Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
  • A61K36/18—Magnoliophyta (angiosperms)
  • A61K36/185—Magnoliopsida (dicotyledons)
  • A61K36/45—Ericaceae or Vacciniaceae (Heath or Blueberry family), e.g. blueberry, cranberry or bilberry
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
  • A61K36/18—Magnoliophyta (angiosperms)
  • A61K36/185—Magnoliopsida (dicotyledons)
  • A61K36/28—Asteraceae or Compositae (Aster or Sunflower family), e.g. chamomile, feverfew, yarrow or echinacea
• • A—HUMAN NECESSITIES
  • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
  • A23L—FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES, NOT OTHERWISE PROVIDED FOR; PREPARATION OR TREATMENT THEREOF
  • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
  • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
  • A23L33/105—Plant extracts, their artificial duplicates or their derivatives
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
  • A61K31/19—Carboxylic acids, e.g. valproic acid
  • A61K31/192—Carboxylic acids, e.g. valproic acid having aromatic groups, e.g. sulindac, 2-aryl-propionic acids, ethacrynic acid 
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/33—Heterocyclic compounds
  • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
  • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
  • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
  • A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
  • A61K36/18—Magnoliophyta (angiosperms)
  • A61K36/185—Magnoliopsida (dicotyledons)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
  • A61K36/18—Magnoliophyta (angiosperms)
  • A61K36/185—Magnoliopsida (dicotyledons)
  • A61K36/35—Caprifoliaceae (Honeysuckle family)
  • A61K36/355—Lonicera (honeysuckle)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
  • A61K36/18—Magnoliophyta (angiosperms)
  • A61K36/185—Magnoliopsida (dicotyledons)
  • A61K36/73—Rosaceae (Rose family), e.g. strawberry, chokeberry, blackberry, pear or firethorn
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators

Definitions

• 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.
• 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.
• SARS-CoV-2 infection can progress from asymptomatic infection to a critical form, including death.
• symptoms of COVID-19 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].
• the medications used for upper respiratory tract infections aim at symptomatic treatment, targeting the reduction of inflammation and swelling.
• 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.
• 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.
• viruses such as: Epstein-Barr, enteroviruses, herpes simplex (HSV), influenza, and respiratory syncytial virus (RSV) [Annunziata et al., 2020], which cause respiratory system infections.
• 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].
• LPS lipopolysaccharide
• 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].
• 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].
• 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.
• HMPC Herbal Medicinal Products Committee
• 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 ] .
• the subject of the invention is a pharmaceutical composition supporting the immune system, which comprises:
• 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.
• composition contains a pharmaceutical excipient or diluent or carrier.
• 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.
• the composition has immunomodulatory properties.
• the composition has antioxidant properties, reducing oxidative stress.
• the composition has a protective properties against hypoxia.
• the composition has antiviral properties, against coronaviruses in particular.
• 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.
• FIG.2 the effect of the composition on IL-8 production by A549 cells (A) and IL-8, IL- 6 production by EpiAirwayTM 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).
• 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 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 2 AACt .
• GAPDH Glyceraldehyde-3-Phosphate Dehydrogenase
• 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.
• 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.
• GPDH Glyceraldehyde-3-Phosphate Dehydrogenase
• FIG.6 the effect of the combination (250 pg/mL) on HIF-la protein levels in A549 cells.
• 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.
• 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.
• 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.
• 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.
• 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.
• 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%.
• the extracts are in liquid, semi-solid, or solid form.
• 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%.
• composition 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.”
• 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.
• PBMC Peripheral blood mononuclear cells
• TNF-a tumor necrosis factor alpha
• PHA-L Phytohemagglutinin-L
• 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.
• 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.
• 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.
• FBS fetal bovine serum
• the EpiAirwayTM tissues (MatTek, Corp., Ashland, MA, USA) were used as a three-dimensional model of human respiratory epithelial tissue.
• 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.
• fresh medium AIR-100-MM; MatTek, Ashland, MA, USA
• LPS lipopolysaccharide
• NF-KB nuclear factor kappa B
• 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 EpiAirwayTM. 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.
• 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
• Results for A549 are presented as a percentage of the positive control, and for EpiAirwayTM as a percentage of the baseline cytokine production for each tissue.
• 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 BiosystemsTM).
• 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.
• PBMC Peripheral blood mononuclear cells
• ROS reactive oxygen species
• 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
• 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.
• FBS fetal bovine serum
• penicillin 100 U/ml penicillin
• streptomycin 100 pg/ml streptomycin
• 0.25 pg/mL amphotericin B 0.25 pg/mL amphotericin B.
• EpiAirwayTM 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.
• Lipopolysaccharide 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 BiosystemsTM ).
• LPS Lipopolysaccharide
• 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.
• composition 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.
• 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.
• FBS fetal bovine serum
• penicillin 100 U/ml penicillin
• streptomycin 100 pg/ml streptomycin
• 0.25 pg/mL amphotericin B 0.25 pg/mL amphotericin B.
• 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 StudioTM Lite Quantification Software. Under hypoxic conditions, there is an inhibition of HIF-la degradation, leading to its accumulation in cells.
• HIF-la hypoxia-inducible transcription factor la
• HIF-la protein levels under hypoxia 1% O2
• hypoxia 1% O2
• the composition reduces HIF-la levels in cells incubated for 6h under hypoxia (reduction to 28.41% relative to 1% O2).
• 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.
• PBMC Peripheral blood mononuclear cells
• 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.
• 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.
• SARS-CoV2 RBD receptor-binding domain
• hACE2 human angiotensinconverting enzyme 2 binding inhibition
• COVID- 19 Spike- ACE2 kit CoV-SACE2-l, RayBiotech Inc, https://www.raybiotech.com/covid-19-spike-ace2- binding-test-kit/
• 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.
• 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.
• HCT-8 human colorectal cancer cell line (ATCC® CCL-244).
• Culture medium RPMI 1640 10 % FBS.
• Test virus
• HCoV-OC43 - human coronavirus (Betacoronavirus 1; ATCC® VR-1558TM, Coronaviridae) (messenger RNA)
• CTE cytotoxic effects
• 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 10 5 cells/ml. The assessment of antiviral activity of the combination was performed in three experimental variants:
• 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).
• 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%).
• 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.
• IFN Positive control - interferon
• 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.
• 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.
• MPV Positive control -molnupivir
• the composition reduced virus titre by approximately 76 %.
• the IC50 concentration of the composition was 101.78 pg/mL.
• CCso cytotoxic concentration
• SI (selectivity index)- selectivity index CC50/IC50.

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• Medicines Containing Plant Substances (AREA)
• Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)

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