permit no. 2021/6-1361-835 to conduct a biomedical study with the SARS-CoV-2 virus. The study is carried out with viable SARS-CoV-2 virus obtained from nasopharyngeal samples stored in Vilnius Santaras Clinics Biobank and human lung epithelial HTB-55 cell cultures. The study was conducted with the delta strain of the SARS-CoV-2 virus.

The concentrations of the product components selected for the study: 68-99% black seed oil (Nigella sativa), 0.1-5.0% tea tree (Melaleuca alternifolia) leaf essential oil, 0.5-5.0% medicinal thyme (Thymus vulgaris). leaf/flower essential oil, 0.1-5.0% laurel leaf (Laurus nobilis) essential oil, 0.410.0% vitamin E (α-tocopherol) dissolved in sunflower (Helianthus annuus) seed oil, 0.1-7.0 % variegated phytosome (modified soy lecithin with variegated (Silybum marianum) extract).

The study was not conducted directly with human samples, but with viruses that infected them. Up to 10 nasopharyngeal samples were requested from Vilnius Santaras Clinic Biobank for the research. The test requires this number of samples due to processing conditions and different strains of the virus. It was important for the study to purify the most relevant strains of the virus. Table 1 shows the samples obtained for the study.

Table 1. Specimens for testing for viable virus.

EIL NO. CODING CT VALUES VARIETY 1 NP03-IN0004371 ORF1ab Ct(21,92); N Ct(23,27); S Ct(22.80) delta 2 NP03-IN0004376 ORF1ab Ct(16,80); N Ct(19.05); S Ct(19,17) delta 3 NP03-IN0004385 ORF1ab Ct(17,81); N Ct(18,30); S Ct(17,34) delta 4 NP03-IN0004451 ORF1ab Ct(14,18); N Ct(14,24); S Ct(14,36) delta 5 NP03-IN0004464 ORF1ab Ct(14,10); N Ct(15.33); S Ct(15,09) delta 6 NP03-IN0004582 ORF1ab Ct(22,23); N Ct(22.45) micron 7 NP03-IN0004573 ORF1ab Ct(18,94); N Ct(18.65) micron 8 NP03-IN0004586 ORF1ab Ct(16.53); N Ct(15.96) micron 9 NP03-IN0004594 ORF1ab Ct(15,61); N Ct(15.98) micron 10 NP03-IN0004569 ORF1ab Ct(17.95); N Ct(19,19) micron

Evaluation criteria: human epithelial cell viability must be at least 80% after direct exposure, virus viability must be at least 50% after direct exposure.

According to the study, the optimal viability of human epithelial cells and the virus was achieved at the following component concentrations: 98.4% black seed oil (Nigella sativa), 0.2% tea tree (Melaleuca alternifolia) leaf essential oil, 0.6% medicinal thyme (Thymus vulgaris). leaf/flower essential oil, 0.1% bay leaf (Laurus nobilis) essential oil, 0.5% vitamin E (α-tocopherol) dissolved in sunflower (Helianthus annuus) seed oil, 0.2% variegated phytosome (modified soy lecithin with (Silybum marianum) extract).

The following drug concentrations were used for the study:

The undiluted preparation was used to evaluate the effects of their direct contact with cells.

Undiluted preparation added to cell culture medium to final concentrations: 10%, 5%, 1%.

% preparation solution in DMSO, which was diluted with medium to final concentrations: 0.05%, 0.025%, 0.0125% and 0.00625%.

Control samples used for the study, which were studied together with the effect of the preparation:

Control (K) - cell culture medium.

Negative control (NK) - phosphate buffer (PBS), pH 7.4 (Gibco).

The positive control (PC), sodium dodecyl sulfate (SDS, Sigma-Aldrich), was dissolved in sterile deionized water to make a 0.05 g/ml solution. Before the test, it was diluted with culture medium to a concentration of 0.1 mg/ml.

Solvent Control (TiK) - cell culture medium supplemented with 0.01% or 0.0005% DMSO.

Control samples were tested in parallel with the cytotoxicity of the test preparation in order to confirm the suitability of the applied system to perform this test. The effect of the test substance, concentrations and exposure time on the viability of A549-hACE2TMPRSS2 line cells was evaluated by MTT and crystal violet tests.

In the initial phase of the study, the effects of undiluted preparation 1 and a 5% solution of preparation 1 in DMSO at various concentrations on the viability of A549-hACE2-TMPRSS2 were evaluated by MTT assay. Duration of effect ranged from 2 to 24 hours. A total of 5 exposure times were evaluated - 2, 4, 6, 16 and 24 hours. (Figure 1).

The data presented show that when the undiluted formulation is used at a final medium concentration of 0.5% and below, cell viability is not significantly different from the control after 24 hours of exposure.

The investigational product would be used undiluted for its intended purpose. Therefore, the next phase of research is evaluating how the preparation affects cell viability of the A549-hACE2-TMPRSS2 line when it is directly applied to the cell monolayer. The volume of material used is 156.25 pl/cm ² of cell monolayer. The duration of the effect is selected from 5 min. up to 30 min. Vitality is assessed every 5 min (Figure 2).

The obtained results showed that after exposure to the test preparation (undiluted), cell viability decreases by an average of 27% compared to the control. The effect of the preparation does not depend on the exposure time.

Infection experiments were performed with SARS-CoV-2 isolate NP03-IN0004464 (Delta) and isolate NP03-IN0004594 (Omikron) under biosafety level three conditions with appropriate respiratory protection. A549-hACE2TMPRSS2 cells were seeded at a density of 100,000 cells/ml in 96-well plates (TPP Techno Plastic Produktai AG). 24 hours after seeding, cells were incubated for 5, 15 and 20 min. were exposed to the undiluted preparation (6 repetitions), washed with cell growth medium 3 times after the specified exposure time, filled with 200 μl of cell growth medium and infected with 1 x 10 ⁴ virus copies up to a concentration of 50 copies/μl. After 24 hours of incubation, 1 μl of medium was taken from each well for real-time reverse transcription-PCR evaluation. Quantitative results for affected cells are compared with 20 replicates of unaffected cells and 6 replicates of each virus isolate placed in a cell-free medium well.

Data presented in Figure 3. and Figure 4. shows that the test preparation strongly limits the infectivity of A549-hACE2-TMPRSS2 cell culture with SARS-CoV-2 Delta and Omikron virus strains. The obtained results of the affected and unaffected A549-hACE2-TMPRSS2 culture cells show assured protection against infection with SARS-CoV-2 Delta and Omikron virus variants.

Based on the obtained results, the concentrations of the product components with the highest antiviral efficiency were determined, as well as the composition was determined for further safety and efficacy studies.

CYTOTOXICITY TEST

The aim of the cytotoxicity test is to assess the possible toxicity of the tested substances to the cell line. The study was performed in vitro in mammalian cell culture (L929 mouse fibroblast line). The study was conducted based on the requirements of ISO 10993-5: 2009 guidelines "Test for in vitro cytotoxicity".

Studies have been conducted with a composition consisting of: 98.4% black seed oil (Nigella sativa), 0.2% tea tree (Melaleuca alternifolia) leaf essential oil, 0.6% thyme (Thymus vulgaris) leaf/flower essential oil, 0.1% bay leaf (Laurus nobilis) essential oil, 0.5% vitamin E (α-tocopherol) dissolved in sunflower (Helianthus annuus) seed oil, 0.2% variegated phytosome (modified soy lecithin with variegated (Silybum marianum) extract) ).

The undiluted product was tested by two methods: direct contact and penetration through agar. An isotonic (0.9%) sodium chloride solution was used as a negative control, and a 5% sodium dodecyl sulfate (SDS) solution in sterile deionized water was used as a positive control. Control samples were tested in parallel to evaluate the cytotoxicity of the test substance in order to confirm the suitability of the applied system to perform this test.

Using the direct contact method, sterile pieces of filter paper 1.2 cm in diameter (1.13 cm ² surface area) are aseptically coated with the test sample and placed with tweezers in the center of each well. In this way, the filter covers about 10% of the area of the well, as regulated by the guidelines ISO 10993-5:2009(E). Control filter pieces are exposed to negative and positive control solutions, respectively. The plate with L929 cells was incubated at 37 ± 1 °C in a 5.0% CO2 atmosphere for 20-24 h.

Using the agar penetration method, sterile pieces of filter paper 2 cm in diameter (3.14 cm ² surface area) are aseptically coated with the test sample and placed with tweezers on the rigid agarose in the center of each well. Control filter pieces are exposed to negative and positive control solutions, respectively. The cells in the plate are incubated with the samples for 4 hours. and 24 ± 1 h. At 37 ± 1 °C, 5.0% CO2/95% atmosphere.

The cells were incubated with the samples after the incubation medium was replaced with fresh cell nutrient medium and the MTT reduction test was applied by measuring the absorbance values of the solutions at 570 nm and 630 nm wavelengths with a microplate reader (Figure 5). 200 μl of isopropanol (reference solution) is also added to individual wells. Three technical repetitions of each sample were performed, three independent experiments were performed, and the average of the obtained results was evaluated.

Table 2. Calculated relative viability of samples by the direct contact method (24-hour exposure):

Relative Viability (%) YOU&OIL spray Negative control (NaCl) Positive control (SDS) 64.14 100 0.64 93.63 100 1.12 86,81 100 0.68 Average 81.53 100 0.82 SD 15.44 0 0.27

Table 3. Calculated relative viability of samples by agar penetration method (4-hour exposure):

Relative Viability (%) YOU&OIL spray Negative control (NaCl) Positive control (SDS) 78.33 100 15.53 94.02 100 15.00 92,94 100 12.30 Average 88.43 100 14.28 SD 8.76 0 1.73

Table 4. Calculated relative viability of samples by the agar penetration method (24-hour exposure):

Relative Viability (%) YOU&OIL spray Negative control (NaCl) Positive control (SDS) 78.40 100 1.70 70.96 100 0.12 57,52 100 3.38 Average 68.96 100 1.73 SD 10.58 0 1.63

The absorbance values and absorbance deviations of the control samples showed the appropriate application of the test and its sufficient sensitivity. After the assay, L929 cell culture after 24 h. cell viability decreased to 81.53 ± 15.44% when exposed to the direct contact product (Table 2, Figure 6). In the agar penetration assay, cell viability decreased to 88.43 ± 8.76% after 4 hours of exposure to the product (Table 3, Figure 7), and after 24 hours, cell viability decreased to 81.51 ± 9.86% (4 table, figure 7). Based on the obtained results, it can be concluded that the effect of the product on L929 cell culture in the study by agar penetration and direct contact methods was insignificant, i.e. the product is non-cytotoxic and safe to use.

SENSITIZATION STUDY

The purpose of the sensitization test is to assess whether the product is safe to use directly on the skin. The study is carried out with short-haired guinea pigs of the family Caviidae, using the guinea pig maximization method (GPMT). In order to carry out the research, the permit of the Lithuanian Experimental Animal Use Ethics Commission under the State Food and Veterinary Service is required to carry out an animal test project.

Studies have been conducted with a composition consisting of: 98.4% black seed oil (Nigella sativa), 0.2% tea tree (Melaleuca alternifolia) leaf essential oil, 0.6% thyme (Thymus vulgaris) leaf/flower essential oil, 0.1% bay leaf (Laurus nobilis) essential oil, 0.5% vitamin E (α-tocopherol) dissolved in sunflower (Helianthus annuus) seed oil, 0.2% variegated phytosome (modified soy lecithin with variegated (Silybum marianum) extract ).

In the study, healthy young guinea pigs are randomized and assigned to exposure and control groups. Before dosing, the hair in the scapular area is removed by clipping or shaving, taking care not to damage the skin. Use 10 animals in the exposure group and at least 5 animals in the control group. During the study, the surface of the skin of guinea pigs is exposed to the test product and all possible skin sensitivity reactions are observed.

Based on the results obtained, the test product did not cause any adverse skin irritation reactions compared to the control group of your test pigs. Based on the obtained results, it can be concluded that the effect of the product on guinea pigs during the sensitization test by the GPMT method was insignificant, i.e. the product does not cause skin irritation and is safe to use.

ORAL MUCOSA IRRITATION TEST

The purpose of the acute oral mucosa irritation test is to assess whether the product is safe to use directly on the oral mucosa, to make sure that the product is safe to use. The study is performed on a specially prepared SkinEthic ^TM HOE model of human oral mucosal epithelial cells. The SkinEthicTM HOE model consists of TR146 cells (derived from buccal mucosal squamous cell carcinoma) grown on an inert polycarbonate filter at the air-liquid interface in a chemically defined medium. This model forms an epithelial tissue that does not contain a stratum corneum, histologically resembling the mucosa of the oral cavity.

Studies have been conducted with a composition consisting of: 98.4% black seed oil (Nigella sativa), 0.2% tea tree (Melaleuca alternifolia) leaf essential oil, 0.6% thyme (Thymus vulgaris) leaf/flower essential oil, 0.1% bay leaf (Laurus nobilis) essential oil, 0.5% vitamin E (α-tocopherol) dissolved in sunflower (Helianthus annuus) seed oil, 0.2% variegated phytosome (modified soy lecithin with variegated (Silybum marianum) extract) ).

The purpose of the study is to evaluate the possible irritating effect of the substance under investigation on the oral mucosa. The research methodology was adapted based on valid guidelines and methodologies for evaluating the irritant effect of substances on the skin, since there are no specific guidelines for evaluating the irritant effect on the oral mucosa. YOU&OIL nasal and oral spray was tested in vitro in the cell culture of the reconstructed oral mucosa epithelium, according to the modified protocol of CellSystems Biotechnologie Vertrieb GmbH, Germany and 2021. in June guidelines no. 439 "In Vitro Skin irritation: Reconstructed Human Epidermis Test Methods" The test used meets the requirements of the OECD guideline for the testing of chemicals in vitro skin irritation: Reconstructed human epidermis test method' (2010).

The undiluted product (nasal and oral spray) was tested. Phosphate buffer, pH 7.4 (Gibco) was used as a negative control (NC). A 5% sodium dodecyl sulfate (SDS) solution in sterile deionized water was used as a positive control (PC). Three replicates of each sample (test material, TK and NK) were performed. The in vitro culture of the oral mucosa was incubated with the test material for 30 min. and 2 o'clock The MTT reduction test was applied to assess tissue viability, measuring the absorbance values of the solutions at a wavelength of 570 nm. Area of influence 0.5 cm ² .

Table 5. Calculated relative viability of the samples during the oral mucosa irritation test (10 min, 1 h and 3 h exposure):

Relative Viability (%) YOU&OIL spray 30 minutes YOU&OIL spray 120 hours Negative control (NaCl) Positive control (SDS) 53.67 33.03 100.91 2.15 49.95 34.87 100.13 2.24 51.39 34.90 98.96 2.08 Average 51.67 34,27 100.00 2.16

The absorbance values of the controls and the deviations of absorbance of all samples were within the guidelines of guideline no. 439 "In Vitro Skin irritation: Reconstructed Human Epidermis Test Methods" and the CellSystems INVITTOX protocol, the used test system met the requirements for its sensitivity and correct evaluation of the results.

The effect of the test substance on the viability of human oral mucosal epithelial cultures was small after 30 min. contact (viability decreased to 51.67 ± 1.88%).

Test material after 120 min. direct contact with human oral mucosal epithelial cultures reduced cell viability to 34.27 ± 1.07%.

Based on the obtained results, it can be concluded that YOU&OIL mouth and nose spray has a slight irritating effect after 30 minutes. of direct contact with human oral mucosal cultures in vitro.

BIOMEDICAL RESEARCH

After completing the pre-clinical research stage, the product was further biomedically tested. The main goal of this study is to transform an effect known from scientific literature (i.e. a certain property) into a health claim (i.e. a benefit). Considering the specific aspects of the statement - i.e. based on the relationship between the product with an antiviral effect and health/reducing the risk of getting sick, the formulated research hypothesis will be confirmed and the biomedical research protocol will be prepared, which will be submitted to the Lithuanian Bioethics Committee.

According to the provisions of the Law and Regulation on the Ethics of Biomedical Research of the Republic of Lithuania, biomedical research is conducted with healthy volunteers or "patients at risk" (i.e. those who are at risk of developing a certain disease, but do not yet have a confirmed diagnosis). 120 subjects who voluntarily agreed to participate in the study were selected for the study and divided into two groups: control and exposure groups. The control group is not given the product, and the exposure group is given the research product, with consumption recommendations. An antigen test is performed every 7 days to analyze the results and evaluate the effectiveness of the product. The examination is carried out three months from the day of the first visit.

Studies have been conducted with a composition consisting of: 98.4% black seed oil (Nigella sativa), 0.2% tea tree (Melaleuca alternifolia) leaf essential oil, 0.6% thyme (Thymus vulgaris) leaf/flower essential oil, 0.1% bay leaf (Laurus nobilis) essential oil, 0.5% vitamin E (α-tocopherol) dissolved in sunflower (Helianthus annuus) seed oil, 0.2% variegated phytosome (modified soy lecithin with variegated (Silybum marianum) extract) ).

Based on the obtained results, it can be concluded that the product is effective when comparing the control group with the investigated exposure group. Subjects in the exposure group were more than 50% less likely to develop SARS-CoV-2 viral infection.

EXAMPLE OF IMPLEMENTATION OF THE INVENTION

Nasal and oral spray composition consisting of 68-99% Nigella sativa seed oil, 0.1-5.0% tea tree (Melaleuca alternifolia) leaf essential oil, 0.5-5.0% thyme ( Thymus vulgaris) leaf/flower essential oil, 0.1-5.0% laurel leaf (Laurus nobilis) essential oil, 0.410.0% vitamin E (α-tocopherol) dissolved in sunflower (Helianthus annuus) seed oil, 0.1- 7.0% marigold phytosome (modified soy lecithin with marigold (Silybum marianum) extract).

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[5] A. Garozzo, R. Timpanaro, B. Bisignano, P.M. Furneri, G. Bisignano, A. Castro, In vitro antiviral activity of Melaleuca alternifolia essential oil, Lett. Appl. Microbiol. 49 (2009). https://doi.org/10.1111/j.1472-765X.2009.02740.x.

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Effect of Aromatic Plants and Essential Oils against COVID-19: Review of Their Antiviral Activity, J. Complement. Alternate Med. Res. (2020). https://doi.org/10.9734/jocamr/2020/v11i130175.

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[20] WO2020080682A1

[21] CN109111405B

[22] CN111184707B

[23] KR102185947B1

[24] KR102185946B1

[25] KR102185945B1

[26] CN111184805B

Claims (6)

1. An oil composition for the daily prevention of viral disease transmitted through the external respiratory tract, comprising more than one herbal component, characterized by the presence of 68-99% Nigella sativa seed oil, 0.1-5.0% Melaleuca alternifolia leaves essential oil, 0.5-5.0% Thymus vulgaris leaf/flower essential oil, 0.1-5.0% Laurus nobilis essential oil, 0.4-10.0% vitamin E dissolved in Helianthus annuus seed oil and 0.1-7, 0% Silybum marianum phytosomes. 2. The oil composition according to claim 1, wherein the viral infection is an infection caused by SARS-CoV-2. 3. Oil composition according to claim 1, wherein the composition is administered by nasal spray. 4. The oil composition according to claim 1, wherein the composition is used by spraying in the mouth. 5. Oil composition according to points 1-4, where the composition is used every 2-3 hours. 6. Oil composition according to points 1-5, where the composition is used no more than 5 times a day.