Pharmaceutical composition to support the immune system before and during upper respiratory tract infections

Abstract

The subject of the invention is a pharmaceutical composition that supports the immune system before and during upper respiratory tract infections, as well as its use in the production of dietary supplements for the prevention and treatment of upper respiratory tract infections.

Description

[0001] PHARMACEUTICAL COMPOSITION TO SUPPORT THE IMMUNE SYSTEM BEFORE AND DURING UPPER RESPIRATORY TRACT INFECTIONS

[0002] The object of the invention is a pharmaceutical composition for supporting the immune system before and during upper respiratory tract infection, and its use in the production of dietary supplements for use in the prevention and treatment of upper respiratory tract infections.

[0003] Upper respiratory tract infections impose a significant economic burden on society, due to healthcare costs, absence from work, school or the need to care for a sick family member. A cold or flu most often results from an upper respiratory tract infection. The common cold is the term widely used to describe a mild infection of the upper respiratory tract. Typical symptoms of a cold include a blocked nose and runny nose, sneezing, sore throat and cough. Although the term suggests that there is a single cause of illness, the common cold is actually a heterogeneous group of diseases caused by numerous viruses belonging to several different families [Heikkinen et al., 2003]. Seasonal flu is a common cause of respiratory tract infections, often mild, but can also be severe and a significant cause of mortality in high-risk groups. Seasonal flu usually presents with fever, cough and congestion of the nasal mucosa [Carina et al., 2021]. With the outbreak of the pandemic, SARS-CoV-2 infection has been added to the group of frequent respiratory infections. SARS-CoV-2 infection can progress from asymptomatic infection to a critical form, including death. In addition to loss of smell or taste, symptoms of COVID-19 (a disease caused by SARS-CoV-2) can occur in the course of flu. Loss of smell or taste is characteristic of COVID-19 and is rarely seen in other viral infections [Dhar Chowdhury et al, 2020].

[0004] The medications used for upper respiratory tract infections aim at symptomatic treatment, targeting the reduction of inflammation and swelling. In terms of pharmacotherapy, doctors have a limited arsenal of antiviral active substances at their disposal, in addition to which these substances have limited efficacy and numerous side-effects. With the outbreak of the COVID-19 pandemic, there is a growing demand for new products that contain highly bioavailable, natural ingredients with immunomodulatory and antiviral effects. The selection of plant extracts rich in polyphenols is driven by the documented effects of these compounds against viruses such as: Epstein-Barr, enteroviruses, herpes simplex (HSV), influenza, and respiratory syncytial virus (RSV) [Annunziata et al., 2020], which cause respiratory system infections.

[0005] Lonicera caerulea L. var. Kamtschatica Sevast. commonly known as blue honeysuckle, honey berry, honeysuckle berry, edible honeysuckle, Kamchatka berry [Ochmian et al., 2012], is a shrub found mainly in Russia (Kamchatka Peninsula), north-east Asia and Japan, but also in Europe [Svarcova et al., 2007]. Its dark blue to purple fruits were considered by the indigenous Japanese to be the 'elixir of life' due to their extensive medicinal properties [Celli et al., 2014]. The antioxidant activity of Kamchatka berry is comparable to that of blackberries, raspberries, blueberries, strawberries, hawthorn and black currants [Khattab et al., 2016]. Rupasinghe and colleagues demonstrated that Kamchatka berry extract significantly inhibits the expression of pro -inflammatory factors such as IL-6, TNF-a and PGE2, as well as the COX-2 enzyme in lipopolysaccharide (LPS)-stimulated human macrophages [Rupasinghe et al., 2015]. Similarly, Wu et al. showed that serum levels of pro-inflammatory biomarkers including IL-6, TNF-a and NO were significantly reduced in rats with adjuvant-induced arthritis fed Kamchatka berry extract [Wu et al., 2015]. The extract significantly reduced immune cell infiltration and levels of NO, TNF-a and PGE2 in the ocular aqueous fluid of mice with LPS-induced uveitis. Additionally, NO, TNF- a and PGE2 levels were reduced in the supernatants of RAW264.7 cells stimulated by LPS [Xue-Hai et al., 2006]. In a mouse model of LPS- induced oedema, oral administration of polyphenols isolated from Kamchatka berry was found to significantly reduce serum levels of RANTES, MCP-1, KC, IL- 10, MIP-la, IL- 6, IL-lp, IL-4, IL-12p70, IL-2, TNF-a and IL-3 [Wu et al., 2017],

[0006] In the case of chokeberry (Aronia melanocarpd) fruit, there are also reports indicating its immunomodulatory effects. In a rat model of ocular choroidal inflammation, intravenous administration of chokeberry extract was shown to reduce the number of inflammatory cells, decrease NO, PGE2 and TNF-a levels in the ocular aqueous fluid. In this study, aronia extract was also shown to inhibit LPS-induced iNOS and COX-2 expression in RAW 264.7 cells [Ohgami et al., 2005]. In in vitro studies on immune cells, chokeberry concentrate was found to inhibit TNF-a, IL- 6 and IL-8 production in human monocytes and NF-kB activation in macrophages of the RAW 264.7 line [Apple et al., 2015]. Furthermore, it was found that the bioactive fraction of chokeberry (ABF®) inhibits the production of pro-inflammatory cytokines and oxidative stress in LPS -stimulated BEAS- 2B lung cells, suggesting potential therapeutic effects in various inflammatory respiratory diseases [Jang et al., 2020]. In vitro models also demonstrated that chokeberry juice exhibits activity against various strains of the influenza virus, including oseltamivir- resistant strains [Park et al., 2013]. The antiviral effect is further supported by the fact that the combination product Bioaron C, which contains chokeberry extract, also showed antiviral activity against a broad spectrum of viruses responsible for upper respiratory tract infections [Glatthaar-Saalmuller et al., 2015].

[0007] The effects of echinacea purpurea (Echinacea purpurea (L.) Moench) are extensively described in scientific conclusions developed by the Herbal Medicinal Products Committee (HMPC) regarding the medicinal uses of echinacea purpurea. The HMPC stated that oral medications containing echinacea herb can be used short-term to prevent and treat common colds, and topical medications can be used to treat minor superficial wounds. Numerous in vivo studies on the effects of echinacea suggest that its administration enhances non-specific immunity by activating neutrophils, macrophages, granulocytes and NK cells. Echinacea has antiviral activity against some viruses depending on the time of administration [Jawad et al., 2012]. Administering echinacea extract in acute upper respiratory tract infections may be beneficial in the early stages of the disease [Cohen et al., 2004]. The combination product Immumax, which contains echinacea, was shown to help reducing the duration and severity of the common cold [ Y akoot et al, 2011 ] .

[0008] To date, there is no compound preparation on the market that contains standardized extracts of Kamchatka berry, chokeberry, echinacea root and herb, with proven immunomodulatory and antiviral effects. Therefore, it is justified to pursue actions aimed at obtaining a combination of these extracts with appropriate standardization. These efforts will enable the use of a smaller portion while maintaining an optimal concentration of the desired active compounds contained in these extracts, resulting in a potentiated antioxidant, immunomodulatory and antiviral effect.

[0009] The subject of the invention is a pharmaceutical composition supporting the immune system, which comprises:

[0010] - a mixture of chokeberry and blueberry fruit extracts and echinacea root and herb extract,

[0011] - where a mixture of chokeberry and Kamchatka berry fruit extract and echinacea root and herb extract contains anthocyanins 10-30% and polyphenols 15-50%,

[0012] - where echinacea root and echinacea herb extract is standardized to a minimum of 2% cichoric acid content, - the extract content in the composition is: 20-50% chokeberry fruit extract, 20-50% Kamchatka berry extract, 5-15% echinacea root extract and 5-15% echinacea herb extract.

[0013] The composition contains a pharmaceutical excipient or diluent or carrier.

[0014] The composition is for oral administration, preferably tablets, capsules, solution, powder. The composition defined above is intended for use in the manufacture of dietary supplements for use in supportive therapy for the treatment of viral upper respiratory tract diseases.

[0015] The composition has immunomodulatory properties.

[0016] The composition has antioxidant properties, reducing oxidative stress.

[0017] The composition has a protective properties against hypoxia.

[0018] The composition has antiviral properties, against coronaviruses in particular.

[0019] Figure description:

[0020] FIG.l - the effect of the composition (10 pg / mL ) on TNF-a production by PBMC cells stimulated with PHA-L (5 pg / mL). A 5 h incubation of cells with PHA-L was preceded by a 4 h pre-incubation of cells with the composition. Cytokines were determined in supernatants taken from cultures of cells in the presence of the composition and from cultures of control cells without the composition. Results are the average of three independent experiments. Results presented as percentage of positive control ± SD (PHA- L treated cells). Statistically significant changes were determined for p < 0.05.

[0021] FIG.2 - the effect of the composition on IL-8 production by A549 cells (A) and IL-8, IL- 6 production by EpiAirway™ tissues (B). A 24 h incubation of cells / tissues with LPS (1 pg / mL) was preceded by 4 or 2 h pre-incubation with the combination (250 pg / mL cell, 50 pg / mL tissue). Results presented for cells as percentage of positive control ± SD (LPS- treated cells) and for tissue as percentage of baseline cytokine production for each tissue. Statistically significant changes were determined for p < 0.05.

[0022] FIG.3 - the effect of the composition (250 pg / mL) on RELA and IL1B gene expression in A549 cells. Control: untreated cells, LPS: positive control, i.e. cells incubated with the inflammatory inducer, LPS at a concentration of 1 pg / mL. A549 stimulation was preceded by a 2 h pre-incubation of cells with the composition, followed by LPS activation for an additional 2 h. Normalized relative mRNA expression was determined relative to the reference gene GAPDH (Glyceraldehyde-3-Phosphate Dehydrogenase), and relative quantification was determined using the method 2AACt. Statistically significant changes were determined for p < 0.05.

[0023] FIG.4 - the effect of the composition (10 pg / mL) on the level of ROS in PBMC blood cells. Control: untreated cells, H2O2 (250pM): positive control, cells incubated with the ROS inducer, i.e. H2O2 at a concentration of 250 pM. The results represent the average of four independent replicates. Results are presented as the relative value to the positive control ± SD (cells treated with an oxidative stress inducer). Statistically significant changes were determined for p < 0.05.

[0024] FIG.5 - (A) the effect of the combination (250 pg / mL) on CAT, HM0X1, SOD1 gene expression in A549 cells. Control: untreated cells, LPS: positive control, i.e. cells incubated with the inflammatory inducer, LPS at a concentration of 1 pg / mL. Stimulation of A549 for 4 h was preceded by 2 h pre-incubation of cells with the composition, followed by LPS activation for an additional 2 h. Normalized relative mRNA expression was determined relative to the Glyceraldehyde-3-Phosphate Dehydrogenase (GAPDH) reference gene, and relative quantification was determined using the 2-AACt method. Statistically significant changes were determined for p < 0.05. (B) shows the effect of the combination (50 pg / mL) on SOD activity in EpiAirway™ tissues. Control: untreated cells, LPS: cells incubated with LPS at a concentration of 1 pg / mL. A 24 h incubation of tissues with LPS (1 pg / mL) was preceded by a 2 h pre-incubation with the combination (50 pg / mL). Results are presented as SOD activity in units / mL for each tissue. Statistically significant changes were determined for p < 0.05.

[0025] FIG.6 - the effect of the combination (250 pg / mL) on HIF-la protein levels in A549 cells. Normoxia - cells cultured under 21% O2 conditions ; hypoxia - cells cultured under 1% O2 conditions. The 6 h stimulation of A549 was carried out as co-incubation of cells with the composition. Graphs show results of densitometric analysis normalized against P-actin and represent the average of three independent biological replicates. Results are presented as a percentage of positive control ± SD (cells cultured under 1% O2 conditions). Statistically significant changes are indicated for p < 0.05.

[0026] FIG.7 - the effect of the composition (10 pg / mL) on IFN-a production by PBMC cells stimulated with R848 (1 pg / mL). A 20 h incubation of cells with R848 was preceded by a 4 h pre-incubation of cells with the combination. IFN-a was measured in supernatants collected from cell cultures in the presence of the combination and from cultures of control cells. Results are the mean of three independent experiments. Results are presented as a percentage of positive control ± SD (cells treated with R848). Statistically significant changes were determined for p < 0.05.

[0027] FIG.8 - the effect of the composition according to the invention on the binding of the recombinant hACE2 protein to the SARS-CoV2 S virus protein S receptor binding domain (RBD) in a competitive ELISA. Statistically significant changes were determined for p < 0.05.

[0028] FIG.9 - OPTION 1- the CPE for HCoV-OC43 in HCT-8 cells after 24 h pre-incubation of cells with the composition according to the invention. OPTION 2- the CPE for HCoV- OC43 in HCT-8 cells after simultaneous incubation of the cells with the composition according to the invention. OPTION 3- the CPE for HCoV-OC43 in HCT-8 cells - incubation of cells with the composition after virus infection. For all conditions, the results represent the mean of three independent experiments with three replicates and a 95% confidence interval.

[0029] The invention is illustrated by the following examples of implementation, which do not constitute a limitation of the invention.

[0030] COMPOSITION OF NATURAL PLANT EXTRACTS CONSISTING OF CHOKEBERRY FRUIT EXTRACT, KAMCHATKA BERRY EXTRACT AND ECHINACEA ROOT AND HERB EXTRACTS

[0031] The subject of the invention is a composition consisting of four natural extracts rich in polyphenolic compounds. The extracts have been isolated from the following parts of plants: the fruit of Kamchatka berry (Lonicera caerulea L. var. Kamtschatica Sevast.), chokeberry fuits (Aronia melanocarpa), roots and herb of purple coneflower (Echinacea purpurea (L.) Moench). The raw materials used to produce the extracts come from Polish and European cultivation. The extracts used to produce the composition according to the invention are obtained by standard methods known in the state of the art.

[0032] The extract from Kamchatka berry fruits, which can be used to implement the invention, is characterized by a polyphenol content ranging from 10% to 50% and anthocyanin content from 5% to 30%. The extract from aronia fruits, which can be used to implement the invention, is characterized by a polyphenol content ranging from 15% to 65% and anthocyanin content from 10% to 40%. The extracts from the root and herb of purple coneflower, which can be used to implement the invention, are characterized by a chicoric acid content from 2% to 6%. Advantageously, the extracts are in liquid, semi-solid, or solid form.

[0033] The composition according to the invention is a mixture of 20-50% chokeberry fruit extract, 20-50% Kamchatka berry extract, 5-15% echinacea root extract and 5-15% echinacea herb extract. The composition, which is a mixture of extracts, is standardized to contain anthocyanins from 10% to 30% and polyphenols from 15% to 50%.

[0034] The quantitative and qualitative composition of the composition was selected based on research conducted on in vitro cellular models as part of the project entitled "Natural support of the body's immune system as a way to prevent the effects of SARS-CoV-2 virus action."

[0035] The combination of plant extracts in the proportions mentioned above allows for the creation of a basic composition of the preparation characterized by multifaceted action, including immunomodulatory, antioxidant, antiviral, and protective against hypoxia effects.

[0036] Example 1

[0037] ANTI-INFLAMMATORY EFFECT OF THE COMPOSITION ACCORDING TO THE INVENTION ON PERIPHERAL BLOOD MONONUCLEAR CELLS (PBMC)

[0038] RESEARCH METHODOLOGY

[0039] Composition

[0040] The composition according to the invention contains a standardized extract of chokeberry, Kamchatka berry, echinacea root, and echinacea herb. The primary solution of the composition at a concentration of 50 mg / mL was prepared in 40% DMSO, and then brought to a concentration of 10 pg / mL in the culture medium.

[0041] Cells used for experiments

[0042] Peripheral blood mononuclear cells (PBMC). PBMCs were isolated from buffy coats of healthy blood donors according to the standard procedure (Bulent et al., 2018) using a density gradient with Ficoll (1.077 g / L). The cells were cultured in RPML1640 medium with the addition of 10% FBS (fetal bovine serum) and 100 U / ml penicillin, 100 pg / ml streptomycin. Evaluation of anti-inflammatory activity

[0043] The level of tumor necrosis factor alpha (TNF-a) was measured in the supernatants collected from PBMC cultures. Phytohemagglutinin-L (PHA-L), a lectin with strong agglutinating and mitogenic activity and high affinity for lymphocyte surface receptors, was used to induce T lymphocytes. The control consisted of untreated cells, and the positive control consisted of cells stimulated with PHA-L (5 pg / mL) for 5 hours. PBMCs were incubated for 9 hours in the presence of the composition (10 pg / mL) or preincubated with the composition (10 pg / mL) for 4 hours, followed by the addition of PHA- L for 5 hours. A commercially available ELISA kit - DuoSet® ELISA Development Systems, R&D - was used to assess TNF-a levels.

[0044] RESULTS

[0045] Analysis of the supernatants showed a 28.7% [95% CI 13.19 - 44.22] reduction in TNF- a production by PBMCs after incubation with the composition according to the invention compared to the positive control cells incubated in the presence of PHA-L.

[0046] (FIG.l)

[0047] Example 2

[0048] ANTI-INFLAMMATORY EFFECT OF THE COMPOSITION ACCORDING TO THE INVENTION ON THE ALVEOLAR EPITHELIAL CELLS

[0049] RESEARCH METHODOLOGY

[0050] Composition

[0051] The composition according to the invention contains a standardized extract of chokeberry, Kamchatka berry, echinacea root, and echinacea herb. The primary solution of the composition at a concentration of 50 mg / mL was prepared in 40% DMSO, and then brought to a concentration of 250 pg / mL in the culture medium.

[0052] Cells and tissues used for experiments

[0053] The A549 cell line was used as a type II alveolar epithelial cell model. The cells were cultured in F12 / K culture medium supplemented with 10% FBS (fetal bovine serum), 100 U / ml penicillin, 100 pg / ml streptomycin and 0.25 pg / mL amphotericin B. Cells at passages 8-12, at 70-90% confluency, were used in the experiments. The EpiAirway™ tissues (MatTek, Corp., Ashland, MA, USA) were used as a three-dimensional model of human respiratory epithelial tissue. Upon receipt, tissues were transferred to 6-well culture plates containing 1 ml of fresh medium (AIR-100-MM; MatTek, Ashland, MA, USA) and placed in a culture incubator at 37°C, 5% CO2 for 18-24 h prior to the experiment.

[0054] Stimulant

[0055] A549 cells and EpiAirway™ tissues were stimulated with lipopolysaccharide (LPS), which specifically activates toll-like receptor 4, leading to activation of the nuclear factor kappa B (NF-KB) signaling pathway and secretion of pro-inflammatory cytokines and chemokines such as interleukin (IL)-8, IL-6 or IL-ip.

[0056] Assessment of cytokine production

[0057] IL-8 was measured in the supernatants collected from A549 cell cultures, and IL-6 and IL-8 were measured in the basal layer of EpiAirway™. Untreated cells / tissues served as the control, and cells / tissues stimulated with LPS (1 pg / mL) for 24 h served as the positive control. Cells / tissues were incubated for 26 or 28 h in the presence of the composition (250 pg / mL for cells and 50 pg / mL for tissues) and pre-incubated with the composition for 4 h (cells) or 2 h (tissues), followed by the addition of LPS for 24 h. A commercially available ELISA kit, DuoSet® ELISA Development Systems, R&D, was used to assess IL-8 levels. Results for A549 are presented as a percentage of the positive control, and for EpiAirway™ as a percentage of the baseline cytokine production for each tissue.

[0058] Evaluation of IL1B and RELA gene expression

[0059] A549 cells were pre-incubated with extracts (250 pg / mL; 2 h) and stimulated with LPS (1 pg / mL, 2 h). Then, cell pellets were collected and RNA was isolated (Total RNA Mini; A&A Biotechnology ©). The obtained RNA was transcribed into cDNA using RT-PCR method (High-Capacity cDNA Reverse Transcription Kit; Applied Biosystems™). Normalized relative expression of the given gene was determined using qPCR (PerfeCTa SYBR® Green FastMix Reagent; Quantabio©), relative to the reference gene GAPDH (glyceraldehyde-3 -phosphate dehydrogenase), and relative quantification was determined using the method 2’AACt.

[0060] RESULTS

[0061] Analysis of the supernatants showed a 27.88% decrease in IL-8 production [95% CI 13.93

[0062] - 41.84] by A549 cells after incubation with the composition according to the invention compared to untreated control cells. There was also a 50.02% [95% CI 36.07 - 63.97] decrease in IL-8 production by cells treated with LPS in the presence of the composition relative to positive control cells treated with LPS alone. Analysis of the basal layer material from EpiAirway™ showed a decrease in IL-6 production compared to the positive control [LPS 95% CI 178.0 - 508.7 vs. composition + LPS 95% CI 178.0- 508.7]. Genetic material analysis showed reduced expression of the RELA and lLlf> genes, encoding the NF-KB subunit and IL-ip, respectively, in A549 cells cultured in the presence of the combination.

[0063] (FIG.2-3)

[0064] Example 3

[0065] REDUCTION OF REACTIVE OXYGEN SPECIES (ROS) LEVELS UNDER THE INFLUENCE OF THE COMPOSITION ACCORDING TO THE INVENTION

[0066] RESEARCH METHODOLOGY

[0067] Composition

[0068] The composition according to the invention contains standardized extracts of chokeberry, Kamchatka berry, echinacea root, and echinacea herb. The primary solution of the composition at a concentration of 50 mg / mL was prepared in 40% DMSO, and then brought to a concentration of 10 pg / mL in the culture medium.

[0069] Cells used for experiments

[0070] Peripheral blood mononuclear cells (PBMC). PBMCs were isolated from buffy coats of healthy blood donors according to the standard procedure (Bulent et al., 2018) using a density gradient with Ficoll (1.077 g / L). The cells were cultured in RPML1640 medium with the addition of 10% FBS (fetal bovine serum) and 100 U / ml penicillin, 100 pg / ml streptomycin.

[0071] Assessment of ROS production

[0072] The production of reactive oxygen species (ROS) was measured in PBMCs stimulated for 2 hours with the ROS inducer H2O2 (250 pM) in the presence of the composition (10 pg / mL), as well as in positive control cells (only in the presence of the ROS inducer) and negative control cells (cells not treated with the inducer). To assess ROS production, a 30-minute labeling with the DCFDA probe (2,7-dichlorodihydrofluorescein diacetate) was used. The labeled cells were evaluated using flow cytometry.

[0073] RESULTS Flow cytometric analysis of PBMCs showed a 27.2 % [95% CI 12.12 - 53.29] decrease in ROS production by PBMCs after incubation with the composition according to the invention compared to the positive control PBMCs (incubated only with the ROS inducer).

[0074] (FIG.4)

[0075] Example 4

[0076] ANTIOXIDANT EFFECT OF THE COMPOSITION ACCORDING TO THE INVENTION ON ALVEOLAR EPITHELIAL CELLS

[0077] RESEARCH METHODOLOGY

[0078] Composition

[0079] The composition according to the invention contains a standardized extract of chokeberry, Kamchatka berry, echinacea root, and echinacea herb. The primary solution of the composition at a concentration of 50 mg / mL was prepared in 40% DMSO, and then brought to a concentration of 250 pg / mL in the culture medium

[0080] Cells and tissues used for experiments

[0081] The A549 cell line was used as a type II alveolar epithelial cell model. Cells were cultured in F12 / K culture medium supplemented with 10% FBS (fetal bovine serum), 100 U / ml penicillin, 100 pg / ml streptomycin and 0.25 pg / mL amphotericin B. Cells at passage 8- 12, at 70-90% confluence were used.

[0082] EpiAirway™ tissues (MatTek, Corp., Ashland, MA, USA) were used as a 3D model of human airway epithelial tissue. Once obtained, tissues were transferred to 6-well culture plates containing 1 ml of fresh medium (AIR-100-MM; MatTek, Ashland, MA, USA) and placed in a culture incubator at 37°C, 5% CO2 for 18-24 h prior to the experiment.

[0083] Assessment of CAT, HM0X1, SOD1 gene expression

[0084] Lipopolysaccharide (LPS), which in addition to inducing inflammation also leads to the development of oxidative stress in cells, was used as a stimulant for A549 cells. Cells were pre-incubated with the composition (250 pg / mL; 2 h) and stimulated with LPS (1 pg / mL, 2 h). Cell pellets were then harvested and RNA was isolated ( Total RNA Mini; A&A Biotechnology © ). The obtaind RNA was transcribed into cDNA using RT-PCR method (High-Capacity cDNA Reverse Transcription Kit; Applied Biosystems™ ). Normalized relative expression was determined using qPCR (PerfeCTa SYBR® Green FastMix Reagent; Quantabio©), relative to the reference gene GAPDH (glyceraldehyde- 3-phosphate dehydrogenase), and relative quantification was determined using the 2AACtmethod. The genes analyzed encode enzymes involved in the removal of reactive oxygen species from cells.

[0085] Assessment of SOD activity

[0086] Lipopolysaccharide (LPS), which in addition to inducing inflammation also leads to the development of oxidative stress in cells, was used as the EpiAirway™ tissue stimulant. Tissues were pre-incubated with the composition (50 pg / mL; 2 h) and stimulated with LPS (1 pg / mL, 24 h). The basal layer was harvested and SOD activity was determined using a commercially available Superoxide Dismutase, SOD, Activity Assay Kit (Sigma- Aldrich).

[0087] RESULTS

[0088] The increase in expression of the CAT, HM0X1 and SOD1 genes, encoding catalase, heme oxygenase 1, and superoxide dismutase 1 respectively, in cells pre-incubated with the composition compared to the LPS control, indicates a strong antioxidant effect of the composition. Analysis of SOD activity showed that in tissues incubated with the composition and LPS, there is a significant increase in the activity of this enzyme compared to the LPS control, which also demonstrates the antioxidant effect of the combination.

[0089] (FIG.5)

[0090] Example 5

[0091] PROTECTIVE EFFECT AGAINST HYPOXIA OF THE COMPOSITION ACCORDING TO THE INVENTION ON THE ALVEOLAR EPITHELIAL CELLS

[0092] RESEARCH METHODOLOGY

[0093] Composition The composition according to the invention contains a standardized extracts of chokeberry, Kamchatka berry, echinacea root, and echinacea herb. The basic solution of the composition at a concentration of 50 mg / mL was prepared in 40% DMSO, and then brought to a concentration of 250 pg / mL in the culture medium.

[0094] Cells used for experiments

[0095] The A549 cell line was used as a type II alveolar epithelial cell model. Cells were cultured in F12 / K culture medium supplemented with 10% FBS (fetal bovine serum), 100 U / ml penicillin, 100 pg / ml streptomycin and 0.25 pg / mL amphotericin B. Cells at passage 8- 12, at 70-90% confluence were used.

[0096] Assessment of HIFl-a protein levels

[0097] A549 cells were cultured under normoxic conditions (21% O2), hypoxia (1% O2) and with the addition of 250 pg / mL of the composition under hypoxia (1% O2). Cell cultures were terminated after 6 h under the aforementioned conditions, and pellets were then harvested. After lysate preparation, protein levels were measured using the Bradford method, and appropriate dilutions were made. The Western blot technique was used to assess the level of the endogenous hypoxia marker, the transcription factor HIF-la (hypoxia-inducible transcription factor la), relative to the level of P-actin protein as a loading control. The obtained image was subjected to densitometric analysis using Image Studio™ Lite Quantification Software. Under hypoxic conditions, there is an inhibition of HIF-la degradation, leading to its accumulation in cells.

[0098] RESULTS

[0099] The increase in HIF-la protein levels under hypoxia (1% O2) demonstrates the validity of the in vitro model. The composition reduces HIF-la levels in cells incubated for 6h under hypoxia (reduction to 28.41% relative to 1% O2).

[0100] (FIG.6)

[0101] Example 6

[0102] ENHANCEMENT OF THE NON-SPECIFIC ANTIVIRAL RESPONSE BY THE COMPOSITION ACCORDING TO THE INVENTION

[0103] RESEARCH METHODOLOGY

[0104] Composition

[0105] The composition according to the invention contains a standardized extracts of chokeberry, Kamchatka berry, echinacea root, and echinacea herb. The basic solution of the composition at a concentration of 50 mg / mL was prepared in 40% DMSO, and then brought to a concentration of 10 pg / mL in the culture medium.

[0106] Cells used for experiments

[0107] Peripheral blood mononuclear cells (PBMC). PBMCs were isolated from buffy coats of healthy blood donors according to the standard procedure (Bulent et al., 2018) using a density gradient with Ficoll (1.077 g / L). The cells were cultured in RPMI-1640 medium with the addition of 10% FBS (fetal bovine serum) and 100 U / ml penicillin, 100 pg / ml streptomycin.

[0108] Assessment of non-specific antiviral response

[0109] The level of alpha interferon (IFN-a) was determined in the supernatants collected from cultured PBMC cells. R848 was used to induce antiviral response, acting as a toll-like receptor (TLR7) ligand mimicking viral single -stranded ribonucleic acid (ssRNA). Positive control consisted of cells stimulated with R848 for 20 h (1 pg / mL). The experimental group comprised PBMC cells pre-incubated with the composition for 4 h (10 pg / mL), after which R848 was added for 20 h. Results for supernatants from PBMC cells without R848 stimulation were below the detection level. Commercially available ELISA kit, DuoSet® ELISA Development Systems, R&D, was used to assess the concentration of IFN- a.

[0110] RESULTS

[0111] Analysis of the supernatants showed a 90.14 % increase [95% CI 42.17 - 138.14] in IFN- a production by PBMC cells after incubation with the composition according to the invention compared to positive control cells stimulated with R848.

[0112] (FIG.7)

[0113] Example 7

[0114] INHIBITORY EFFECT OF THE COMPOSITION ACCORDING TO THE INVENTION ON ACE2 AND SARS-COV-2 RBD BINDING IN VITRO)

[0115] RESEARCH METHODOLOGY

[0116] Composition

[0117] The composition according to the invention contains standardized extracts of chokeberry, Kamchatka berry, echinacea root, and echinacea herb. A stock solution of the composition at a concentration of 50 mg / mL was prepared in 40% DMSO and then brought to a concentrations ranging from 100-2000 pg / mL in the analysis buffer. The Evaluation of the inhibitory effect of the composition according to the invention on the binding of ACE2 and SARS-CoV2 RBD in vitro

[0118] SARS-CoV2 RBD (receptor-binding domain) and hACE2 (human angiotensinconverting enzyme 2) binding inhibition was assessed using the COVID- 19 Spike- ACE2 kit (CoV-SACE2-l, RayBiotech Inc, https: / / www.raybiotech.com / covid-19-spike-ace2- binding-test-kit / ) according to the protocol provided by the manufacturer. The composition according to the invention was prepared in four concentrations (100, 500, 1000, 2000 pg / mL), and the inhibitory potential for each concentration was assessed in duplicate. Analyzed extracts were mixed with recombinant hACE2 protein, added to an ELISA plate coated with recombinant RBD protein S of SARS-CoV-2, and incubated overnight at 4°C. Unbound ACE2 was removed by washing, and the binding was evaluated based on the reaction of HRP-conjugated anti-ACE2 antibody with 3, 3', 5,5'- tetramethylbenzidine (TMB). Absorbance at 450 nm was measured using a PerkinElmer reader.

[0119] RESULTS

[0120] The analysis of the ACE2-SARS-CoV-2 RBD binding assay in vitro demonstrated a concentration-dependent inhibitory effect of the composition according to the invention. (FIG. 8)

[0121] Example 8

[0122] THE ACTIVITY OF THE COMPOSITION ACCORDING TO THE INVENTION AGAINST BETACORONAVIRUSES

[0123] RESEARCH METHODOLOGY

[0124] Composition

[0125] A composition according to the invention contains a standardized extract of chokeberry, Kamchatka berry, echinacea root, and echinacea herb. An initial concentration of 10 mg / mL of the composition was prepared in MiliQ water at 50° C. The solutions were prepared fresh before each experiment and filtered on a Millipore 0.22 pm filter before being brought to the appropriate concentrations in the culture medium.

[0126] Cells used for experiments

[0127] HCT-8 human colorectal cancer cell line (ATCC® CCL-244). Culture medium: RPMI 1640 10 % FBS. Test virus

[0128] HCoV-OC43 - human coronavirus (Betacoronavirus 1; ATCC® VR-1558™, Coronaviridae) (messenger RNA)

[0129] Cell viability test (CTE method, evaluation of cytotoxic effects)

[0130] To determine non-toxic concentrations of the composition relative to HCT-8 cells, a microscopic assessment method of cytotoxic effects (CTE), i.e., changes in cell morphology under the influence of the composition, was utilized according to the scale provided in Table 1. 24-hour cultures of HCT-8 cells with >90% confluence were incubated with the composition at concentrations ranging from 5-2000 pg / mL for 72 hours at 37°C and 5% CO2. Dilution series were then performed in appropriate culture medium. The control consisted of cells treated with culture medium. Morphological changes in cells were observed daily under an inverted microscope, and cell viability was calculated based on the CTE scale. The cytotoxic concentration reducing cell viability by 50% (CC50) was estimated.

[0131] Table 1. Cytotoxic effects (CTE)

[0132] Antiviral assays (AVA)

[0133] Antiviral activity (AVA) tests of selected, non-toxic concentrations of the compositions against the test virus were conducted on monolayer, 24-hour HCT-8 cells at a density of 3 x 105cells / ml. The assessment of antiviral activity of the combination was performed in three experimental variants:

[0134] • Incubation of cells with the combination prior to viral infection (OPTION 1)

[0135] • Incubation of cells with the combination and viruses simultaneously (OPTION 2)

[0136] • Incubation of cells with the combination after viral infection (OPTION 3)

[0137] Cytopathic effects (CPE) of the virus were determined under an inverted microscope according to the scale in Table 2. The negative control comprised the test virus alone and uninfected cells incubated only in culture medium. The positive control consisted of antiviral drugs molnupirovir (50 pM) and interferon (0.01 pg / mL).

[0138] Table 2. Cytopathic effects (CPE).

[0139] RESULTS

[0140] Determination of non-toxic concentrations of composition according to the invention The composition according to the invention showed complete lack of toxicity or slight cytotoxicity (viability >85%) towards HCT-8 cells in the concentration ranging from 1- 200 pg / ml. Starting from a concentration of 500 pg / ml, the composition showed high toxicity towards the tested cells. The cytotoxic concentration for 50% of the HCT-8 cells (CC50 ) was 447 pg / ml. To study the effect of the composition on tested virus replication, nontoxic concentrations were selected (cell viability did not fall below 95%).

[0141] Evaluation of antiviral activity

[0142] OPTION 1. The composition was incubated for 24 hours with HCT-8 cells before infection with the tested virus. Positive control - interferon (IFN) at a concentration of 0.01 pg / mL. The composition exhibited antiviral activity against human HCoV-OC43 coronavirus when administered to cells 24 hours prior to the virus infection. At the highest non-toxic concentration of 150 pg / mL, it reduced the virus titer by approximately 60 %. The IC50 concentration of the composition was 110.54 pg / mL.

[0143] OPTION 2. The composition was incubated for 24 hours with HCT-8 cells simultaneously with the tested virus. Positive control - interferon (IFN) at a concentration of 0.01 pg / mL. The composition showed antiviral activity against human coronavirus HCoV-OC43 when administered simultaneously with the virus. At the highest non-toxic concentration of 150 pg / mL, it reduced the virus titer by approximately 88 %. The IC50 concentration of the composition was 76.69 pg / mL. OPTION 3. The composition was added to already infected with the tested virus HCT-8 cells. HCT-8 cells were incubated for 90 minutes with HCoV-OC43 virus, then the residual unabsorbed virus was washed off, and the composition was applied. Positive control -molnupivir (MPV) at a concentration of 50 pM.

[0144] At the highest non-toxic concentration of 150 pg / mL, the composition reduced virus titre by approximately 76 %. The IC50 concentration of the composition was 101.78 pg / mL. (FIG.9)

[0145] Determination of selectivity indices

[0146] The selectivity indices of the compositions against HCoV-OC43 virus (antiviral activity) are shown in Table 3.

[0147] Table 3. Antiviral activity of the composition according to the invention against HCoV- OC43 virus.

[0148] CCso (cytotoxic concentration)-concentration of the composition that is toxic to 50% of the cells in the culture compared to the control.

[0149] ICso (inhibitory concentration)-concentration of the composition that inhibits viral replication by 50% compared to the viral control.

[0150] SI (selectivity index)- selectivity index =CC50 / IC50.

Claims

Patent Claims1. A pharmaceutical composition supporting the immune system, characterized in that it contains:A mixture of extracts from chokeberry fruits and haskap berry fruits, and extracts from the root and herb of echinacea,The mixture of chokeberry and haskap berry fruit extracts and echinacea root and herb extracts contains 10-30% anthocyanins and 15-50% polyphenols,Where the echinacea root and herb extract is standardized to a minimum of 2% cichoric acid content,The extract content in the composition is: 20-50% chokeberry fruit extract, 20-50% haskap berry fruit extract, 5-15% echinacea root extract, and 5-15% echinacea herb extract.

2. The composition according to claim 1, characterized in that it contains a pharmaceutical excipient or diluent or carrier.

3. The composition according to claim 1, characterized in that it is for oral administration, preferably in the form of a tablet, capsule, or solution.

4. The composition defined above for use in the production of dietary supplements for supportive therapy in the treatment of viral upper respiratory tract infections.

5. The composition according to claim 4, characterized in that it has immunomodulatory application.

6. The composition according to claim 4, characterized in that it has antioxidant application, reducing oxidative stress.

7. The composition according to claim 4, characterized in that it has protective application against hypoxia.

8. The composition according to claim 4, characterized in that it has antiviral application, particularly against coronaviruses.

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