Compositions for use in the treatment or prevention of infections caused by enveloped viruses, comprising a Zymocarpus extract

Abstract

To provide a well-tolerated composition that is effective for the treatment and / or prevention of a disease caused by a coronavirus or an influenza virus, and / or at least one symptom thereof. [Solution] The present invention relates to a composition comprising or corresponding to an extract obtained from Zymocarpus longan for use in the treatment and / or prevention of diseases caused by, for example, enveloped viruses, in particular coronaviruses such as SARS-CoV, MERS-CoV and SARS-CoV-2, or influenza viruses, such as influenza virus type A and influenza virus type B, and / or at least one symptom thereof.

Description

[Technical field]

[0001] The present invention relates to a composition comprising or corresponding to an extract obtained from a plant of the Sapindaceae family, in particular fruits of the genus Dimocarpus or Litchi, for use in the treatment and / or prevention of a disease caused by an enveloped virus such as a coronavirus, for example an influenza virus, a respiratory syncytial virus (RSV), a human parainfluenza virus (HPIV), a human metapneumovirus (HPMV), a rhinovirus or a coronavirus (CoV), in particular a coronavirus such as SARS-CoV, MERS-CoV and SARS-CoV-2, or an influenza virus, such as influenza virus type A or influenza virus type B, and / or at least one symptom thereof.

[0002] In particular, the present invention relates to a composition comprising or corresponding to an extract of Dimocapus longan Lour. for use in the treatment or prevention of at least one symptom of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection and / or coronavirus disease-19 (COVID-19).

[0003] Furthermore, the present invention particularly relates to a composition comprising or corresponding to an extract of Dimocarpus longan Lour for use in the treatment or prevention of influenza A virus infection (subtypes A(H1N1), A(H1N1)pdm09 and A(H3N2)) or influenza B virus infection and / or at least one symptom thereof. [Background technology]

[0004] Dimocarpus is a genus in the Sapindaceae family, also known as the soapberry family of angiosperms (division Angiosperms), which also includes lychee, rambutan, guarana, koran, pitongba, mamonchiro, and ackee. The main characteristics of the genus are that it is a tree or shrub that can grow to 25-40 meters tall and has pinnate leaves. The flowers are arranged in large panicles. The edible fruits are 3-5 cm long and contain a single seed surrounded by a layer of pulp. Dimocarpus is distributed mainly in tropical South and Southeast Asia, from Sri Lanka and India to East Malaysia and Australia. The edible fruit from this genus, known as "longan", is produced from Dimocarpus longan Lour, especially the commercial longan, Dimocarpus longan ssp. longan var. longan.

[0005] In the present invention, the term "Dimocarpus" refers to various species of this genus, such as, for example, Dimocarpus australicus, Dimocarpus dentatus, Dimocarpus foveolatus, Dimocarpus gardneri, Dimocarpus fumatus, Dimocarpus confinis, Dimocarpus leichhardtii and Dimocarpus yunnanesis, but in particular Dimocarpus longan Lour and its subspecies Dimocarpus longan ssp. longan), Dimocarpus longan ssp malesianus and its varieties.

[0006] Further genera of the Sapindaceae family that can be used in the present invention are the genus Litchi, of which the only species is Litchi chinensis (Litchi), or the genus Nephelium, such as Nephelium lappaceum (Rambutan).

[0007] Longan has historically been cultivated for its edible properties, but it can also be used medicinally. Longan fruit contains high amounts of bioactive substances such as proteins, carbohydrates, vitamin C, polysaccharides, polyphenols such as corilagin and ellagic acid and their conjugates, 4-O-methyl gallic acid, flavone glycosides, quercetin and kaempferol glycosides, ethyl gallate-1-β-O-galloyl-d-glucopyranose, grevifolin, 4-O-α-l-rhamnopyranosyl-ellagic acid, GABA, and tannic acid. The fruit has been used in traditional Chinese medicine formulations to help relieve neuralgia and swelling (Non-Patent Document 1). Longan fruit is consumed in various forms, including dried longan pulp, longan juice, longan jelly, longan wine, and canned longan syrup. Recent reports have shown that the polyphenols and polysaccharides contained in the pulp and peel of longan contribute to antioxidant, anti-glycation, anti-tyrosinase, strong immunomodulatory, and anti-cancer effects (Non-Patent Document 2). A review of the bioactive substances and physiological activities of longan pulp is published in Non-Patent Document 3. Longan fruit is approximately 83% water, 15% carbohydrate, and 1% protein.

[0008] The family of enveloped viruses includes many of the most dangerous pathogenic viruses for humans and livestock, such as human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HBC), influenza virus (e.g., Non-Patent Document 4 or Non-Patent Document 5), as well as severe acute respiratory syndrome coronavirus 1 (SARS-CoV-1), Middle East respiratory syndrome coronavirus (MERS-CoV), and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 is a positive single-stranded RNA virus that belongs to the family Betacoronavirus (Non-Patent Document 6). Phylogenetically, it is 97% related to bat coronaviruses, 79% to severe acute respiratory syndrome coronavirus-1 (SARS-CoV-1), and 50% to Middle East respiratory syndrome coronavirus (MERS-CoV) (Non-Patent Document 7, Non-Patent Document 8). Coronaviruses are enveloped, positive-positive, single-stranded RNA viruses that are widely distributed among humans and can cause respiratory, enteric, hepatic, and neurological diseases, most commonly causing mild respiratory infections in humans. Betacoronavirus (β-CoV or Beta-CoV) is one of four genera of coronaviruses belonging to the family Coronaviridae, subfamily Orthocoronavirus, order Nidovirales. They are enveloped, positive, single-stranded RNA viruses, mostly of zoonotic origin. The genome of betacoronaviruses encodes four structural proteins known as S (spike), E (envelope), M (membrane) and N (nucleocapsid) proteins. The N protein holds the RNA genome, while the S, E and M proteins together constitute the viral envelope. The spike protein is the major glycoprotein on the surface of coronaviruses, responsible for the virus attachment and fusion with the membrane of the host cell. The spike protein forms a crown-like structure on the surface of coronaviruses.

[0009] The emergence of the novel SARS-CoV virus causing COVID-19 in late 2019 has resulted in an unprecedented global public health emergency with significant social and economic impacts. On January 31, 2020, following a rapid increase in patients worldwide, the World Health Organization (WHO) declared Severe Acute Respiratory Syndrome Virus-2 (official name: SARS-CoV-2) and its infectious disease COVID-19 a pandemic (Non-Patent Document 9). Due to the highly infectious nature and rapid spread of SARS-CoV-2, the WHO declared the ongoing pandemic COVID-19 a global emergency in March 2020. As of June 1, 2021, there have been more than 171 million confirmed cases of SARS-CoV-2 worldwide, with more than 3.55 million deaths.

[0010] SARS-CoV-2 has a high similarity to SARS-CoV and MERS-CoV, and in fact, these viruses use similar receptors to enter cells, allowing them to replicate in similar tissues (Non-Patent Document 10). So far, the structural spike (S) glycoprotein has been shown to have a very high affinity for the angiotensin-converting enzyme 2 (ACE2) receptor. This receptor is ubiquitously expressed in the nasal epithelium, lungs, heart, kidney, and intestine, but is rarely found on immune cells (Non-Patent Document 11, Non-Patent Document 12). Early events occurring immediately after SARS-CoV-2 infection of respiratory tissues may affect the outcome in terms of disease severity. In some patients, infection with COVID-19 leads to excessive activation of immune responses in the epithelial / immune barrier, generating an inflammatory environment (Non-Patent Document 13, Non-Patent Document 14). The development of cytokine storm and acute lung injury leading to acute respiratory distress syndrome (ARDS) is a possible undesirable outcome of this disease. ARDS with systemic coagulopathy is an important aspect of morbidity and mortality in COVID-19 (Non-Patent Document 15, Non-Patent Document 16). These excessive immune responses caused by the invading virus can cause widespread tissue destruction in severe cases, resulting in tissue damage and multiple organ failure (Non-Patent Document 17, Non-Patent Document 18). Complement deposition and high serum levels of anaphylatoxins have been reported in severe / critical patients, suggesting that complement may be one of the factors causing immune hyperactivation (Non-Patent Document 19).

[0011] Recently, the complement system was one of the most significantly upregulated intracellular pathways after SARS-CoV-2 infection, as shown by transcriptome analysis of bronchoalveolar lavage fluid from patients (Non-Patent Document 20, Non-Patent Document 21). Furthermore, the transcriptome of primary normal human bronchial epithelial (NHBE) cells infected in vitro with SARS-CoV-2 revealed enrichment of complement signatures (Non-Patent Document 20, Non-Patent Document 21). More recently, Ramlall et al. identified a robust involvement of the complement and coagulation systems after SARS-CoV-2 infection, in addition to IFN- and IL-6-dependent inflammatory responses (Non-Patent Document 22).

[0012] Influenza viruses of the Orthomyxoviridae family are enveloped, negative-stranded RNA viruses with segmented genomes. Of the two genera, one includes influenza virus type A and influenza virus type B, and the other is influenza virus type C. The three viruses differ in host range and pathogenicity. Type A and B viruses have eight separate gene segments, each encoding at least one protein. Influenza viruses are covered with three protein projections: hemagglutinin (HA), neuraminidase (NA), and matrix 2 (M2). Each influenza RNA segment is encapsulated by a nucleoprotein, forming a ribonucleotide-nucleoprotein complex. Influenza type B and C viruses are almost always isolated only from humans. However, influenza type A viruses, all of which circulate in or originate from avian reservoirs, can infect a variety of warm-blooded animals, including pigs, horses, dogs, cats, and other mammals, as well as humans. Waterfowl are the natural reservoir for all known subtypes of influenza virus type A and are likely the ultimate source of human pandemic influenza strains. Influenza A viruses are subdivided according to the serological or genetic characteristics of the HA and NA surface glycoproteins that protrude from the virion. Sixteen types of HA (or “H”) and nine types of NA (or “N”) are known, abbreviated as H1-H16 and N1-N9 (Non-Patent Document 23). Seasonal influenza A and B viruses are particularly relevant to humans. Influenza A subtypes A(H1N1)pdm09, A(H3N2) and B viruses have been circulating in human populations since 2009. The influenza A(H1N1) viruses circulating before the 2009 pandemic have since been completely replaced by the A(H1N1)pdm09 viruses. Influenza A(H3N2) viruses infect birds and mammals. Symptoms of influenza infection include, for example, body aches, fever, chills, fatigue, diarrhea and vomiting. Adults over 65 years old, children under 5 years old, pregnant women, people with underlying conditions such as asthma, diabetes, or heart disease, and people with weakened immune systems due to drug therapy (steroids, chemotherapy) or chronic illnesses (HIV, leukemia) are at higher risk of developing severe influenza.

[0013] A research group at the Institute of Hygiene and Medical Microbiology, Medical University of Innsbruck, Austria, recently described in primary NHBE cells using a standardized human 3D respiratory model (Non-Patent Document 24, Non-Patent Document 25) that SARS-CoV-2 infection leads to widespread tissue destruction, which is associated with intracellular complement activation in epithelial cells and massive anaphylatoxin production (Non-Patent Document 26). [Prior art documents] [Non-patent literature]

[0014] [Non-Patent Document 1] Yang et al., Food Research International 44(7):1837-1842 (2011); Zhang et al., Food Science and Human Wellness 9: 95-102 (2020) [Non-Patent Document 2] N. Nuengchamnong & K. Ingkaninan, Food Chem 118: 147-152 (2010); Khan et al., J. Food Sci. Technol.55: 4782-4791 (2018) [Non-Patent Document 3] Zhang et al., Food Science and Human Wellness 9: 95-102 (2020) [Non-Patent Document 4] Rey FA et al., Cell 172(6):1319-34(2018) [Non-Patent Document 5] Vaney MC et al., Cell Microbiol 13(10):1451-9 (2011)

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Non-Patent Document 23

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Non-Patent Document 25

Non-Patent Document 26

Summary of the Invention

Problems to be Solved by the Invention

[0015] Symptoms of SARS-CoV-2 infection are nonspecific. Common symptoms early in the infection are fever, weakness, and dry cough. Less common symptoms include headache, myalgia, fatigue, chest tightness, dyspnea, sputum, diarrhea, confusion, sore throat, rhinorrhea, chest pain, nausea, and vomiting. Up to 50% of patients experience shortness of breath. Severe COVID-19 is characterized by acute respiratory distress syndrome (ARDS) and widespread damage to the alveoli of the lung parenchyma. The proportion of patients who require treatment for ARDS is approximately 10% of those who are hospitalized and have symptoms. It is known that some patients are asymptomatic carriers of infection who do not show clinical symptoms. Critically ill patients are usually elderly and have chronic diseases, and the most common associated diseases among critically ill patients are hypertension and cardiovascular disease.

[0016] Thus, there remains a need for effective, well-tolerated therapeutic agents for the prevention and / or treatment of COVID-19 and secondary symptoms caused by the disease, which is addressed by the present invention. [Means for solving the problem]

[0017] The present invention therefore relates to a composition comprising or corresponding to a Ganoderma lucidum extract or a Zymocarpus extract for use in the treatment and / or prevention of at least one symptom of SARS-CoV-2 infection and / or COVID-19.

[0018] The present invention particularly relates to a composition comprising or corresponding to a Zymocarpus extract for use in the treatment and / or prevention of at least one symptom of SARS-CoV-2 infection and / or COVID-19.

[0019] The present invention also relates to a method for the treatment and / or prevention of at least one symptom of SARS-CoV-2 infection and / or COVID-19, which comprises administering to a subject a therapeutically or prophylactically effective amount of a composition comprising or corresponding to a Ganoderma lucidum extract or a Zymocarpus extract, the subject in this regard generally being a human being.

[0020] The present invention particularly relates to a method for the treatment and / or prevention of at least one symptom of SARS-CoV-2 infection and / or COVID-19, which comprises administering to a subject a therapeutically or prophylactically effective amount of a composition comprising or corresponding to an extract of Zymocarpus. In this regard, the subject is generally a human being.

[0021] Specifically, the extract is Dimocarpus longan Lour extract.

[0022] In a further embodiment, the present invention relates to a method for the treatment and / or prevention of influenza virus infection and / or at least one symptom of influenza virus infection, comprising administering to a subject a therapeutically or prophylactically effective amount of a composition comprising or corresponding to a Zymocarpus extract, the subject in this regard generally being a human being. In particular, the influenza virus is selected from influenza virus type A and influenza virus type B. Influenza virus type A is in particular influenza virus subtype H3N2.

[0023] Specifically, the extract is Dimocarpus longan Lour extract. [Brief description of the drawings]

[0024] [Figure 1]Schematic of the standardized human 3D respiratory model used in the in vitro studies of the examples. The human in vitro model closely replicates the entire upper airway epithelium. Cells are cultured on a permeable filter support at the air-liquid interface, where they can obtain nutrients from the bottom of the dish with culture medium (as they would in vivo access the blood stream). At the top, the cells are exposed to air, which stimulates cell differentiation and produces mucus to protect them from drying. The culture contains mucus-producing goblet cells, ciliated epithelial cells, and basal cells with stem cell properties. The culture system can therefore be used and manipulated from both sides in the study. For example, it can be infected or treated from the upper (apical) side, and culture medium can be harvested from the lower (basal) side for marker analysis. [Diagram 2] Figure 2a-c: Transepithelial Electrical Resistance (TEER) study in 3D cultures of NHBE cells exposed to SARS-CoV-2 Zymocarpus extract. TEER measurements are performed by applying an alternating electrical signal between electrodes placed on either side of a cell monolayer on a permeable filter support and measuring the voltage and current to calculate the electrical resistance of the barrier. Figure 2a: Effect of exposure to 0.1% and 1% extract on the apical and basolateral sides Figure 2b: TEER measurements 1 day post infection (d1PI); 0.1%, 1% and 2% extract (w / w) apical side Figure 2c: TEER measurements 2 days post infection (d2PI); 0.1%, 1% and 2% extract (w / w) apical side See Example 2 [Diagram 3] SARS-CoV-2 innate immune response C3a reduced complement expression See Example 3 [Figure 4] SARS-CoV-2 Reduction of infection by Zymocarpus extract in NHBE primary monolayers See Example 4 [Diagram 5] SARS-CoV-2 Reduction of infection by Zymocarpus extract in 3D cultures of NHBE cells See Example 4 [Figure 6] FIG. 1 is a schematic diagram showing the preparation process of Zymocarpus extract for use in the present invention. [Figure 7]Figures 7a-8d: Transepithelial electrical resistance (TEER) studies in 3D cultures of NHBE cells exposed to influenza virus Zymocarpus extract. TEER measurements are performed by applying an alternating electrical signal between electrodes placed on either side of a cell monolayer on a permeable filter support and measuring the voltage and current to calculate the electrical resistance of the barrier. See Example 5. Figure 7a: TEER measurements 1 day post-infection (d1PI); Influenza A(H3N2) added at MOI 0.05; 1% extract (w / w) apical side. Figure 7b: TEER measurements 2 days post-infection (d2PI); Influenza A(H3N2) added at MOI 0.05; 1% extract (w / w) apical side. Figure 7c: TEER measurements 1 day post-infection (d1PI); Influenza B added at MOI 0.05; 1% extract (w / w) apical side. Figure 7d: TEER measurements 2 days post-infection (d2PI); Influenza B added at MOI 0.05; 1% extract (w / w) apical side. [Figure 8a] TEER measurements 1 day post-infection (d1PI); influenza A (H3N2) added at MOI 0.005; 1% extract (w / w) apical side [Figure 8b] TEER measurements 1 day post-infection (d1PI); influenza A (H3N2) added at MOI 0.005; 1% extract (w / w) apical side [Figure 8c] TEER measurements Day 1 post-infection (d1PI); influenza B added at MOI 0.005; 1% extract (w / w) apical side [Figure 8d] TEER measurements Day 2 post-infection (d2PI); influenza B added at MOI 0.005; 1% extract (w / w) apical side [Figure 9a] Figures 9a to 10c: Study of influenza virus particles released from the apical and basolateral sides using analysis by reverse transcription polymerase chain reaction (RT-PCR) See Example 6. RT-PCR apical side, influenza A (H3N2) added at MOI 0.005; 1 day post infection (d1PI) [Figure 9b]RT-PCR apical side, influenza A(H3N2) added at MOI 0.005; 2 days post-infection (d2PI) [Figure 9c] RT-PCR apical side, influenza B added at MOI 0.005; 1 day post infection (d1PI) [Figure 9d] RT-PCR apical side, influenza B was added at an MOI of 0.005, 2 days post-infection (d2PI) [Figure 10a] RT-PCR basal side, influenza A(H3N2) added at MOI 0.005, 1 day post infection (d1PI) [Figure 10b] RT-PCR basal side, influenza A(H3N2) added at MOI 0.005, 2 days post-infection (d2PI) [Figure 10c] RT-PCR basal, influenza B added at MOI 0.005; day 2 post-infection (d2PI) DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0025] In the context of the present invention, it has surprisingly been found that ingredients derived from plants of the family Sapindaceae, such as the genus Ganoderma lucidum and the genus Zymocarpus, in particular the genus Zymocarpus, and more particularly Zymocarpus longan Lour, prevent / inhibit tissue damage, inflammation and infection caused by the SARS-CoV-2 virus, as well as tissue damage, inflammation and infection caused by the influenza virus.

[0026] In particular, it was surprisingly found that topical administration of a composition containing or corresponding to a Zymocarpus extract enhanced mucociliary clearance (MCC) in SARS-CoV-2-infected NHBE cells. MCC is the main innate defense mechanism in the lungs. Its functional components are the protective mucus layer, the airway surface liquid layer, and the cilia on the surface of ciliated cells. Cilia are specialized organelles that repeatedly bend to generate metachronal waves to propel pathogens and inhaled particles trapped in the mucus layer out of the airways.

[0027] Moreover, it was surprisingly found that topical administration of a composition consisting of or corresponding to a Zymocarpus extract stabilized the transepithelial electrical resistance (TEER) in SARS-CoV-2-infected NHBE cells. TEER measurements are used to assess the integrity and stability of the epithelial cell layer (i.e., barrier function).

[0028] Moreover, it was surprisingly found that topical administration of a composition consisting of a Zymocarpus extract or a corresponding composition inhibited complement activation and downregulated the expression of inflammatory markers and chemoattractants of immune cells in SARS-CoV-2-infected NHBE cells.

[0029] Moreover, surprisingly, it was found to suppress infection with SARS-CoV-2.

[0030] Moreover, it has been surprisingly found that topical administration of a composition consisting of a Zymocarpus extract, or a corresponding composition, stabilizes the transepithelial electrical resistance (TEER) in influenza A(H3N2) and influenza B infected NHBE cells. TEER measurements are used to assess the integrity and stability of epithelial cell layers (i.e., barrier function).

[0031] Furthermore, it was surprisingly found that the intracellular formation and excretion of virus particles of the novel influenza A (H3N2) virus and influenza B virus were inhibited.

[0032] It has surprisingly been found that glycoproteins on the viral surface of the lipid bilayer act as access keys, allowing the virus to enter the cell, but that the components of the Zymocarpus extract contained in the composition for use according to the invention cover the viral surface proteins and prevent the virus from entering the cell.

[0033] Thus, the present invention provides, inter alia: (1) A composition comprising an extract of Zymocarpus or a composition corresponding to said extract for use in the treatment and / or prevention of a respiratory infection due to an enveloped virus, an enveloped single-stranded virus, an enveloped positive single-stranded RNA virus (+ssRNA) virus, influenza or coronavirus and / or at least one symptom thereof. (2) The composition for use according to (1) in the treatment of a respiratory infection caused by an enveloped virus, an enveloped single-stranded virus, an enveloped positive single-stranded RNA virus (+ssRNA) virus, influenza or coronavirus and / or at least one symptom thereof. (3) enveloped virus, enveloped single-stranded virus, enveloped positive single-stranded RNA virus (+ssRNA) virus, influenza or coronavirus The composition for use according to (1) in the prevention of respiratory infections and / or at least one symptom thereof. (4) The composition for use according to any one of (1) to (3), wherein the respiratory infection is an upper respiratory tract infection and / or a lower respiratory tract infection. (5) The composition for use according to any one of (1) to (4), wherein the Dimocarpus extract is an extract of Dimocarpus longan Lour. (6) The composition for use according to any one of (1) to (5) above, wherein the virus is selected from influenza virus, respiratory syncytial virus (RSV), human parainfluenza virus (HPIV), human metapneumovirus (HPMV), rhinovirus or coronavirus (CoV). (7) The composition for use according to any one of (1) to (6), wherein the virus is selected from SARS-CoV, MERS-CoV and SARS-CoV-2. (8) The composition for use according to any one of (1) to (7), wherein the infectious disease is SARS-CoV-2 infection (COVID-19). (9) The composition for use according to any one of (1) to (6), wherein the virus is selected from subtypes of influenza virus type A (particularly type A(H3N2)) or influenza virus type B. (10) The composition for use according to any one of (1) to (6) and (9), wherein the infectious disease is an influenza virus A(H3N2) or influenza virus B infection. (11) The composition for use according to any one of (1) to (10), wherein the Zymocarpus extract comprises one or more of vitamin C; one or more of polyphenols selected from corilagin, gallic acid, ellagic acid, ellagic acid conjugates, (-)-epicatechin, quercetin, kaempferol, tannic acid (tannin) and chlorogenic acid; protocatechuic acid; brevifolin; gamma-aminobutyric acid (GABA); carbohydrates and water. (12) The composition for use according to any one of (1) to (11), wherein the Zymocarpus extract comprises vitamin C; one or more polyphenols selected from corilagin, gallic acid, ellagic acid, ellagic acid conjugates, (-)-epicatechin, quercetin, kaempferol, tannic acid (tannin), and chlorogenic acid; protocatechuic acid; brevifolin; gamma-aminobutyric acid (GABA); carbohydrates, and water. (13) The composition for use according to any one of (1) to (12), wherein the Zymocarpus extract comprises vitamin C, corilagin, gallic acid, ellagic acid, ellagic acid conjugates of tannic acid, GABA, sucrose, glucose, fructose, polysaccharides and water.

[0034] In the present invention, the Zymocarpus extract contains 100-1000 mg / kg, preferably 105-760 mg / kg, or 400-1000 mg / kg, more preferably 600-760 mg / kg, and most preferably 720 mg / kg of vitamin C.

[0035] In the present invention, the Zymocarpus extract contains 750-2000 mg / kg, 750-1800 mg / kg, preferably 1188-1880 mg / kg, more preferably 1200-1500 mg / kg, and most preferably 1250 mg / kg corilagin.

[0036] In the present invention, the Zymocarpus extract contains 200-600 mg / kg, preferably 340-428 mg / kg, more preferably 380-430 mg / kg, and most preferably 409 mg / kg of gallic acid.

[0037] In the present invention, the Zymocarpus extract contains 600 to 1250 mg / kg, 600 to 1200, preferably 1010 to 1230 mg / kg, more preferably 1010 to 1100 mg / kg, and most preferably 1050 mg / kg of ellagic acid (including ellagic acid conjugates).

[0038] In the present invention, the Zymocarpus extract contains 200-700 mg / kg, preferably 420-510 mg / kg, more preferably 450-480 mg / kg, and most preferably 430 mg / kg of tannic acid.

[0039] In the present invention, the Zymocarpus extract contains 1200-2000 mg / kg, preferably 1133-1896 mg / kg, more preferably 1150-1700 mg / kg, and most preferably 1638.30 mg / kg GABA.

[0040] In the present invention, the Zymocarpus extract contains 30-50%, preferably 30-45%, more preferably 30-40%, and most preferably 42.4% (w / w) of total extracted saccharose (sucrose).

[0041] In the present invention, the Zymocarpus extract contains 5-25%, preferably 7.5-23%, more preferably 10-20%, and most preferably 11.7% (w / w) total extracted glucose.

[0042] In the present invention, the Zymocarpus extract contains 10-20%, preferably 10-18%, more preferably 10-15%, and most preferably 13.3% (w / w) of total extracted fructose.

[0043] In the present invention, the Zymocarpus extract contains 50-85 g / kg, preferably 50-80 g / kg, more preferably 50-70 g / kg, and most preferably 66 g / kg of polysaccharides.

[0044] In the present invention, the Zymocarpus extract contains 15-25%, preferably 18-24%, more preferably 19-23%, and most preferably 21% (w / w) of water.

[0045] In the present invention, the total phenol content of the Zymocarpus extract is 2950-7600 mg / kg, preferably 3500-7600 mg / kg, more preferably 4000-6500 mg / kg, and most preferably 7565 mg / kg.

[0046] In the present invention, the total carbohydrate content is 700-800 g / kg, preferably 742 g / kg.

[0047] In the master's thesis “Mono, Oligo- and Polysaccharide analysis of a beverage obtained from the fruit of Dimocarpus longan” by Tobias Schlappack, September 16, 2020, Institute of Analytical Chemistry and Radiochemistry, Leopold-Franzens University Innsbruck, the sugar profile in terms of free sugars, oligosaccharides, polysaccharides, total carbohydrates and water content of a beverage obtained from the fruit of Dimocarpus longan is disclosed.

[0048] According to the invention, the composition used has a sugar content of about 74-84°Brix, more preferably a sugar content of about 76-82°Brix, most preferably a sugar content of about 78-80°Brix.

[0049] In one embodiment, the present invention provides a composition for use according to any one of (1) to (13), wherein the Dimocarpus extract, in particular the Dimocarpus longan Lour extract, comprises:

[0050] [Table 1]

[0051] The present invention specifically provides: (14) A composition for use according to any one of (1) to (13), wherein the Dimocarpus extract, in particular the Dimocarpus longan Lour extract, comprises:

[0052] [Table 2]

[0053] (15) A composition for use according to any one of (1) to (13), wherein the Dimocarpus extract, in particular the Dimocarpus longan Lour extract, comprises:

[0054] [Table 3]

[0055] (16) A composition for use according to any one of (1) to (14), wherein the Dimocarpus extract, in particular the Dimocarpus longan Lour extract, comprises:

[0056] [Table 4]

[0057] (17) A composition for use according to any one of (1) to (16), wherein the Dimocarpus extract, in particular the Dimocarpus longan Lour extract, comprises:

[0058] [Table 5]

[0059] (18) The composition for use according to any one of (1) to (17), wherein the extract is produced from whole dried or fresh fruit, from the pericarp, seeds, arils, pulp of dried or fresh fruit, or from a combination of at least two of the pericarp, seeds, arils, pulp of fresh fruit. It is particularly preferred to use whole fresh fruit as starting material.

[0060] (19) A composition for use according to any one of (1) to (18), wherein the Dimocarpus extract, in particular the Dimocarpus longan Lour extract, is obtainable by a method comprising the steps of: (a) Extraction of zymocarpus juice from fresh whole fruits, followed by (b) Separation of solids from the resulting raw solution (c) concentrating the liquid obtained in step (b) to obtain a sugar concentration of about 74-84°Brix, preferably 76-82°Brix, most preferably 78-80°Brix; and (d) Aseptic filling.

[0061] (20) The composition for use according to any one of (1) to (19), wherein the Zymocarpus extract can be obtained by a method comprising the following steps: (a) Fresh whole Zymocarpus fruits crushed; (b) Extraction of juice from the crushed whole fruit obtained in (a); (c) preparing the fresh fruit juice obtained in (b) by rapidly heating the fresh fruit juice to about 95-98°C, maintaining the fresh fruit juice at about 95-98°C, and then rapidly cooling the fresh fruit juice to about 5-15°C; (d) Separation of the supernatant from the product of (c) by centrifugation and microfiltration; (e) concentrating the supernatant obtained in step (d), preferably by evaporation under reduced pressure, to obtain a sugar concentration of about 74-84°Brix; (f) microfiltration of the concentrate obtained in step (e); and (g) Aseptic filling.

[0062] (21) The composition for use according to any one of (1) to (20), wherein the composition is administered locally via the ocular, nasal, or (naso)pharyngeal route, i.e., for topical administration to the conjunctival epithelium of the eye and the epithelium of the upper respiratory tract. (22) The composition for use according to any one of (1) to (21), which is in the form of a mouthwash, a gargle, a nasal drop, a nasal spray / aerosol, a throat nasal drop, or a throat spray / aerosol. (23) The composition according to any one of (1) to (21), which is in the form of a nasal spray / aerosol or a throat spray / aerosol.

[0063] In the present invention, "Zymocarpus" (also referred to herein as "Zymocarpus species") may be selected from the group of, for example, Dimocarpus longan, Dimocarpus australicus, Dimocarpus dentatus, Dimocarpus foveolatus, Dimocarpus fumatus, Dimocarpus gardneri, Dimocarpus confinis, Dimocarpus leichhardtii, and Dimocarpus yunnanesis.

[0064] The extract may be an extract obtained from any of the Zymocarpus species / subspecies, or an extract obtained from a mixture of at least two of the above. In a preferred embodiment of the composition of the present invention, the Dimocarpus extract is an extract of Dimocarpus longan Lour.

[0065] In a more preferred embodiment of the present invention, the extract is prepared from Dimocarpus longan Lour and is used for the prevention of COVID-19 in the form of a nasal spray, throat spray or lung spray, most preferably in the form of a nasal spray.

[0066] In a further preferred embodiment of the present invention, the extract is prepared from Dimocarpus longan Lour and is used in the form of a nasal spray, throat spray or lung spray for the prevention of influenza virus infections, particularly influenza virus A(H3N2) or influenza virus B infections, most preferably in the form of a nasal spray.

[0067] Compositions according to the invention and their preparation

[0068] The term "extract" refers to a mixture of substances, derivative products or components that can be obtained from Zymocarpus (Zymocarpus extract) by any suitable method known to a person skilled in the art.

[0069] The extract can be obtained from all components of the whole plant, including leaves, bark, flowers, seeds, pericarp, fruit, pulp, arils, stems, branches, stalks, roots, wood and parts thereof. Fresh or dried fruits can be used. Different Zymocarpus components / parts can be used individually or together. It is especially preferable to use whole fresh fruit.

[0070] Solid parts / particles such as the fruit shell, arils, pericarp and solid components of the pulp are separated from the liquid juice during the process by means of squeezing / pressing, sedimentation / centrifugation and (micro)filtration. In particular, the method for producing Zymocarpus extract according to the present invention comprises the steps as disclosed in (19) and (20) above.

[0071] As used herein, the term "juice extraction" refers to the process of separating the liquid portion of the fruit from the solid portion.

[0072] Preferably, the whole longan fruit is comminuted prior to extraction by means known to those skilled in the art, such as by a hammer mill.

[0073] Thus, techniques include solvent extraction, but also (cold) pressed juice (fresh juice) obtained from fresh plant material by means other than solvent extraction, for example by methods known to those skilled in the art, such as using hydraulic presses (e.g. standard hydraulic cold pressing techniques with vertical pressing beds), roll mills or double screw juicers, or juice extractors, especially industrial scale juice extractors. Particularly preferred is the use of industrial scale juice extractors. Concentration methods include direct heating, steam heating, vacuum evaporation, with vacuum evaporation being particularly preferred.

[0074] According to a preferred embodiment of the present invention, the components obtained from Zymocarpus are obtained by a method comprising the following steps:

[0075] (1) Crush fresh whole Jimmocarpus longan (longan fruit), preferably using a hammer mill, and optionally passing through a 32 mm diameter sieve to remove initial solids and lumps to obtain a homogeneous powder; (2) extracting fresh fruit juice from the pulverized fruit obtained in step (1) using a fruit juice extractor, preferably for about 5 minutes under a pressure of about 0.8 to 4.5 bar; (3) discarding the paste obtained after extraction, and rapidly heating the fresh fruit juice obtained in step (2) to about 95°C, maintaining it at a temperature of 95°C to 98°C for at least 45 minutes, more preferably at 98°C for at least 45 minutes, and then rapidly cooling it to a temperature of about 5.0 to 15.0°C, more preferably about 12°C; (4) subjecting the cooled product of step (3) to a precipitation separation step at least once at 4000 rpm to 7200 rpm, preferably 7200 rpm, and discarding the residue; (5) subjecting the supernatant obtained in step (4) to precipitation filtration (removal at 0.2 microns) at a temperature of 45° C. or less, preferably at a temperature of about 15-40° C., more preferably at a temperature of about 12° C., to obtain a turbidity of 40 NTU or less; (6) subjecting the filtrate obtained in step (5) to an evaporation step under reduced pressure, in which the filtrate is heated to a temperature of about 83.0-87°C, preferably heated to about 85°C under reduced pressure at least once, and then cooled stepwise to about 35-45°C, preferably 40°C, to obtain a sugar concentration of about 74-84°Brix, preferably about 76-82°Brix, most preferably about 78-80°Brix; (7) optionally stirring the concentrate obtained in step (6) at a temperature of about 35.0°C to about 45.0°C to obtain a Brix of about 76°C to about 78°C; (8) filtration of the concentrate (removal of 1.0 mm or less, preferably about 0.15 mm); and (9) Aseptically filling and cooling to a temperature below 40°C to obtain Zymocarpus extract for use according to the present invention.

[0076] In a particularly preferred embodiment, the Zymocarpus extract is obtained / obtainable by the method disclosed in Thai Petty Patent Application No. TH2103000091 (a flow chart of which is shown in FIG. 6 for illustrative purposes).

[0077] The extract can be obtained from all components of the whole plant, including leaves, bark, flowers, seeds, peels, fruits, stems, branches, stalks, roots, wood and parts thereof. Fresh or dried fruits can be used. Different Zymocarpus components / parts can be used individually or together. It is particularly preferred to use fresh whole fruit as starting material.

[0078] Exemplary Methods It is particularly preferred that a composition comprising or corresponding to a Zymocarpus longan extract for use according to the present invention is prepared by the following method, as shown in FIG. 6 and disclosed in TH2103000091.

[0079] Properties of Zymocarpus extract The qualitative characterization of the components of the Zymocarpus extract of the present invention was carried out using ultra-high performance liquid chromatography quadrupole time-of-flight mass spectrometry (UHPLC-UV-HR-QTOF-MS). The method used was as follows: A Thermo Scientific Dionex UltiMate 3000 coupled with a Bruker Daltonics maxis Impact Ultra High Resolution TOF-MS was used for the analysis. An Agilent RRHD Zorbax C18 (2.1 x 100 mm, 1.8 μm) column was used for the chromatographic separation of gallic acid, ellagic acid (and conjugates) and corilagin. The mobile phase consisted of acetonitrile (B) and 0.2% formic acid (FA) in water (A). The flow rate was 0.4 mL / min and the injection volume was 2 μL. The column oven was set at 45 °C. The sampler was set at 20 °C. The LC gradient was as follows: Min / B%: 0 / 0, 2 / 0, 17 / 50, 19 / 100, 20.5 / 100, 21 / 0, 23 / 0. The eluate from the LC was introduced directly into the mass spectrometer, using electrospray in positive mode with a mass scan of 50-2000 m / z and a spectral rate of 4 Hz. The mass accuracy was verified before each run by comparison with the sodium formate adduct. The mass accuracy was rounded to 1 mDa and the corresponding retention times to 0.05 min. UV spectra were recorded at 270 nm. Bruker® Compass Data Analysis 4.2 was used for the interpretation of the mass signals. Each sample was run in triplicate. The above instrument parameters and LC column were also used for the quantification of tannins (as gallic acid equivalents) with minor adjustments to the LC method as follows: Min / B%: 0 / 3, 15 / 50, 18 / 80, 19 / 100, 21 / 100, 21.5 / 0, 23 / 0. The autosampler was kept at 4°C.

[0080] A Thermo Scientific AccucoreTM HILIC silica column (2.1x150 mm, 2.6 μm) was used for the analysis of vitamin C. The mobile phase consisted of acetonitrile (B) and 0.01% formic acid (FA) in water (A). The flow rate was 0.4 mL / min and the injection volume was 1 μL. The column oven was set at 25 °C. The sampler was set at 20 °C. The LC gradient was as follows: Min / B%: 0 / 95, 5 / 95, 5.5 / 60, 8 / 80, 9 / 15, 13 / 15, 14 / 95, 17 / 95. The eluate from the LC was directly introduced into the mass spectrometer (ESI negative) with a mass scan of 50-800 m / z and a spectral rate of 4 Hz. XIC (m / z 173) was used for the quantification of vitamin C. For GABA analysis, an Eppendorf BioSpectrometer® basic (Eppendorf, Hamburg, Germany) operating at 340 nm was used.

[0081] In a preferred embodiment, the Zymocarpus extract of the present invention comprises the ingredients shown in Table 1 below.

[0082] [Table 6]

[0083] Pharmaceutical formulations of the present invention Compositions containing or corresponding to the Zymocarpus extract according to the invention can be administered by any means that brings said extract into contact with the site of action in the mammalian body, preferably the human body, and in the form of pharmaceutical preparations containing them. The composition / formulation is preferably an aqueous composition.

[0084] According to the invention, pharmaceutical formulations for topical administration and topical administration of the formulations according to the invention are preferred. In particular, topical administration to the epithelial lining of the upper respiratory tract or to the eyes is preferred, and thus the compositions are preferably administered to a subject via the ocular, nasal or pharyngeal route in the form of eye drops, mouthwash, gargle, nasal spray / aerosol, nasal drops, throat spray / aerosol or throat drops. Nasal or pharyngeal administration is particularly preferred, and therefore the aqueous composition is particularly preferably provided in the form of a mouthwash, a gargle, a nasal spray / aerosol, a nasal drop, a throat spray / aerosol or a throat nasal drop.

[0085] Administration to the epithelial lining of the upper respiratory tract is most preferred, and therefore administration is most preferred in the form of a mouthwash, gargle, nasal spray / aerosol, nasal drops, throat spray / aerosol, or throat drops. Of the aforementioned dosage forms, a nasal spray or throat spray is most preferred.

[0086] Therefore, the present invention further provides a pharmaceutical formulation / composition suitable for topical administration comprising the above-mentioned Zymocarpus extract according to the present invention.

[0087] The compositions can be formulated according to techniques known to those skilled in the art, for example, those described in "Remington: The Science and Practice of Pharmacy", Pharmaceutical Press, 22nd edition and Bauer et al., Pharmazeutische Technologie, 5. Edt. Govi-Verlag Frankfurt, 1997; Rudolf Voigt, and Pharmazeutische Technologie, 9. Edt., Deutscher Apotheker Verlag Stuttgart, 2000).

[0088] In particular, the composition can be formulated as a dosage form for the nasal cavity or pharynx (e.g., through the mouth and / or nose).Dosage forms for nasal administration include, for example, nasal sprays (e.g., nasal pump sprays) and nasal drops.Dosage forms for pharynx administration include, for example, pharyngeal sprays and pharyngeal nasal drops.

[0089] The Zymocarpus extract of the present invention is used in the pharmaceutical formulations of the present invention at a pharma- ceutical effective concentration to obtain the desired effect.

[0090] As a rule, the formulations / compositions according to the invention may contain 0.1-10% (w / w), preferably the compositions according to the invention may contain 0.5-7% (w / w), more preferably 1-5% (w / w), most preferably 2-4% (w / w) of the Zymocarpus extract according to the invention, based on the total weight of the formulation, preferably as a solution in a liquid physiologically / pharmacologically acceptable vehicle. Preferably, the vehicle is aqueous.

[0091] The excipient / diluent may be selected from the group consisting of water (such as aqua ad injectabilia, aqua bidist and aqua purificata); saline, phosphate buffered saline (PBS); other physiologically / pharmacologically acceptable buffer systems such as Sorensen's buffer, sodium citrate / citric acid, glycerol, sorbitol and mixtures thereof; oil-based excipients such as sesame oil. The use of aqueous excipients is preferred.

[0092] It is particularly preferred to use water for injection, double distilled water, purified water, physiological saline and PBS as excipients or mixtures thereof, and most preferred to use water for injection or purified water as excipient / diluent.

[0093] When formulating an aqueous product to be administered to the epithelial lining, it is important to control properties such as viscosity, pH, buffering capacity, and osmolality.

[0094] In addition to physiologically acceptable excipients well known to those skilled in the art, the pharmaceutical formulations / compositions of the present invention may contain additional components such as osmolality / tonicity adjusters such as NaCl, sorbitol, glucose and dextrose; pH adjusters such as citric acid, sodium hydroxide, hydrochloric acid, sulfuric acid, etc.; buffer components such as sodium citrate, sodium phosphate, etc.; viscosity enhancing excipients such as hydroxyethylcellulose, tragacanth, sodium hyaluronate, xanthan, etc.

[0095] In particular, for intranasal / upper respiratory tract administration, the pH should be adjusted to within the range of 3.5 to 7.5, more preferably 4 to 7.5, and most preferably 4.5 to 6.5. The pH of the ophthalmic formulation should preferably be adjusted to within the range of 6-8. The pH of the aqueous composition can be adjusted (eg, to any of the pH ranges or values ​​described above) using, for example, sodium hydroxide or hydrochloric acid and / or other suitable pH adjusting agents.

[0096] The aqueous composition can have, for example, an osmotic pressure of about 200 mOsm / kg to about 800 mOsm / kg, preferably an osmotic pressure of about 250 mOsm / kg to about 500 mOsm / kg, and more preferably an osmotic pressure of about 280 to 500 mOsm / kg. The osmolarity of the aqueous composition can be adjusted (eg, to any of the osmolarity ranges or values ​​set forth above) using, for example, sodium chloride and / or other suitable osmolarity adjusting agents.

[0097] Unless otherwise specified, all properties, parameters referred to herein, including pH values ​​and amounts / concentrations (e.g., expressed in mg / ml, % w / v or % v / v), are preferably measured at standard ambient temperature and pressure conditions, in particular at a temperature of 25° C. (298.15 K) and an absolute pressure of 101.325 kPa (1 atm). Thus, the pH indicated herein is preferably measured at a temperature of 25° C., more preferably at a temperature of 25° C. and an absolute pressure of 1 atm.

[0098] As used herein, unless expressly indicated otherwise or contradicted by context, the terms "a," "an," and "the" are used interchangeably with "one or more" and "at least one." Thus, for example, a composition that includes "an" excipient can be interpreted as referring to a composition that includes "one or more" excipients.

[0099] Where numerical ranges are provided / disclosed herein, it is to be understood that all values ​​and subranges contained within each numerical range are meant to be included within the scope of the invention, and thus the invention specifically and individually relates to each value that falls within the numerical ranges disclosed herein, and each subrange that falls within the numerical ranges disclosed herein.

[0100] In the present invention, the term "total phenolic content" refers to all phenolic compounds measured by the Folin-Ciocalteu method, including but not limited to those specifically identified phenolic compounds.

[0101] In the present invention, the term "total carbohydrate" refers to all carbohydrate compounds, including polysaccharides, as determined by the phenol-sulfuric acid method, including, but not limited to, specifically identified carbohydrate compounds. More suitable dosage forms of the antiviral pharmaceutical formulation of the present invention are oral dosage forms such as hard and soft candies, dragees, pastilles, (throat) lozenges and (medicinal) chewing gums, which allow the Zymocarpus longan extract of the present invention to come into direct contact with the epithelial lining of the upper respiratory tract / oral cavity, the portal of entry for pathogens.

[0102] Thus, in a further embodiment, the Jimmocarpus longan extract of the present invention can be formulated into soft or hard candies, dragees, pastilles, (throat) lozenges or (medicinal) chewing gum using techniques and carriers, excipients and additives well known to those skilled in the art.

[0103] The following formulation examples are for illustrative purposes only and are not intended to limit the scope of the present invention.

[0104] Prescription Examples In the present invention, the following compositions / preparations are particularly preferred for use:

[0105] (a) 0.1-10% (w / w) of the Zymocarpus longan extract according to the invention, particularly as described in Table 1 Glycerin 32% (w / w) Sorbitol aqueous solution (70% (w / w)) 39.1-50% (w / w) Purified water 15%(w / w)

[0106] (b) Zymocarpus longan extract according to the invention, particularly as described in Table 1, 0.1-10% (w / w) Propolis 0.1% (w / w) Licorice powder 0.13%(w / w) Peppermint powder 0.13% (w / w) Glycerin 1.97% (w / w) Citric acid 0.99% (w / w) Disodium ethylenediaminetetraacetate 0.013% (w / w) Hydroxypropyl cellulose 0.99% (w / w) Sodium chloride 0.66% (w / w) Purified water 85.017%-94.917% (w / w) (i.e., make the volume to 100% (w / w))

[0107] The term 100% (w / w) in the present invention refers to the addition of purified water to reach the expected final total weight of the formulation.

[0108] (c) 0.1-10% (w / w) of the Zymocarpus longan extract according to the invention, particularly as described in Table 1 In aquavist or saline

[0109] The above formulations can in particular be used for administration via the pharyngeal and nasal routes, such as in the form of nasal drops, nasal spray / aerosol, pharyngeal drops or pharyngeal spray / aerosol.

[0110] As mentioned above, the more suitable dosage forms of the antiviral pharmaceutical preparation of the present invention are oral dosage forms such as hard and soft candies, dragees, pastilles, (throat) troches and (medicinal) chewing gums, which allow the Zymocarpus longan extract of the present invention to come into direct contact with the epithelial lining of the upper respiratory tract / oral cavity, which is the portal of entry for pathogens.

[0111] Thus, in a further embodiment, the Zymocarpus longan extract of the present invention can be incorporated into soft or hard candies, dragees, pastilles, (throat) troches or (medicinal) chewing gums using techniques and carriers, excipients and additives well known to those skilled in the art. Such dosage forms according to the present invention can contain the Zymocarpus longan extract of the present invention (i.e., active ingredient) in an amount of about 0.1 to about 20% (w / w) and a base and further carriers, excipients and additives (inactive ingredients) in an amount of about 80 to about 99.9% (w / w) based on the total weight of the formulation.

[0112] Exemplary formulations for candies, dragees, pastilles or troches include the following: :

[0113] Zymocarpus longan extract 0.1-20%(w / w) Sugar or sugar substitutes 0-98% (w / w) Bulking agent 0-98%(w / w) Gum arabic 0-15%(w / w) Water 0.1-15% (w / w) Fat 0-15%(w / w) Natural or artificial flavors 0.01-10% (w / w)

[0114] Exemplary formulations for (medicated) chewing gum include the following:

[0115] Zymocarpus longan extract 0.1-20%(w / w) Polyisobutylene 0-50%(w / w) Polyvinyl acetate 0-50%(w / w) Natural rubber such as chicle 0-50% (w / w) Sugar or sugar substitute 20-80% (w / w) Bulking agent 0-98%(w / w) Water 0-15% (w / w) Fat 0-15%(w / w) Natural or artificial flavors 0.01-10% (w / w)

[0116] In a preferred embodiment, the Zymocarpus longan extract of the present invention is a Zymocarpus longan extract as shown in Table 1 above.

[0117] Indications In the present invention, the pharmaceutical preparation according to the present invention can be used in a therapeutic method for treating and / or preventing a respiratory infection due to an enveloped virus, preferably an enveloped single-stranded virus, more preferably an enveloped positive single-stranded RNA virus (+ssRNA) virus, even more preferably influenza or coronavirus and / or at least one symptom thereof.

[0118] In the present invention, the enveloped virus is particularly selected from influenza virus, respiratory syncytial virus (RSV), human parainfluenza virus (HPIV), human metapneumovirus (HPMV), rhinovirus or coronavirus (CoV).

[0119] With regard to infections by coronaviruses, the treatment and / or prevention of infections by SARS-CoV, MERS-CoV and SARS-CoV-2 is particularly preferred.

[0120] The present invention therefore provides a composition comprising or corresponding to an extract of Zymocapus for use in the treatment and / or prevention of at least one symptom of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infection and / or Coronavirus Disease-19 (COVID-19), among others.

[0121] In a further particularly preferred embodiment, the present invention provides a composition comprising or corresponding to a Zymocapus extract for use in the treatment of at least one symptom of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infection and / or COVID-19.

[0122] In another particularly preferred embodiment, the present invention provides a composition comprising or corresponding to a Zymocapus extract for use in the prevention of at least one symptom of Severe Acute Respiratory Syndrome Coronavirus-2 (SARS-CoV-2) infection and / or Coronavirus Disease-19 (COVID-19).

[0123] The COVID-19 symptoms listed above include one or more of the following: fever, chills, (dry) cough, stuffy or runny nose, fatigue, muscle or body aches, sore throat, diarrhea, nausea or vomiting, conjunctivitis, headache, loss of taste or smell, discolouration of the fingers or toes or skin rash, difficulty breathing or shortness of breath, constant chest pain or pressure, abdominal pain, speech or movement problems, sudden confusion, or bluish lips or face.

[0124] More severe symptoms and manifestations of COVID-19 include pneumonia, severe pneumonia, pulmonary fibrosis, (acute) lung injury, and acute respiratory syndromes such as Severe Acute Respiratory Syndrome (SARS) and Acute Respiratory Distress Syndrome (ARDS).

[0125] Extrapulmonary manifestations / manifestations of COVID-19, including hematological and / or immune system related manifestations, include lymphopenia (also known as lymphocytopenia), leukocytosis, neutrophilia, blood coagulation disorders, thrombocytopenia, pulmonary embolism, disseminated intravascular coagulation, deep vein thrombosis, and prothrombotic states; cardiovascular manifestations of COVID-19 include myocardial injury, acute cardiac injury, acute coronary syndrome (ACS), cardiomyopathy, acute cor pulmonale, heart failure (including new onset atrial fibrillation, heart attack, heart block, ventricular arrhythmias), cardiogenic syndrome, and pulmonary embolism. COVID-19 manifestations include acute kidney injury (AKI), proteinuria and hematuria and may be characterized by electrolyte abnormalities (e.g., hyperkalemia, hyponatremia and / or hypernatremia); COVID-19 gastrointestinal (GI) manifestations include diarrhea, nausea, vomiting, abdominal pain, anorexia, anosmia, dysgeusia, as well as hepatobiliary (liver) manifestations of COVID-19, endocrinological manifestations of COVID-19, neurological and ophthalmological manifestations of COVID-19, and dermatological manifestations of COVID-19.

[0126] With regard to influenza virus infections, treatment and / or prevention of infections with influenza virus type A (such as A(H3N2)) and influenza virus type B is particularly preferred.

[0127] Thus, the present invention inter alia provides a composition comprising or corresponding to an extract of Zymocapus for use in the treatment and / or prevention of influenza virus infection and / or at least one symptom of influenza virus infection.

[0128] In a further particularly preferred embodiment, the present invention provides a composition comprising or corresponding to an extract of Dimocapus for use in the treatment of influenza virus infection and / or at least one symptom thereof.

[0129] In another particularly preferred embodiment, the present invention provides a composition comprising or corresponding to an extract of Zymocapus for use in the prevention of influenza virus infection and / or at least one symptom thereof.

[0130] Symptoms of influenza virus infection mentioned above include one or more of the following: fever, chills, cough, stuffy or runny nose, fatigue, general weakness, muscle or body aches, sore throat, diarrhea, nausea or vomiting, headache, and sweating.

[0131] More severe manifestations of influenza virus infection include primary influenza virus pneumonia, superimposed bacterial pneumonia (e.g., pneumococcal, staphylococcal, or Haemophilus influenzae), and exacerbation of chronic lung disease (e.g., COPD). In addition, multiple organ involvement can lead to myositis, rhabdomyolysis, encephalitis, and myocarditis.

[0132] The subject or patient treated in the present invention may be an animal (e.g., a non-human animal). Preferably, the subject / patient is a mammal. More preferably, the subject / patient is a human (e.g., a male or female human) or a non-human mammal (e.g., a guinea pig, hamster, rat, mouse, rabbit, dog, cat, horse, monkey, ape, marmoset, baboon, gorilla, chimpanzee, orangutan, gibbon, sheep, cow, pig, or mink). Most preferably, the subject / patient treated in the present invention is a human.

[0133] As used herein, the term "treatment" of a disorder or disease (e.g., "treatment" of COVID-19) is well known in the art.

[0134] "Treating" a disorder or disease means that a patient / subject is suspected of or has been diagnosed with a disorder or disease. A patient / subject suspected of having a disorder or disease will typically exhibit certain clinical and / or pathological symptoms that can be readily ascribed to a particular pathological state by one of ordinary skill in the art (i.e., the disorder or disease can be diagnosed).

[0135] "Treatment" of a disorder or disease may, for example, result in the cessation of the progression of the disorder or disease (e.g., no worsening of symptoms) or the slowing of the progression of the disorder or disease (if the cessation of progression is transient). "Treatment" of a disorder or disease may also result in partial remission (e.g., improvement of symptoms) or complete remission (e.g., disappearance of symptoms) of the subject / patient suffering from the disorder or disease. Thus, "treatment" of a disorder or disease may also refer to the amelioration of the disorder or disease, for example, leading to the cessation of the progression of the disorder or disease or the slowing of the progression of the disorder or disease. Such partial or complete remission may be followed by relapse. It is understood that a subject / patient may experience a wide range of responses to treatment (such as the exemplary responses as described herein above). Treatment of a disorder or disease includes, inter alia, curative treatment (preferably leading to a complete response and ultimately a cure of the disorder or disease) and palliative treatment (including the alleviation of symptoms).

[0136] The term "prevention" of a disorder or disease as used herein (e.g., "prevention" of COVID-19) is also well known in the art. For example, a patient / subject suspected of being susceptible to a disorder or disease may particularly benefit from prevention of the disorder or disease. The subject / patient may have a susceptibility or predisposition to the disorder or disease, including but not limited to a genetic predisposition. Such predisposition may be measured by standard methods and tests, for example, using genetic markers or phenotypic indicators. It is understood that the disorder or disease to be prevented in the present invention has not been diagnosed or cannot be diagnosed in the patient / subject (e.g., the patient / subject does not exhibit clinical or pathological symptoms). Thus, the term "prevention" consists of the use of a compound of the present invention before clinical and / or pathological symptoms are diagnosed or determined by the attending physician or before they can be diagnosed or determined.

[0137] Testing of compositions containing Zymocarpus extract according to the present invention The present invention uses the standardized human 3D respiratory model described in Zaderer et al. Cells 2019;8:1292 and Chandorkar et al. Sci Rep 2017;7:11644 as a screening platform / in vitro model to evaluate the effect of topical administration of Zymocarpus extracts / compositions according to the present invention.

[0138] The model consists of in vitro reconstituted primary human normal bronchial epithelial or small airway epithelial cells (NHBE, SAE) from the upper and lower airways. Cells are seeded onto collagen or cellulose scaffolded transwell filters and cultured at the air-liquid interface (ALI).

[0139] Normal human bronchial epithelial (NHBE, Lonza, catalog number CC-2540S) cells were cultured at the air-liquid interface (ALI) as described previously in Zaderer et al., Cells 2019;8:1292 (Patent Document 24) and Chandorkar et al., Sci Rep 2017;7:11644 (Patent Document 25). Briefly, cells were cultured in T75 flasks for 2-4 days until reaching 80% confluence. Cells were trypsinized and plated on GrowDexT (UPM)-coated 0.33 cm 2 1 × 10 cells per transwell (Costar, Corning, NY, USA) were placed in a porous (0.4 μm) polyester membrane insert. 5 Cells were seeded at a seeding density of 100 μg / ml. Cells were grown to near confluence in liquid culture for 2–3 days using specific epithelial cell growth medium according to the manufacturer's instructions. Cultures were maintained in a humidified atmosphere of 5% CO2 at 37°C before being transferred to ALI cultures. Epithelia were grown and differentiated using Stem Cell's airway medium. Days of development were counted as day 0, the day ALI culture was initiated. MucilAir nasal cells were obtained from Epicelix, Geneva, Switzerland, and cultured according to the manufacturer's protocol.

[0140] Please refer to FIG. 1 for explanation. Using this in vitro model, the following parameters were specifically analyzed: - Mucociliary Clearance (MCC) - Ciliary Beat Frequency - Apical cytokine release - Transepithelial Electrical Resistance (TEER) - Innate immune response (C3a) - Detect infection rates through images EXAMPLES

[0141] All experimental data was generated using Zymocarpus extracts according to the present invention, specifically those disclosed in Table 1 above, diluted to the desired concentrations with sterile double-distilled water or saline. [Example 1] Mucociliary transport / clearance (prevention) Study on the effect of Zymocarpus extract on ciliary motility and mucociliary clearance in NHBE three-dimensional cultures

[0142] Assessment of ciliary motility and mucociliary clearance after administration of D-PBS spray (control) or 1% Zymocarpus extract spray.

[0143] Mucus in the respiratory system is moved across the mucosa by ciliary beating, an important non-specific defense mechanism called mucociliary clearance (MCC). The MCC is the main self-cleaning system of the nasal cavity and paranasal sinuses and is crucial as a nonspecific defense against organic and inorganic pollutants constantly carried by the air. It functions by trapping particles and microorganisms in the mucus and transporting them along the mucus membrane to the pharynx, where they are either coughed up or swallowed.

[0144] method D-PBS (control) or 1% (w / w) Zymopcarpus extract in D-PBS was sprayed onto fully differentiated 3D NHBE cultures (passage 2, day 92 at the air-liquid interface) (see Figure 1 ). Ciliary motility was assessed by brightfield observation using HCS on an Operetta CLS (PerkinElmer). Mucociliary clearance was monitored after adding fluorescently labeled beads (Invitrogen) to fully differentiated 3D NHBE cultures (passage 2, day 92 at the air-liquid interface) (control or pretreated with 1% Zymocarpus extract) and tracking the fluorescently labeled beads using Harmony 4.8 software (PerkinElmer) with Ready Made Solution (RMS) Bead Tracking (modified from RMS Cell Migration). Short videos were recorded.

[0145] result The results are summarized in Tables 2 and 3 below.

[0146] [Table 7]

[0147] [Table 8]

[0148] conclusion The above data demonstrate that apical administration of Zymocarpus extract to epithelial cells increases cilia speed and motility, thereby clearing viruses from mucosa and reducing viral load on mucosa, suggesting that Zymocarpus extract may be particularly useful / important in preventing viral infections via mucosa.

[0149] [Example 2] Transepithelial Electrical Resistance (TEER) Study of transepithelial electrical resistance (TEER) in three-dimensional cultures of NHBE cells exposed to Zymocarpus extract

[0150] Transepithelial / transendothelial electrical resistance (TEER) is a widely accepted quantitative technique to measure the integrity of tight junction dynamics in cell culture models of epithelial and endothelial monolayers. TEER values ​​are a strong indicator of cell barrier integrity prior to evaluating drug or chemical transport. TEER measurements can be performed in real time without damaging cells and are generally based on measuring resistance or impedance over a wide frequency spectrum. TEER measurements for various cell types have been reported using commercially available measurement systems, but we used an EVOM2 Volt-Ohmmeter (World Precision Instruments, WPI). Measurement of transepithelial electrical resistance is a simple and convenient technique that provides information on the uniformity of the Caco-2 cell layer on the filter support and the integrity of the tight junctions formed between polarized cells. Thus, TEER measurements can be used to study epithelial barrier function.

[0151] [Example 2(a)] Effect of different concentrations of Zymocarpus extract on the TEER of uninfected cells (control)

[0152] Testing Procedure: Zymocarpus longan spray (0.1% or 1% w / w in sterile double distilled water, approximately 50 μl equivalent) was applied to the apical or basal side of fully differentiated epithelium (3D NHBE cultures), respectively. Transepithelial electrical resistance (TEER) values ​​were measured in ALI cultures using an EVOM volt-ohmmeter with STX-2 chopstick electrodes (World Precision Instruments, Stevenage, UK). For these measurements, 0.1 ml and 0.7 ml of medium were added to the apical and basolateral sides, respectively. Before measuring the TEER, the cells were allowed to equilibrate. The reported TEER values ​​were corrected for the resistance and surface area of ​​the transwell filters. The results in Table 4 show that the extracts have little effect on the TEER of uninfected NHBE (see also Figure 2(a)).

[0153] [Table 9]

[0154] [Examples 2(b) and (c)] Effect of different concentrations of Zymocarpus extract on the TEER of uninfected cells (day 1 postinfection (b) and day 2 postinfection (c)).

[0155] Testing Procedure: Prior to infection with SARS-CoV-2, the apical side of fully differentiated epithelia (3D NHBE cultures) was sprayed with a single squirt of Zymocarpus spray (0.1%, 1% or 2% w / w in sterile double-distilled water, approximately 50 μl equivalent). The apical application was performed carefully to avoid mechanically disrupting the epithelial surface.

[0156] Transepithelial electrical resistance (TEER) values ​​were measured using an EVOM volt-ohmmeter with STX-2 chopstick electrodes (World Precision Instruments, Stevenage, UK). Measurements of SARS-CoV-2-infected and uninfected ALI cultures were performed immediately before changing the medium. For measurements, 0.1 ml and 0.7 ml of medium were added to the apical and basolateral sides, respectively. Cells were allowed to equilibrate before measuring TEER. Reported TEER values ​​were corrected for the resistance and surface area of ​​the transwell filter. TEER was measured at day 1 post-infection (d1pI) and day 2 post-infection (d2pI).

[0157] In SARS-CoV-2-infected epithelium, significantly lower TEER values ​​were measured in d1pI and d2pI compared to UI and Zymocarpus extract / UI. The results in Tables 5 and 6 below (see also Figures 2(b) and (c)) show that Zymocarpus extract was able to preserve the TEER values ​​of infected cultures at all concentrations tested, at d1pI (see Figure 2b), and also at d2pI when administered as a 0.1% spray (see Figure 2c). However, Zymocarpus extract significantly reduced the TEER values ​​of infected epithelium when administered as a 2% solution, indicating infection and destruction of the epithelium.

[0158] [Table 10]

[0159] [Table 11]

[0160] [Example 3] Production of the cytokine and anaphylotoxin known as complement component C3a

[0161] This analysis allows each biomarker to be identified by laser and the amount in the sample to be quantified. The levels of IL-1a, IL-1ra, IL-6, IL-10, GM-CSF, IP-10, MCP-1, RANTES, TSLP, and TNF-α cytokines were measured using a dual-laser, flow sorting and detection platform, FLEXMAP-3D (Luminex, Austin, TX). Supernatants of HAE cells treated with C5aR and / or SARS-CoV-2 were analyzed using the Magnetic Luminex Multiplex Assay (LXSAHM) from R&D Systems (Minneapolis, MN) according to the manufacturer's instructions. Final data calculations and analysis were performed in Excel. C3a secretion in the HAE tissue model was detected using the BD OptEIA Human C3a ELISA Kit (BD Biosciences) according to the manufacturer's instructions.

[0162] [Example 3a] Decreased expression of complement C3a in natural immune response Study on the effect of Zymocarpus extract on anaphylatoxin production (C3adesArg, C5adesArg) in SARS-CoV-2 infected 3D NHBE cultures

[0163] Early events occurring immediately after SARS-CoV-2 infection of respiratory tissues may affect the outcome in terms of disease severity. In some patients, COVID-19 infection leads to hyperactivation of immune responses in the epithelial / immune barrier, generating a proinflammatory environment. The development of cytokine storm and acute lung injury leading to acute respiratory distress syndrome (ARDS) are possible undesirable outcomes of this disease. ARDS with systemic coagulopathy is an important aspect of morbidity and mortality in COVID-19. Such excessive immune responses triggered by the invading virus can cause widespread tissue destruction in severe cases, resulting in tissue injury and multiple organ failure. Complement deposition and high serum levels of anaphylatoxins have been reported in severe / critically ill patients, suggesting that complement may be one of the factors driving immune hyperactivation.

[0164] Activation of the classical, alternative, and lectin complement pathways results in the production of the C3a anaphylatoxin. C3a has been shown to be a multifunctional inflammatory mediator. As such, C3a has been shown to increase vascular permeability, exhibit spasmodic and chemotactic properties, and induce the release of pharmacologically active mediators from many cell types. C3a production in vivo can also cause, promote, or exacerbate inflammatory responses. Once formed in plasma or serum, the nascent C3a anaphylatoxin is rapidly cleaved to the C3a-desArg form by the endogenous serum carboxypeptidase N enzyme. Thus, quantification of C3a-desArg in plasma or experimental samples should provide a reliable measure of the degree of complement activation that has occurred in the test sample under investigation.

[0165] Testing Procedure: Supernatants from uninfected and SARS-CoV-2-infected samples were collected after apical pretreatment with 0.1%, 1% and 2% Zymocarpus extract, as well as from control cells (uninfected NHBE UI, or OV infected with IBK isolate, NHBE-OV) collected at 2 days post infection (d2pI), detergent treated (2% Ipegal) for virus inactivation and stored at -20°C.

[0166] Therefore, the following samples were analyzed: 1_UI (uninfected) / EtOH, 2_INF (infected with cell culture isolate (isolate OV) dilution of COVID19 positive patient 1 / 1000) / EtOH, 3_UI / 1% (control), 4_INF / 0.1%, 5_INF / 1%, 6_INF / 2%. For Luminex analysis, harvested supernatants were warmed to room temperature and 50 μl of each sample was processed according to the manufacturer's protocol.

[0167] C3a in the samples was quantified using the BD OptEIA Human C3a ELISA Kit (catalog no. 550499), which quantifies human C3a-desArg in human EDTA plasma, serum and other biological samples in vitro, according to the manufacturer's protocol.

[0168] As expected from TEER and image analysis, an inflammatory response was induced in SARS-CoV-2-infected epithelium, which was completely prevented by pre-treating the epithelium with a composition containing 1% Zymocarpus extract prior to infection. C3a levels were also low in epithelia pre-treated with sprays containing 0.1% and 2% Zymocarpus extract (see Figure 3).

[0169] [Example 3b] Decreased expression of inflammatory markers and immune cell chemoattractants Cytokine release (inflammatory response) in primary normal human bronchial epithelial (NHBE) cells

[0170] Testing Procedure: The expression of 10 inflammatory cytokines / biomarkers (MCP-1, IP-10, IL-α, IL-6, TNF-α, RANTES, GM-CSF, IL-1ra, IL-10, TSLP) was monitored using the Human Magnetic Luminex Assay 10-plex human 2STD (R&D Systems). This assay allows each biomarker to be identified by laser and the amount in the sample to be quantified. The levels of all biomarkers in each sample were analyzed using the Luminex FLEXMAP 3D platform (SN-:FM3DD12269001), a dual laser, flow sorting and detection platform.

[0171] Supernatants from uninfected and SARS-CoV-2-infected samples were harvested after apical or basolateral pretreatment with 0.1% Zymocarpus extract, as well as from control cells (uninfected NHBE UI, or OV infected with IBK isolate, NHBE-OV) harvested 2 days post-infection (d2pI), detergent-treated (2% Ipegal) for viral inactivation, and stored at -20°C. For Luminex analysis, harvested supernatants were warmed to room temperature and 50 μl of each sample was processed according to the manufacturer's protocol.

[0172] The results are summarized in Table 7 below, which shows that anti-inflammatory activity was observed. The release of MCP-1, RANTES and IL-6 was reduced in virus-infected tissues treated with Zymocarpus extract compared to virus-infected tissues not treated with Zymocarpus extract.

[0173] [Table 12]

[0174] [Example 4] Visualization of SARS-CoV-2 infection and inhibition of infection by Zymocarpus extract

[0175] To visualize SARS-CoV-2 infection in monolayers and 3D tissue models, cells were infected with clinical specimens of SARS-CoV-2 and analyzed for characteristic markers in either 2 h binding experiments or 3 days post-infection (d3pI) infection experiments. After SARS-CoV-2 exposure, 3D cell cultures were fixed with 4% paraformaldehyde. Intracellular staining was performed using 1× Intracellular Staining Permeabilization Wash Buffer (10×; BioLegend, San Diego, CA, USA). Antibodies were used to stain the cell surface (wheat germ agglutinin [WGA-680]; Thermo Fisher Scientific, Waltham, MA, USA), nuclei (Hoechst 33342; Cell Signaling Technologies, Danvers, MA, USA), actin (phalloidin-Alexa 647; Cell Signaling Technologies, Danvers, MA, USA), and complement C3 (C3-fluorescein isothiocyanate [FITC]; Agilent Technologies, Santa Clara, CA, USA). Intracellular SARS-CoV-2 was detected using Alexa 594-labeled SARS-CoV-2 antibodies against S1 and N (both from Sino Biological, Beijing, China). The Alexa 594 labeling kit was purchased from Abcam, Cambridge, UK. After staining, the 3D cultures were mounted on Mowiol. To study these complex models using primary cells cultured in 3D and generate detailed phenotypic fingerprints at high throughput to gain deeper biological insights, the Operetta CLS system (PerkinElmer, Waltham, MA, USA) was applied. Spot analysis and absolute quantification of SARS-CoV-2-containing cells (Harmony software) was performed on more than 1,200 cells per condition.

[0176] At D2pI, cells were fixed (Cytofix, BD Biosciences, overnight) and permeabilized with Perm / Wash buffer for intracellular staining (BD Biosciences, cat#554723), then stained with nuclear counterstains Hoechst·Hoechst33342 (Molecular Probes, H-3570, 1 / 1000), C3-FITC (Dako / Agilent, cat#F020102-2, 1 / 50), SARS-CoV-2 spike antibodies conjugated to Alexa488 or Alexa594 (Rabbit Mab, Sinobiological cat#40150-R007, 1 / 50), and phalloidin-iFluor Alexa647 (abcam, ab176759, 1 / 1000) for 3 h. After staining, cells were washed with D-PBS and transferred to slides (Mowiol, 4-88, Carlsbad Biosciences, cat#554723). Roth, #0718) and dried overnight at room temperature. Imaging was performed using an Operetta CLS NHS (PerkinElmer) with a 40x or 63x water objective. Images confirmed that SARS-CoV-2 infection had already destroyed the respiratory epithelium at d2pI compared to UI and Zymocarpus / UI, as shown by TEER measurements.

[0177] Imaging demonstrated high infection rates and high innate immune activation (intracellular C3 induction) from SARS-CoV-2-infected respiratory epithelium. Zymocarpus extract was able to preserve epithelial integrity at 1% and also prevented intracellular C3 production (innate immune activation), but when applied to the respiratory epithelium as a 2% solution, it also caused epithelial destruction and C3 induction, exacerbating SARS-CoV-2 infection.

[0178] [Example 4(a)] Study on the effect of different concentrations of Zymocarpus extract on SARS-CoV-2 infection in NHBE primary monolayers

[0179] Primary normal human bronchial epithelial (NHBE) cells (passage 3) p3 (25.000 cells / 100 μl) were seeded in Operetta Cell Carrier Ultra 96-well plates.

[0180] After 3 days, 80% confluent NHBE cells were either infected for 5 days (5dpi) with various cell culture isolates from COVID19 positive patients or left uninfected (control); isolate dilution: 1 / 1000; cells were further treated with Zymocarpus extract (0.5%-0.25%-0.1%) or vehicle.

[0181] Cells were stained with Hoechst (nuclei, blue), C3 (green) to detect intracellular complement formation, SARS-CoV-2-spike 1 (S1) and nucleocapsid (N) (red) to detect productively infected cells, and WGA (recognizes cell surface / intracellular sugars, orange) and bright field (BF) images were also taken.

[0182] Imaging was performed using an Operetta CLS HCS (PerkinElmer) and a 63x water objective, and images were analyzed using Harmony 4.8 software (PerkinElmer).

[0183] SARS-CoV-2 infection was significantly reduced by treatment with 0.5% Zymocarpus extract (see Figure 4).

[0184] [Example 4(b)] Imaging reduced SARS-CoV-2 infection of NHBE cells in fully differentiated human 3D cultures by immunofluorescence

[0185] Infection of Zymocarpus extract-treated NHBE cells

[0186] 3D cultures of NHBE cells were grown and differentiated at the air-liquid interface (ALI) for at least 40 days. The extracts were diluted with DPBS to final concentrations of 0.5%, 0.25%, and 0.1%, respectively. The cells were incubated at 37°C for 30 min under 5% CO2 with the extract diluted as above, except for DPBS, which served as a control. To infect cells, 50 μl of virus dilution was added to the apical side of each transwell. For untreated controls, the same volume of RPMI was added to the cells. Cells were then cultured at 37°C, 5% CO2 for the desired period (overnight / 1–3 days). Infection rates were measured by confocal staining as described in Posch W, et al., J. Allergy Clin Immunol. 2021 Jun;147(6):2083-2097 and Posch W, et al. 2021 Apr 27;12(2): e00904-21 using Operetta CLS (PerkinElmer) and Harmony Software for image analysis (also PerkinElmer).

[0187] SARS-CoV-2 infection was significantly reduced by treatment with 0.5% Zymocarpus extract (see Figure 5).

[0188] [Example 5] Measuring tissue integrity by transepithelial electrical resistance

[0189] As described above in Example 2, transepithelial / transendothelial electrical resistance (TEER) is a widely accepted quantitative method to measure the integrity of tight junction dynamics in cell culture models of endothelial and epithelial monolayers.

[0190] [Example 5(a) and (b)] Effect of 1% Zymocarpus extract on TEER of cells infected with influenza virus A (H3N2); MOI 0.05; 1 day postinfection (a) and 2 days postinfection (b).

[0191] [Example 5(c) and (d)] Effect of 1% Zymocarpus extract on TEER of cells infected with influenza virus type B; MOI 0.05; 1 day postinfection (c) and 2 days postinfection (d).

[0192] Experimental procedure A single puff of Zymocarpus spray (1% diluted in double distilled water) equivalent to approximately 50 μl was applied to the apical side of fully differentiated epithelial cultures (3D NHBE cells, 80 days in ALI culture) before infection with influenza virus type A (H3N2) and influenza virus type B at a multiplicity of infection (MOI) of 0.05, respectively. Apical application was performed carefully to avoid mechanically disrupting the epithelial surface. Cells were cultured for 1 h before infection with influenza viruses (influenza A and influenza B).

[0193] Transepithelial electrical resistance (TEER) values ​​were measured using an EVOM volt-ohmmeter with STX-2 chopstick electrodes (World Precision Instruments, Stevenage, UK). Measurements of influenza virus-infected and uninfected ALI cultures (UI) were performed immediately before changing the medium. For measurements, 0.1 ml and 0.7 ml of medium were added to the apical and basolateral sides, respectively. Cells were allowed to equilibrate before measuring TEER. Reported TEER values ​​were corrected for the resistance and surface area of ​​the transwell filter. TEER was measured at day 1 post-infection (d1pI) and day 2 post-infection (d2pI).

[0194] In epithelia infected with influenza A(H3N2) and influenza B, significantly lower TEER values ​​were measured at d1pI and d2pI compared to Zymocarpus extract / UI. The results shown in Figures 7a)–d) indicated that Zymocarpus extract was able to preserve the TEER values ​​of infected cultures at d1pI (see Figures 7a and c) and d2pI (see Figures 7b and d) at a test concentration of 1%.

[0195] [Example 5(e) and Example 5(f)] Examples 5(e) and 5(f) Effect of 1% Zymocarpus extract on TEER of cells infected with influenza virus type A (H3N2); MOI 0.005; 1 day postinfection (e) and 2 days postinfection (f).

[0196] [Example 5(g) and Example 5(h)] Effect of 1% Zymocarpus extract on TEER of cells infected with influenza virus type B; MOI 0.005; 1 day postinfection (g) and 2 days postinfection (h)

[0197] Experimental procedure A single puff of Zymocarpus spray (1% diluted in double distilled water) equivalent to approximately 50 μl was applied to the apical side of fully differentiated epithelial culture cells (3D NHBE cells, 80 days in ALI culture) before infection with influenza virus type A (H3N2) and influenza virus type B at a multiplicity of infection (MOI) of 0.005, respectively. The apical application was performed carefully to avoid mechanically disrupting the epithelial surface. The cells were incubated for 1 h before infection with influenza viruses (influenza A and influenza B).

[0198] Transepithelial electrical resistance (TEER) values ​​were measured using an EVOM volt-ohmmeter with STX-2 chopstick electrodes (World Precision Instruments, Stevenage, UK). Measurements of influenza virus-infected and uninfected ALI cultures (UI) were performed immediately before changing the medium. For measurements, 0.1 ml and 0.7 ml of medium were added to the apical and basolateral sides, respectively. Cells were allowed to equilibrate before measuring TEER. Reported TEER values ​​were corrected for the resistance and surface area of ​​the transwell filter. TEER was measured at day 1 postinfection (d1pI) and day 2 postinfection (d2pI).

[0199] In epithelia infected with influenza A(H3N2) and influenza B, significantly lower TEER values ​​were measured at d1pI and d2pI compared to Zymocarpus extract / UI. The results shown in Figures 8a)-d) indicated that Zymocarpus extract was able to preserve the TEER values ​​of infected cultures at d1pI (see Figures 8a and c) and d2pI (see Figures 8b and d) at a test concentration of 1%.

[0200] [Example 6] RT-PCR of apically and basolaterally released influenza virus particles to analyze the effect of Zymocarpus extract on viral load in epithelial cells Measurement of the effect of 1% Zymocarpus extract on apically released influenza A (H3N2) particles using RT-PCR; 1 day post-infection (a) and 2 days post-infection (b). Measurement of the effect of 1% Zymocarpus extract on apically released influenza B virus particles using RT-PCR; 1 day postinfection (c) and 2 days postinfection (d).

[0201] Measurement of the effect of 1% Zymocarpus extract on basolaterally released influenza A (H3N2) particles using RT-PCR; day 1 postinfection (e) and day 2 postinfection (f). Measurement of the effect of 1% Zymocarpus extract on basolaterally released influenza B virus particles using RT-PCR; day 1 of infection (g) and day 2 of infection (h).

[0202] Experimental procedure A single puff of Zymocarpus spray (1% diluted in double distilled water) equivalent to approximately 50 μl was applied to the apical side of fully differentiated epithelial cell cultures (3D NHBE, 80 days of ALI culture) before infection with influenza virus type A (H3N2) and influenza virus type B at a multiplicity of infection (MOI) of 0.005, respectively. Apical application was performed carefully to avoid mechanically disrupting the epithelial surface. Cells were cultured for 1 h before infection with influenza viruses (influenza A and influenza B). Viral particle release (apical and basolateral, respectively) on days 1 (d1pI) and 2 (d2pI) postinfection was measured by RT-PCR.

[0203] RNA isolation: For RNA isolation from viral particles, we used the FavorPrep Viral RNA / Viral Nucleic Acid Mini Kit (#FAVNK001-2) (Favorgen Biotech). According to the manufacturer's instructions (user manual), 140 μl of sample was mixed with 560 μl of VNE lysis buffer, and the protocol was carried out according to the manufacturer's instructions. To prepare samples for evaluation of influenza infection (detection and quantification of viral RNA by R-PCR), 140 μl samples were taken from the basolateral medium chamber of the transwell under ALI conditions. Media was collected from TEER measurements to generate apical-derived RT-PCR samples (see Examples 2, 7, and 8).

[0204] RNA detection and quantification by RT-PCR PCR was performed using the LUNA Universal Probe One-Step RT-qPCR Kit E3006G (New England Biolabs) with the following primers and probe (Metabion, Planegg, Germany) according to the manufacturer's instructions:

[0205] H1N1 / H3N2 Metabion ○MP-39-37-F(N2) -F CCM AGG TCG AAA CGT AYG TTC TCT CTA TC ○MP 183 153 R(N2) -R TGA CAG RAT YGG TCT TGT CTT TAG CCA YTC CA Probe -P 6-Fam-ATYTCG GCT TTG AGG GGG CCT BHQ (probe)

[0206] Type B Victoria Metabion Type B-Vic-F F CCT GTT ACA TCT GGG TGC TTT CCT ATA ATG Type B-Vic-R R GTT GAT ARC CTG ATA TGT TCG TAT CCT CKG Type B Vic-P P 6-FAM TTA GAC AGC TGC CTA ACC BHQ 1(Probe) PCR STD copy number 10 8 -10 4

[0207] [Table 13]

[0208] PCR results were analyzed using Bio-Rad CFX Manager or Bio-Rad Maestro Software.

[0209] Significantly lower copy numbers were measured apically in d1pI and d2pI of epithelia infected with influenza A(H3N2) and influenza B in cultures treated with Zymocarpus extract compared with untreated infected cultures.

[0210] The results shown in Figure 9a) to 9d) show that Zymocarpus extract was able to reduce the apical viral load / number of apically excreted viral particles from infected cultures at the tested concentration of 1% in d1pI (see Figures 9a and 9c) and d2pI (see Figures 9b and 9d).

[0211] Significantly lower copy numbers were also measured basolaterally in d1pI and d2pI of influenza A(H3N2)- and influenza B-infected epithelia in Zymocarpus-treated cultures compared with untreated infected cultures.

[0212] The results shown in Figures 10a-10c show that Zymocarpus extract was able to reduce the basolateral viral load / number of virus particles excreted at the basolateral side from infected cultures in d1pI (see Figure 10a) and d2pI (see Figures 10b and 10c) at a test concentration of 1%. The copy number of influenza B virus particles excreted at the basolateral side of d1pP was below the detection limit of 50 copies / μl.

[0213] In conclusion, topical administration of the Zymocarpus extract of the present invention protected tissue integrity against influenza A and B virus infection and prevented the intracellular formation and excretion of new virus particles.

[0214] Thus, the inventors have been able to demonstrate that topical administration of a composition according to the invention comprising a Zymocarpus extract: - Exhibits antiviral activity, reducing infections. - Prevents enveloped viruses such as SARS-CoV-2 from entering the lining of (upper) respiratory epithelial cells by inhibiting binding of enveloped viruses to mucosal epithelial surfaces. - It has the effect of reducing the expression of inflammatory cytokines and regulating the innate immune system, preventing cytokine storms and C3a-induced destruction of lung tissue, and preventing tissue damage after infection. - It has a favorable effect on the transport of (viral) particles by ciliary movement and rhythmic beating, stimulating ciliary movement and promoting MCC. - Has a moisturizing effect.

[0215] Although the invention is described herein with reference to specific embodiments, variations and modifications obvious to those skilled in the art are within the scope of the invention. The contents of all documents cited in this application, both patent and other documents, are hereby incorporated by reference in their entirety.

Claims

1. A composition comprising a Zymocarpus extract or a composition corresponding to said extract for use in the treatment or prevention of a respiratory infection due to an enveloped virus, an enveloped single-stranded virus, an enveloped positive single-stranded RNA virus (+ssRNA) virus, influenza or coronavirus and / or at least one symptom thereof.

2. 2. The composition for use according to claim 1 in the treatment of a respiratory infection caused by an enveloped virus, an enveloped single-stranded virus, an enveloped positive single-stranded RNA virus (+ssRNA) virus, influenza or coronavirus and / or at least one symptom thereof.

3. 2. The composition for use according to claim 1 in the prevention of respiratory infections due to enveloped viruses, enveloped single-stranded viruses, enveloped positive single-stranded RNA viruses (+ssRNA) viruses, influenza or coronavirus and / or at least one symptom thereof.

4. The composition for use according to any one of claims 1 to 3, wherein the respiratory infection is an upper respiratory tract infection and / or a lower respiratory tract infection.

5. The composition for use according to any one of claims 1 to 4, wherein the extract of Dimocarpus is an extract of Dimocarpus longan Lour.

6. The composition for use according to any one of claims 1 to 5, wherein the virus is selected from influenza virus, respiratory syncytial virus (RSV), human parainfluenza virus (HPIV), human metapneumovirus (HPMV), rhinovirus or coronavirus.

7. The composition for use according to any one of claims 1 to 6, wherein the virus is selected from SARS-CoV, MERS-CoV and SARS-CoV-2.

8. The composition for use according to any one of claims 1 to 7, wherein the infectious disease is SARS-CoV-2 infection (COVID-19).

9. The composition for use according to any one of claims 1 to 6, wherein the virus is selected from the influenza virus subtypes A and B.

10. The composition for use according to any one of claims 1 to 6 and 9, wherein the infectious disease is an influenza A (H3N2) or influenza B infection.

11. 11. A composition for use according to any one of claims 1 to 10, wherein the Zymocarpus extract comprises vitamin C; one or more polyphenols selected from corilagin, gallic acid, ellagic acid, ellagic acid conjugates, (-)-epicatechin, quercetin, kaempferol, tannic acid (tannin) and chlorogenic acid; protocatechuic acid; brevifolin; gamma-aminobutyric acid (GABA); carbohydrates and water.

12. The composition for use according to any one of claims 1 to 11, wherein the Zymocarpus extract comprises Vitamin C, Corilagin, Gallic Acid, Ellagic Acid, Ellagic Acid Conjugates Tannic Acid, GABA, Sucrose, Glucose, Fructose, Polysaccharides and Water.

13. A composition for use according to any one of claims 1 to 12, wherein the Zymocarpus extract comprises: 【Table 1】

14. A composition for use according to any one of claims 1 to 13, wherein the Zymocarpus extract comprises: 【Table 2】

15. A composition for use according to any one of claims 1 to 14, wherein the Zymocarpus extract comprises: 【Table 3】

16. A composition for use according to any one of claims 1 to 15, wherein the Zymocarpus extract comprises: 【Table 4】

17. A composition for use according to any one of claims 1 to 16, wherein the extract has a sugar content of 74 to 84°Brix, preferably 76 to 82°Brix, most preferably 78 to 80°Brix.

18. A composition for use according to any one of claims 1 to 17, wherein the extract is prepared from fresh whole fruit.

19. A composition for use according to any one of claims 1 to 18, wherein the Zymocarpus extract is obtainable by a process comprising the following steps: (a) Extraction of zymocarpus juice from fresh whole fruits, followed by (b) Separation of solids from the resulting raw solution (c) concentrating the liquid obtained in step (b) to obtain a sugar concentration of about 74-84, preferably 76-82, most preferably 78-80°Brix; and (d) Aseptic filling.

20. A composition for use according to any one of claims 1 to 17, wherein the Zymocarpus extract is obtainable by a process comprising the following steps: (1) Crush fresh whole Zymocarpus fruits; (2) extracting fresh fruit juice from the crushed fruit obtained in step (1); (3) preparing the fresh fruit juice obtained in step (2) by rapidly heating the fresh fruit juice to about 95-98°C, maintaining the fresh fruit juice at about 95-98°C, and then rapidly cooling the fresh fruit juice to about 5-15°C; (4) separating the supernatant from the product of step (3) by centrifugation and microfiltration; (5) concentrating the supernatant obtained in step (4), preferably by evaporation under reduced pressure, to obtain a sugar concentration of about 74-84°Brix; (6) microfiltration of the concentrate obtained in step (5); and (7) Optionally aseptic filling.

21. The composition for use according to any one of claims 1 to 18, wherein the composition is applied topically, via the ophthalmic, nasal or pharyngeal route.

22. The composition for use according to any one of claims 1 to 19, which is in the form of an eye drop, a mouthwash, a gargle, a nose drop, a nose spray / aerosol, a throat drop, or a throat spray / aerosol.

23. The composition according to any one of claims 1 to 20, which is in the form of a nasal drop, a nasal spray / aerosol, a throat nasal drop, or a throat spray / aerosol.

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