WO2023141673A1 - Immunomodulatory combination comprising plant and animal extracts having antiviral activity - Google Patents

Immunomodulatory combination comprising plant and animal extracts having antiviral activity Download PDF

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Publication number
WO2023141673A1
WO2023141673A1 PCT/AU2023/050043 AU2023050043W WO2023141673A1 WO 2023141673 A1 WO2023141673 A1 WO 2023141673A1 AU 2023050043 W AU2023050043 W AU 2023050043W WO 2023141673 A1 WO2023141673 A1 WO 2023141673A1
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Prior art keywords
extract
immunomodulatory composition
combination
lps
cordifolia
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PCT/AU2023/050043
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French (fr)
Inventor
Helder Marcal
Nico Wanandy
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PT Agrapana Damayanti Indobiotek
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Priority claimed from AU2022900137A external-priority patent/AU2022900137A0/en
Application filed by PT Agrapana Damayanti Indobiotek filed Critical PT Agrapana Damayanti Indobiotek
Publication of WO2023141673A1 publication Critical patent/WO2023141673A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/12—Ketones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/12—Ketones
    • A61K31/122—Ketones having the oxygen directly attached to a ring, e.g. quinones, vitamin K1, anthralin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A61K31/375—Ascorbic acid, i.e. vitamin C; Salts thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/26—Iron; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/30—Zinc; Compounds thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/56—Materials from animals other than mammals
    • A61K35/60—Fish, e.g. seahorses; Fish eggs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/185—Magnoliopsida (dicotyledons)
    • A61K36/59—Menispermaceae (Moonseed family), e.g. hyperbaena or coralbead
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/886—Aloeaceae (Aloe family), e.g. aloe vera
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
    • A61K36/18—Magnoliophyta (angiosperms)
    • A61K36/88—Liliopsida (monocotyledons)
    • A61K36/906—Zingiberaceae (Ginger family)
    • A61K36/9066—Curcuma, e.g. common turmeric, East Indian arrowroot or mango ginger
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/06—Tripeptides
    • A61K38/063—Glutathione
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/38—Albumins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0087—Galenical forms not covered by A61K9/02 - A61K9/7023
    • A61K9/0095—Drinks; Beverages; Syrups; Compositions for reconstitution thereof, e.g. powders or tablets to be dispersed in a glass of water; Veterinary drenches
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2300/00—Mixtures or combinations of active ingredients, wherein at least one active ingredient is fully defined in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/0012—Galenical forms characterised by the site of application
    • A61K9/0053—Mouth and digestive tract, i.e. intraoral and peroral administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/141—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers
    • A61K9/148—Intimate drug-carrier mixtures characterised by the carrier, e.g. ordered mixtures, adsorbates, solid solutions, eutectica, co-dried, co-solubilised, co-kneaded, co-milled, co-ground products, co-precipitates, co-evaporates, co-extrudates, co-melts; Drug nanoparticles with adsorbed surface modifiers with compounds of unknown constitution, e.g. material from plants or animals

Definitions

  • This invention relates generally to compositions comprising a combination of active ingredients and methods of using such compositions. More particularly, the present invention relates to compositions comprising a combination of extracts for use as an immunomodulatory agent.
  • Antiviral agents may be used to treat some viral infections, usually by targetting key, virus-specific processes that selectively inhibit viral entry into cells and/or the viral replication pathway. This may mediate reduction in viral load, or prevention of an increase in viral load, and may assist the host immune response to reduce or eliminate the virus from the body.
  • cytokine storm may result in the overproduction of proinflammatory cytokines such as tumour necrosis factor (TNF), Interleukin (IL)-6, IL- 1(3 and/or IL-8; which may mediate acute lung injury (ALI), or in its more severe form, acute respiratory distress syndrome (ARDS).
  • ALI and/or ARDS may be associated with vascular hyperpermeability, multiple organ failure and may lead to death.
  • ALI and/or ARDS are sometimes observed during infection with influenza virus, Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV or SARS-CoV-1, the causative agent of SARS), or Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2, the causative agent of Coronavirus Infectious Disease (COVID)-19).
  • SARS-CoV or SARS-CoV-1 Severe Acute Respiratory Syndrome Coronavirus
  • SARS-CoV-2 Severe Acute Respiratory Syndrome Coronavirus 2
  • COVID Coronavirus Infectious Disease
  • COVID-19 is associated with significant mortality, particularly in at-risk groups with poor prognostic features at hospital admission.
  • the spectrum of disease is broad; however, pneumonia, sepsis, respiratory failure, and acute respiratory distress syndrome (ARDS) are among symptoms frequently encountered in hospitalised COVID-19 patients, accompanied the overproduction of early response proinflammatory cytokines (eg. tumour necrosis factor (TNF), IL-6, and IL- lg) leading to a cytokine storm.
  • cytokines eg. tumour necrosis factor (TNF), IL-6, and IL- lg
  • Therapeutic strategies targeting the overactive cytokine response with anti-cytokine therapies or immunomodulators may assist, but this must be balanced with maintaining an adequate inflammatory response for pathogen clearance.
  • the present inventors have realized that a combination of naturally derived active agents or extracts may have an antiviral effect, and may alternatively or additionally modulate the immune system to mediate an effective antiviral immune response while minimising or avoiding host-induced pathology.
  • the present disclosure relates to an immunomodulatory composition
  • an immunomodulatory composition comprising a combination of agents consisting of a curcuminoid, an emodin, bioavailable zinc and an at least one active agent obtained from Tinospora cordifolia.
  • the immunomodulatory composition further comprises an agent selected from the list consisting of an albumin, a glutathione, ascorbic acid and bioavailable iron.
  • at least one of the agents are provided as extracts derived from plants and/or animals.
  • the at least one active agent obtained from T. cordifolia is provided by a Tinospora cordifolia extract.
  • the curcuminoid is provided by a Curcuma spp. extract.
  • the emodin is provided by an Aloe spp. extract.
  • the bioavailable zinc is provided by a moringa extract.
  • the albumin is provided by a Channa striata (snakehead fish) extract.
  • the present disclosure relates to an antiviral composition
  • an antiviral composition comprising a combination of agents consisting of a curcuminoid, an emodin, bioavailable zinc and an at least one active agent obtained from Tinospora cordifolia.
  • the antiviral composition further comprises an agent selected from the list consisting of an albumin, a glutathione, ascorbic acid and bioavailable iron.
  • at least one of the agents are provided as extracts derived from plants and/or animals.
  • the at least one active agent obtained from T. cordifolia is provided by a Tinospora cordifolia extract.
  • the curcuminoid is provided by a Curcuma spp. extract.
  • the emodin is provided by an Aloe spp. extract.
  • the bioavailable zinc is provided by a moringa extract.
  • the albumin is provided by a Channa striata (snakehead fish) extract.
  • the present disclosure relates to an immunomodulatory composition
  • an immunomodulatory composition comprising a combination of agents consisting of a Tinospora cordifolia extract, a Curcuma xanthorrhiza extract, an aloe extract and a moringa extract.
  • the immunomodulatory composition of claim 10 further comprising a Channa striata (snakehead fish) extract.
  • the present disclosure relates to an immunomodulatory composition
  • an immunomodulatory composition comprising a combination of agents consisting of a Tinospora cordifolia extract, a Curcuma xanthorrhiza extract, an aloe extract, a moringa extract and a Channa striata extract.
  • the immunomodulatory composition is formulated for oral delivery.
  • the immunomodulatory composition comprises a dried powder.
  • the present disclosure relates to the immunomodulatory composition of the present disclosure when used to treat a viral infection.
  • the viral infection is a coronavirus infection.
  • the viral infection is a SARS-Cov-2 infection.
  • the present disclosure relates to the use of the immunomodulatory composition of the present disclosure to treat a viral infection.
  • the present disclosure relates to the use of the immunomodulatory composition of the present disclosure in the manufacture of a medicament for the treatment of a viral infection.
  • the viral infection is a coronavirus infection.
  • the viral infection is COVID-19.
  • the present disclosure relates to a method of treating a viral infection comprising administering to a to a subject in need thereof a therapeutically effective amount of the immunomodulatory composition of the present disclosure.
  • the viral infection is a coronavirus infection.
  • the viral infection is COVID-19.
  • Figure 1 shows a graphical representation of interferon (IFN)-y levels of culture supernatant from peripheral blood monocytes (PBMC) stimulated with LPS and with various concentrations (1, 5, 25, 50, 100 or 200 pg/mL) of an immunomodulatory composition containing a combination of extracts from Moringa olifera (38.46%), T. cordifolia (3.84%), Aloe vera (38.46%) and Curcuma xanthorrhiza (19.23%); with LPS only stimulation as a negative control and LPS stimulation followed by dexamethasone treatment as a positive anti-inflammatory control.
  • **p ⁇ 0.05 for DMSO+LPS a graphical representation of interferon (IFN)-y levels of culture supernatant from peripheral blood monocytes (PBMC) stimulated with LPS and with various concentrations (1, 5, 25, 50, 100 or 200 pg/mL) of an immunomodulatory composition containing
  • FIG. 2 shows a graphical representation of interleukin (IL)-ip levels of culture supernatant from peripheral blood monocytes (PBMC) stimulated with LPS and with various concentrations (1, 5, 25, 50, 100 or 200 pg/mL) of an immunomodulatory composition containing a combination of extracts from Moringa olifera (38.46%), T. cordifolia (3.84%), Aloe vera (38.46%) and Curcuma xanthorrhiza (19.23%); with LPS only stimulation as a negative control and LPS stimulation followed by dexamethasone treatment as a positive anti-inflammatory control.
  • **p ⁇ 0.05 for DMSO+LPS a graphical representation of interleukin (IL)-ip levels of culture supernatant from peripheral blood monocytes (PBMC) stimulated with LPS and with various concentrations (1, 5, 25, 50, 100 or 200 pg/mL) of an immunomodul
  • FIG. 3 shows a graphical representation of IL-2 levels of culture supernatant from peripheral blood monocytes (PBMC) stimulated with LPS and with various concentrations (1, 5, 25, 50, 100 or 200 pg/mL) of an immunomodulatory composition containing a combination of extracts from Moringa olifera (38.46%), T. cordifolia (3.84%), Aloe vera (38.46%) and Curcuma xanthorrhiza (19.23%); with LPS only stimulation as a negative control and LPS stimulation followed by dexamethasone treatment as a positive anti-inflammatory control.
  • PBMC peripheral blood monocytes
  • Figure 4 shows a graphical representation of IL-4 levels of culture supernatant from peripheral blood monocytes (PBMC) stimulated with LPS and with various concentrations (1, 5, 25, 50, 100 or 200 pg/mL) of an immunomodulatory composition containing a combination of extracts from Moringa olifera (38.46%), T. cordifolia (3.84%), Aloe vera (38.46%) and Curcuma xanthorrhiza (19.23%); with LPS only stimulation as a negative control and LPS stimulation followed by dexamethasone treatment as a positive anti-inflammatory control.
  • PBMC peripheral blood monocytes
  • Figure 5 shows a graphical representation of IL-8 (also known as chemokine (C-X-C motif) ligand (CXCL)-8) levels of culture supernatant from peripheral blood monocytes (PBMC) stimulated with LPS and with various concentrations (1, 5, 25, 50, 100 or 200 pg/mL) of an immunomodulatory composition containing a combination of extracts from Moringa olifera (38.46%), T.
  • PBMC peripheral blood monocytes
  • FIG. 6 shows a graphical representation of monocyte chemoattractant protein (MCP)-l (also known as chemokine (C-C motif) ligand (CCL)-2) levels of culture supernatant from peripheral blood monocytes (PBMC) stimulated with LPS and with various concentrations (1, 5, 25, 50, 100 or 200 pg/mL) of an immunomodulatory composition containing a combination of extracts from Moringa olifera (38.46%), T. cordifolia (3.84%), Aloe vera (38.46%) and Curcuma xanthorrhiza (19.23%); with LPS only stimulation as a negative control and LPS stimulation followed by dexamethasone treatment as a positive antiinflammatory control.
  • MCP monocyte chemoattractant protein
  • PBMC peripheral blood monocytes
  • FIG. 7 shows a graphical representation of macrophage inflammatory protein (MIP)-lo (also known as CCL-3) levels of culture supernatant from peripheral blood monocytes (PBMC) stimulated with LPS and with various concentrations (1, 5, 25, 50, 100 or 200 pg/mL) of an immunomodulatory composition containing a combination of extracts from Moringa olifera (38.46%), T. cordifolia (3.84%), Aloe vera (38.46%) and Curcuma xanthorrhiza (19.23%); with LPS only stimulation as a negative control and LPS stimulation followed by dexamethasone treatment as a positive anti-inflammatory control.
  • **p ⁇ 0.05 for DMSO+LPS **p ⁇ 0.05 for DMSO+LPS.
  • Figure 8 shows a graphical representation of changes in levels of analytes MIP-lo, MCP-1, IL-8, IL-4, IL-2, IL-13 and IFN-y detected in culture supernatant of PBMCs stimulated with LPS and 5 pg/mL of an immunomodulatory composition containing a combination of extracts from Moringa olifera (38.46%), T. cordifolia (3.84%), Aloe vera (38.46%) and Curcuma xanthorrhiza (19.23%) compared to either LPS stimulation alone (negative control; upper bar) or LPS stimulation plus dexamethasone treatment (positive anti-inflammatory control; lower bar).
  • Figure 9 shows a graphical representation of tumor necrosis factor-o (TNF-o) levels of culture supernatant from peripheral blood monocytes (PBMC) stimulated with LPS and with various concentrations (1, 5, 25, 50, 100 or 200 pg/mL) of an immunomodulatory composition containing a combination of extracts from Moringa olifera (38.46%), T. cordifolia (3.84%), Aloe vera (38.46%) and Curcuma xanthorrhiza (19.23%); with LPS only stimulation as a negative control and LPS stimulation followed by dexamethasone treatment as a positive anti-inflammatory control.
  • PBMC peripheral blood monocytes
  • FIG 10 shows a graphical representation of Eotaxin-1 (also known as CCL-11) levels of culture supernatant from peripheral blood monocytes (PBMC) stimulated with LPS and with various concentrations (1, 5, 25, 50, 100 or 200 pg/mL) of an immunomodulatory composition containing a combination of extracts from Moringa olifera (38.46%), T. cordifolia (3.84%), Aloe vera (38.46%) and Curcuma xanthorrhiza (19.23%); with LPS only stimulation as a negative control and LPS stimulation followed by dexamethasone treatment as a positive anti-inflammatory control.
  • PBMC peripheral blood monocytes
  • FIG 11 shows a graphical representation of interferon gammainduced protein 10 (IP-10) (also known as C-X-C motif chemokine ligand 10 (CXCL-10)) levels of culture supernatant from peripheral blood monocytes (PBMC) stimulated with LPS and with various concentrations (1, 5, 25, 50, 100 or 200 pg/mL) of an immunomodulatory composition containing a combination of extracts from Moringa olifera (38.46%), T. cordifolia (3.84%), Aloe vera (38.46%) and Curcuma xanthorrhiza (19.23%); with LPS only stimulation as a negative control and LPS stimulation followed by dexamethasone treatment as a positive antiinflammatory control.
  • IP-10 interferon gammainduced protein 10
  • CXCL-10 CX-C motif chemokine ligand 10
  • FIG 12 shows a graphical representation of macrophage inflammatory protein (MIP)-ip (also known as CCL-3) levels of culture supernatant from peripheral blood monocytes (PBMC) stimulated with LPS and with various concentrations (1, 5, 25, 50, 100 or 200 pg/mL) of an immunomodulatory composition containing a combination of extracts from Moringa olifera (38.46%), T. cordifolia (3.84%), Aloe vera (38.46%) and Curcuma xanthorrhiza (19.23%); with LPS only stimulation as a negative control and LPS stimulation followed by dexamethasone treatment as a positive anti-inflammatory control.
  • MIP macrophage inflammatory protein
  • Figure 13 shows a graphical representation of changes in levels of analytes MIP-ip, MIP-lo, MCP-1, IP-10, IL-8, Eotaxin-1, TNF-o, IL-4, IL-2, IL- ip and IFN-y detected in culture supernatant of PBMCs stimulated with LPS and extracts from Moringa olifera (38.46%), T. cordifolia (3.84%), Aloe vera (38.46%) and Curcuma xanthorrhiza (19.23%) compared to either LPS stimulation alone (negative control; upper bar) or LPS stimulation plus dexamethasone treatment (positive anti-inflammatory control; lower bar).
  • the present disclosure relates to an immunomodulatory composition
  • a combination of agents consists of a combination of natural active agents or extracts that have an antiviral effect.
  • the antiviral combination mediates immunomodulation of the host immune response, for example, by stimulating cytokine expression rhythm that assists with recovery from viral infection without inducing host-derived pathology such as that seen during a cytokine storm.
  • active as used to describe “active agents” or similar herein is referring to chemicals having medicinal benefits.
  • antiviral as used herein is the ability of a drug or substance to suppress or reduce the ability of a virus to enter host cells and/or replicate.
  • bioavailable is ability of a drug or other substance to be absorbed and used by the body. Orally bioavailable means that a drug or other substance that is taken by mouth can be absorbed and used by the body.
  • natural as used in the context of "natural extract” or “natural agent” etc as used herein is referring to an extract or agent that can be derived from a natural source, such as a plant or animal, or alternatively from other natural sources such as yeast, bacteria, etc.
  • the "therapeutically effective amount” will be any suitable amount that will elicit a beneficial or therapeutic effect in the subject.
  • the articles “a” and “an” are used herein to refer to one or to more than one (/.e., to at least one) of the grammatical object of the article.
  • an element means one element or more than one element.
  • Immune responses induced by viral infection include cytotoxic cells that can kill virus infected cells; antibodies that can neutralise the virus by binding and blocking viral surface proteins that mediate binding to host cellular receptors, or antibodies that can agglutinate virions, opsonise virions and/or induce the innate immune system to phagocytose virions; and a variety of interferons and inflammatory cytokines that may intensify the antiviral immune response.
  • Many antiviral drugs target key, virus-specific processes that selectively inhibit viral entry into cells and/or the viral replication pathway. This may mediate reduction in viral load, or prevention of an increase in viral load, and may assist the host immune response to reduce or eliminate the virus from the body.
  • targets include viral proteins involved with fusion of the virus with the host cell (for example, attachment, entry); uncoating of the virus; replication of the viral genome (for example, reverse transcription (reverse transcriptase) or DNA integration (integrase) for viruses that utilise these steps in their replication cycle; or viral polymerase); protein synthesis and assembly of viral components (for example, transcription, translation, protease); or release of new viruses from the host cell (for example, viral budding).
  • cytokine storm a cytokine storm may result in the overproduction of proinflammatory cytokines such as tumour necrosis factor (TNF), Interleukin (IL)-6, IL- ip and/or IL-8; and may be associated with acute lung injury (ALI), or in its more severe form, acute respiratory distress syndrome (ARDS).
  • TNF tumour necrosis factor
  • IL-6 Interleukin-6
  • IL- ip and/or IL-8 an acute respiratory distress syndrome
  • ALI and/or ARDS may be associated with vascular hyperpermeability, multiple organ failure and may lead to death. ALI and/or ARDS are sometimes observed during infection with influenza virus, Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV or SARS-CoV- 1, the causative agent of SARS), or Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2, the causative agent of Coronavirus Infectious Disease (COVID)-19).
  • SARS-CoV or SARS-CoV- 1 the causative agent of SARS
  • SARS-CoV-2 Severe Acute Respiratory Syndrome Coronavirus 2
  • Severe influenza infections caused by may be characterized by overinduction of proinflammatory cytokines, TNF, IL-1 p, IL-8, and IL-6 and monocyte chemotactic protein-1 (MCP-1). This may eventually mediate multiple organ dysfunction and failure and increased vascular hyperpermeability.
  • Immunomodulation of the cytokine response during acute viral respiratory disease may assist the cytokine rhythm to attain in a balanced equilibrium wherein the host produces an effective anti-viral immune response while minimising or avoiding host-induced harm.
  • immunomodulators that assist with treating the symptoms associated with a cytokine storm in response to a particular cause are likely to have broad action against cytokine storm with alternative causes, because it is the host immune response that is modulated rather than the virus (for example) per se.
  • the immunomodulatory compositions of the invention enhance or stimulate an immune response.
  • Coronaviruses are an extensive family of RNA viruses that can cause disease in both animals and humans, and coronavirus strains that infect animals can evolve and become infectious to humans.
  • the current classification of coronaviruses recognizes 39 species in 27 subgenera that belong to the family Coronaviridae. They are divided into alphacoronaviruses and betacoronaviruses which infect mammals; and gammacoronaviruses and deltacoronaviruses, which primarily infect birds. From those, at least 7 coronaviruses are known to cause respiratory infections in humans. Four of these coronaviruses produce symptoms that are generally mild, and can cause common cold-like symptoms:
  • HoV human coronavirus
  • HCoV HKU1 a beta coronavirus identified in 2004.
  • MERS-CoV Middle East Respiratory Syndrome Coronavirus
  • SARS-CoV Severe Acute Respiratory Syndrome Coronavirus
  • SARS-CoV-1 Severe Acute Respiratory Syndrome Coronavirus
  • SARS-CoV-2 Severe Acute Respiratory Syndrome Coronavirus 2
  • beta coronavirus a beta coronavirus identified in 2019.
  • COVID-19 severe acute respiratory syndrome
  • MERS Middle East respiratory syndrome
  • COVID-19 coronavirus disease during 2019
  • Coronaviruses are a group of enveloped positive-sense, single stranded RNA viruses with roughly spherical or moderately pleomorphic virions of approximately 60 to 140 nm in diameter. Coronaviruses have four major structural proteins, namely the Spike (S) protein, envelope (E) protein, membrane (M) protein (a type III transmembrane glycoprotein), and nucleocapsid (N) protein; and a number of non-structural proteins (NSP).
  • S Spike
  • E envelope
  • M membrane protein
  • N nucleocapsid
  • NSP non-structural proteins
  • the viral envelope also known as a membrane
  • the membrane contains the surface (or spike) (S) glycoprotein that forms the peplomers on the virion surface, giving the virus
  • Infection with coronavirus involves the binding of the spike protein of a virion with a complementary host receptor. Entry to a host cell involves a host cell protease cleaving and activates the receptor-attached spike protein.
  • the virus may enter the host cell by endocytosis or direct fusion of the viral envelop with the host membrane. Upon entry into the host cell, the virus particle may be uncoated.
  • the coronavirus RNA genome may be directly translated by the ribosomes of a host cell.
  • the produced viral polypeptides may be cleaved into viral peptides by viral proteases PL pro (NSP3) and main protease (M pro ), also called 3CL pro (NSP5).
  • a number of the nonstructural proteins coalesce to form a multiprotein replicase-transcriptase complex.
  • the main replicase-transcriptase protein is the RNA-dependent RNA polymerase (RdRp).
  • RdRp is directly involved in the replication and transcription of RNA from an RNA strand.
  • Other nonstructural proteins in the complex assist in the replication and transcription process.
  • the exoribonuclease nonstructural protein for example, provides extra fidelity to replication by providing a proofreading function which the RNA-dependent RNA polymerase lacks.
  • S glycoprotein spike glycoprotein
  • M pro pro and RNA-dependent RNA-polymerase (RdRp) being of particular interest.
  • RdRp RNA-dependent RNA-polymerase
  • the S glycoprotein of HCoV-NL63, SARS-CoV, and SARS-CoV-2 viruses binds with human angiotensin-converting enzyme 2 (hACE2), a cellular receptor, which mediates entry of the virus into human cells.
  • hACE2 human angiotensin-converting enzyme 2
  • ACE2 is a membrane protein present in cells of most organs, including lung, heart, kidney and intestines/ ACE2 is expressed by type II alveolar cells within the lung, enterocytes of the small intestine, arterial and venous endothelial cells and arterial smooth muscle cells in most organs. ACE2 mRNA expression has also been found in the cerebral cortex, striatum, hypothalamus, and brainstem.
  • the host type II transmembrane serine protease TMPRSS2 cleaves the ACE-2 receptor and primes (ie activates) the S protein, which in turns facilitates entry of the virus into host cells via endocytosis.
  • the receptor binding domain (RBD) region of the S proteins of SARS-CoV-2 and SARS-CoV are highly homologous, with approximately 73%- 76% sequence similarity (Huang et al., 2020b).
  • RBD receptor binding domain
  • the pathophysiology of SARS-CoV-2-induced ARDS may have similarities to that of severe community-acquired pneumonia caused by other viruses.
  • the overproduction of early response proinflammatory cytokines (tumour necrosis factor (TNF), IL-6, and IL- ip) may mediate a cytokine storm, leading to an increased risk of vascular hyperpermeability, multiorgan failure, and eventually death when the high cytokine concentrations are unabated over time.
  • TNF tumor necrosis factor
  • IL-6 interleukin-6
  • IL- ip early response proinflammatory cytokines
  • COVID-19 may also been linked to cardiovascular complications, with a high inflammatory burden that can induce vascular inflammation, myocarditis, and cardiac arrhythmias (Madjid et al,. 2020).
  • Age, viral load, lung injury score, and blood biochemistry indexes, albumin, CRP, lactate dehydrogenase, lympopenia lymphocyte (%), and neutrophil (%), may be predictors of disease severity.
  • Angiotensin II level in the plasma sample from SARS-CoV-2 infected patients may be markedly elevated and linearly associated to viral load and lung injury (Liu et al., 2020).
  • the immunomodulatory composition comprises an at least one active agent obtained from Tinospora cordifolia. In some embodiments, the immunomodulatory composition comprises a Tinospora cordifolia extract.
  • T. cordifolia also known also as Guduchi, Amritaballi, Brotowali, Chakralakshanika, or Gurcha, is a medicinal plant that is used to treat a broad range of medical ailments. T.
  • cordifolia extract has been used in traditional medicine in response to a range of medical ailments including diabetes, high cholesterol, allergic rhinitis (hay fever), upset stomach, gout, lymphoma and other cancers, rheumatoid arthritis (RA), hepatitis, peptic ulcer disease (PUD), fever, gonorrhea, syphilis, and to boost the immune system. It is believed to possess antioxidant, antiseptic and analgesic and immunomodulatory properties.
  • the Tinospora cordifolia extract is selected from an extract selected from the group consisting of a leaf extract, a stem extract and a root extract.
  • T. cordifolia extract has immunomodulatory effects.
  • T. cordifolia has antiviral effects.
  • oral administration of T. cordifolia stem aqueous extract has been shown to result in significant increase in the IFN-y, IL-2, IL-4, and IL-1 levels in the peripheral blood mononuclear cells (PBMCs) (p ⁇ 0.05) of chickens in the treatment groups following infection with Infectious Bursal Disease (IBD) virus (IBDV), a non-enveloped, double stranded RNA virus (Sachan et al., 2019).
  • IBD Infectious Bursal Disease
  • IBDV Infectious Bursal Disease
  • IBD Infectious Bursal Disease
  • IBDV immunosuppressive disease caused by
  • cordifolia have been reported to possess protease inhibitor activity against HIV, with anti-HIV effects revealed by reduction in eosinophil count, stimulation of B lymphocytes, macrophages and polymorphonuclear leucocytes and hemoglobin percentage indicating a role in management of the disease (Saha and Ghosh, 2012).
  • T. cordifolia An aqueous extract of T. cordifolia has also been shown to activate macrophages as well as mediate an antiviral affect against chicken infectious anemia virus (CIAV) infection in poultry (Latheef et al., 2017). Further, T. cordifolia extract supplementation in chicks may increase the Escherichia coli specific antibody titer and lymphocyte proliferation response in chicks infected with E. coli. Moreover, the roots of T. cordifolia may contain a protease inhibitor that inhibits HIV virus. The anti-HIV activity of T.
  • CMV chicken infectious anemia virus
  • cordifolia extract is reflected in the reduced number of eosinophil, on the other hand, the stimulation of B lymphocytes, macrophages, polymorphonuclear leucocytes and percentages of haemoglobin were observed in the study, therefore T. cordifolia extract for immune system management in the HIV patients shows promising potential (Saha et al., 2012). Phytochemicals from T. cordifolia are also reported be effective inhibitors of Influenza strain H1N1 viral proteins (Saikia et al., 2019).
  • T. cordifolia extract may increase expression of the proinflammatory cytokine IL-1.
  • IL-1 is known to facilitate a branch of immunity by inhibiting an immune-evasive viral replication (Orzalli et al, 2018).
  • Interleukin-ip signaling in dendritic cells has been shown to cause the cells to release antiviral interferon (Aarreberg et al, 2018).
  • T. cordifolia extract may stimulate the body to release cytokine IL-2 and IL-4 (Sachan et al, 2019).
  • IL-2 and IL-4 may stimulate long-term immunity to modulate the immune system to stimulate B cells to mature and release antibody type IgG, and may also enhance the memory B cells and memory T cell response, which may provide long-term immunity against the virus (Rang et al., 2003).
  • IL-4 is an antiinflammatory cytokine, nonetheless IL-4 also the stimulates the priming of B Cells (IgG antibody) (Chatterjee et al, 2014). Accordingly, a T. cordifolia extract may have useful immunomodulatory effects.
  • T. cordifolia extract contains a number of phytochemicals.
  • the at least one active agent obtained from Tinospora cordifolia is selected from the list consisting of terpenoids (eg. tinosporide, furanolactone diterpene, furanolactone clerodane diterpene, furanoid diterpene, tinosporaside, ecdysterone makisterone and several glucosides isolated as polyacetate, phenylpropene disaccharides cordifolioside A, B and C, cordifoliside D and E, tinocordioside, cordioside, palmatosides C and F, sesquiterpene glucoside tinocordifolioside, sesquiterpene tinocordifolin); alkaloids (eg.
  • tinosporine magnoflorine, berberine, choline, jatrorrhizine, 1,2-substituted pyrrolidine, jatrorrhizine, palmatine, beberine, tembeterine
  • lignans eg. 3 (a, 4-dihydroxy- 3-methoxybenzyl)-4-(4- hydroxy-3-methoxybenzyl
  • steroids eg.
  • the at least one active agent obtained from Tinospora cordifolia is selected from the list consisting of tinosporin, tinocordiside, arabinogalactan, isocolumbin and magnoflorine.
  • G1-4A an arabinogalactan polysaccharide isolated from the stem of T. cordifolia may have immunomodulatory potential.
  • Administration of G1-4A in mice may lead to splenomegaly and an increase in the numbers of T cells, B cells and macrophages (Raghu et al., 2009).
  • This increase in spleen cellularity was believed to due to in vivo proliferation of lymphocytes and upregulation of anti- apoptotic genes. It may act by activating the B cells polyclonally, via an increase in CD69 expression in lymphocytes. It may have potential in protecting mice from septic shock by modulating pro-inflammatory cytokines (Raghu etal., 2009).
  • TLR4 on B lymphocytes and macrophages may act as a receptor for G1-4A polysaccharide, activating these immune cells in a TLR4/MyD88 dependent manner (Gupta et al., 2017).
  • G1-4A may lead to enhanced antigen presentation from dendritic cells (DC), indicating it may be useful in maturing dendritic cells, and further activation of cytotoxic T cells (Pandey et al., 2012). It may also be a useful adjuvant in immunotherapy (Pandey et al., 2012).
  • active agents from a T. cordifolia extract have immunomodulatory effects.
  • some of the phytochemicals of T. cordifolia may have good binding energetics with the target proteins in SARS-CoV and/or SARS-CoV-2, as shown in Table 1.
  • the at least one active agent obtained from Tinospora cordifolia is selected from the phytochemicals listed in Table 1.
  • Berberine, isocolumbin, magnoflorine and tinocordiside showed high binding efficacy against four key SARS-CoV-2 targets, that is, S glycoprotein, including the receptor binding domain, RNA dependent RNA polymerase and main protease (Sagar and Kuman, 2020).
  • Tinocordiside and Isocolumbin showed IC50 value of ⁇ 1 pM against both main protease and S glycoprotein (Sagar and Kuman, 2020).
  • a T. cordifolia extract comprises a plurality of phytochemicals that have an antiviral effect.
  • the at least one active agent obtained from Tinospora cordifolia is selected from the list consisting of tinosporin, tinocordiside, arabinogalactan, isocolumbin and magnoflorine.
  • At least one of the active agents provided within T. cordifolia extract may be a useful antiviral agent.
  • an active agent provided within T. cordifolia extract is an antiviral agent for the treatment of coronaviruses, for example, SARS-CoV and/or SARS-CoV-2.
  • a T. cordifolia extract is a useful antiviral agent in the treatment of coronaviruses, for example, SARS-CoV and/or SARS- CoV-2.
  • the at least one active agents obtained from T is obtained from T.
  • cordifolia is selected from the group consisting of corydine, cordioside, cordiofolioside A, tinosporin, berberine, syringin, larch arabinogalactan, Gl-4 arabinogalactan, magnoflorine, tinocordiside, and isocolumbin.
  • the immunomodulatory composition comprises a curcuminoid.
  • a curcuminoid may be a linear diarylheptanoid such as curcumin (diferuloylmethane) or derivatives of curcumin, including bisdemethoxycurcumin, demethoxycurcumin and/or tetrahydrocurcumin.
  • the curcuminoid is selected form the group consisting of curcumin, bisdemethoxycurcumin, demethoxycurcumin and tetrahydrocurcumin. These compounds are natural phenols. Curcumin is a bright yellow chemical that provides turmeric its colour.
  • curcumin may have anti-inflammatory antioxidant, and/or cardiovascular protective properties. Curcumin has been shown to inhibit acute vascular inflammation via the activation of heme oxygenase-1 (HO-1) expression (Xiao et al., 2018). Overexpression of HO-1 may inhibit atherosclerosis, inflammation, and oxidative stress (Xiao et al., 2018). As COVID-19 may be associated with a high inflammatory burden that can induce vascular inflammation, myocarditis, and cardiac arrhythmias, curcumin may mediate the reduction of cardiovascular symptoms of COVID-19 patients (Madjid et al., 2020). Curcumin has been found also as an HDAC inhibitor (Hassan et al.,
  • Curcumin has been reported to have antiviral activity against diverse viruses including dengue virus (serotype 2), herpes simplex virus, HIV, influenza (including an H5N1 strain), human papilloma virus (HPV), Hepatitis C virus (HCV), Zika virus and Chikungunya virus among others (Pandit and Latha,
  • curcumin has been shown to reduce the yield of influenza virus by over 90% in cell culture (Chen et al., 2009). Curcumin may inhibit SARS-CoV replication, possibly by binding to the SARS-CoV 3-chymotrypsin-like protease (3CL protease or 3CL pro ) (Barnard and Kumaki, 2011). The 3CL pro of SARS-CoV-2 (encoded by NSP-5) is an interest as a target for antiviral therapy (Suwannarach et al., 2020). Curcumin has been shown to have significant binding activity with the SARS-Cov-2 spike glycoprotein (Maurya et al., 2020).
  • curcumin may be a useful antiviral agent.
  • curcumin may be a useful antiviral agent in the treatment of coronaviruses, for example, SARS-CoV and/or SARS-CoV-2.
  • curcumin is produced by plants such as Curcuma spp. including Curcuma longa, Curcuma xanthorrhiza, Curcuma domestica Curcuma zedoaria, Curcuma aromatica, Curcuma raktakanta, Curcuma ecalcarata, etc.
  • the curcuminoid is provided by a Curcuma spp. extract.
  • the curcuminoid is provided by a Curcuma xanthorrhiza extract.
  • C. xanthorrhiza is also known as Curcuma zanthorrhiza, temulawak, Java ginger, Javanese ginger, or Javanese turmeric.
  • the curcuminoid is provided by a Curcuma longa extract.
  • the immunomodulatory composition comprises a Curcuma spp. tuber (that is, root) extract.
  • C. xanthorrhiza is believed to possess antidiuretic, antiinflammatory, antioxidant, antihypertensive, antihepatotoxic, antibacterial, and antifungal effects.
  • Phytochemicals found in C. xanthorrhiza include including xanthorrhizol and curcumene (including Ar-Curcumene, 0-Curcumene, y- Curcumene) all of which are bisabolene-type sesquiterpenoids; and a number of curcuminoids selected from the group consisting of curcumin, bisdemethoxycurcumin, demethoxycurcumin and tetra hydrocurcumin. The presence of xanthorrhizol in C.
  • xanthorrhiza differentiates this plant from other Curcuma species.
  • Xanthorrhizol has been reported to encompass a wide range of biological activities such as antibacterial, antiseptic, and antibiotic, and is a strong antioxidant (Jantan et al., 2012).
  • a C. xanthorrhiza extract comprises a plurality of phytochemicals that have an antiviral effect.
  • some of the phytochemicals found in C. xanthorrhiza extract have good binding energetics with the target proteins in SARS-CoV and/or SARS-CoV-2, as shown in Table 2.
  • the at least one active agent obtained from the C. xanthorrhiza extract is selected from the phytochemicals listed in Table 2.
  • At least one of the active agents provided within Curcuma spp. extract may be a useful antiviral agent.
  • at least one active agent provided within Curcuma spp. extract is an antiviral agent for the treatment of coronaviruses, for example, SARS-CoV and/or SARS-CoV-2.
  • a Curcuma spp. extract is an antiviral agent for the treatment of coronaviruses, for example, SARS-CoV and/or SARS-CoV-2.
  • the Curcuma spp. is Curcuma xanthorrhiza.
  • the immunomodulatory composition comprises a Moringa spp. extract.
  • the moringa extract is a Moringa oleifera extract. Moringa oleifera is also known as kelor, horseradish tree, Mulangay, Mlonge, Benzolive, Drumstick tree, Sajna, Kelor, Saijihan and Marango.
  • a Moringa extract may be obtained from the leaves, stems, seeds, root bark, flower and/or pods of moringa. Accordingly, in some embodiments, the Moringa extract is selected from the group consisting of a leaf extract, a seed extract, a root bark extract, a flower extract and a pod extract. In some embodiments, the immunomodulatory composition comprises a Moringa spp. leaf extract.
  • Moringa is high in essential amino acids, iron, zinc, Vitamin C (ascorbic acid), carotenoids (including p-carotene and vitamin A), calcium, potassium and protein (Gopalakrishnan et al., 2016; Shija et al., 2019; Sena et al., 1998). Moringa also contains vitamin Bl, Vitamin B2, Vitamin B3, folic acid, pyridoxine and nicotinic acid, vitamin D and/or vitamin E, phosphorous, copper, magnesium and sulphur (Gopalakrishnan et al., 2016). For example, a Moringa oleifera leaf extract was found to contain 25.5 ⁇ 2.6 mg zinc and 28.7 ⁇ 2.8 mg iron per 100 gram dried moringa leaf (Barminas et al., 1998).
  • Moringa oleifera has been shown to have an antiviral effect against Hepatitis B virus (HBV) in Huh7 cells in vitro, with reduced HBV antigen secreted into the supernatant of Huh7 following treatment (Feustel et al., 2017). It has been reported that Moringa oleifera extract has an antiviral effect on Foot and Mouth Disease virus (FMDV), a picornavirus (Younus et al., 2015) and influenza virus (Ashraf et al., 2017).
  • the beneficial effects include an antioxidant effect, an antiviral effect and/or an antiinflammatory effect.
  • At least one of the phytochemicals found in Moringa extract may have good binding energetics with the target proteins in SARS-CoV-2, as shown in Table 3.
  • the at least one active agent obtained from the Moringa extract is selected from the phytochemicals listed in Table 3
  • At least one active agents provided within a Moringa extract may be a useful antiviral agent.
  • an active agent provided within a Moringa extract is an antiviral agent for the treatment of coronaviruses, for example, SARS-CoV and/or SARS- CoV-2.
  • a moringa extract has an antiviral effect.
  • the at least one active agents obtained from moringa extract is selected from the group consisting of bioactive zinc, bioactive iron, ascorbic acid, Gallic acid, Catechin, Chlorogenic acid, Ellagic acid, Epicatechin, Rutin, Isoquercitrin, Quercetin, Kaempferol, Astragalin, and isothiocyanate.
  • a Moringa extract comprises a plurality of phytochemicals that have an antiviral effect.
  • the immunomodulatory composition comprises an emodin.
  • Emodin may be isolated from a number of plants including rhubarb, buckthorn, Japanese knotwood (Reynoutria japonica synonym Polygonum cuspidatum ⁇ , many species of fungi, including members of the genera Aspergillus, Pyrenochaeta, and Pestalotiopsis, Acalypha australis, Cassia occidentalis, Cassia siamea, Frangula alnus, Glossostemon bruguieri, Kalimeris indica, Polygonum hypoleucum, Reynoutria japonica (synonym Fallopia japonica, or Polygonum cuspidatum), Rhamnus alnifolia, the alderleaf buckthorn, Rhamnus cathartica, the common buckthorn, Rheum palmatum, Rumex nepalensis, Senna obtusifolia (synonym Cassia
  • the emodin is an aloe-emodin.
  • Aloe emodin is an anthraquinone compound found in Aloe vera and other species of the Asphodelaceae and the Polygonaceae families. Aloe-emodin is found in the gel, sap or leaves of aloe vera, the socotrine aloe, Barbados aloe, and Zanzibar aloes, the bark of Frangula Rhamnus frangula and cascara sagrada Rhamnus purshiana ⁇ , the leaves of Senna (Cassia angusti folia ⁇ , and the rhizome of rhubarb (Rheum rhaponticum ⁇ .
  • An emodin is a phytochemical that is believed to have antiviral, antibacterial and/or anti-inflammatory effects.
  • An emodin, an anthraquinone has also been implicated in cardiac protection.
  • an emodin is an antiviral agent.
  • emodin obtained from a plant extract has been shown to inactivate enveloped virus (Sydiskis et al., 1991).
  • Emodin isolated from Rheum palmatum has been shown to have potent inhibitory effects against Coxsackie virus (CV) and respiratory syncytial virus (RSV) in cell culture (Liu et al., 2015).
  • CV Coxsackie virus
  • RSV respiratory syncytial virus
  • emodin is an immunomodulatory agent.
  • Anthraquinone derivatives aloe-emodin, emodin and chrysophanol have been shown to exhibit antiviral activity against highly pathogenic influenza A viruses H7N9 and H5N1 (Li et al., 2014).
  • the antiviral activity of aloe-emodin against influenza A virus is via the up-regulation of galectin-3 (Li et al, 2014).
  • an emodin derived from genus Rheum and Polygonum has been shown to blocks the interaction between SARS coronavirus spike protein of SARS-Cov and angiotensin-converting enzyme in a dose-dependent manner (Ho et al. 2007). Emodin may inhibit the activity of histone deacetylase (HDAC); the inhibition of histone deacetylase (HDAC) enzymes may attenuate pathological cardiac hypertrophy in vitro and in vivo. (Evans et al., 2020), in a similar manner to curcumin. Accordingly, in some embodiments, an emodin is an antiviral agent. In some embodiments, an emodin is an antiviral agent for the treatment of coronaviruses, for example, SARS-CoV and/or SARS-CoV-2.
  • the emodin is provided by an aloe extract.
  • the aloe extract is a stem extract.
  • the aloe extract is an Aloe barbadensis extract.
  • A. barbadensis is also known as Aloe vera.
  • A. barbadensis has been used medicinally for healing and therapeutic purposes.
  • the biologically active components of an aloe extract include anthraquinones, anthrones, chromanes, alkaloids, flavonoids, terpenes, minerals, carbohydrates and pyrans.
  • Aloe-emodin levels in plant extracts and commercial formulations can be determined by HPLC with tandem UV absorption and fluorescence detection, as described in Mandrioli et al., 2011.
  • An Aloe vera extract has been found to be have an antiviral effect against a broad range of viruses, especially causing the infections of the upper respiratory tract (Pandit et al., 2020).
  • Anthraquinones isolated from Aloe vera has shown to inhibit herpes simplex virus-2, hepatitis virus, Influenza virus (including Influenza A) and HIV (Pandit et al., 2020).
  • an aloe extract comprises a plurality of phytochemicals that may have an antiviral effect.
  • at least one of the phytochemicals found in Aloe barbadensis extract may have good binding energetics with the target proteins in SARS-CoV-2, as shown in Table 4.
  • the at least one active agent obtained from the Aloe extract is selected from the phytochemicals listed in Table 4.
  • At least one active agents provided within A. barbadensis extract may be a useful antiviral agent.
  • an active agent provided within A. barbadensis extract is an antiviral agent for the treatment of coronaviruses, for example, SARS-CoV and/or SARS-CoV-2.
  • an A. barbadensis extract has an antiviral effect.
  • a A. barbadensis extract comprises a plurality of phytochemicals that have an antiviral effect.
  • a significant reduction of serum albumin levels and an increase in C-reactive protein may be associated with inflammation.
  • albumin is the most abundant plasma protein with a concentration ranging from 35 to 50 g/L.
  • Albumin production may be inhibited by proinflammatory mediators such as IL-1, IL-6 and TNF.
  • hypoalbuminemia is defined as a serum albumin concentration ⁇ 35 g/l.
  • hypoalbuminemia is defined as a serum albumin concentration ⁇ 30 g/l.
  • hypoalbuminemia is defined as a serum albumin concentration ⁇ 25 g/l.
  • Critical illness and/or mortality may be associated with hypoalbuminemia in a wide range of diseases.
  • a decrease in the serum albumin concentration is found in sepsis and septic shock (Gounden et al., 2020), which may be mediated by reduced synthesis/production of serum albumin, an increase in utilisation of albumin within the patient, and/or an increase in transcapillary leak from blood vessels associated with an increase of vascular permeability.
  • Albumin is a carrier molecule within blood that may bind and transport iron and copper ions, and/or other endogenous and exogenous substances, such as hormones, vitamins (e.g., Vitamin D), folate, fatty acids, arachidonic acid, glucose, nitric oxide, electrolytes (e.g., Calcium, Magnesium), cortisol, thyroxine, amino acids, enzymes, bilirubin and/or various drugs (NSAIDs, sedatives, anti-epileptic, digoxin, anticoagulants, and/or antibiotics) (Vincent et al., 2014).
  • vitamins e.g., Vitamin D
  • folate fatty acids
  • arachidonic acid e.g., arachidonic acid
  • glucose e.g., nitric oxide
  • electrolytes e.g., Calcium, Magnesium
  • cortisol e.g., calcium, Magnesium
  • cortisol e.g.,
  • Serum albumin is also a zinc carrier, with most blood plasma zinc bound with albumin (Mustafa et al., 2012), Lu et al., 2008), Handing et al., 2016; Blaundauer et al., 2009). Serum albumin may also be involved in maintaining intravascular volume. For example, albumin may be involved in the endothelial surface layer and associated with maintaining vascular barrier competence. Albumin may act as an antioxidant, free radical scavenger, able to interact with or trap reactive oxygen or nitrogen species, including nitric oxide, a key mediator in many conditions including sepsis. Albumin may also have anticoagulant effects.
  • Changes in albumin concentrations and structure during critical illness can therefore potentially have marked effects on normal homeostasis and metabolism and on drug delivery and efficacy.
  • Maintaining serum albumin levels in the blood during inflammation may be beneficial in that it may facilitate effective transport of substances to cells, and/or to assist with the effects of inflammation.
  • Albumin therapy may be useful in the treatment of a number of serious conditions. Studies indicate that orally delivered albumin increases serum albumin levels (Rosyidi et al., 2019; Mustafa et al., 2012). In some embodiments, albumin therapy may be useful to treat viral infection including COVID-19 (Huang et al., 2020; Liu et al., 2020). Albumin downregulates the expression of the ACE2 receptors (Liu et al., 2009) and has been shown to improve the ratio of arterial partial pressure of oxygen/fraction of inspired oxygen in patients with acute respiratory distress syndrome as soon as 24 hours after treatment and with an effect that persisted for at least seven days (Uhlig et al., 2014).
  • the immunomodulatory composition comprises an albumin.
  • the albumin is provided by a plant and/or animal extract. More than 60% of the protein fraction of the Channa striata (snakehead fish) extract comprises albumin (Rosyidi et al., 2019), accordingly snakehead fish extract is an excellent source of albumin. Rosyidi et al. (2019) found that in neurosurgery patients treated with two capsules of snakehead extract three times per day for up to three weeks post-surgery, serum albumin levels increased significantly.
  • albumin is provided by egg whites.
  • the albumin is provided by a snakehead fish (e.g., Channa striata) extract.
  • the snakehead fish extract is a flesh extract.
  • the snakehead fish is also known as "ikan gabus".
  • Snakehead fish extract is accordingly rich in zinc, copper and iron, and is also an excellent source of protein and amino acids including essential amino acids (Rosmawati et al., 2018; Gam et al., 2006).
  • Albumin may be provided by any species of the Channa genus. Suitable species include C. micropeltes, C. lucius, C. gachua, C. bankanensis, C. cyanospilos, C. marulioides, C. melanoptera, C. melasoma, C. pleurophthalma, and C. argus.
  • the albumin is provided by a Channa striata extract.
  • Zinc is an essential trace element that assists effective immune function. Zinc has direct antiviral activities against a variety of viruses and enhances antiviral immunity (Read et al., 2019). For example, zinc has been shown to mediate direct antiviral effects against influenza and assists in generating both innate and acquired (humoral) antiviral responses. Studies indicate that zinc plays an inhibitory role on almost every aspect of herpes simplex virus (HSV)-l and HSV-2 viral life cycle including viral polymerase function, protein production and processing, and free virus inactivation, corresponding to a significant reduction of HSV infection and disease burden (Read et al., 2019). Zinc is known to mediate antiviral activity against rhinovirus and shorten the duration rhinovirus infection.
  • HSV herpes simplex virus
  • the immunomodulatory composition comprises bioavailable zinc.
  • Bioavailable zinc may be provided by any suitable source.
  • the bioavailable zinc is provided by a plant or animal extract.
  • the bioavailable zinc is provided by a moringa extract.
  • the bioavailable zinc is provided by a Channa striata (snakehead fish) extract.
  • the bioavailable zinc is provided by a moringa extract and a Channa striata (snakehead fish) extract.
  • curcumin may chelate iron.
  • the immunomodulatory composition may further comprise an agent comprising bioavailable iron.
  • Bioavailable iron may be provided by any suitable source. A morninga extract is high in iron. Accordingly, in some embodiments, inclusion of a moringa extract in the immunomodulatory composition may also counteract at least some of the iron chelating activity of curcumin.
  • the bioavailable iron is provided by a moringa extract. In some embodiments, the bioavailable iron is provided by a snakehead fish extract. In some embodiments, the bioavailable iron is provided by a moringa extract and a snakehead fish extract.
  • bioavailable iron could be provided by other known commercial sources and/or alternative natural extracts known to have high levels of bioavailable iron such as Fenugreek (Trigonella foenum-graecum), Jute (Corchorus olitorius), Common Guava (Psidium guajava'), Spinach Spinacia oleracea), leaf of winged bean (Psophocarpus tetragonolobus), morel mushroom / true Mmrels (Morchella), Amaranthus spinosus, Cajanus cajan, Hoslundia opposite, Imperata cylindrical, Justicia secunda, Khaya senegalensis, Milicia excels, Ricinus communis, Stylosanthes erecta, Tectona grandis, Thalia geniculate, animal livers, sardines, kangaroo, and/or beef etc.
  • Fenugreek Trigonella foenum-graecum
  • Jute Chorus olitori
  • Glutathione is an antioxidant found in plants, animals, fungi, and some bacteria and archaea. GSH, including L-Glutathione, can also be ontained from commercial sources (e.g., LUCKERKONG Biotech Co., Ltd; Guangdong, China). Glutathione defends against oxidative damage of cells from reactive oxygen species (ROS) and is also involved in the regulation of various metabolic pathways essential for whole body homeostasis. Several studies indicate that higher levels of glutathione may improve an individual's responsiveness to viral infections. Glutathione may protect host immune cells through its antioxidant mechanism and mediate optimal functioning of a variety of cells that are part of the immune system. Evidence indicates that glutathione inhibits replication of various viruses at different stages of the viral life cycle, and this antiviral property of GSH seems to prevent increased viral loads and the subsequent massive release of inflammatory cells into the lung mediated by a "cytokine storm".
  • ROS reactive oxygen species
  • NAC N-acetylcysteine
  • glutathione is an antiviral agent.
  • pathophysiological conditions such as lung cell injury and inflammation in patients with severe ARDS were identified as the targets of NAC treatment.
  • glutathione deficiency is the postulated to mediate serious manifestation and death in COVID-19 patients (Polonikov, 2020).
  • the immunomodulatory composition comprises glutathione.
  • the immunomodulatory agent is an antiviral agent.
  • an immunomodulatory composition comprising a combination of natural agents may offer therapeutic antiviral effects during a viral infection.
  • some active agents within natural extracts mediate an enhanced antiviral response within a subject suffering from a viral infection, for example, by mediating immunomodulation of the host immune response.
  • Some active agents stimulate a cytokine rhythm that assists with recovery from viral infection.
  • the antiviral combination comprises at least one active agent that that down-regulates viral replication.
  • the antiviral combination comprises at least one active agent that enhances the antiviral immune response of a subject suffering from a viral infection.
  • the antiviral combination comprises at least one active agent that binds with viral components and down- regulates viral replication. In some embodiments, the antiviral combination enhances the antiviral immune response of a subject suffering from a viral infection and comprises at least one active agent that binds with viral components that inhibits viral replication.
  • SARS-CoV and SARS-Cov-2 viruses have a high degree of similarity, and both bind to the same host receptor, ACE-2, to gain entry into the subject's cells. Accordingly, an agent that is active against SARS-CoV may also be active against SARS-CoV-2.
  • the immunomodulatory composition of the present disclosure may have at least one of the following benefits: Increasing serum albumin by oral consumption of an albumin containing extract (e.g., from snakehead fish); modulating cytokine levels and balance; increasing the distribution capability of the body to deliver nutrition mediated by active agents of plant extracts (e.g., C. xanthorrhiza, T. cordifolia and aloe-emodin); and increasing capacity of mineral distribution in the body, particularly zinc.
  • cytokine balance is enhanced, and the inflammation becomes a moderate, appropriate antiviral inflammation.
  • access of SARS-CoV-2 virus into the cells, particularly the lung cells is reduced by the use of ACE-2 blockers (e.g., aloe vera extract / Emodin), TMPRSS2 down regulator (C. xanthorrhiza extract / Curcumin).
  • ACE-2 blockers e.g., aloe vera extract / Emodin
  • TMPRSS2 down regulator C. xanthorrhiza extract / Curcumin
  • the immunomodulatory composition slows down the replication of SARS-CoV-2 virus in the human body via the interruption of 3CL Protease (encoded by NSP-5) by C. xanthorrhiza extract/Curcumin, and/or interruption of RdRP (encoded by NSP-12) by zinc from snakehead and/or dried moringa leaf extracts.
  • a T. cordifolia extract within the immunomodulatory composition modulates the immune system toward the adaptive immune response route. This may enhance antibody production, and/or stimulate the maturity of memory T and B cells. In turn, this may mediate longterm immunity to the viral infection.
  • the immunomodulatory composition of the present disclosure comprises a combination of agents consisting of a curcuminoid, an emodin, and bioavailable zinc.
  • the immunomodulatory composition of the present disclosure comprising a combination of agents consisting of a curcuminoid, an emodin, bioavailable zinc and an at least one active agent obtained from Tinospora cordifolia.
  • the at least one active agent obtained from Tinospora cordifolia is selected from the list consisting of Corydine, Cordioside, Cordiofolioside A, Tinosporin, Berberine, Syringin, Larch arabinogalactan, Gl-4 arabinogalactan, Magnoflorine, Tinocordiside, and Isocolumbin, any of which can be extracted from Tionospora cordifolia using standard techniques known to those skilled in the art.
  • the immunomodulatory composition further comprises an agent selected from the list consisting of an albumin, a glutathione, ascorbic acid and bioavailable iron.
  • At least one of the agents are provided as extracts derived from plants and/or animals.
  • the at least one active agent obtained from T. cordifolia is provided by a Tinospora cordifolia extract.
  • the curcuminoid is provided by a Curcuma spp. extract
  • the curcuminoid is provided by a Curcuma xanthorrhiza extract.
  • the emodin is provided by an aloe extract.
  • the emodin is provided by an Aloe barbadensis extract.
  • the bioavailable zinc is provided by a moringa extract.
  • the bioavailable zinc is provided by a Channa striata (snakehead fish) extract. In some embodiments, the bioavailable zinc is provided by a moringa oleifera extract and a Channa striata (snakehead fish) extract. In some embodiments, the albumin is provided by a Channa striata (snakehead fish) extract. In some embodiments, glutathione is provided as L- glutathione by a commercial source, e.g., LUCKERKONG Biotech Co., Ltd, China. In some embodiments, the ascorbic acid is provided by a moringa oleifera extract. In some embodiments, the bioavailable iron is provided by a moringa extract.
  • the bioavailable iron is provided by a Channa striata (snakehead fish) extract. In some embodiments, the bioavailable iron is provided by a moringa oleifera extract and a Channa striata (snakehead fish) extract.
  • the immunomodulatory composition of the present disclosure comprises a combination of agents consisting of a Tinospora cordifolia extract, a Curcuma xanthorrhiza extract, an aloe extract and a moringa extract.
  • the percentage of each of the extracts within the immunomodulatory composition may vary.
  • the Tinospora cordifolia extract may comprise between 0.1% and 15% of the immunomodulatory composition, or between 1% and 12%, or between 2% and 12%, or between 3% and 5% of the antiviral combination, etc.
  • the Curcuma xanthorrhiza extract may comprise between 5% and 40% of the immunomodulatory composition, or between 10% and 30%, or between 12% and 25%, or between 15% and 22% of the antiviral combination, etc.
  • the aloe extract may comprise between 10% and 60% of the immunomodulatory composition, or between 20% and 50%, or between 25% and 45%, or between 30% and 40% of the antiviral combination, etc.
  • the Moringa extract may comprise between 10% and 60% of the immunomodulatory composition, or between 20% and 50%, or between 25% and 45%, or between 30% and 40% of the antiviral combination, etc.
  • the antiviral combination comprises 38.46% Moringa extract, 3.84% T.
  • Cordifolia extract 38.46% Aloe vera extract, and 19.23% Curcuma xanthorrhiza extract.
  • the combined dried extracts are packed into sachets containing 1000 mg Moringa extract, 100 mg T. Cordifolia extract, 1000 mg Aloe vera extract, 500 mg Curcuma xanthorrhiza extract.
  • sachets containing 1000 mg Moringa extract, 100 mg T. Cordifolia extract, 1000 mg Aloe vera extract, 500 mg Curcuma xanthorrhiza extract.
  • ratios of the extracts may fall within the scope of the invention.
  • the plant and/or animal extracts may be extracted from raw material using any suitable extraction method providing the resulting extract is suitable for oral delivery.
  • the immunomodulatory composition of the present disclosure further comprises a Channa striata (snakehead fish) extract.
  • the immunomodulatory composition of the present disclosure comprises a combination of agents consisting of a Tinospora cordifolia extract, a Curcuma xanthorrhiza extract, an aloe extract, a moringa extract and a Channa striata (snakehead fish) extract.
  • the antiviral combination comprises 32.5% Moringa extract, 3.22% T. Cordifolia extract, 32.25% Aloe vera extract, 16.13% Curcuma xanthorrhiza extract, and 16.13% Channa striata extract.
  • the Channa striata extract may comprise between 5% and 40% of the immunomodulatory composition, or between 10% and 30%, or between 12% and 25%, or between 15% and 20% of the antiviral combination, etc.
  • the combined dried extracts are packed into sachets containing 1000 mg Moringa extract, 100 mg T. Cordifolia extract, 1000 mg Aloe vera extract, 500 mg Curcuma xanthorrhiza extract, and 500 mg Channa striata extract.
  • sachets containing 1000 mg Moringa extract, 100 mg T. Cordifolia extract, 1000 mg Aloe vera extract, 500 mg Curcuma xanthorrhiza extract, and 500 mg Channa striata extract.
  • persons skilled in the art will appreciate that other ratios of the extracts may fall within the scope of the invention.
  • the extracts of the present disclosure can be extracted from the raw materials using any suitable extraction technique known to persons skilled in the art, providing the resulting extract is suitable for oral delivery.
  • Such extraction methods may be selected from supercritical carbon dioxide extraction, aqueous extraction, alcohol extraction, solvent extraction, distillation method, pressing and sublimation according to the extraction principle etc, using methods known to those skilled in the art.
  • the extraction method is supercritical carbon dioxide extraction.
  • the immunomodulatory composition can be formulated to be in a liquid form, for example, a ready to drink product; or a powdered form, which can be formulated as a sachet, tablet, pill or capsule. Solid extract preparation before mixing will involve hammer mill grinder and sifter to obtain certain particle size. For tablet, pills, and capsules form may utilise a granulator to support the production.
  • the immunomodulatory composition is formulated for oral delivery
  • the extracts may be mixed using standard tank with agitator according to the formulation.
  • the liquid mixture may then be UHT system sterilized before bottling to accommodate long shelf life product.
  • single dosages can be provided in sachets such that the physician (or subject suffering from a viral infection) can readily administer a dose.
  • One sachet can be dissolved in water, for example 150 mL of cold water.
  • Associated with such dosages(s) can be various written materials such as instructions for use, or a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
  • the immunomodulatory composition of the present disclosure can be used in combination with other drug(s) or antiviral treatment(s).
  • the components can be administered in the same formulation or in separate formulations. If administered in separate formulations, the immunomodulatory composition of the present disclosure may be administered sequentially or simultaneously with the other drug(s) or antiviral treatment(s).
  • the immunomodulatory composition may be combined with a filler, carrier, diluent and/or excipient that is suitable for oral administration.
  • the immunostimulatory composition of the present disclosure can be formulated with a number of pharmaceutically acceptable carriers, excipients and diluents, as well known in the art.
  • the composition may be formulated one or more of an anti-caking agent/stabilising agent, sweeter, polysaccharide, fibre, anti-oxidant, and/or flavouring agent.
  • Anti-caking agents/stabilisers are typically present in the formulation at a concentration of between about 0.1 to about 3.0% (w/w). Suitable anti-caking agents/stabilisers may be selected from the the group comprising silicon dioxide, dextrin, tricalcium phosphate, calcium phosphate, sodium bicarbonate, magnesium trisilicate, diatomaceous earth, lecithin, or a combination thereof.
  • Sweeteners are typically present in the formulation at a concentreation of between about 0.1% and 3.0% (w/w).
  • Suitable anti-caking agents/stabilisers may be selected from the the group comprising dextrose monohydrate, saccharine-Na, polyols (e.g., sucralose, sorbitol, maltitol, xylitol, erythritol), stevioside, inulin, monk fruit sweetener, or a combination thereof.
  • polyols e.g., sucralose, sorbitol, maltitol, xylitol, erythritol
  • stevioside e.g., sucralose, sorbitol, maltitol, xylitol, erythritol
  • inulin monk fruit sweetener, or a combination thereof.
  • Polysaccharides and fibre are typically present in the fomaultion at a combined concentration of between about 0.1% and 35% (w/w).
  • Suitable polysaccharides and fibre composnentes may be selected from the group comprising maltodextrin, resistant dextrin (e.g., wheat dextrin), collagen, polydextrose, gums, polyols, oligosaccharides and starches (e.g., tapioca starch, maize starch, potato starch, glucomannan), or a combination thereof.
  • Anti-oxidants are typically present in the formulation at a concentration of between about 0.1% and 6.0% (w/w). Suitable anti-oxidants may be selected from the group comprising glutathione, N -acetylcysteine, ascorbic acid, citric acid, lactic acid, malic acid, or a combination thereof.
  • Flavouring agents are typically present in the formulation at a concentration of between about 0.1% to 0.8% (or quantum suffic/at).
  • Suitable flavours envisaged for use with the composition include apple, strawberry, raspberry, green apple, passionfruit, mangosteen, pomegranate, blueberry, orange, mandarin, peach, nectarine, plum, lemon, lemongrass, or a combination thereof.
  • additiona additives may be added, including glutathione (at around 0.02 g per 10 g dose), polydextrose (at around 1.93 g per 10 g dose), dextrose monohydrate (at around 3.15 g per 10 g dose), silicon dioxide/silicium dioxide (at around 0.23 g per 10 g dose), ascorbic acid (at around 0.48 g per 10 g dose), and fish collagen (at around 0.97 g per 10 g dose).
  • the moringa extract may be administered to subjects at a dose between about 0.2 g and 4 g per day, or between about 1 and 3 g per day, or between about 1.5 and 2.5 g per day.
  • the T. cordifolia extract may be administered to subjects at a dose between about 0.01 mg and 4 g per day, or between about 0.1 and 1 g per day, or between about 0.3 and 0.9 g per day, or between about 0.1 and 0.4 g per day, or between about 0.4 and 0.75 g per day.
  • T. cordifolia extract is bitter and may be sweetened.
  • cordifolia extract may be administered to subjects at a dose three times per day (e.g., 3 x lOOmg per day).
  • Aloe vera extract may be administered to subjects at a dose between about 0.2 g and 5 g per day, or between about 0.5 to 4 g per day, or between about 1.0 g to 3 g per day, or between about 1.5 g to 2.0 g per day.
  • the Curcuma xanthorrhiza extract may be administered to subjects at a dose between 0.05 mg and 4 g per day, or between about 0.2 g and 3 g per day, or between about 0.5 g to 2 g per day, or between about 1.0 g to 1.5 g per day.
  • the Curcuminoid may be administered to subjects at a dose between about 0.05 mg and 4 g per day, or between about 0.2 g to 3 g per day, or between about 0.5 to 2 g per day, or between about 1.0 g to 1.5 g per day or between about 1.8 g to 2.2 g per day.
  • albumin from Channa striata (snakehead fish) extract may be administered to subjects at a dose between about 0.05 mg and 4 g per day, or between about 0.2 g to 3 g per day, between about 0.5 g to 2 g per day, or between about 1.0 g to 1.5 g per day, or between about 1.8 to 2.2 g per day.
  • the antiviral combination may be administered once, twice, three, or four times daily.
  • the daily dose of the immunomodulatory composition is 1000 mg Moringa extract, 100 mg T. cordifolia extract, 1000 mg Aloe vera extract, 500 mg Curcuma xanthorrhiza extract, and optionally 500 mg Channa striata extract.
  • the daily dose of the immunomodulatory composition is 2000 mg Moringa extract, 200 mg T. cordifolia extract, 2000 mg Aloe vera extract, 1000 mg Curcuma xanthorrhiza extract, and optionally 1000 mg Channa striata extract.
  • the daily dose of the immunomodulatory composition is 3000 mg Moringa extract, 300 mg T. cordifolia extract, 3000 mg Aloe vera extract, 1500 mg Curcuma xanthorrhiza extract, and, optionally, 1500 mg Channa striata extract.
  • the daily dose of the immunomodulatory composition is 4000 mg Moringa extract, 400 mg T. cordifolia extract, 4000 mg Aloe vera extract, 2000 mg Curcuma xanthorrhiza extract, and optionally 2000 mg Channa striata extract. It is to be understood that dosages between those specified above also fall with the daily dosages of the present disclosure.
  • the Curcuma xanthorrhiza extract is at half the dosage indicated above, that is, each sachet would contain 1000 mg Moringa extract, 100 mg T. cordifolia extract, 1000 mg Aloe vera extract, 250 mg Curcuma xanthorrhiza extract, and optionally 500 mg Channa striata extract.
  • the daily dose for the Curcuma xanthorrhiza extract would be 250 mg, 500 mg, 750 mg, 1000 mg, respectively, in the daily dose of the immunomodulatory composition described above.
  • the method may be suitable for the treatment of non-human primates and other mammals such as livestock (including race horses), exotic animals (e.g., tigers and elephants) and companion animals (e.g., dogs and cats); however, typically the subject will be a human.
  • the present disclosure is directed to the immunomodulatory composition when used to treat a viral infection.
  • the present disclosure is directed to the use of the immunomodulatory composition in the manufacture of a medicament for the treatment of a viral infection.
  • the present disclosure is directed to the use of the immunomodulatory composition to treat a viral infection.
  • the present disclosure is directed to a method of treating a viral infection comprising administering to a to a subject in need thereof a therapeutically effective amount of the immunomodulatory composition.
  • the treatment is the oral administration of the antiviral combination of the present invention.
  • the oral combination may be dissolved in water or sweetened beverages or similar. Multiple doses of the antiviral combination may be administered per day.
  • the virus infection is a respiratory virus infection including influenza infection or coronavirus infection.
  • the virus is SARS-Cov or SARS-Cov-2.
  • the virus infection is COVID-19.
  • the immunomodulatory composition is tested in peripheral blood mononuclear cells (PBMCs) from healthy subjects cultured in Roswell Park Memorial Institute 1640 Medium (RPMI 1640) culture medium supplemented with Glutamax (commercially available from sources such as ThermoFisher Scientific) with IX PenStrep and 2.5 pg/mL Plasmocin (commercially available from sources such as InvivoGen) ⁇ 10% heat inactivated fetal bovine serum (FBS), containing approximately 5 mg/mL bovine serum albumin (BSA; Francis, 2010). Initially, the PBMC were cultured in RPMI with fetal bovine serum (FBS).
  • FBS fetal bovine serum
  • the cells will cultured in RPMI supplemented with Gutamax, PenStrep and Plasmocin as above, but in the absence of FBS, in either the presence or absence of 5 mg/mL BSA. This will assess the impact of cell health and viability as well as observe the effects of the active ingredients of the extracts.
  • Extracts derived from supercritical extraction procedure described above are concentrated as follows. 250 g of each extract to be used is added to 1000 mL of 80% methanol (v/v) dissolved in Mill-Q (MQ)-H2O and stirred using a magnetic stirrer for 3 hours at room temperature in a sealed container. The solution is then filtered using Buchner funnel with Whatman filter paper No. l (125 mm). The filtrate is transferred to a round-bottom flask and connected to a rotary evaporator (Rotavap). The round bottom flask is submerged in a water bath at 50°C under vacuum. The concentrate is further dried using freeze drying method as above and re-ground to fine powder when required. The concentrated powder of each extract is dissolved in 100% dimethyl sulfoxide (DMSO) to produce a stock solution of 40 mg/mL of extracts for use in in vitro studies.
  • DMSO dimethyl sulfoxide
  • PBMCs are thawed and resuspended in culture medium (with or without BSA). 100 pL PBMC containing 2.5 x 10 5 cells are plated into 96-well plates (2.5 x 10 5 cells/well) and incubated overnight in 37°C in a 5% CO2 incubator (approximately 16-18 hours). Cells are washed, supernatant discarded and resuspended in 100 pL fresh culture medium (with or without BSA).
  • each extract is added per well, at one of six different concentrations as shown in Table 6, tested in triplicate, and incubated at 37°C/5% CO2 for 30 mins (150 pl in each well), with DMSO vehicle negative controls.
  • Cells are stimulated with 10 pl lipopolysaccharide (LPS) at 50 ng/mL in phosphate buffered saline (PBS) (final well volume 160 pL) and incubated at 37°C/5% CO2 for 24 hours to simulate inflammation in triplicate and compared to vehicle control (DMSO with no LPS or extract), LPS only control or LPS plus dexamethasone.
  • 80 pL of each supernatants from triplicate test conditions are pooled (240 pL per condition).
  • 120 pL of each condition extract concentrations is tested by a Human 45-Plex ProcartaPlex Panel (eBioScience; Cat # EPX450-12171-901) to analyse cytokine production in accordance with manufacturer's instructions, with each condition being compared to vehicle control, LPS control or LPS plus dexamethasone control. Remaining pooled supernatants are stored at -80°C for further analysis.
  • CellTitre-Blue is used to assess cell viability in accordance with manufacturer's instructions. Briefly, CellTitre Blue reagent is added to cells remaining in 96 well plates and incubated for further 6 hours. Absorbance is read (570 nm; ref: 620 nm) at 1, 2, 4 and 6 hour timepoints. Viability of each condition compared to vehicle control, LPS control and LPS and dexamtheasone control.
  • EXTRACTS TESTED - 4 PLANT COMBINATION + SNAKEHEAD FISH EXTRACT [0133]
  • the PBMCs in this example will provide data relating to a change of production levels of various cytokines. It is expected that the extracts will produce a reduction in the level of a number of cytokines associated with inflammation, for example, interleukin (IL)-6, IL-18, Tumour Necrosis Factor (TNF), monocyte chemoattractant protein (MCP)-1/C-C motif ligand (CCL)2, monocyte inflammatory protein (MIP)-l alpha/CCL3, CCL5/ Regulated upon Activation, Normal T Cell Expressed and Presumably Secreted (RANTES), and/or granulocyte macrophage-colony stimulating factor (GM-CSF); and a moderate production of IL-ip and IL-2.
  • IL interleukin
  • TNF Tumour Necrosis Factor
  • MCP
  • cytokines such as IL-10, IL- 4, interferon (IFN)-o, and IFN-y interleukin is anticipated. This would indicate that the extracts have antiviral properties. Asssessment of PBMCs in the presence of LPS and/or dexamethasone (an anti-inflammatory compound) will be investigated.
  • Extracts or raw materials are commercially sourced from a variety of suppliers such as PT. Tri Rahardja (Javaplant), Indonesia, PT. Indesso Aroma, Indonesia, PT. Insular Multi Natural, Indonesia, PT. Industri Jamu dan Farmasi Sido Muncul, Indonesia, PJ Herbal Nusantara, Indonesia, PT. Merpati Mahardika, Indonesia or Herbal Hills, India, in either dried, powder or liquid form.
  • Raw material from plant an animal matter for extraction were obtained as follows: T. cordifolia (obtained from PT. Insular Multi Natural, Indonesia, listed under alternative name Brotowali extract); Curcuma xanthorrhiza (obtained from PT. Industri Jamu dan Farmasi Sido Muncul, Indonesia, lised under alternative name Sari Temulawak Sido Muncul; Channa striata (obtained from PJ Herbal Nusantara, Indonesia, under name Channa); Aloe barbadensis (Aloe vera; obtained from PT. Merpati Mahardika, Indonesia, listed under alternative name Lidah Buaya extract; Moringa oleifera (obtained from PT. Industri Jamu dan Farmasi Sido Muncul, Indoneisa, listed under alternative name Sari Daun Kelor Sido Muncul).
  • Raw material including Moringa leaves, Curcuma xanthorrhiza tubers/roots, T. cordifolia leaves, stems and roots, Aloe vera stems and Channa striata flesh were obtained from commercial sources as described herein.
  • Curcuma xanthorrhiza, Moringa oleifera, and Tinospora cordyfolia 1 kg of dried materials produced around 4% - 7% liquid extract.
  • the raw materials individually were extracted using supercritical carbon dioxide extraction as follows. The dried plant material was ground to 50 mesh particle size using a commercial grinding machine. Then, the ground dried material was inserted into the supercritical material chamber of a commercial supercritical CO2 extractor.
  • the temperature on the CO2 tanks was set to 30°C to reach 900 -1100 psi, and then the extraction operating timer was set for for 2 hours and 15 minutes with solenoid valve timer releasing every 2 seconds. Accordingly, every 2 seconds, the extract was collected in the collector chamber until the operation was completed.
  • aloe vera For aloe vera, the same process was followed, except that the material was not dried and ground.
  • the inner gel inside aloe vera leaves was sliced into approximately 3 mm thickness and continuouslyinserted into the supercritical material chamber of the supercritical CO2 extractor using the same operating procedure as above. A higher yield of 5 - 8% liquid aloe vera extract was collected.
  • the liquid extract was collected and freeze dried using a standard freeze dry chamber within 18 hours to 24 hours to produce a solid powder material.
  • the solid powder extracts were obtained using the supercritical carbon dioxide extraction method described above or commercially obtained.
  • the solid powder extracts were combined using a tumbler mixer until homogenous.
  • the solid powder extracts were combined to form an immunomodulatory composition mixture combined in the following percentages:
  • the mixture is milled to a preferred particle size using a hammer mill, for example, 180 - 200 mesh powder, and then sifted using an industrial sifter and dust collector.
  • the combined dried extracts are packed into sachets containing:
  • each of the extracts is individually prepared as above (i.e., without combination), and packaged into sachets such that the sachets contain the same amount of an individual extract as that extract in combination, that is:
  • IFN-y The IFN-y system is associated with antiviral defence. IFN-y can downregulate virus replication and activate cytokine production by T cells, augmenting cytotoxic T lymphocyte killing activity (Levy and Garcia -Sastre, 2001). However, persistent high levels of IFN-y can aggravate systemic inflammation and increase tissue injury and organ failure (Yin et al., 2005). Low levels of plasma IFN-y have been reported to be associated with lung fibrosis in COVID-19 patients, with two-fold lower plasma interferon-y (IFN-y) levels at discharge observed in patients with fibrosis in the lung compared to those without fibrosis (p > 0.05; Hu et al., 2020).
  • IFN-y plasma interferon-y
  • cordifolia (3.84%), Aloe vera (38.46%) and Curcuma xanthorrhiza (19.23%) contained increasedd IFN-y levels compared to culture supernatant stimulated with LPS in the absence of the combination of extracts (negative control) or the culture supernatant stimulated with LPS and dexamethasone (anti-inflammatory control; p ⁇ 0.05).
  • p ⁇ 0.05 a significant increase of 12% (p ⁇ 0.05) and 26% (p ⁇ 0.05), respectively, of IFN-y was observed compared to the LPS negative control and the LPS+ dexamethasone control.
  • the increase of IFN-y in the in vitro study indicates the potential of the immunomodulatory composition containing the combination of extracts to enhance beneficial antiviral activity without inducing very high levels associated with a cytokine storm/ cytokine release syndrome (CRS).
  • CRS cytokine storm/ cytokine release syndrome
  • IL-1B is a pro-inflammatory cytokine
  • high IL-1B levels in addition to IL-18 and IL-33 levels, is considered to play a central role in CRS (Shimabukuro-Vornhagen etal., 2018), and reduction of IL- 13 levels is considered useful in treating COVID-associated CRS (Shi et al., 2020).
  • IL-1 receptor antagonists have been shown to reduce the cytokine storm associated with severe infection induced inflammation and has been shown to significantly improve the 28-day survival rate of patients with severe sepsis (Shakoory et al., 2016), indicating that reduced IL- 13 levels assist in reducing or avoiding a cytokine storm response.
  • IL-2 is a pleiotropic cytokine that is a key regulator of T cell metabolic programs. Amongst other functions, it influences effector T cell differentiation and is a critical determinant of the fate decisions of antigen receptor-activated T cells. When activated, both CD4+ T and cytotoxic CD8+ T cells exert protective immunity on both chronic and acute viral infection (Zhou et al., 2012; Sant and McMichael, 2012). IL-2 is also released during CRS (Eastwood et al., 2013), and can stimulate IL-6 release (Yiu et al., 2012).
  • IL-4 is an anti-inflammatory pleiotropic cytokine.
  • IL-4 drives CD4+ T cell polarization toward the Th2 phenotype and down regulates IFN-y- producing Thl cells.
  • IL-4 also supports the activation, differentiation and maturation of B lymphocytes, controlling the specificity of the immunoglobulin G (IgG) class switching and the development of memory B cells.
  • IgG immunoglobulin G
  • IL-4 can enhance the recruitment of T cells and eosinophils, rather than granulocytes, into the site of inflammation.
  • IL-8/CXCL8 is a pro-inflammatory chemoattractant chemokine for T-lymphocytes, mast cells, monocytes/macrophages, granulocytes, keratinocytes, and endothelial cells and particularly recruits neutrophils.
  • the recruitment of neutrophils in inflammatory pneumonia was found to correlate with the development and mortality of Adult Respiratory Distress Syndrome (ARDS) associated with the cytokine storm.
  • ARDS Adult Respiratory Distress Syndrome
  • High level IL-8/CXCL-8 is found in the CRS in SARS patients.
  • MCP-lo/CCL-2 is one of the key chemokines that regulate migration and infiltration of monocytes/macrophages, and central in CRS in SARS patients, severity of COVID-19 and increased plasma levels in ICU vs. non-ICU patients (Huang et al., 2020c; Mehta et al., 2020).
  • Macrophage Inflammatory Protein-lo (MIP-lo/CCL-3) is a chemoattractant chemokine and serves as biomarker for detection of several inflammatory diseases including lung conditions including ARDS.
  • MIP-lo/CCL-3 is linked to severity of COVID-19 (Mehta et al., 2020) and plays a significant role in promoting inflammation in response to viral infections.
  • this plasma levels of MIP-lo/CCL-3 has been found to be increased in ICU vs. non-ICU COVID-19 patients (Huang et al., 2020c).
  • PBMC samples obtained from healthy subjects as described above were frozen and thawed and resuspended in RPMI medium (supplemented with glutamax) with 1 x PenStrep and 2.5 pg/mL plasmocin ⁇ 10% heat inactivated FBS containing approximatey 5 mg/mL BSA at 2.6 x 10 6 cells/mL.
  • 100 pL of cells (2.5 x 10 5 cells) were platedout into 96 well plates and cultured overnight (approximately 16-18 hours) in a 37°C/5% CO2 incubator. Then, the PBMCs were washed, removing supernatants and re-suspended in 100 pl fresh culture medium in the presence of FBS (BSA).
  • each condition was tested in triplicate. Briefly, 50 pL of the combined extract at a concentration of 1, 5, 25, 50, 100 or 200 pg/mL was added to the appropriate wells. For the negative controls, 50 pL of the extract diluent was added instead, and for the positive control, 50 pL of DMSO was added and the samples were incubated for a further 30 minutes in a 37°C/5% CO2 incubator. 10 pL of LPS/PBS (final concentration of LPS was 50 ng/mL) was added to each well to induce inflammation. The plates were then incubated in a 37°C/5% CO2 incubator for 24 hours.
  • PBMC peripheral blood monocytes
  • an immunomodulatory composition containing 5 pg/mL of a combination of extracts from Moringa olifera (38.46%), T. cordifolia (3.84%), Aloe vera (38.46%) and Curcuma xanthorrhiza (19.23%) modulates the levels of at least IFN-y, IL-ip, IL- 2, IL-4, IL-8, MCP-1 and MIP-lo compared to samples stimulated with either LPS alone or LPS and dexamethasone.
  • PBMC peripheral blood monocytes
  • Immunomodulatory compositions were prepared and packaged into sachets as described above. Each sachet contained 1000 mg Moringa extract, 100 mg T. Cordifolia extract, 1000 mg Aloe vera extract, 500 mg Curcuma xanthorrhiza and 500 mg Channa striata extract.
  • the patient was administered two sachets per day on the day of his positive COVID-19 test (Day 0 and Day 1); three sachets per day for six days (Day 2 to Day 7); and two sachets per day for seven days (Day 8 to Day 14).
  • Day 7 the patient took a rapid test and tested non-reactive.
  • Day 11 the patient took a second COVID-19 PCR test and tested negative. At that time, his symptoms were completely resolved.
  • this result is from a single patient, it indicates that the antiviral combination is safe in humans and is associated with a quick recovery from COVID-19.
  • SARS-CoV-2 PCR positive patients in Indonesia are enrolled into a clinical trial that is currently underway.
  • Groups of 20 patients with mild to moderate COVID-19 symptoms are orally administered 3 sachets dissolved in water per day for at least 14 days, the sachets containing extracts in the following doses:
  • Placebo e.g., starch/sugar-based composition.
  • PCR for SARS-CoV-2 is performed at day 1, 14 and preferably also day 20. At days 1, 5, 10 and 14, 20, the parameters described below are examined using standard laboratory techniques.
  • Antisera IgG and IgM
  • ALB albumin
  • CRP C reactive protein
  • LDH lactate dehydrogenase
  • AST aspartate aminotransferase
  • ALT alanine aminotransferase
  • Haematology Haemoglobin, red blood cells (RBC), Haematocrit MCV, MCH, MCHC, RDW, ABO Blood Group, Rhesus (+/-);
  • White blood cell count Neutrophils, lymphocytes, monocytes, eosinophils, Basophils, nucleated red blood cells (NRBC), platelets;
  • the COVID-19 patients receiving the antiviral combination are expected to show improved blood markers for COVID-19 more rapidly than the patients receiving the negative and placebo control.
  • albumin, lymphopenia, lymphocyte (%), LDH, neutrophil (%), and CRP levels are highly correlated to acute lung injury.
  • Age, viral load, lung injury score, and blood biochemistry indexes, albumin, CRP, lactate dehydrogenase, lympopenia lymphocyte (%), and neutrophil (%), may be predictors of disease severity.
  • COVID-19 patients taking the antiviral combination will return to health more rapidly that patients receiving the negative control and placebo. Accordingly, it is anticipated that COVID-19 patients receiving the antiviral combination may have an enhanced return, for example, more rapid return on average, to a COVID-19 PCR negative result than those patients receiving the negative/placebo control.
  • SARS-CoV-2 PCR positive patients are enrolled into a clinical trial. Groups of 20 patients with mild to moderate COVID-19 symptoms are orally administered 3 sachets dissolved in water per day for at least 14 days, the sachets containing extracts in the following doses:
  • the trial is conducted and assessed using the protocol as outlined in Example 3 (Ongoing in vivo trial). The trial assesses the effectiveness of the antiviral combination with and without the inclusion of the Channa striata extract.
  • Serum cytokine levels will be assessed to determine whether the antiviral combination induces cytokine levels that assist with recovery from viral infection without inducing host-derived pathology such as that seen during a cytokine storm.
  • SARS-CoV-2 PCR positive patients are enrolled into a clinical trial. Groups of 5-20 patients with mild to moderate COVID-19 symptoms are orally administered the following combinations of antiviral agents daily for at least 14 days:
  • T. cordifolia extract 500 mg albumin, 500 mg glutathione, and 5 mg iron
  • T. cordifolia extract 500 mg albumin, 500 mg glutathione, 250 mg ascorbic acid and 5 mg iron;
  • TNFs contribute significantly to acute lung injury and impair the T cell response in SARS-CoV- challenged mice.
  • neutralization of TNF activity or loss of the TNF receptor provides protection against SARS-CoV-induced morbidity and mortality (Channappanavar et al., 2016; and McDermott et al., 2016).
  • TNF-a plays a central role in CRS. By blocking TNF-a potentially beneficial for patients with severe inflammation in the lung due to CRS (Huang et al., 2020).
  • cordifolia (3.84%), Aloe vera (38.46%) and Curcuma xanthorrhiza (19.23%) contained significantly decreased TNF-o levels as compared to the culture supernatant stimulated with LPS and dexamethasone (anti-inflammatory positive control).
  • dexamethasone anti-inflammatory positive control.
  • an decrease of 25.92% in TNF level was observed as compared to the LPS+ dexamethasone control. This result indicates that the extract combination offers potential to downregulate TNF-o help combat the SARS-CoV-2 virus.
  • Neurologic complication associated with the SARS-CoV-2 infection in COVID-19 patients and survivors comprise symptoms including anxiety, depression, sleep impairment, headaches, dizziness, muscle pain, fatigue, myopathies and anosmia/hyposmia have been reported, this issue can prolonged for months and have been reported Goertz et al., 2020; Heneka et al., 2020; Seeano-Castro et al., 2020; Mei Junhua et al., 2020).
  • Eotaxin-1 plays roles in physiosomatic and neuroinflammation, analyzing the level of this chemokine in COVID-19 patients during hospitalization and to predicting post-COVID-19-related neurologic complications may be worthwhile. It is thought that, using chemokine modulators may be helpful in lessening the neurologic complications in such patients (Nazarinia et al., 2022).
  • the extract combination appears to downregulate the Eotaxin-1 level reflected by the obtained result.
  • Eotaxin-1 levels appears to be lower when using extract combination particularly in the range of 5 - 200 pg/mL. Particularly at 5 pg/mL, the Eotaxin-1 is significantly down-regulated (-13.99% fold) than the negative control DMSO+LPS (**p ⁇ 0.05).
  • the lowering capability of Eotaxin-1 secretion at 5 pg/mL comparable to the dexamethasone+LPS control with a small difference of -2.79% fold (* >0.05).
  • the result demonstrated that the extract combination an act as chemokine modulators and do have potential as neuron protection as it lowers the amount of Eotaxin-1 significantly when compared towards the DMSO+LPS control.
  • IP-10/CXCL-10 pro-inflammatory cytokines and chemokines
  • IP-10/CXCL-10 circulating concentration was significantly higher in patients requiring admission to intensive care units as compared to patients experiencing a less severe clinical course (Huang et al., 2020c).
  • a study by Zhicheng et. al. corroborates this. The result revealed significant lower levels of immune cells (CD3 + T, CD4 + T, CD8 + T, B and NK cells).
  • significantly higher levels of chemokines including IP-10/CXCL-10) in severe cases compared with mild cases of COVID-19 patients were also observed (Zhicheng et al., 2020).
  • the IP-10/CXCL-10 is lower (-8.29% fold) than the negative control DMSO+LPS (**p ⁇ 0.05).
  • the lowering capability of IP-10 secretion is more noticeable than the dexamethasone+LPS control with a small difference of - 6.64% fold (*p>0.05).
  • MIP-ip/CCL-4 is a CC type Chemokine.
  • chemokine milieu induced by one pathogen, by specifically recruiting T cells in the infected tissue, underpins a crucial role in determining the nature of the immune response Sokol et al., 2015; Kaiko et al., 2008).
  • PBMC peripheral blood mononuclear cells
  • MIP- ip/CCL-4 In the sample treated with LPS and 5 pg/mL of the combination of extracts, of extract combination to the LPS-stimulated cells, the level of MIP- ip/CCL-4 has been down-regulated significantly by -27.87% folds and -38.75% folds when compared to dexamethasone+LPS (*p ⁇ 0.05) and DMSO+LPS (**p ⁇ 0.05) controls respectively. Both MIP-lo/CCL-3 and MIP-ip/CCL-4 are chemoattractant. When dysregulated and highly secreted by the macrophages/monocytes will call upon various immune cells and may lead to CRS.
  • the extract combination significantly down-regulate the MIP- ip/CCL-4 secretion even lower than the dexamethasone+LPS control.
  • cordifolia (3.84%), Aloe vera (38.46%) and Curcuma xanthorrhiza (19.23%) modulates the levels of at least IFN-y, IL-ip, IL- 2, IL-4, TNF-o, Eotaxin-l/CCL-11, IL-8/CXC-10, MCP-l/CCL-2, MIP-lo/CCL-3, and MIP-ip/CCL-4 compared to samples stimulated with either LPS alone or LPS and dexamethasone.
  • the combination of extracts may assist to modulate immune responses associated with cytokine storms, such as those seen in severe inflammatory lung disease such as COVID-19, SARS, MERS and influenza, etc.
  • HDAC histone deacetylase
  • Circulatory zinc transport is controlled by distinct interdomain sites on mammalian albumins. Chem. Sci. 7: 6635-6648.
  • Coronavirus disease 2019 (COVID-19): first indication of efficacy of Gene-Eden-VIR/Novirin in SARS-CoV-2 infection. International journal of antimicrobial agents. 55(6): 105971-105971.
  • Polonikov 2020. Endogenous Deficiency of Glutathione as the Most Likely Cause of Serious Manifestations and Death in COVID-19 Patients ACS Infect Dis. 2020 May 28.
  • Tinospora cordifolia One plant, many roles. Ancient science of life. 31(4): 151-159.
  • Serum albumin level as a predictor of intensive respiratory or vasopressor support in influenza A (H1N1) virus infection. IntJ Clin Pract 68(2): 222-9.

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Abstract

The present invention relates to an immunomodulatory composition comprising a combination of agents consisting of a curcuminoid, an emodin, bioavailable zinc and an at least one active agent obtained from Tinospora cordifolia, as well as a composition comprising a combination of agents consisting of a Tinospora cordifolia extract, a Curcuma xanthorrhiza extract, an aloe extract and a moringa extract. Said composition may further comprising an agent selected from an albumin, a glutathione, ascorbic acid and bioavailable iron. Said composition is preferable formulated for oral delivery, such as a dried powder, and used in a method of treating a viral infection such as coronavirus or SARS-CoV-2 infection.

Description

TITLE OF THE INVENTION
IMMUNOMODULATORY COMBINATION COMPRISING PLANT AND ANIMAL EXTRACTS HAVING ANTIVIRAL ACTIVITY
RELATED APPLICATIONS
[0001] This application claims priority to Australian Provisional Application No. 2022900137 entitled "Immunomodulatory Combination" filed on 25 January 2022, the contents of which are incorporated herein by reference in their entirety.
FIELD OF THE INVENTION
[0002] This invention relates generally to compositions comprising a combination of active ingredients and methods of using such compositions. More particularly, the present invention relates to compositions comprising a combination of extracts for use as an immunomodulatory agent.
BACKGROUND OF THE INVENTION
[0003] Antiviral agents may be used to treat some viral infections, usually by targetting key, virus-specific processes that selectively inhibit viral entry into cells and/or the viral replication pathway. This may mediate reduction in viral load, or prevention of an increase in viral load, and may assist the host immune response to reduce or eliminate the virus from the body.
[0004] Additionally, mammalian host organisms have evolved an array of immune responses that usually are successful in eliminating the virus from the body. However, during acute viral respiratory disease, a "cytokine storm" may result in the overproduction of proinflammatory cytokines such as tumour necrosis factor (TNF), Interleukin (IL)-6, IL- 1(3 and/or IL-8; which may mediate acute lung injury (ALI), or in its more severe form, acute respiratory distress syndrome (ARDS). ALI and/or ARDS may be associated with vascular hyperpermeability, multiple organ failure and may lead to death.
[0005] ALI and/or ARDS are sometimes observed during infection with influenza virus, Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV or SARS-CoV-1, the causative agent of SARS), or Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2, the causative agent of Coronavirus Infectious Disease (COVID)-19).
[0006] COVID-19 is associated with significant mortality, particularly in at-risk groups with poor prognostic features at hospital admission. The spectrum of disease is broad; however, pneumonia, sepsis, respiratory failure, and acute respiratory distress syndrome (ARDS) are among symptoms frequently encountered in hospitalised COVID-19 patients, accompanied the overproduction of early response proinflammatory cytokines (eg. tumour necrosis factor (TNF), IL-6, and IL- lg) leading to a cytokine storm. Therapeutic strategies targeting the overactive cytokine response with anti-cytokine therapies or immunomodulators may assist, but this must be balanced with maintaining an adequate inflammatory response for pathogen clearance.
[0007] Traditional medicinal plant and animal extracts have been used in response to a range of medical ailments, including immunomodulatory conditions and viral infections. A variety of naturally derived active agents have been shown to have antiviral effects in humans, and others may modulate the immune response.
[0008] The present inventors have realized that a combination of naturally derived active agents or extracts may have an antiviral effect, and may alternatively or additionally modulate the immune system to mediate an effective antiviral immune response while minimising or avoiding host-induced pathology.
SUMMARY OF THE INVENTION
[0009] In a first aspect, the present disclosure relates to an immunomodulatory composition comprising a combination of agents consisting of a curcuminoid, an emodin, bioavailable zinc and an at least one active agent obtained from Tinospora cordifolia. In some embodiments, the immunomodulatory composition further comprises an agent selected from the list consisting of an albumin, a glutathione, ascorbic acid and bioavailable iron. In some embodiments, at least one of the agents are provided as extracts derived from plants and/or animals.
[0010] In some embodiments, the at least one active agent obtained from T. cordifolia is provided by a Tinospora cordifolia extract. In some embodiments, the curcuminoid is provided by a Curcuma spp. extract. In some embodiments, the emodin is provided by an Aloe spp. extract. In some embodiments, the bioavailable zinc is provided by a moringa extract. In some embodiments, the albumin is provided by a Channa striata (snakehead fish) extract.
[0011] In a second aspect, the present disclosure relates to an antiviral composition comprising a combination of agents consisting of a curcuminoid, an emodin, bioavailable zinc and an at least one active agent obtained from Tinospora cordifolia. In some embodiments, the antiviral composition further comprises an agent selected from the list consisting of an albumin, a glutathione, ascorbic acid and bioavailable iron. In some embodiments, at least one of the agents are provided as extracts derived from plants and/or animals.
[0012] In some embodiments, the at least one active agent obtained from T. cordifolia is provided by a Tinospora cordifolia extract. In some embodiments, the curcuminoid is provided by a Curcuma spp. extract. In some embodiments, the emodin is provided by an Aloe spp. extract. In some embodiments, the bioavailable zinc is provided by a moringa extract. In some embodiments, the albumin is provided by a Channa striata (snakehead fish) extract.
[0013] In a third aspect, the present disclosure relates to an immunomodulatory composition comprising a combination of agents consisting of a Tinospora cordifolia extract, a Curcuma xanthorrhiza extract, an aloe extract and a moringa extract. In some embodiments, the immunomodulatory composition of claim 10 further comprising a Channa striata (snakehead fish) extract.
[0014] In a fourth aspect, the present disclosure relates to an immunomodulatory composition comprising a combination of agents consisting of a Tinospora cordifolia extract, a Curcuma xanthorrhiza extract, an aloe extract, a moringa extract and a Channa striata extract.
[0015] In some embodiments, the immunomodulatory composition is formulated for oral delivery. In some embodiments, the immunomodulatory composition comprises a dried powder.
[0016] In a fifth aspect, the present disclosure relates to the immunomodulatory composition of the present disclosure when used to treat a viral infection. In some embodiments, the viral infection is a coronavirus infection. In some embodiments, the viral infection is a SARS-Cov-2 infection.
[0017] In a sixth aspect, the present disclosure relates to the use of the immunomodulatory composition of the present disclosure to treat a viral infection. In a fifth aspect, the present disclosure relates to the use of the immunomodulatory composition of the present disclosure in the manufacture of a medicament for the treatment of a viral infection. In some embodiments, the viral infection is a coronavirus infection. In some embodiments, the viral infection is COVID-19.
[0018] In a seventh aspect, the present disclosure relates to a method of treating a viral infection comprising administering to a to a subject in need thereof a therapeutically effective amount of the immunomodulatory composition of the present disclosure. In some embodiments, the viral infection is a coronavirus infection. In some embodiments, the viral infection is COVID-19.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] Figure 1 shows a graphical representation of interferon (IFN)-y levels of culture supernatant from peripheral blood monocytes (PBMC) stimulated with LPS and with various concentrations (1, 5, 25, 50, 100 or 200 pg/mL) of an immunomodulatory composition containing a combination of extracts from Moringa olifera (38.46%), T. cordifolia (3.84%), Aloe vera (38.46%) and Curcuma xanthorrhiza (19.23%); with LPS only stimulation as a negative control and LPS stimulation followed by dexamethasone treatment as a positive anti-inflammatory control. *p <0.05 for LPS + dexamethasone; **p <0.05 for DMSO+LPS
[0020] Figure 2 shows a graphical representation of interleukin (IL)-ip levels of culture supernatant from peripheral blood monocytes (PBMC) stimulated with LPS and with various concentrations (1, 5, 25, 50, 100 or 200 pg/mL) of an immunomodulatory composition containing a combination of extracts from Moringa olifera (38.46%), T. cordifolia (3.84%), Aloe vera (38.46%) and Curcuma xanthorrhiza (19.23%); with LPS only stimulation as a negative control and LPS stimulation followed by dexamethasone treatment as a positive anti-inflammatory control. *p <0.05 for LPS + dexamethasone; **p < 0.05 for DMSO+LPS
[0021] Figure 3 shows a graphical representation of IL-2 levels of culture supernatant from peripheral blood monocytes (PBMC) stimulated with LPS and with various concentrations (1, 5, 25, 50, 100 or 200 pg/mL) of an immunomodulatory composition containing a combination of extracts from Moringa olifera (38.46%), T. cordifolia (3.84%), Aloe vera (38.46%) and Curcuma xanthorrhiza (19.23%); with LPS only stimulation as a negative control and LPS stimulation followed by dexamethasone treatment as a positive anti-inflammatory control. *p <0.05 for LPS + dexamethasone; **p <0.05 for DMSO+LPS
[0022] Figure 4 shows a graphical representation of IL-4 levels of culture supernatant from peripheral blood monocytes (PBMC) stimulated with LPS and with various concentrations (1, 5, 25, 50, 100 or 200 pg/mL) of an immunomodulatory composition containing a combination of extracts from Moringa olifera (38.46%), T. cordifolia (3.84%), Aloe vera (38.46%) and Curcuma xanthorrhiza (19.23%); with LPS only stimulation as a negative control and LPS stimulation followed by dexamethasone treatment as a positive anti-inflammatory control. *p <0.05 for LPS + dexamethasone; **p <0.05 for DMSO+LPS [0023] Figure 5 shows a graphical representation of IL-8 (also known as chemokine (C-X-C motif) ligand (CXCL)-8) levels of culture supernatant from peripheral blood monocytes (PBMC) stimulated with LPS and with various concentrations (1, 5, 25, 50, 100 or 200 pg/mL) of an immunomodulatory composition containing a combination of extracts from Moringa olifera (38.46%), T. cordifolia (3.84%), Aloe vera (38.46%) and Curcuma xanthorrhiza (19.23%); with LPS only stimulation as a negative control and LPS stimulation followed by dexamethasone treatment as a positive anti-inflammatory control. *p <0.05 for LPS + dexamethasone; **p <0.05 for DMSO+LPS
[0024] Figure 6 shows a graphical representation of monocyte chemoattractant protein (MCP)-l (also known as chemokine (C-C motif) ligand (CCL)-2) levels of culture supernatant from peripheral blood monocytes (PBMC) stimulated with LPS and with various concentrations (1, 5, 25, 50, 100 or 200 pg/mL) of an immunomodulatory composition containing a combination of extracts from Moringa olifera (38.46%), T. cordifolia (3.84%), Aloe vera (38.46%) and Curcuma xanthorrhiza (19.23%); with LPS only stimulation as a negative control and LPS stimulation followed by dexamethasone treatment as a positive antiinflammatory control. *p <0.05 for LPS + dexamethasone; **p <0.05 for DMSO+LPS.
[0025] Figure 7 shows a graphical representation of macrophage inflammatory protein (MIP)-lo (also known as CCL-3) levels of culture supernatant from peripheral blood monocytes (PBMC) stimulated with LPS and with various concentrations (1, 5, 25, 50, 100 or 200 pg/mL) of an immunomodulatory composition containing a combination of extracts from Moringa olifera (38.46%), T. cordifolia (3.84%), Aloe vera (38.46%) and Curcuma xanthorrhiza (19.23%); with LPS only stimulation as a negative control and LPS stimulation followed by dexamethasone treatment as a positive anti-inflammatory control. *p <0.05 for LPS + dexamethasone; **p <0.05 for DMSO+LPS.
[0026] Figure 8 shows a graphical representation of changes in levels of analytes MIP-lo, MCP-1, IL-8, IL-4, IL-2, IL-13 and IFN-y detected in culture supernatant of PBMCs stimulated with LPS and 5 pg/mL of an immunomodulatory composition containing a combination of extracts from Moringa olifera (38.46%), T. cordifolia (3.84%), Aloe vera (38.46%) and Curcuma xanthorrhiza (19.23%) compared to either LPS stimulation alone (negative control; upper bar) or LPS stimulation plus dexamethasone treatment (positive anti-inflammatory control; lower bar). [0027] Figure 9 shows a graphical representation of tumor necrosis factor-o (TNF-o) levels of culture supernatant from peripheral blood monocytes (PBMC) stimulated with LPS and with various concentrations (1, 5, 25, 50, 100 or 200 pg/mL) of an immunomodulatory composition containing a combination of extracts from Moringa olifera (38.46%), T. cordifolia (3.84%), Aloe vera (38.46%) and Curcuma xanthorrhiza (19.23%); with LPS only stimulation as a negative control and LPS stimulation followed by dexamethasone treatment as a positive anti-inflammatory control.
[0028] Figure 10 shows a graphical representation of Eotaxin-1 (also known as CCL-11) levels of culture supernatant from peripheral blood monocytes (PBMC) stimulated with LPS and with various concentrations (1, 5, 25, 50, 100 or 200 pg/mL) of an immunomodulatory composition containing a combination of extracts from Moringa olifera (38.46%), T. cordifolia (3.84%), Aloe vera (38.46%) and Curcuma xanthorrhiza (19.23%); with LPS only stimulation as a negative control and LPS stimulation followed by dexamethasone treatment as a positive anti-inflammatory control.
[0029] Figure 11 shows a graphical representation of interferon gammainduced protein 10 (IP-10) (also known as C-X-C motif chemokine ligand 10 (CXCL-10)) levels of culture supernatant from peripheral blood monocytes (PBMC) stimulated with LPS and with various concentrations (1, 5, 25, 50, 100 or 200 pg/mL) of an immunomodulatory composition containing a combination of extracts from Moringa olifera (38.46%), T. cordifolia (3.84%), Aloe vera (38.46%) and Curcuma xanthorrhiza (19.23%); with LPS only stimulation as a negative control and LPS stimulation followed by dexamethasone treatment as a positive antiinflammatory control.
[0030] Figure 12 shows a graphical representation of macrophage inflammatory protein (MIP)-ip (also known as CCL-3) levels of culture supernatant from peripheral blood monocytes (PBMC) stimulated with LPS and with various concentrations (1, 5, 25, 50, 100 or 200 pg/mL) of an immunomodulatory composition containing a combination of extracts from Moringa olifera (38.46%), T. cordifolia (3.84%), Aloe vera (38.46%) and Curcuma xanthorrhiza (19.23%); with LPS only stimulation as a negative control and LPS stimulation followed by dexamethasone treatment as a positive anti-inflammatory control.
[0031] Figure 13 shows a graphical representation of changes in levels of analytes MIP-ip, MIP-lo, MCP-1, IP-10, IL-8, Eotaxin-1, TNF-o, IL-4, IL-2, IL- ip and IFN-y detected in culture supernatant of PBMCs stimulated with LPS and
Figure imgf000007_0001
extracts from Moringa olifera (38.46%), T. cordifolia (3.84%), Aloe vera (38.46%) and Curcuma xanthorrhiza (19.23%) compared to either LPS stimulation alone (negative control; upper bar) or LPS stimulation plus dexamethasone treatment (positive anti-inflammatory control; lower bar).
DETAILED DESCRIPTION OF THE INVENTION
[0032] The present disclosure relates to an immunomodulatory composition comprising a combination of agents. In some embodiments, the combination of agents consists of a combination of natural active agents or extracts that have an antiviral effect. In an alternative or additional embodiment, the antiviral combination mediates immunomodulation of the host immune response, for example, by stimulating cytokine expression rhythm that assists with recovery from viral infection without inducing host-derived pathology such as that seen during a cytokine storm.
Definitions
[0033] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described. For the purposes of the present invention, the following terms are defined below.
[0034] The term "active" as used to describe "active agents" or similar herein is referring to chemicals having medicinal benefits.
[0035] The term "antiviral" as used herein is the ability of a drug or substance to suppress or reduce the ability of a virus to enter host cells and/or replicate.
[0036] The term "bioavailable" as used herein is ability of a drug or other substance to be absorbed and used by the body. Orally bioavailable means that a drug or other substance that is taken by mouth can be absorbed and used by the body.
[0037] The term "natural" as used in the context of "natural extract" or "natural agent" etc as used herein is referring to an extract or agent that can be derived from a natural source, such as a plant or animal, or alternatively from other natural sources such as yeast, bacteria, etc.
[0038] The "therapeutically effective amount" will be any suitable amount that will elicit a beneficial or therapeutic effect in the subject. [0039] The articles "a" and "an" are used herein to refer to one or to more than one (/.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.
[0040] As used herein, "and/or" refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (or).
[0041] Throughout this specification, unless the context requires otherwise, the words "comprise," "comprises" and "comprising" will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements. Thus, use of the term "comprising" and the like indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present. By "consisting of" is meant including, and limited to, whatever follows the phrase "consisting of". Thus, the phrase "consisting of" indicates that the listed elements are required or mandatory, and that no other elements may be present. By "consisting essentially of" is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase "consisting essentially of" indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they affect the activity or action of the listed elements.
[0042] It will be appreciated that the terms used herein and associated definitions are used for the purpose of explanation only and are not intended to be limiting.
1.1 Immunomodulatory agents and antiviral immune response.
[0043] Immune responses induced by viral infection include cytotoxic cells that can kill virus infected cells; antibodies that can neutralise the virus by binding and blocking viral surface proteins that mediate binding to host cellular receptors, or antibodies that can agglutinate virions, opsonise virions and/or induce the innate immune system to phagocytose virions; and a variety of interferons and inflammatory cytokines that may intensify the antiviral immune response.
[0044] Many antiviral drugs target key, virus-specific processes that selectively inhibit viral entry into cells and/or the viral replication pathway. This may mediate reduction in viral load, or prevention of an increase in viral load, and may assist the host immune response to reduce or eliminate the virus from the body. Such targets include viral proteins involved with fusion of the virus with the host cell (for example, attachment, entry); uncoating of the virus; replication of the viral genome (for example, reverse transcription (reverse transcriptase) or DNA integration (integrase) for viruses that utilise these steps in their replication cycle; or viral polymerase); protein synthesis and assembly of viral components (for example, transcription, translation, protease); or release of new viruses from the host cell (for example, viral budding).
[0045] Symptoms of some viral infections may be worsened by an inappropriate or overactive host immune response. Many diverse viral infections (for example, cytomegalovirus, some Herpes viruses including Epstein-Barr virus, variola virus, some strains of the influenza virus, and some strains of coronavirus) in some hosts may induce a "cytokine storm". During acute viral respiratory disease, a cytokine storm may result in the overproduction of proinflammatory cytokines such as tumour necrosis factor (TNF), Interleukin (IL)-6, IL- ip and/or IL-8; and may be associated with acute lung injury (ALI), or in its more severe form, acute respiratory distress syndrome (ARDS). ALI and/or ARDS may be associated with vascular hyperpermeability, multiple organ failure and may lead to death. ALI and/or ARDS are sometimes observed during infection with influenza virus, Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV or SARS-CoV- 1, the causative agent of SARS), or Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2, the causative agent of Coronavirus Infectious Disease (COVID)-19).
[0046] These pathogens disrupt the delicate balance of a suitable inflammatory response, tipping it from being beneficial to destructive by causing large amounts of positive feedback in immune cells and upregulation of proinflammatory markers, in particular cytokines TNF, IL-1 p, IL-8, and IL-6. This can result in symptoms such as hypotension, fever, and edema and can eventually cause organ dysfunction and death. The influenza A virus, including the highly virulent subtypes such as H1N1 (which caused the 1918 pandemic) and H5N1 (which caused the 2008 "bird flu"), may be associated with cytokine storms. Severe influenza infections caused by may be characterized by overinduction of proinflammatory cytokines, TNF, IL-1 p, IL-8, and IL-6 and monocyte chemotactic protein-1 (MCP-1). This may eventually mediate multiple organ dysfunction and failure and increased vascular hyperpermeability.
[0047] Immunomodulation of the cytokine response during acute viral respiratory disease may assist the cytokine rhythm to attain in a balanced equilibrium wherein the host produces an effective anti-viral immune response while minimising or avoiding host-induced harm. Moreover, immunomodulators that assist with treating the symptoms associated with a cytokine storm in response to a particular cause are likely to have broad action against cytokine storm with alternative causes, because it is the host immune response that is modulated rather than the virus (for example) per se.
[0048] In preferred embodiments of the invention the immunomodulatory compositions of the invention enhance or stimulate an immune response.
2. Coronaviruses
[0049] Coronaviruses (CoVs) are an extensive family of RNA viruses that can cause disease in both animals and humans, and coronavirus strains that infect animals can evolve and become infectious to humans. The current classification of coronaviruses recognizes 39 species in 27 subgenera that belong to the family Coronaviridae. They are divided into alphacoronaviruses and betacoronaviruses which infect mammals; and gammacoronaviruses and deltacoronaviruses, which primarily infect birds. From those, at least 7 coronaviruses are known to cause respiratory infections in humans. Four of these coronaviruses produce symptoms that are generally mild, and can cause common cold-like symptoms:
• human coronavirus (HCoV) 229E, an alpha coronavirus identified in 1965;
• HCoV NL63, an alpha coronavirus identified in 2003;
• HCoV OC43, a beta coronavirus identified in 1967; and
• HCoV HKU1, a beta coronavirus identified in 2004.
[0050] Three human corona viruses are known that may produce symptoms that are potentially severe and/or fatal:
• Middle East Respiratory Syndrome Coronavirus (MERS-CoV), a beta coronavirus identified in 2012;
• Severe Acute Respiratory Syndrome Coronavirus (SARS-CoV or SARS- CoV-1), a beta coronavirus identified in 2003; and
• Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), a beta coronavirus identified in 2019.
[0051] These viruses are responsible for causing severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS) and the most recently discovered coronavirus disease during 2019 (COVID-19), respectively. COVID-19 was declared a global pandemic by the World Health Organization on 11th March 2020. It is a severe, highly infectious disease, and there is presently no approved treatment.
[0052] Coronaviruses are a group of enveloped positive-sense, single stranded RNA viruses with roughly spherical or moderately pleomorphic virions of approximately 60 to 140 nm in diameter. Coronaviruses have four major structural proteins, namely the Spike (S) protein, envelope (E) protein, membrane (M) protein (a type III transmembrane glycoprotein), and nucleocapsid (N) protein; and a number of non-structural proteins (NSP). The viral envelope (also known as a membrane) contains the surface (or spike) (S) glycoprotein that forms the peplomers on the virion surface, giving the virus a "corona" (crown-like) morphology that can be visualized with an electron microscope. The membrane
(M) glycoprotein and the envelope (E) protein provide the envelope structure. Within the virion interior lies a helical nucleocapsid comprised of the nucleocapsid
(N) protein complexed with a positive-sense single stranded RNA genome of about 26 to 32 kb in length and comprising a 5' terminal methylated cap structure and a 3' polyadenylated tail.
[0053] Infection with coronavirus involves the binding of the spike protein of a virion with a complementary host receptor. Entry to a host cell involves a host cell protease cleaving and activates the receptor-attached spike protein. The virus may enter the host cell by endocytosis or direct fusion of the viral envelop with the host membrane. Upon entry into the host cell, the virus particle may be uncoated. The coronavirus RNA genome may be directly translated by the ribosomes of a host cell. The produced viral polypeptides may be cleaved into viral peptides by viral proteases PLpro (NSP3) and main protease (Mpro), also called 3CLpro (NSP5).
[0054] A number of the nonstructural proteins coalesce to form a multiprotein replicase-transcriptase complex. The main replicase-transcriptase protein is the RNA-dependent RNA polymerase (RdRp). RdRp is directly involved in the replication and transcription of RNA from an RNA strand. Other nonstructural proteins in the complex assist in the replication and transcription process. The exoribonuclease nonstructural protein, for example, provides extra fidelity to replication by providing a proofreading function which the RNA-dependent RNA polymerase lacks.
[0055] A number of proteins in coronaviruses have been targeted as potential targets for antiviral drugs, with the spike (S) glycoprotein, Mpro and RNA- dependent RNA-polymerase (RdRp) being of particular interest. The S glycoprotein of HCoV-NL63, SARS-CoV, and SARS-CoV-2 viruses binds with human angiotensin-converting enzyme 2 (hACE2), a cellular receptor, which mediates entry of the virus into human cells. ACE2 is a membrane protein present in cells of most organs, including lung, heart, kidney and intestines/ ACE2 is expressed by type II alveolar cells within the lung, enterocytes of the small intestine, arterial and venous endothelial cells and arterial smooth muscle cells in most organs. ACE2 mRNA expression has also been found in the cerebral cortex, striatum, hypothalamus, and brainstem. Following the binding of the spike SI protein of SARS-CoV and SARS-CoV-2 to the enzymatic domain of ACE2 on the surface of cells, the host type II transmembrane serine protease TMPRSS2 cleaves the ACE-2 receptor and primes (ie activates) the S protein, which in turns facilitates entry of the virus into host cells via endocytosis.
[0056] The receptor binding domain (RBD) region of the S proteins of SARS-CoV-2 and SARS-CoV are highly homologous, with approximately 73%- 76% sequence similarity (Huang et al., 2020b). Nine ACE-2-contacting residues in SARS-CoV RBD are fully conserved in SARS-Cov-2, and four are partially conserved (Huang et al., 2020b). Analysis of the receptor-binding motif (RBM), a portion of RBD making direct contacts with ACE-2 of SARS-CoV and SARS-CoV-2, revealed that most residues essential for ACE-2 binding in the SARS-CoV S protein are conserved in the SARS-CoV-2 S protein (Huang et al., 2020b). Accordingly, In some embodiments, agents that bind SARS-CoV S protein are likely to also bind SARS-CoV-2 S protein.
3. COVID-19
[0057] Most COVID-19 patients have mild to moderate symptoms, but a subgroup may become severely ill. Common symptoms of a person infected with SARS-CoV-2 include fever, cough, nasal congestion, shortness of breath, and dyspnea. In more severe cases, the infection can cause pneumonia, sepsis, acute respiratory distress syndrome (ARDS), kidney failure, cardiac issues, and death due to multiple organ failure. Factors associated with ICU admission and death include older age, comorbid conditions, elevated body mass index, lymphopenia, and elevated transaminases, LDH, D-dimer, ferritin, and soluble IL-2 receptor (sIL-2R). These symptoms may be associated with a cytokine storm syndrome, in which hyperinflammation and multi-organ disease arise through excessive cytokine release from uncontrolled immune activation.
[0058] The pathophysiology of SARS-CoV-2-induced ARDS may have similarities to that of severe community-acquired pneumonia caused by other viruses. The overproduction of early response proinflammatory cytokines (tumour necrosis factor (TNF), IL-6, and IL- ip) may mediate a cytokine storm, leading to an increased risk of vascular hyperpermeability, multiorgan failure, and eventually death when the high cytokine concentrations are unabated over time. In addition to the respiratory symptoms, COVID-19 may also been linked to cardiovascular complications, with a high inflammatory burden that can induce vascular inflammation, myocarditis, and cardiac arrhythmias (Madjid et al,. 2020).
[0059] Biochemically, severe COVID-19 symptoms may be marked by hypoalbuminemia, lymphopenia, decreased percentage of lymphocytes and neutrophils, elevated C-reactive protein (CRP) and lactate dehydrogenase (LDH), and decreased CD8 count. The viral load of SARS-CoV-2 detected from patient respiratory tracts may be positively linked to lung disease severity. Albumin, lymphopenia, lymphocyte (%), LDH, neutrophil (%), and CRP levels are highly correlated to acute lung injury (Liu et al., 2020). Age, viral load, lung injury score, and blood biochemistry indexes, albumin, CRP, lactate dehydrogenase, lympopenia lymphocyte (%), and neutrophil (%), may be predictors of disease severity. Angiotensin II level in the plasma sample from SARS-CoV-2 infected patients may be markedly elevated and linearly associated to viral load and lung injury (Liu et al., 2020).
[0060] No treatment or vaccine for COVID-19 has yet been approved. Poor outcomes in COVID-19 and other viral infections may correlate with clinical and laboratory features of cytokine storm syndrome. In some embodiments, targeted anti-inflammatory therapy may prevent or reduce severe immunopathology.
4. Natural active agents and extracts
[0061] Traditional medicinal plants have been used in response to a range of medical ailments, including viral infection. It is believed that 70- 80 % of the human population in developing countries utilise medicinal plants for their primary healthcare. A variety of phytochemicals derived from the plants have been shown to have antiviral effects in humans. Other phytochemicals have been shown to be immunomodulatory.
4.1 Tinospora cordi folia
[0062] In some embodiments, the immunomodulatory composition comprises an at least one active agent obtained from Tinospora cordifolia. In some embodiments, the immunomodulatory composition comprises a Tinospora cordifolia extract. [0063] T. cordifolia, also known also as Guduchi, Amritaballi, Brotowali, Chakralakshanika, or Gurcha, is a medicinal plant that is used to treat a broad range of medical ailments. T. cordifolia extract has been used in traditional medicine in response to a range of medical ailments including diabetes, high cholesterol, allergic rhinitis (hay fever), upset stomach, gout, lymphoma and other cancers, rheumatoid arthritis (RA), hepatitis, peptic ulcer disease (PUD), fever, gonorrhea, syphilis, and to boost the immune system. It is believed to possess antioxidant, antiseptic and analgesic and immunomodulatory properties. In some embodiments, the Tinospora cordifolia extract is selected from an extract selected from the group consisting of a leaf extract, a stem extract and a root extract.
[0064] In some embodiments, T. cordifolia extract has immunomodulatory effects. In some embodiments, T. cordifolia has antiviral effects. For example, oral administration of T. cordifolia stem aqueous extract has been shown to result in significant increase in the IFN-y, IL-2, IL-4, and IL-1 levels in the peripheral blood mononuclear cells (PBMCs) (p < 0.05) of chickens in the treatment groups following infection with Infectious Bursal Disease (IBD) virus (IBDV), a non-enveloped, double stranded RNA virus (Sachan et al., 2019). Infectious Bursal Disease (IBD) is an acute, highly contagious, and immunosuppressive disease caused by (IBDV). IBD particularly affects chicks of 3 to 6 weeks age and is of global economic importance. Oral administration of T. cordifolia stem aqueous extract significantly reduced the mortality rate in chickens infected with a very virulent IBDV (vvIBDV) strain (Sachan et al., 2019). Roots from T. cordifolia have been reported to possess protease inhibitor activity against HIV, with anti-HIV effects revealed by reduction in eosinophil count, stimulation of B lymphocytes, macrophages and polymorphonuclear leucocytes and hemoglobin percentage indicating a role in management of the disease (Saha and Ghosh, 2012).
[0065] An aqueous extract of T. cordifolia has also been shown to activate macrophages as well as mediate an antiviral affect against chicken infectious anemia virus (CIAV) infection in poultry (Latheef et al., 2017). Further, T. cordifolia extract supplementation in chicks may increase the Escherichia coli specific antibody titer and lymphocyte proliferation response in chicks infected with E. coli. Moreover, the roots of T. cordifolia may contain a protease inhibitor that inhibits HIV virus. The anti-HIV activity of T. cordifolia extract is reflected in the reduced number of eosinophil, on the other hand, the stimulation of B lymphocytes, macrophages, polymorphonuclear leucocytes and percentages of haemoglobin were observed in the study, therefore T. cordifolia extract for immune system management in the HIV patients shows promising potential (Saha et al., 2012). Phytochemicals from T. cordifolia are also reported be effective inhibitors of Influenza strain H1N1 viral proteins (Saikia et al., 2019).
[0066] Consumption of a T. cordifolia extract may increase expression of the proinflammatory cytokine IL-1. However, the increase of IL-1 is not significant and is inconsistent (Henderson et al., 2020). Further, IL-1 is known to facilitate a branch of immunity by inhibiting an immune-evasive viral replication (Orzalli et al, 2018). Interleukin-ip signaling in dendritic cells has been shown to cause the cells to release antiviral interferon (Aarreberg et al, 2018). T. cordifolia extract may stimulate the body to release cytokine IL-2 and IL-4 (Sachan et al, 2019). IL-2 and IL-4 may stimulate long-term immunity to modulate the immune system to stimulate B cells to mature and release antibody type IgG, and may also enhance the memory B cells and memory T cell response, which may provide long-term immunity against the virus (Rang et al., 2003). Although IL-4 is an antiinflammatory cytokine, nonetheless IL-4 also the stimulates the priming of B Cells (IgG antibody) (Chatterjee et al, 2014). Accordingly, a T. cordifolia extract may have useful immunomodulatory effects.
[0067] T. cordifolia extract contains a number of phytochemicals. In some embodiments, the at least one active agent obtained from Tinospora cordifolia is selected from the list consisting of terpenoids (eg. tinosporide, furanolactone diterpene, furanolactone clerodane diterpene, furanoid diterpene, tinosporaside, ecdysterone makisterone and several glucosides isolated as polyacetate, phenylpropene disaccharides cordifolioside A, B and C, cordifoliside D and E, tinocordioside, cordioside, palmatosides C and F, sesquiterpene glucoside tinocordifolioside, sesquiterpene tinocordifolin); alkaloids (eg. tinosporine, magnoflorine, berberine, choline, jatrorrhizine, 1,2-substituted pyrrolidine, jatrorrhizine, palmatine, beberine, tembeterine); lignans (eg. 3 (a, 4-dihydroxy- 3-methoxybenzyl)-4-(4- hydroxy-3-methoxybenzyl); steroids (eg. giloinsterol, (S), B-Sitosterol, 20a- Hydroxy ecdysone); and other phytochemicals including columbin, Corydine, giloin, tinosporan acetate, tinosporal acetate, tinosporidine, tinosporic acid, heptacosanol, octacosanol, sinapic acid, tinosponone, tinosporal, tinosporon, two phytoecdysones, and arabinogalactan. In some embodiments, the at least one active agent obtained from Tinospora cordifolia is selected from the list consisting of tinosporin, tinocordiside, arabinogalactan, isocolumbin and magnoflorine.
[0068] G1-4A, an arabinogalactan polysaccharide isolated from the stem of T. cordifolia may have immunomodulatory potential. Administration of G1-4A in mice may lead to splenomegaly and an increase in the numbers of T cells, B cells and macrophages (Raghu et al., 2009). This increase in spleen cellularity was believed to due to in vivo proliferation of lymphocytes and upregulation of anti- apoptotic genes. It may act by activating the B cells polyclonally, via an increase in CD69 expression in lymphocytes. It may have potential in protecting mice from septic shock by modulating pro-inflammatory cytokines (Raghu etal., 2009). TLR4 on B lymphocytes and macrophages may act as a receptor for G1-4A polysaccharide, activating these immune cells in a TLR4/MyD88 dependent manner (Gupta et al., 2017). Similarly, G1-4A may lead to enhanced antigen presentation from dendritic cells (DC), indicating it may be useful in maturing dendritic cells, and further activation of cytotoxic T cells (Pandey et al., 2012). It may also be a useful adjuvant in immunotherapy (Pandey et al., 2012).
[0069] Accordingly, In some embodiments, active agents from a T. cordifolia extract have immunomodulatory effects. Moreover, In some embodiments, some of the phytochemicals of T. cordifolia may have good binding energetics with the target proteins in SARS-CoV and/or SARS-CoV-2, as shown in Table 1. In some embodiments, the at least one active agent obtained from Tinospora cordifolia is selected from the phytochemicals listed in Table 1. Berberine, isocolumbin, magnoflorine and tinocordiside showed high binding efficacy against four key SARS-CoV-2 targets, that is, S glycoprotein, including the receptor binding domain, RNA dependent RNA polymerase and main protease (Sagar and Kuman, 2020). Tinocordiside and Isocolumbin showed IC50 value of < 1 pM against both main protease and S glycoprotein (Sagar and Kuman, 2020). In some embodiments, a T. cordifolia extract comprises a plurality of phytochemicals that have an antiviral effect. In some embodiments, the at least one active agent obtained from Tinospora cordifolia is selected from the list consisting of tinosporin, tinocordiside, arabinogalactan, isocolumbin and magnoflorine.
TABLE 1
Phytochemicals Derived from T. cordifolia and Potential Antiviral Applications and Binding Potential for SARS-CoV-2 or SARS-CoV Targets
Figure imgf000017_0001
Figure imgf000018_0001
[0070] Accordingly, In some embodiments, at least one of the active agents provided within T. cordifolia extract may be a useful antiviral agent. In some embodiments, an active agent provided within T. cordifolia extract is an antiviral agent for the treatment of coronaviruses, for example, SARS-CoV and/or SARS-CoV-2. In some embodiments, a T. cordifolia extract is a useful antiviral agent in the treatment of coronaviruses, for example, SARS-CoV and/or SARS- CoV-2. In some embodiments, the at least one active agents obtained from T. cordifolia is selected from the group consisting of corydine, cordioside, cordiofolioside A, tinosporin, berberine, syringin, larch arabinogalactan, Gl-4 arabinogalactan, magnoflorine, tinocordiside, and isocolumbin.
4.2Curcuminoids
[0071] In some embodiments, the immunomodulatory composition comprises a curcuminoid. A curcuminoid may be a linear diarylheptanoid such as curcumin (diferuloylmethane) or derivatives of curcumin, including bisdemethoxycurcumin, demethoxycurcumin and/or tetrahydrocurcumin. In some embodiments, the curcuminoid is selected form the group consisting of curcumin, bisdemethoxycurcumin, demethoxycurcumin and tetrahydrocurcumin. These compounds are natural phenols. Curcumin is a bright yellow chemical that provides turmeric its colour.
[0072] Studies indicate that curcumin may have anti-inflammatory antioxidant, and/or cardiovascular protective properties. Curcumin has been shown to inhibit acute vascular inflammation via the activation of heme oxygenase-1 (HO-1) expression (Xiao et al., 2018). Overexpression of HO-1 may inhibit atherosclerosis, inflammation, and oxidative stress (Xiao et al., 2018). As COVID-19 may be associated with a high inflammatory burden that can induce vascular inflammation, myocarditis, and cardiac arrhythmias, curcumin may mediate the reduction of cardiovascular symptoms of COVID-19 patients (Madjid et al., 2020). Curcumin has been found also as an HDAC inhibitor (Hassan et al.,
2019).
[0073] Curcumin has been reported to have antiviral activity against diverse viruses including dengue virus (serotype 2), herpes simplex virus, HIV, influenza (including an H5N1 strain), human papilloma virus (HPV), Hepatitis C virus (HCV), Zika virus and Chikungunya virus among others (Pandit and Latha,
2020). 30 pM curcumin has been shown to reduce the yield of influenza virus by over 90% in cell culture (Chen et al., 2009). Curcumin may inhibit SARS-CoV replication, possibly by binding to the SARS-CoV 3-chymotrypsin-like protease (3CL protease or 3CLpro) (Barnard and Kumaki, 2011). The 3CLpro of SARS-CoV-2 (encoded by NSP-5) is an interest as a target for antiviral therapy (Suwannarach et al., 2020). Curcumin has been shown to have significant binding activity with the SARS-Cov-2 spike glycoprotein (Maurya et al., 2020). Accordingly, In some embodiments, curcumin may be a useful antiviral agent. In some embodiments, curcumin may be a useful antiviral agent in the treatment of coronaviruses, for example, SARS-CoV and/or SARS-CoV-2.
4.3 Curcuma SOD.
[0074] Many phytochemical studies on the extracts and essential oils of several Curcuma species have identified curcuminoids and sesquiterpenoids as the major active components, and these compounds have been identified as the major groups of antioxidants in the plants. In some embodiments, curcumin is produced by plants such as Curcuma spp. including Curcuma longa, Curcuma xanthorrhiza, Curcuma domestica Curcuma zedoaria, Curcuma aromatica, Curcuma raktakanta, Curcuma ecalcarata, etc. In some embodiments, the curcuminoid is provided by a Curcuma spp. extract. In some embodiments, the curcuminoid is provided by a Curcuma xanthorrhiza extract. C. xanthorrhiza is also known as Curcuma zanthorrhiza, temulawak, Java ginger, Javanese ginger, or Javanese turmeric. In some embodiments, the curcuminoid is provided by a Curcuma longa extract. In some embodiments, the immunomodulatory composition comprises a Curcuma spp. tuber (that is, root) extract.
[0075] C. xanthorrhiza is believed to possess antidiuretic, antiinflammatory, antioxidant, antihypertensive, antihepatotoxic, antibacterial, and antifungal effects. Phytochemicals found in C. xanthorrhiza include including xanthorrhizol and curcumene (including Ar-Curcumene, 0-Curcumene, y- Curcumene) all of which are bisabolene-type sesquiterpenoids; and a number of curcuminoids selected from the group consisting of curcumin, bisdemethoxycurcumin, demethoxycurcumin and tetra hydrocurcumin. The presence of xanthorrhizol in C. xanthorrhiza differentiates this plant from other Curcuma species. Xanthorrhizol has been reported to encompass a wide range of biological activities such as antibacterial, antiseptic, and antibiotic, and is a strong antioxidant (Jantan et al., 2012).
[0076] In some embodiments, a C. xanthorrhiza extract comprises a plurality of phytochemicals that have an antiviral effect. In some embodiments, some of the phytochemicals found in C. xanthorrhiza extract have good binding energetics with the target proteins in SARS-CoV and/or SARS-CoV-2, as shown in Table 2. In some embodiments, the at least one active agent obtained from the C. xanthorrhiza extract is selected from the phytochemicals listed in Table 2.
TABLE 2
Phytochemicals Derived From C. xanthorrhiza and Potential Antiviral Applications and Binding Potential for SARS-CoV-2 or SARS-CoV Targets
Figure imgf000021_0001
[0077] Accordingly, In some embodiments, at least one of the active agents provided within Curcuma spp. extract, such as curcumin or derivatives of curcumin, may be a useful antiviral agent. In some embodiments, at least one active agent provided within Curcuma spp. extract is an antiviral agent for the treatment of coronaviruses, for example, SARS-CoV and/or SARS-CoV-2. In some embodiments, a Curcuma spp. extract is an antiviral agent for the treatment of coronaviruses, for example, SARS-CoV and/or SARS-CoV-2. In some embodiments, the Curcuma spp. is Curcuma xanthorrhiza.
4.4 Moringa SDD.
[0078] In some embodiments, the immunomodulatory composition comprises a Moringa spp. extract. In some embodiments, the moringa extract is a Moringa oleifera extract. Moringa oleifera is also known as kelor, horseradish tree, Mulangay, Mlonge, Benzolive, Drumstick tree, Sajna, Kelor, Saijihan and Marango.
[0079] A Moringa extract may be obtained from the leaves, stems, seeds, root bark, flower and/or pods of moringa. Accordingly, In some embodiments, the Moringa extract is selected from the group consisting of a leaf extract, a seed extract, a root bark extract, a flower extract and a pod extract. In some embodiments, the immunomodulatory composition comprises a Moringa spp. leaf extract.
[0080] Moringa is high in essential amino acids, iron, zinc, Vitamin C (ascorbic acid), carotenoids (including p-carotene and vitamin A), calcium, potassium and protein (Gopalakrishnan et al., 2016; Shija et al., 2019; Sena et al., 1998). Moringa also contains vitamin Bl, Vitamin B2, Vitamin B3, folic acid, pyridoxine and nicotinic acid, vitamin D and/or vitamin E, phosphorous, copper, magnesium and sulphur (Gopalakrishnan et al., 2016). For example, a Moringa oleifera leaf extract was found to contain 25.5 ± 2.6 mg zinc and 28.7 ± 2.8 mg iron per 100 gram dried moringa leaf (Barminas et al., 1998).
[0081] An aqueous leaf extract of Moringa oleifera has been shown to have an antiviral effect against Hepatitis B virus (HBV) in Huh7 cells in vitro, with reduced HBV antigen secreted into the supernatant of Huh7 following treatment (Feustel et al., 2017). It has been reported that Moringa oleifera extract has an antiviral effect on Foot and Mouth Disease virus (FMDV), a picornavirus (Younus et al., 2015) and influenza virus (Ashraf et al., 2017). In some embodiments, the beneficial effects include an antioxidant effect, an antiviral effect and/or an antiinflammatory effect. In some embodiments, at least one of the phytochemicals found in Moringa extract may have good binding energetics with the target proteins in SARS-CoV-2, as shown in Table 3. In some embodiments, the at least one active agent obtained from the Moringa extract is selected from the phytochemicals listed in Table 3
TABLE 3
Phytochemicals Derived From M. oleifera and Potential Antiviral Applications and Binding Potential for SARS-CoV-2 or SARS-CoV Targets
Figure imgf000022_0001
Figure imgf000023_0001
[0082] Accordingly, In some embodiments, at least one active agents provided within a Moringa extract may be a useful antiviral agent. In some embodiments, an active agent provided within a Moringa extract is an antiviral agent for the treatment of coronaviruses, for example, SARS-CoV and/or SARS- CoV-2. In some embodiments, a moringa extract has an antiviral effect. In some embodiments, the at least one active agents obtained from moringa extract is selected from the group consisting of bioactive zinc, bioactive iron, ascorbic acid, Gallic acid, Catechin, Chlorogenic acid, Ellagic acid, Epicatechin, Rutin, Isoquercitrin, Quercetin, Kaempferol, Astragalin, and isothiocyanate. In some embodiments, a Moringa extract comprises a plurality of phytochemicals that have an antiviral effect.
4.5 Emodin
[0083] In some embodiments, the immunomodulatory composition comprises an emodin. Emodin may be isolated from a number of plants including rhubarb, buckthorn, Japanese knotwood (Reynoutria japonica synonym Polygonum cuspidatum}, many species of fungi, including members of the genera Aspergillus, Pyrenochaeta, and Pestalotiopsis, Acalypha australis, Cassia occidentalis, Cassia siamea, Frangula alnus, Glossostemon bruguieri, Kalimeris indica, Polygonum hypoleucum, Reynoutria japonica (synonym Fallopia japonica, or Polygonum cuspidatum), Rhamnus alnifolia, the alderleaf buckthorn, Rhamnus cathartica, the common buckthorn, Rheum palmatum, Rumex nepalensis, Senna obtusifolia (synonym Cassia obtusifolia), Thielavia subthermophila, Ventilago madraspatana.
[0084] In some embodiments, the emodin is an aloe-emodin. Aloe emodin is an anthraquinone compound found in Aloe vera and other species of the Asphodelaceae and the Polygonaceae families. Aloe-emodin is found in the gel, sap or leaves of aloe vera, the socotrine aloe, Barbados aloe, and Zanzibar aloes, the bark of Frangula Rhamnus frangula and cascara sagrada Rhamnus purshiana}, the leaves of Senna (Cassia angusti folia}, and the rhizome of rhubarb (Rheum rhaponticum}. [0085] An emodin is a phytochemical that is believed to have antiviral, antibacterial and/or anti-inflammatory effects. An emodin, an anthraquinone, has also been implicated in cardiac protection. In some embodiments, an emodin is an antiviral agent. For example, emodin obtained from a plant extract has been shown to inactivate enveloped virus (Sydiskis et al., 1991). Emodin isolated from Rheum palmatum has been shown to have potent inhibitory effects against Coxsackie virus (CV) and respiratory syncytial virus (RSV) in cell culture (Liu et al., 2015). Studies indicate that aloe-emodin induced interferon (IFN) in cell culture and upregulated an IFN regulated antiviral response against Japanese encephalitis virus (JEV) and enterovirus 71 (EV71) (Lin et al, 2008). Accordingly, in some embodiments, emodin is an immunomodulatory agent. Anthraquinone derivatives aloe-emodin, emodin and chrysophanol have been shown to exhibit antiviral activity against highly pathogenic influenza A viruses H7N9 and H5N1 (Li et al., 2014). The antiviral activity of aloe-emodin against influenza A virus is via the up-regulation of galectin-3 (Li et al, 2014). Moreover, emodin derived from genus Rheum and Polygonum has been shown to blocks the interaction between SARS coronavirus spike protein of SARS-Cov and angiotensin-converting enzyme in a dose-dependent manner (Ho et al. 2007). Emodin may inhibit the activity of histone deacetylase (HDAC); the inhibition of histone deacetylase (HDAC) enzymes may attenuate pathological cardiac hypertrophy in vitro and in vivo. (Evans et al., 2020), in a similar manner to curcumin. Accordingly, In some embodiments, an emodin is an antiviral agent. In some embodiments, an emodin is an antiviral agent for the treatment of coronaviruses, for example, SARS-CoV and/or SARS-CoV-2.
4.6Aloe
[0086] In some embodiments, the emodin is provided by an aloe extract. In some embodiments, the aloe extract is a stem extract. In some embodiments, the aloe extract is an Aloe barbadensis extract. A. barbadensis is also known as Aloe vera. A. barbadensis has been used medicinally for healing and therapeutic purposes. The biologically active components of an aloe extract include anthraquinones, anthrones, chromanes, alkaloids, flavonoids, terpenes, minerals, carbohydrates and pyrans. Aloe-emodin levels in plant extracts and commercial formulations can be determined by HPLC with tandem UV absorption and fluorescence detection, as described in Mandrioli et al., 2011. An Aloe vera extract has been found to be have an antiviral effect against a broad range of viruses, especially causing the infections of the upper respiratory tract (Pandit et al., 2020). Anthraquinones isolated from Aloe vera has shown to inhibit herpes simplex virus-2, hepatitis virus, Influenza virus (including Influenza A) and HIV (Pandit et al., 2020). It has also been reported that consumption of Aloe vera might be helpful to HIV-infected individuals since it enhances the CD4 count and thereby improves the functioning of the immune system. [0087] In some embodiments, an aloe extract comprises a plurality of phytochemicals that may have an antiviral effect. In some embodiments, at least one of the phytochemicals found in Aloe barbadensis extract may have good binding energetics with the target proteins in SARS-CoV-2, as shown in Table 4. In some embodiments, the at least one active agent obtained from the Aloe extract is selected from the phytochemicals listed in Table 4.
TABLE 4
Phytochemicals derived from A. barbadensis and Potential Antiviral Applications and Binding Potential for SARS-CoV-2 or SARS-CoV Targets
Figure imgf000025_0001
Rhein | | 3CLPro | Farabi et a/., 2008 |
[0088] Accordingly, In some embodiments, at least one active agents provided within A. barbadensis extract may be a useful antiviral agent. In some embodiments, an active agent provided within A. barbadensis extract is an antiviral agent for the treatment of coronaviruses, for example, SARS-CoV and/or SARS-CoV-2. In some embodiments, an A. barbadensis extract has an antiviral effect. In some embodiments, a A. barbadensis extract comprises a plurality of phytochemicals that have an antiviral effect.
4.7 Albumin
[0089] A significant reduction of serum albumin levels and an increase in C-reactive protein may be associated with inflammation. In human adults, albumin is the most abundant plasma protein with a concentration ranging from 35 to 50 g/L. Albumin production may be inhibited by proinflammatory mediators such as IL-1, IL-6 and TNF. In some embodiments, hypoalbuminemia is defined as a serum albumin concentration <35 g/l. In some embodiments, hypoalbuminemia is defined as a serum albumin concentration <30 g/l. In some embodiments, hypoalbuminemia is defined as a serum albumin concentration <25 g/l. Critical illness and/or mortality may be associated with hypoalbuminemia in a wide range of diseases. For example, a decrease in the serum albumin concentration is found in sepsis and septic shock (Gounden et al., 2020), which may be mediated by reduced synthesis/production of serum albumin, an increase in utilisation of albumin within the patient, and/or an increase in transcapillary leak from blood vessels associated with an increase of vascular permeability.
[0090] Albumin is a carrier molecule within blood that may bind and transport iron and copper ions, and/or other endogenous and exogenous substances, such as hormones, vitamins (e.g., Vitamin D), folate, fatty acids, arachidonic acid, glucose, nitric oxide, electrolytes (e.g., Calcium, Magnesium), cortisol, thyroxine, amino acids, enzymes, bilirubin and/or various drugs (NSAIDs, sedatives, anti-epileptic, digoxin, anticoagulants, and/or antibiotics) (Vincent et al., 2014). Serum albumin is also a zinc carrier, with most blood plasma zinc bound with albumin (Mustafa et al., 2012), Lu et al., 2008), Handing et al., 2016; Blaundauer et al., 2009). Serum albumin may also be involved in maintaining intravascular volume. For example, albumin may be involved in the endothelial surface layer and associated with maintaining vascular barrier competence. Albumin may act as an antioxidant, free radical scavenger, able to interact with or trap reactive oxygen or nitrogen species, including nitric oxide, a key mediator in many conditions including sepsis. Albumin may also have anticoagulant effects. Changes in albumin concentrations and structure during critical illness can therefore potentially have marked effects on normal homeostasis and metabolism and on drug delivery and efficacy. Maintaining serum albumin levels in the blood during inflammation may be beneficial in that it may facilitate effective transport of substances to cells, and/or to assist with the effects of inflammation.
[0091] A number of studies have indicated that serum albumin level is decreased during viral infection. For example, a decrease in albumin in early Human Immunodeficiency Virus (HIV) type 1 infection in human adults has been shown to predict subsequent disease progression and/or survival (Mehta et al., 2006; Graham et al., 2007; Feldman et al., 2000; Feldman et al., 2003); and a decrease in albumin in both early and chronic Simian Immunodeficiency Virus (SIV) infection in pig-tailed macaques is related to viral pathogenicity (Graham et al., 2009); lowered serum albumin levels independently predict the need for intensive respiratory or vasopressor support (IRVS) in patients with 2009 H1N1 influenza A (Wi and Peck, 2014). Significantly decreased albumin levels have been observed in severe COVID-19 (Zhou et al., 2020; Zhang et al, 2020) and low serum albumin levels have been independently associated with mortality in COVID-19 (Violi et al., 2020) or independently predictive for mortality in COVID-19.
[0092] Albumin therapy may be useful in the treatment of a number of serious conditions. Studies indicate that orally delivered albumin increases serum albumin levels (Rosyidi et al., 2019; Mustafa et al., 2012). In some embodiments, albumin therapy may be useful to treat viral infection including COVID-19 (Huang et al., 2020; Liu et al., 2020). Albumin downregulates the expression of the ACE2 receptors (Liu et al., 2009) and has been shown to improve the ratio of arterial partial pressure of oxygen/fraction of inspired oxygen in patients with acute respiratory distress syndrome as soon as 24 hours after treatment and with an effect that persisted for at least seven days (Uhlig et al., 2014).
[0093] In some embodiments, the immunomodulatory composition comprises an albumin. In some embodiments, the albumin is provided by a plant and/or animal extract. More than 60% of the protein fraction of the Channa striata (snakehead fish) extract comprises albumin (Rosyidi et al., 2019), accordingly snakehead fish extract is an excellent source of albumin. Rosyidi et al. (2019) found that in neurosurgery patients treated with two capsules of snakehead extract three times per day for up to three weeks post-surgery, serum albumin levels increased significantly. Oral administration of snakehead extract obtained from 2 kg of snakehead fish daily for five consecutive days in patients with hypoalbuminemia has been shown to increase serum albumin level from 1.8 g/lOOmL to normal condition, > 3.5 g/100 mL (Mustafa et al., 2012).
[0094] In some embodiments, albumin is provided by egg whites.
4.8Channa
[0095] In some embodiments, the albumin is provided by a snakehead fish (e.g., Channa striata) extract. In some embodiments, the snakehead fish extract is a flesh extract. The snakehead fish is also known as "ikan gabus".
[0096] 100 mL of snakehead fish extract has been found to contain 3.36
± 0.29 g protein, 2.17 ± 0.14 g albumin, 0.77 ± 0.66 g total fat, total Glucose 70 ± 20 mg, zinc 3.34 ± 0.8 mg; Cu 2.34 ± 0.98 mg and Fe 0.20 ± 0.09 mg (Mustafa et al., 2012). Snakehead fish extract is accordingly rich in zinc, copper and iron, and is also an excellent source of protein and amino acids including essential amino acids (Rosmawati et al., 2018; Gam et al., 2006).
[0097] Albumin may be provided by any species of the Channa genus. Suitable species include C. micropeltes, C. lucius, C. gachua, C. bankanensis, C. cyanospilos, C. marulioides, C. melanoptera, C. melasoma, C. pleurophthalma, and C. argus. In some embodiments, the albumin is provided by a Channa striata extract.
4.9 Zinc
[0098] Zinc is an essential trace element that assists effective immune function. Zinc has direct antiviral activities against a variety of viruses and enhances antiviral immunity (Read et al., 2019). For example, zinc has been shown to mediate direct antiviral effects against influenza and assists in generating both innate and acquired (humoral) antiviral responses. Studies indicate that zinc plays an inhibitory role on almost every aspect of herpes simplex virus (HSV)-l and HSV-2 viral life cycle including viral polymerase function, protein production and processing, and free virus inactivation, corresponding to a significant reduction of HSV infection and disease burden (Read et al., 2019). Zinc is known to mediate antiviral activity against rhinovirus and shorten the duration rhinovirus infection. Additionally, studies indicate that zinc may interrupt the activity of SARS-CoV RdRP protein (te Velthuis et al., 2010). Accordingly, In some embodiments, zinc is an antiviral agent. In some embodiments, the immunomodulatory composition comprises bioavailable zinc.
[0099] Bioavailable zinc may be provided by any suitable source. In some embodiments, the bioavailable zinc is provided by a plant or animal extract. In some embodiments, the bioavailable zinc is provided by a moringa extract. In some embodiments, the bioavailable zinc is provided by a Channa striata (snakehead fish) extract. In some embodiments, the bioavailable zinc is provided by a moringa extract and a Channa striata (snakehead fish) extract.
4.10 Iron
[0100] In some embodiments, curcumin may chelate iron. Accordingly, In some embodiments, the immunomodulatory composition may further comprise an agent comprising bioavailable iron. Bioavailable iron may be provided by any suitable source. A morninga extract is high in iron. Accordingly, In some embodiments, inclusion of a moringa extract in the immunomodulatory composition may also counteract at least some of the iron chelating activity of curcumin. In some embodiments, the bioavailable iron is provided by a moringa extract. In some embodiments, the bioavailable iron is provided by a snakehead fish extract. In some embodiments, the bioavailable iron is provided by a moringa extract and a snakehead fish extract. In an alternative embodiment, bioavailable iron could be provided by other known commercial sources and/or alternative natural extracts known to have high levels of bioavailable iron such as Fenugreek (Trigonella foenum-graecum), Jute (Corchorus olitorius), Common Guava (Psidium guajava'), Spinach Spinacia oleracea), leaf of winged bean (Psophocarpus tetragonolobus), morel mushroom / true Mmrels (Morchella), Amaranthus spinosus, Cajanus cajan, Hoslundia opposite, Imperata cylindrical, Justicia secunda, Khaya senegalensis, Milicia excels, Ricinus communis, Stylosanthes erecta, Tectona grandis, Thalia geniculate, animal livers, sardines, kangaroo, and/or beef etc.
4.11 Glutathione
[0101] Glutathione (GSH) is an antioxidant found in plants, animals, fungi, and some bacteria and archaea. GSH, including L-Glutathione, can also be ontained from commercial sources (e.g., LUCKERKONG Biotech Co., Ltd; Guangdong, China). Glutathione defends against oxidative damage of cells from reactive oxygen species (ROS) and is also involved in the regulation of various metabolic pathways essential for whole body homeostasis. Several studies indicate that higher levels of glutathione may improve an individual's responsiveness to viral infections. Glutathione may protect host immune cells through its antioxidant mechanism and mediate optimal functioning of a variety of cells that are part of the immune system. Evidence indicates that glutathione inhibits replication of various viruses at different stages of the viral life cycle, and this antiviral property of GSH seems to prevent increased viral loads and the subsequent massive release of inflammatory cells into the lung mediated by a "cytokine storm".
[0102] A six month preventive administration of N-acetylcysteine (NAC, glutathione precursor) significantly reduced the incidence of clinically apparent influenza and influenza-like episodes, especially in elderly high-risk individuals (De Flora et al., 1997). Accordingly, In some embodiments, glutathione is an antiviral agent. In addition, pathophysiological conditions such as lung cell injury and inflammation in patients with severe ARDS were identified as the targets of NAC treatment. In particular, the deficiency of reduced glutathione in the alveolar fluid in ARDS patients was found to enhance lung cell injury by ROS/oxidative stress and inflammation, and this damage could be effectively prevented and treated by the administration of NAC. Moreover, glutathione deficiency is the postulated to mediate serious manifestation and death in COVID-19 patients (Polonikov, 2020). In some embodiments, the immunomodulatory composition comprises glutathione.
[0103] In some preferred embodiments of the invention, the immunomodulatory agent is an antiviral agent.
5. Antiviral combination
[0104] The present inventors have realized that an immunomodulatory composition comprising a combination of natural agents may offer therapeutic antiviral effects during a viral infection. In some embodiments, some active agents within natural extracts mediate an enhanced antiviral response within a subject suffering from a viral infection, for example, by mediating immunomodulation of the host immune response. Some active agents stimulate a cytokine rhythm that assists with recovery from viral infection. In some embodiments, the antiviral combination comprises at least one active agent that that down-regulates viral replication. In some embodiments, the antiviral combination comprises at least one active agent that enhances the antiviral immune response of a subject suffering from a viral infection. In some embodiments, the antiviral combination comprises at least one active agent that binds with viral components and down- regulates viral replication. In some embodiments, the antiviral combination enhances the antiviral immune response of a subject suffering from a viral infection and comprises at least one active agent that binds with viral components that inhibits viral replication.
[0105] Some of these active agents are broadly antiviral against a range of viruses, whereas others are specific for a particular virus or virus family. Moreover, the SARS-CoV and SARS-Cov-2 viruses have a high degree of similarity, and both bind to the same host receptor, ACE-2, to gain entry into the subject's cells. Accordingly, an agent that is active against SARS-CoV may also be active against SARS-CoV-2.
[0106] In some embodiments, the immunomodulatory composition of the present disclosure may have at least one of the following benefits: Increasing serum albumin by oral consumption of an albumin containing extract (e.g., from snakehead fish); modulating cytokine levels and balance; increasing the distribution capability of the body to deliver nutrition mediated by active agents of plant extracts (e.g., C. xanthorrhiza, T. cordifolia and aloe-emodin); and increasing capacity of mineral distribution in the body, particularly zinc. In some embodiments, cytokine balance is enhanced, and the inflammation becomes a moderate, appropriate antiviral inflammation. Additionally, In some embodiments, access of SARS-CoV-2 virus into the cells, particularly the lung cells is reduced by the use of ACE-2 blockers (e.g., aloe vera extract / Emodin), TMPRSS2 down regulator (C. xanthorrhiza extract / Curcumin). Moreover, the immunomodulatory composition slows down the replication of SARS-CoV-2 virus in the human body via the interruption of 3CL Protease (encoded by NSP-5) by C. xanthorrhiza extract/Curcumin, and/or interruption of RdRP (encoded by NSP-12) by zinc from snakehead and/or dried moringa leaf extracts.
[0107] In some embodiments, a T. cordifolia extract within the immunomodulatory composition modulates the immune system toward the adaptive immune response route. This may enhance antibody production, and/or stimulate the maturity of memory T and B cells. In turn, this may mediate longterm immunity to the viral infection.
[0108] In an aspect, the immunomodulatory composition of the present disclosure comprises a combination of agents consisting of a curcuminoid, an emodin, and bioavailable zinc. In some embodiments, the immunomodulatory composition of the present disclosure comprising a combination of agents consisting of a curcuminoid, an emodin, bioavailable zinc and an at least one active agent obtained from Tinospora cordifolia. In some embodiments, the at least one active agent obtained from Tinospora cordifolia is selected from the list consisting of Corydine, Cordioside, Cordiofolioside A, Tinosporin, Berberine, Syringin, Larch arabinogalactan, Gl-4 arabinogalactan, Magnoflorine, Tinocordiside, and Isocolumbin, any of which can be extracted from Tionospora cordifolia using standard techniques known to those skilled in the art. In some embodiments, the immunomodulatory composition further comprises an agent selected from the list consisting of an albumin, a glutathione, ascorbic acid and bioavailable iron.
[0109] In some embodiments, at least one of the agents are provided as extracts derived from plants and/or animals. In some embodiments, the at least one active agent obtained from T. cordifolia is provided by a Tinospora cordifolia extract. In some embodiments, the curcuminoid is provided by a Curcuma spp. extract In some embodiments, the curcuminoid is provided by a Curcuma xanthorrhiza extract. In some embodiments, the emodin is provided by an aloe extract. In some embodiments, the emodin is provided by an Aloe barbadensis extract. In some embodiments, the bioavailable zinc is provided by a moringa extract. In some embodiments, the bioavailable zinc is provided by a Channa striata (snakehead fish) extract. In some embodiments, the bioavailable zinc is provided by a moringa oleifera extract and a Channa striata (snakehead fish) extract. In some embodiments, the albumin is provided by a Channa striata (snakehead fish) extract. In some embodiments, glutathione is provided as L- glutathione by a commercial source, e.g., LUCKERKONG Biotech Co., Ltd, China. In some embodiments, the ascorbic acid is provided by a moringa oleifera extract. In some embodiments, the bioavailable iron is provided by a moringa extract. In some embodiments, the bioavailable iron is provided by a Channa striata (snakehead fish) extract. In some embodiments, the bioavailable iron is provided by a moringa oleifera extract and a Channa striata (snakehead fish) extract.
[0110] In an aspect, the immunomodulatory composition of the present disclosure comprises a combination of agents consisting of a Tinospora cordifolia extract, a Curcuma xanthorrhiza extract, an aloe extract and a moringa extract. In some embodiments, the percentage of each of the extracts within the immunomodulatory composition may vary. For example, In some embodiments, the Tinospora cordifolia extract may comprise between 0.1% and 15% of the immunomodulatory composition, or between 1% and 12%, or between 2% and 12%, or between 3% and 5% of the antiviral combination, etc. In some embodiments, the Curcuma xanthorrhiza extract may comprise between 5% and 40% of the immunomodulatory composition, or between 10% and 30%, or between 12% and 25%, or between 15% and 22% of the antiviral combination, etc. In some embodiments, the aloe extract may comprise between 10% and 60% of the immunomodulatory composition, or between 20% and 50%, or between 25% and 45%, or between 30% and 40% of the antiviral combination, etc. In some embodiments, the Moringa extract may comprise between 10% and 60% of the immunomodulatory composition, or between 20% and 50%, or between 25% and 45%, or between 30% and 40% of the antiviral combination, etc. In some embodiments, the antiviral combination comprises 38.46% Moringa extract, 3.84% T. Cordifolia extract, 38.46% Aloe vera extract, and 19.23% Curcuma xanthorrhiza extract. In some embodiments, the combined dried extracts are packed into sachets containing 1000 mg Moringa extract, 100 mg T. Cordifolia extract, 1000 mg Aloe vera extract, 500 mg Curcuma xanthorrhiza extract. However, persons skilled in the art will appreciate that other ratios of the extracts may fall within the scope of the invention.
[Olli] The plant and/or animal extracts may be extracted from raw material using any suitable extraction method providing the resulting extract is suitable for oral delivery.
[0112] In some embodiments, the immunomodulatory composition of the present disclosure further comprises a Channa striata (snakehead fish) extract. In an aspect, the immunomodulatory composition of the present disclosure comprises a combination of agents consisting of a Tinospora cordifolia extract, a Curcuma xanthorrhiza extract, an aloe extract, a moringa extract and a Channa striata (snakehead fish) extract. In some embodiments, the antiviral combination comprises 32.5% Moringa extract, 3.22% T. Cordifolia extract, 32.25% Aloe vera extract, 16.13% Curcuma xanthorrhiza extract, and 16.13% Channa striata extract. In some embodiments, the Channa striata extract may comprise between 5% and 40% of the immunomodulatory composition, or between 10% and 30%, or between 12% and 25%, or between 15% and 20% of the antiviral combination, etc. In some embodiments, the combined dried extracts are packed into sachets containing 1000 mg Moringa extract, 100 mg T. Cordifolia extract, 1000 mg Aloe vera extract, 500 mg Curcuma xanthorrhiza extract, and 500 mg Channa striata extract. However, persons skilled in the art will appreciate that other ratios of the extracts may fall within the scope of the invention.
6. Extraction and Formulations
[0113] The extracts of the present disclosure can be extracted from the raw materials using any suitable extraction technique known to persons skilled in the art, providing the resulting extract is suitable for oral delivery. Such extraction methods may be selected from supercritical carbon dioxide extraction, aqueous extraction, alcohol extraction, solvent extraction, distillation method, pressing and sublimation according to the extraction principle etc, using methods known to those skilled in the art. In some embodiments, the extraction method is supercritical carbon dioxide extraction. The immunomodulatory composition can be formulated to be in a liquid form, for example, a ready to drink product; or a powdered form, which can be formulated as a sachet, tablet, pill or capsule. Solid extract preparation before mixing will involve hammer mill grinder and sifter to obtain certain particle size. For tablet, pills, and capsules form may utilise a granulator to support the production. In some embodiments, the immunomodulatory composition is formulated for oral delivery
[0114] For liquid form product, the extracts may be mixed using standard tank with agitator according to the formulation. The liquid mixture may then be UHT system sterilized before bottling to accommodate long shelf life product.
[0115] Conveniently, single dosages can be provided in sachets such that the physician (or subject suffering from a viral infection) can readily administer a dose. One sachet can be dissolved in water, for example 150 mL of cold water. Associated with such dosages(s) can be various written materials such as instructions for use, or a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
[0116] The immunomodulatory composition of the present disclosure can be used in combination with other drug(s) or antiviral treatment(s). The components can be administered in the same formulation or in separate formulations. If administered in separate formulations, the immunomodulatory composition of the present disclosure may be administered sequentially or simultaneously with the other drug(s) or antiviral treatment(s). In some embodiments, the immunomodulatory composition may be combined with a filler, carrier, diluent and/or excipient that is suitable for oral administration.
[0117] The immunostimulatory composition of the present disclosure can be formulated with a number of pharmaceutically acceptable carriers, excipients and diluents, as well known in the art. For example, the composition may be formulated one or more of an anti-caking agent/stabilising agent, sweeter, polysaccharide, fibre, anti-oxidant, and/or flavouring agent.
[0118] Anti-caking agents/stabilisers are typically present in the formulation at a concentration of between about 0.1 to about 3.0% (w/w). Suitable anti-caking agents/stabilisers may be selected from the the group comprising silicon dioxide, dextrin, tricalcium phosphate, calcium phosphate, sodium bicarbonate, magnesium trisilicate, diatomaceous earth, lecithin, or a combination thereof. [0119] Sweeteners are typically present in the formulation at a concentreation of between about 0.1% and 3.0% (w/w). Suitable anti-caking agents/stabilisers may be selected from the the group comprising dextrose monohydrate, saccharine-Na, polyols (e.g., sucralose, sorbitol, maltitol, xylitol, erythritol), stevioside, inulin, monk fruit sweetener, or a combination thereof.
[0120] Polysaccharides and fibre are typically present in the fomaultion at a combined concentration of between about 0.1% and 35% (w/w). Suitable polysaccharides and fibre composnentes may be selected from the group comprising maltodextrin, resistant dextrin (e.g., wheat dextrin), collagen, polydextrose, gums, polyols, oligosaccharides and starches (e.g., tapioca starch, maize starch, potato starch, glucomannan), or a combination thereof.
[0121] Anti-oxidants are typically present in the formulation at a concentration of between about 0.1% and 6.0% (w/w). Suitable anti-oxidants may be selected from the group comprising glutathione, N -acetylcysteine, ascorbic acid, citric acid, lactic acid, malic acid, or a combination thereof.
[0122] Flavouring agents are typically present in the formulation at a concentration of between about 0.1% to 0.8% (or quantum suffic/at). Suitable flavours envisaged for use with the composition include apple, strawberry, raspberry, green apple, passionfruit, mangosteen, pomegranate, blueberry, orange, mandarin, peach, nectarine, plum, lemon, lemongrass, or a combination thereof.
[0123] In addition, in order to enhance stability, dissolving properties, and taste, additiona additives may be added, including glutathione (at around 0.02 g per 10 g dose), polydextrose (at around 1.93 g per 10 g dose), dextrose monohydrate (at around 3.15 g per 10 g dose), silicon dioxide/silicium dioxide (at around 0.23 g per 10 g dose), ascorbic acid (at around 0.48 g per 10 g dose), and fish collagen (at around 0.97 g per 10 g dose).
[0124] By way of an illustrative example, some suitable formulations of the invention are included in Table 5.
TABLE 5
Exemplary Formulations of the Immunomodulatory Composition
Figure imgf000035_0001
Figure imgf000036_0001
7. Dosage
[0125] In some embodiments, the moringa extract may be administered to subjects at a dose between about 0.2 g and 4 g per day, or between about 1 and 3 g per day, or between about 1.5 and 2.5 g per day. In some embodiments, the T. cordifolia extract may be administered to subjects at a dose between about 0.01 mg and 4 g per day, or between about 0.1 and 1 g per day, or between about 0.3 and 0.9 g per day, or between about 0.1 and 0.4 g per day, or between about 0.4 and 0.75 g per day. T. cordifolia extract is bitter and may be sweetened. In some embodiments, the T. cordifolia extract may be administered to subjects at a dose three times per day (e.g., 3 x lOOmg per day). Aloe vera extract may be administered to subjects at a dose between about 0.2 g and 5 g per day, or between about 0.5 to 4 g per day, or between about 1.0 g to 3 g per day, or between about 1.5 g to 2.0 g per day. In some embodiments, the Curcuma xanthorrhiza extract may be administered to subjects at a dose between 0.05 mg and 4 g per day, or between about 0.2 g and 3 g per day, or between about 0.5 g to 2 g per day, or between about 1.0 g to 1.5 g per day. In some embodiments, the Curcuminoid may be administered to subjects at a dose between about 0.05 mg and 4 g per day, or between about 0.2 g to 3 g per day, or between about 0.5 to 2 g per day, or between about 1.0 g to 1.5 g per day or between about 1.8 g to 2.2 g per day. In some embodiments, albumin from Channa striata (snakehead fish) extract may be administered to subjects at a dose between about 0.05 mg and 4 g per day, or between about 0.2 g to 3 g per day, between about 0.5 g to 2 g per day, or between about 1.0 g to 1.5 g per day, or between about 1.8 to 2.2 g per day.
[0126] In some embodiments, the antiviral combination may be administered once, twice, three, or four times daily. Accordingly, In some embodiments, the daily dose of the immunomodulatory composition is 1000 mg Moringa extract, 100 mg T. cordifolia extract, 1000 mg Aloe vera extract, 500 mg Curcuma xanthorrhiza extract, and optionally 500 mg Channa striata extract. In some embodiments, the daily dose of the immunomodulatory composition is 2000 mg Moringa extract, 200 mg T. cordifolia extract, 2000 mg Aloe vera extract, 1000 mg Curcuma xanthorrhiza extract, and optionally 1000 mg Channa striata extract. In some embodiments, the daily dose of the immunomodulatory composition is 3000 mg Moringa extract, 300 mg T. cordifolia extract, 3000 mg Aloe vera extract, 1500 mg Curcuma xanthorrhiza extract, and, optionally, 1500 mg Channa striata extract. In some embodiments, the daily dose of the immunomodulatory composition is 4000 mg Moringa extract, 400 mg T. cordifolia extract, 4000 mg Aloe vera extract, 2000 mg Curcuma xanthorrhiza extract, and optionally 2000 mg Channa striata extract. It is to be understood that dosages between those specified above also fall with the daily dosages of the present disclosure. In some embodiments, the Curcuma xanthorrhiza extract is at half the dosage indicated above, that is, each sachet would contain 1000 mg Moringa extract, 100 mg T. cordifolia extract, 1000 mg Aloe vera extract, 250 mg Curcuma xanthorrhiza extract, and optionally 500 mg Channa striata extract. In this case, the daily dose for the Curcuma xanthorrhiza extract would be 250 mg, 500 mg, 750 mg, 1000 mg, respectively, in the daily dose of the immunomodulatory composition described above.
8. Use of the antiviral combination
[0127] The method may be suitable for the treatment of non-human primates and other mammals such as livestock (including race horses), exotic animals (e.g., tigers and elephants) and companion animals (e.g., dogs and cats); however, typically the subject will be a human. In an aspect, the present disclosure is directed to the immunomodulatory composition when used to treat a viral infection. In an aspect, the present disclosure is directed to the use of the immunomodulatory composition in the manufacture of a medicament for the treatment of a viral infection. In an aspect, the present disclosure is directed to the use of the immunomodulatory composition to treat a viral infection. In an aspect, the present disclosure is directed to a method of treating a viral infection comprising administering to a to a subject in need thereof a therapeutically effective amount of the immunomodulatory composition. In some embodiments, the treatment is the oral administration of the antiviral combination of the present invention. The oral combination may be dissolved in water or sweetened beverages or similar. Multiple doses of the antiviral combination may be administered per day.
[0128] In some embodiments, the virus infection is a respiratory virus infection including influenza infection or coronavirus infection. In some embodiments, the virus is SARS-Cov or SARS-Cov-2. In some embodiments, the virus infection is COVID-19.
[0129] In order that the invention may be readily understood and put into practical effect, particular preferred embodiments will now be described by way of the following non-limiting examples.
EXAMPLES
EXAMPLE 1
IN VITRO TESTING
[0130] The immunomodulatory composition is tested in peripheral blood mononuclear cells (PBMCs) from healthy subjects cultured in Roswell Park Memorial Institute 1640 Medium (RPMI 1640) culture medium supplemented with Glutamax (commercially available from sources such as ThermoFisher Scientific) with IX PenStrep and 2.5 pg/mL Plasmocin (commercially available from sources such as InvivoGen) ± 10% heat inactivated fetal bovine serum (FBS), containing approximately 5 mg/mL bovine serum albumin (BSA; Francis, 2010). Initially, the PBMC were cultured in RPMI with fetal bovine serum (FBS). Then, the cells will cultured in RPMI supplemented with Gutamax, PenStrep and Plasmocin as above, but in the absence of FBS, in either the presence or absence of 5 mg/mL BSA. This will assess the impact of cell health and viability as well as observe the effects of the active ingredients of the extracts.
[0131] Extracts derived from supercritical extraction procedure described above are concentrated as follows. 250 g of each extract to be used is added to 1000 mL of 80% methanol (v/v) dissolved in Mill-Q (MQ)-H2O and stirred using a magnetic stirrer for 3 hours at room temperature in a sealed container. The solution is then filtered using Buchner funnel with Whatman filter paper No. l (125 mm). The filtrate is transferred to a round-bottom flask and connected to a rotary evaporator (Rotavap). The round bottom flask is submerged in a water bath at 50°C under vacuum. The concentrate is further dried using freeze drying method as above and re-ground to fine powder when required. The concentrated powder of each extract is dissolved in 100% dimethyl sulfoxide (DMSO) to produce a stock solution of 40 mg/mL of extracts for use in in vitro studies.
[0132] Briefly, extracts are reconcentrated in DMSO as described and filter sterilised. Final concentration of DMSO in experiments is no more than 0.5% (v/v). PBMCs are thawed and resuspended in culture medium (with or without BSA). 100 pL PBMC containing 2.5 x 105 cells are plated into 96-well plates (2.5 x 105 cells/well) and incubated overnight in 37°C in a 5% CO2 incubator (approximately 16-18 hours). Cells are washed, supernatant discarded and resuspended in 100 pL fresh culture medium (with or without BSA). 50 pL of each extract is added per well, at one of six different concentrations as shown in Table 6, tested in triplicate, and incubated at 37°C/5% CO2 for 30 mins (150 pl in each well), with DMSO vehicle negative controls. Cells are stimulated with 10 pl lipopolysaccharide (LPS) at 50 ng/mL in phosphate buffered saline (PBS) (final well volume 160 pL) and incubated at 37°C/5% CO2 for 24 hours to simulate inflammation in triplicate and compared to vehicle control (DMSO with no LPS or extract), LPS only control or LPS plus dexamethasone. 80 pL of each supernatants from triplicate test conditions are pooled (240 pL per condition). 120 pL of each condition extract concentrations is tested by a Human 45-Plex ProcartaPlex Panel (eBioScience; Cat # EPX450-12171-901) to analyse cytokine production in accordance with manufacturer's instructions, with each condition being compared to vehicle control, LPS control or LPS plus dexamethasone control. Remaining pooled supernatants are stored at -80°C for further analysis. CellTitre-Blue is used to assess cell viability in accordance with manufacturer's instructions. Briefly, CellTitre Blue reagent is added to cells remaining in 96 well plates and incubated for further 6 hours. Absorbance is read (570 nm; ref: 620 nm) at 1, 2, 4 and 6 hour timepoints. Viability of each condition compared to vehicle control, LPS control and LPS and dexamtheasone control.
TABLE 6
EXTRACTS TESTED - 4 PLANT EXTRACT COMBINATION
Figure imgf000039_0001
Figure imgf000040_0001
TABLE 7
EXTRACTS TESTED - 4 PLANT COMBINATION + SNAKEHEAD FISH EXTRACT
Figure imgf000040_0002
[0133] The PBMCs in this example will provide data relating to a change of production levels of various cytokines. It is expected that the extracts will produce a reduction in the level of a number of cytokines associated with inflammation, for example, interleukin (IL)-6, IL-18, Tumour Necrosis Factor (TNF), monocyte chemoattractant protein (MCP)-1/C-C motif ligand (CCL)2, monocyte inflammatory protein (MIP)-l alpha/CCL3, CCL5/ Regulated upon Activation, Normal T Cell Expressed and Presumably Secreted (RANTES), and/or granulocyte macrophage-colony stimulating factor (GM-CSF); and a moderate production of IL-ip and IL-2. Increased production of cytokines such as IL-10, IL- 4, interferon (IFN)-o, and IFN-y interleukin is anticipated. This would indicate that the extracts have antiviral properties. Asssessment of PBMCs in the presence of LPS and/or dexamethasone (an anti-inflammatory compound) will be investigated.
MATERIALS AND METHODS
[0134] The extracts of the present invention are sourced and prepared using the following methods.
Extract Sources
[0135] Extracts or raw materials are commercially sourced from a variety of suppliers such as PT. Tri Rahardja (Javaplant), Indonesia, PT. Indesso Aroma, Indonesia, PT. Insular Multi Natural, Indonesia, PT. Industri Jamu dan Farmasi Sido Muncul, Indonesia, PJ Herbal Nusantara, Indonesia, PT. Merpati Mahardika, Indonesia or Herbal Hills, India, in either dried, powder or liquid form.
Extract Preparation
[0136] Raw material from plant an animal matter for extraction were obtained as follows: T. cordifolia (obtained from PT. Insular Multi Natural, Indonesia, listed under alternative name Brotowali extract); Curcuma xanthorrhiza (obtained from PT. Industri Jamu dan Farmasi Sido Muncul, Indonesia, lised under alternative name Sari Temulawak Sido Muncul; Channa striata (obtained from PJ Herbal Nusantara, Indonesia, under name Channa); Aloe barbadensis (Aloe vera; obtained from PT. Merpati Mahardika, Indonesia, listed under alternative name Lidah Buaya extract; Moringa oleifera (obtained from PT. Industri Jamu dan Farmasi Sido Muncul, Indoneisa, listed under alternative name Sari Daun Kelor Sido Muncul).
Supercritical Extraction
[0137] Raw material including Moringa leaves, Curcuma xanthorrhiza tubers/roots, T. cordifolia leaves, stems and roots, Aloe vera stems and Channa striata flesh were obtained from commercial sources as described herein. For Curcuma xanthorrhiza, Moringa oleifera, and Tinospora cordyfolia, 1 kg of dried materials produced around 4% - 7% liquid extract. The raw materials individually were extracted using supercritical carbon dioxide extraction as follows. The dried plant material was ground to 50 mesh particle size using a commercial grinding machine. Then, the ground dried material was inserted into the supercritical material chamber of a commercial supercritical CO2 extractor. The temperature on the CO2 tanks was set to 30°C to reach 900 -1100 psi, and then the extraction operating timer was set for for 2 hours and 15 minutes with solenoid valve timer releasing every 2 seconds. Accordingly, every 2 seconds, the extract was collected in the collector chamber until the operation was completed.
[0138] For aloe vera, the same process was followed, except that the material was not dried and ground. The inner gel inside aloe vera leaves was sliced into approximately 3 mm thickness and continuouslyinserted into the supercritical material chamber of the supercritical CO2 extractor using the same operating procedure as above. A higher yield of 5 - 8% liquid aloe vera extract was collected.
[0139] For all plant extracts, the liquid extract was collected and freeze dried using a standard freeze dry chamber within 18 hours to 24 hours to produce a solid powder material.
[0140] For Channa striata, 1 kg of Channa Striata flesh produced approximately 5% of powder extract. The raw flesh was steamed using a commercial steamer in demineralized water with maximum 1 atm pressure at 70°C for 3 operating cycles. Approximately 200 mL of raw liquid extract was collected from 1 kg raw material. Then, the liquid extract was dried to a solid powder material using a the standard freeze dry chamber within 6 hours to 12 hours, prior to undergoing the supercritical carbon dioxide extraction as described above.
Formulation
[0141] To prepare the immunomodulatory composition, the solid powder extracts were obtained using the supercritical carbon dioxide extraction method described above or commercially obtained. The solid powder extracts were combined using a tumbler mixer until homogenous. The solid powder extracts were combined to form an immunomodulatory composition mixture combined in the following percentages:
• 4 extract combination: 38.46% Moringa extract, 3.84% T. Cordifolia extract, 38.46% Aloe vera extract, 19.23% Curcuma xanthorrhiza extract;
• 5 extract combination: 32.25% Moringa extract, 3.22%T. Cordifolia extract, 32.25% Aloe vera extract, 16.13% Curcuma xanthorrhiza extract, 16.13% Channa striata extract;
[0142] The mixture is milled to a preferred particle size using a hammer mill, for example, 180 - 200 mesh powder, and then sifted using an industrial sifter and dust collector. The combined dried extracts are packed into sachets containing:
• 4 extract combination: 1000 mg Moringa extract, 100 mg T. cordifolia extract, 1000 mg Aloe vera extract, 500 mg Curcuma xanthorrhiza; • 5 extract combination: 1000 mg Moringa extract, 100 mg T. cordifolia extract, 1000 mg Aloe vera extract, 500 mg Curcuma xanthorrhiza, 500 mg Channa striata extract;
• 4 extract combination - low Curcuma xanthorrhiza-. 1000 mg Moringa extract, 100 mg T. cordifolia extract, 1000 mg Aloe vera extract, 250 mg Curcuma xanthorrhiza;
• 5 extract combination - low Curcuma xanthorrhiza-. 1000 mg Moringa extract, 100 mg T. cordifolia extract, 1000 mg Aloe vera extract, 250 mg Curcuma xanthorrhiza, 500 mg Channa striata extract.
[0143] Separately, the same amount of each of the extracts is individually prepared as above (i.e., without combination), and packaged into sachets such that the sachets contain the same amount of an individual extract as that extract in combination, that is:
• 1000 mg Moringa extract;
• 100 mg T. cordifolia extract;
• 1000 mg Aloe vera extract;
• 500 mg (or 250 mg) Curcuma xanthorrhiza; and
• 500 mg Channa striata.
EXAMPLE 2
CYTOKINE RESPONSE TO EXTRACTS IN IN VITRO INFLAMMATION MODEL
[0144] In order to assess the clinical efficacy of the extracts, the present inventors investigated the effect on cellular inflammation induced by LPS.
IFN-v
[0145] The IFN-y system is associated with antiviral defence. IFN-y can downregulate virus replication and activate cytokine production by T cells, augmenting cytotoxic T lymphocyte killing activity (Levy and Garcia -Sastre, 2001). However, persistent high levels of IFN-y can aggravate systemic inflammation and increase tissue injury and organ failure (Yin et al., 2005). Low levels of plasma IFN-y have been reported to be associated with lung fibrosis in COVID-19 patients, with two-fold lower plasma interferon-y (IFN-y) levels at discharge observed in patients with fibrosis in the lung compared to those without fibrosis (p > 0.05; Hu et al., 2020). This indicates that lower circulating IFN-y is an increased risk factor of lung fibrosis in COVID-19. [0146] As shown in Figure 1, in a model of in vitro cellular inflammation, culture supernatant from peripheral blood monocytes (PBMC) stimulated with LPS and then treated with 1, 5 or 25 pg/mL of an immunomodulatory composition containing a combination of extracts from Moringa olifera (38.46%), T. cordifolia (3.84%), Aloe vera (38.46%) and Curcuma xanthorrhiza (19.23%) contained increasedd IFN-y levels compared to culture supernatant stimulated with LPS in the absence of the combination of extracts (negative control) or the culture supernatant stimulated with LPS and dexamethasone (anti-inflammatory control; p < 0.05). In particular, in the sample treated with LPS and 5 pg/mL of the combination of extracts, a significant increase of 12% (p <0.05) and 26% (p <0.05), respectively, of IFN-y was observed compared to the LPS negative control and the LPS+ dexamethasone control. The increase of IFN-y in the in vitro study indicates the potential of the immunomodulatory composition containing the combination of extracts to enhance beneficial antiviral activity without inducing very high levels associated with a cytokine storm/ cytokine release syndrome (CRS).
IL- IB
[0147] IL-1B is a pro-inflammatory cytokine, and high IL-1B levels, in addition to IL-18 and IL-33 levels, is considered to play a central role in CRS (Shimabukuro-Vornhagen etal., 2018), and reduction of IL- 13 levels is considered useful in treating COVID-associated CRS (Shi et al., 2020). IL-1 receptor antagonists have been shown to reduce the cytokine storm associated with severe infection induced inflammation and has been shown to significantly improve the 28-day survival rate of patients with severe sepsis (Shakoory et al., 2016), indicating that reduced IL- 13 levels assist in reducing or avoiding a cytokine storm response.
[0148] As shown in Figure 2, in the in vitro cellular inflammation model, culture supernatant from peripheral blood monocytes (PBMC) stimulated with LPS and then treated with an immunomodulatory composition containing a combination of extracts from Moringa olifera (38.46%), T. cordifolia (3.84%), Aloe vera (38.46%) and Curcuma xanthorrhiza (19.23%), particularly at 5 pg/mL, contained significantly decreased IL-13 levels compared to culture supernatant stimulated with LPS in the absence of the combination of extracts (negative control) or the culture supernatant stimulated with LPS and dexamethasone (antiinflammatory control). In the sample treated with LPS and 5 pg/mL of the combination of extracts, an decrease of 17% (p <0.05) and 8% (p <0.05), respectively, of IL- IB levels was observed compared to the LPS negative control and the LPS+ dexamethasone control. The decrease of IL-1 p levels in this study indicates the potential of the immunomodulatory composition containing the combination of extracts to beneficially reduce the level of this inflammatory cytokine, which may assist in avoiding inducing very high levels of IL-1 p associated with a cytokine storm/ cytokine release syndrome (CRS).
IL-2
[0149] IL-2 is a pleiotropic cytokine that is a key regulator of T cell metabolic programs. Amongst other functions, it influences effector T cell differentiation and is a critical determinant of the fate decisions of antigen receptor-activated T cells. When activated, both CD4+ T and cytotoxic CD8+ T cells exert protective immunity on both chronic and acute viral infection (Zhou et al., 2012; Sant and McMichael, 2012). IL-2 is also released during CRS (Eastwood et al., 2013), and can stimulate IL-6 release (Yiu et al., 2012).
[0150] As shown in Figure 3, in the in vitro cellular inflammation model, culture supernatant from peripheral blood monocytes (PBMC) stimulated with LPS and then treated with an immunomodulatory composition containing some concentrations of a combination of extracts from Moringa olifera (38.46%), T. cordifolia (3.84%), Aloe vera (38.46%) and Curcuma xanthorrhiza (19.23%) contained significantly increased IL-2 levels compared to culture supernatant stimulated with LPS in the absence of the combination of extracts (negative control) or the culture supernatant stimulated with LPS and dexamethasone (antiinflammatory positive control). In the sample treated with LPS and 5 pg/mL of the combination of extracts, an increase of 9% (p <0.05) and 5.5% (p <0.05), respectively, of IL-2 levels was observed compared to the LPS negative control and the LPS+ dexamethasone control. This result indicates that the extract combination offers potential to augment the adaptive immune system to help combat the SARS-CoV-2 virus.
IL-4
[0151] IL-4 is an anti-inflammatory pleiotropic cytokine. IL-4 drives CD4+ T cell polarization toward the Th2 phenotype and down regulates IFN-y- producing Thl cells. IL-4 also supports the activation, differentiation and maturation of B lymphocytes, controlling the specificity of the immunoglobulin G (IgG) class switching and the development of memory B cells. IL-4 can enhance the recruitment of T cells and eosinophils, rather than granulocytes, into the site of inflammation. [0152] As shown in Figure 4, in the in vitro cellular inflammation model, culture supernatant from peripheral blood monocytes (PBMC) stimulated with LPS and then treated with an immunomodulatory composition containing any of the tested concentrations of a combination of extracts from Moringa olifera (38.46%), T. cordifolia (3.84%), Aloe vera (38.46%) and Curcuma xanthorrhiza (19.23%) contained significantly increased IL-4 levels compared to culture supernatant stimulated with LPS in the absence of the combination of extracts (negative control) or the culture supernatant stimulated with LPS and dexamethasone (antiinflammatory control). In the sample treated with LPS and 5 pg/mL of the combination of extracts, an increase of 14% (p <0.05) and 14% (p <0.05), respectively, of IL-4 levels was observed compared to the LPS negative control and the LPS+ dexamethasone control. This result indicates that the extract combination may induce the activation and maturation of B lymphocytes, the development of B cells, augment the control of IgG class switching specificity and/or recruitment of T lymphocytes to the sites of inflammation.
IL-8
[0153] IL-8/CXCL8 is a pro-inflammatory chemoattractant chemokine for T-lymphocytes, mast cells, monocytes/macrophages, granulocytes, keratinocytes, and endothelial cells and particularly recruits neutrophils. The recruitment of neutrophils in inflammatory pneumonia was found to correlate with the development and mortality of Adult Respiratory Distress Syndrome (ARDS) associated with the cytokine storm. High level IL-8/CXCL-8 is found in the CRS in SARS patients.
[0154] As shown in Figure 5, in the in vitro cellular inflammation model, culture supernatant from peripheral blood monocytes (PBMC) stimulated with LPS and then treated with an immunomodulatory composition containing any of the tested concentrations of a combination of extracts from Moringa olifera (38.46%), T. cordifolia (3.84%), Aloe vera (38.46%) and Curcuma xanthorrhiza (19.23%) contained significantly decreased IL-8 levels compared to culture supernatant stimulated with LPS in the absence of the combination of extracts (negative control) or the culture supernatant stimulated with LPS and dexamethasone (antiinflammatory control). In the sample treated with LPS and 5 pg/mL of the combination of extracts, a decrease of 50.5% (p < 0.05) and 49% (p < 0.05), respectively, of IL-8 levels was observed compared to the LPS negative control and the LPS+ dexamethasone control. This result indicates that the extract combination may reduce the cytokine storm associated with the over recruitment of neutrophils. MCP-l/CCL-2
[0155] Migration of monocytes from the blood stream across the vascular endothelium is required for the routine immunological surveillance of tissues, and in response to inflammation. MCP-lo/CCL-2 is one of the key chemokines that regulate migration and infiltration of monocytes/macrophages, and central in CRS in SARS patients, severity of COVID-19 and increased plasma levels in ICU vs. non-ICU patients (Huang et al., 2020c; Mehta et al., 2020).
[0156] As shown in Figure 6, in the in vitro cellular inflammation model, culture supernatant from peripheral blood monocytes (PBMC) stimulated with LPS and then treated with an immunomodulatory composition containing any of the tested concentrations of a combination of extracts from Moringa olifera (38.46%), T. cordifolia (3.84%), Aloe vera (38.46%) and Curcuma xanthorrhiza (19.23%) contained significantly decreased MIP-lo levels compared to culture supernatant stimulated with LPS in the absence of the combination of extracts (negative control) or the culture supernatant stimulated with LPS and dexamethasone (antiinflammatory control). In the sample treated with LPS and 5 pg/mL of the combination of extracts, a decrease of 18% (p <0.05) and 26.5% (p < 0.05), respectively, of MIP-lo levels was observed compared to the LPS negative control and the LPS+ dexamethasone control. This result indicates that the extract combination may reduce serum plasma MCP-l/CCL-2 levels during inflammation induced by COVID-19 infection, leading to a more regulated macrophage recruitment, potentially providing benefits by reducing of symptoms associated with inflammatory cytokine storm associated with severe ARDS such as that seen in SARS, COVID-19, and other lung diseases.
MIP-lo /CCL-3
[0157] Macrophage Inflammatory Protein-lo (MIP-lo/CCL-3) is a chemoattractant chemokine and serves as biomarker for detection of several inflammatory diseases including lung conditions including ARDS. MIP-lo/CCL-3 is linked to severity of COVID-19 (Mehta et al., 2020) and plays a significant role in promoting inflammation in response to viral infections. In addition, this plasma levels of MIP-lo/CCL-3 has been found to be increased in ICU vs. non-ICU COVID-19 patients (Huang et al., 2020c).
[0158] As shown in Figure 7, in the in vitro cellular inflammation model, culture supernatant from peripheral blood monocytes (PBMC) stimulated with LPS and then treated with an antiviral composition containing any of the tested concentrations of a combination of extracts from Moringa olifera (38.46%), T. cordifolia (3.84%), Aloe vera (38.46%) and Curcuma xanthorrhiza (19.23%) contained significantly decreased MIP-lo levels compared to culture supernatant stimulated with LPS in the absence of the combination of extracts (negative control) or the culture supernatant stimulated with LPS and dexamethasone (antiinflammatory control). In the sample treated with LPS and 5 pg/mL of the combination of extracts, a decrease of 58% (p < 0.05) and 24% (p < 0.05), respectively, of MIP-lo levels was observed compared to the LPS negative control and the LPS+ dexamethasone control. This result indicates that the extract combination may reduce the inflammatory cytokine storm associated with severe ARDS such as that seen in SARS, COVID-19, and other lung diseases.
Materials & Methods
[0159] This experiment was performed using methods described above. Briefly, stock concentration containing a combination of Moringa olifera extract (38.46%), T. cordifolia extract (3.84%), Aloe vera extract (38.46%) and Curcuma xanthorrhiza extract was prepared having a concentration of 40 mg/mL. The combination of extracts was then diluted in DMSO such that 50 pL contained either 0, 1, 5, 25, 50, 100 or 200 pg/mL of the combined extract.
[0160] PBMC samples obtained from healthy subjects as described above were frozen and thawed and resuspended in RPMI medium (supplemented with glutamax) with 1 x PenStrep and 2.5 pg/mL plasmocin ± 10% heat inactivated FBS containing approximatey 5 mg/mL BSA at 2.6 x 106 cells/mL. 100 pL of cells (2.5 x 105 cells) were platedout into 96 well plates and cultured overnight (approximately 16-18 hours) in a 37°C/5% CO2 incubator. Then, the PBMCs were washed, removing supernatants and re-suspended in 100 pl fresh culture medium in the presence of FBS (BSA).
[0161] Each condition was tested in triplicate. Briefly, 50 pL of the combined extract at a concentration of 1, 5, 25, 50, 100 or 200 pg/mL was added to the appropriate wells. For the negative controls, 50 pL of the extract diluent was added instead, and for the positive control, 50 pL of DMSO was added and the samples were incubated for a further 30 minutes in a 37°C/5% CO2 incubator. 10 pL of LPS/PBS (final concentration of LPS was 50 ng/mL) was added to each well to induce inflammation. The plates were then incubated in a 37°C/5% CO2 incubator for 24 hours. Supernatants were collected the supernatants from each condition (tested in triplicate) was pooled and stored at -80°C until analysis using a Human Multiplex Panel (Cat # EPX450-12171-901) to analyse cytokines and chemokines production according to the manufacturer's instructions. Controls were PBMC stimulated with LPS in the presence of DMSO, with and without dexamethasone, which was used as an anti-inflammatory control. The final concentration of DMSO in the experiments was kept to 0.5% (v/v).
SUMMARY
[0162] As shown in Figure 8, culture supernatant from peripheral blood monocytes (PBMC) stimulated with LPS and then treated with an immunomodulatory composition containing 5 pg/mL of a combination of extracts from Moringa olifera (38.46%), T. cordifolia (3.84%), Aloe vera (38.46%) and Curcuma xanthorrhiza (19.23%) modulates the levels of at least IFN-y, IL-ip, IL- 2, IL-4, IL-8, MCP-1 and MIP-lo compared to samples stimulated with either LPS alone or LPS and dexamethasone. This accordingly shows that the combination of extracts enables an immunomodulation mechanism that assists to reduce cytokines and chemokines imbalances caused by the LPS stimulation. Moreover, this result indicates that the combination of extracts may assist to modulate immune responses associated with cytokine storms, such as those seen in severe inflammatory lung disease such as COVID-19, SARS, MERS and influenza, etc.
EXAMPLE 3
IN VITRO TESTING OF EXTRACT COMPSITION
[0163] Immunomodulatory compositions were prepared and packaged into sachets as described above. Each sachet contained 1000 mg Moringa extract, 100 mg T. Cordifolia extract, 1000 mg Aloe vera extract, 500 mg Curcuma xanthorrhiza and 500 mg Channa striata extract.
[0164] A 51-year old male tested positive to SARS-CoV-2 in a standard public COVID-19 testing clinic in Udayana University Hospital in Bali, Indonesia. The patient's symptoms were moderate including breathing difficulty, fever, loss sense of taste, and headache. For seven days prior to the positive COVID-19 test, the patient was orally administered one sachet daily duiluted in water. After the test date, he was orally administered two to three satchets daily diluted in water for 14 days. Specifically, the patient was administered two sachets per day on the day of his positive COVID-19 test (Day 0 and Day 1); three sachets per day for six days (Day 2 to Day 7); and two sachets per day for seven days (Day 8 to Day 14). On Day 7 the patient took a rapid test and tested non-reactive. On Day 11, the patient took a second COVID-19 PCR test and tested negative. At that time, his symptoms were completely resolved. Although this result is from a single patient, it indicates that the antiviral combination is safe in humans and is associated with a quick recovery from COVID-19.
Example 4 ONGOING IN VIVO TRIAL
[0165] SARS-CoV-2 PCR positive patients in Indonesia are enrolled into a clinical trial that is currently underway. Groups of 20 patients with mild to moderate COVID-19 symptoms are orally administered 3 sachets dissolved in water per day for at least 14 days, the sachets containing extracts in the following doses:
• 1000 mg Moringa extract, 100 mg T. cordifolia extract, 1000 mg Aloe vera extract, 500 mg Curcuma xanthorrhiza and 500 mg Channa striata extract
[0166] Placebo (e.g., starch/sugar-based composition).
[0167] PCR for SARS-CoV-2 is performed at day 1, 14 and preferably also day 20. At days 1, 5, 10 and 14, 20, the parameters described below are examined using standard laboratory techniques.
• Antisera (IgG and IgM);
• Serum levels of albumin (ALB), C reactive protein (CRP), lactate dehydrogenase (LDH), aspartate aminotransferase (AST), alanine aminotransferase (ALT);
• Haematology: Haemoglobin, red blood cells (RBC), Haematocrit MCV, MCH, MCHC, RDW, ABO Blood Group, Rhesus (+/-);
• White blood cell count: Neutrophils, lymphocytes, monocytes, eosinophils, Basophils, nucleated red blood cells (NRBC), platelets;
• Iron Studies: Iron Transferrin, TIBC (calculated), Saturation, Ferritin;
• Heavy metals: Serum, Zinc;
• Vitamins: D3, Bll and B12;
• Oxygen Saturation, Body Temperature, Heart Rate, Blood Pressure;
• CD4+ and CD8+ levels; and
• Serum cytokine levels.
[0168] Recovery time for patients administered the antiviral combination and the placebo will be assessed.
[0169] It has already been found that the combination is associated with reduced breathing difficulties in COVID-19 patients. It is is expected that the trial results will confirm that the combination assists by reducing breathing difficulties in COIVD-19 patients compared to those receiving the placebo control. Additionally, the antiviral combination has already been found to assist sleeping pattern and rest due to decreased breathing difficulties. It is anticipated that the trial will confirm that the COVID-19 patients administered the antiviral combination of can achieve better quality sleep due to more resting breathing.
[0170] The COVID-19 patients receiving the antiviral combination are expected to show improved blood markers for COVID-19 more rapidly than the patients receiving the negative and placebo control. For example, albumin, lymphopenia, lymphocyte (%), LDH, neutrophil (%), and CRP levels are highly correlated to acute lung injury. Age, viral load, lung injury score, and blood biochemistry indexes, albumin, CRP, lactate dehydrogenase, lympopenia lymphocyte (%), and neutrophil (%), may be predictors of disease severity.
[0171] Additionally, it is anticipated that patients receiving the antiviral combination will produce an enhanced IgG antisera against SARS-CoV-2 compared to those in the placebo/negative control group, indicating B-cell stimulation and increase in the humoral response. The trial will investigate whether the antiviral combination enhances T-cell stimulation (i.e., cellular response. For example, even without a significant increase of the IgG, COVID-19 patients receiving the antiviral combination may recover faster due to enhanced cytotoxic T-cell and/or memory T-cell responses.
[0172] It is anticipated that COVID-19 patients taking the antiviral combination will return to health more rapidly that patients receiving the negative control and placebo. Accordingly, it is anticipated that COVID-19 patients receiving the antiviral combination may have an enhanced return, for example, more rapid return on average, to a COVID-19 PCR negative result than those patients receiving the negative/placebo control.
[0173] There have been documented cases of COVID-19 in which PCR- positive COVID-19 infection has persisted (for example, some cases have been document in which patients have been PCR positive for many weeks, and sometimes months, for example, up to 100 days post infection). In the case of patients with persistent SARS-CoV-2 infection, it is anticipated that the antiviral combination will reduce symptoms.
EXAMPLE 5
SARS CoV-19 IN VIVO TRIAL
[0174] SARS-CoV-2 PCR positive patients are enrolled into a clinical trial. Groups of 20 patients with mild to moderate COVID-19 symptoms are orally administered 3 sachets dissolved in water per day for at least 14 days, the sachets containing extracts in the following doses:
• 1000 mg Moringa extract, 100 mg T. cordifolia extract, 1000 mg Aloe vera extract and 500 mg Curcuma xanthorrhiza
• 1000 mg Moringa extract, 100 mg T. cordifolia extract, 1000 mg Aloe vera extract, 500 mg Curcuma xanthorrhiza and 500 mg Channa striata extract
• No sachet (negative control)
[0175] The trial is conducted and assessed using the protocol as outlined in Example 3 (Ongoing in vivo trial). The trial assesses the effectiveness of the antiviral combination with and without the inclusion of the Channa striata extract.
[0176] Serum cytokine levels will be assessed to determine whether the antiviral combination induces cytokine levels that assist with recovery from viral infection without inducing host-derived pathology such as that seen during a cytokine storm.
[0177] It is anticipated that both of the immunomodulatory compositions tested will enhance outcome for COVID-19 patients by reducing disease symptoms as described above. The components of the Channa striata extract are expected to provide additional benefits due to the presence of at least one of the the antiviral agents albumin, zinc and/or iron found in the Channa striata extract.
EXAMPLE 6
EFFECT OF EXTRACT ON COVID- 19 PATIENTS
[0178] SARS-CoV-2 PCR positive patients are enrolled into a clinical trial. Groups of 5-20 patients with mild to moderate COVID-19 symptoms are orally administered the following combinations of antiviral agents daily for at least 14 days:
• 500 mg curcumin, 500 mg aloe-emodin, 25 mg zinc and 25 mg tinosporin;
• 500 mg curcumin, 500 mg aloe-emodin, 25 mg zinc and 25 mg tinocordiside;
• 500 mg curcumin, 500 mg aloe-emodin, 25 mg zinc and 25 mg arabinogalactan;
• 500 mg curcumin, 500 mg aloe-emodin, 25 mg zinc and 25 mg isocolumbin; • 500 mg curcumin, 500 mg aloe-emodin, 25 mg zinc and 25 mg magnoflorine;
• 500 mg curcumin, 500 mg aloe-emodin, 25 mg zinc and 100 mg T. cordifolia extract;
• 500 mg curcumin, 500 mg aloe-emodin, 25 mg zinc and 100 mg T. cordifolia extract, 500 mg albumin, and 5 mg iron
• 500 mg curcumin, 500 mg aloe-emodin, 25 mg zinc and 100 mg
T. cordifolia extract, 500 mg albumin, 500 mg glutathione, and 5 mg iron
• 500 mg curcumin, 500 mg aloe-emodin, 25 mg zinc and 100 mg
T. cordifolia extract, 500 mg albumin, 500 mg glutathione, 250 mg ascorbic acid and 5 mg iron; and
• Negative control (no supplement).
[0179] All components are readily obtained from commercial sources. The trial is conducted and assessed using the protocol as outlined in Example 3 (Ongoing in vivo trial). The trial assesses the effectiveness of different antiviral combinations. It is anticipated that each of the immunomodulatory compositions tested will enhance outcome for COVID-19 patients by increasing antiviral response, reducing disease symptoms and/or length of illness as described above.
EXAMPLE 7
ADDITIONAL CYTOKINE/CHEMOKINE RESPONSES TO EXTRACT
[0180] In order to further assess the clinical efficacy of the extracts, the present inventors investigated the effect on cellular inflammation induced by LPS.
TFN-g
[0181] Studies in animal models have shown that TNFs contribute significantly to acute lung injury and impair the T cell response in SARS-CoV- challenged mice. In mice, neutralization of TNF activity or loss of the TNF receptor provides protection against SARS-CoV-induced morbidity and mortality (Channappanavar et al., 2016; and McDermott et al., 2016). It is also found that TNF-a plays a central role in CRS. By blocking TNF-a potentially beneficial for patients with severe inflammation in the lung due to CRS (Huang et al., 2020). Whilst inhibition of IL-17A only shows a signal for Candida species but not for viral infection, targeting TNF and IL-6 increases the risk of bacterial infections but has lesser effects on viral infections (except for hepatitis B activation) (Schett et al., 2020). [0182] As shown in Figure 9, in the in vitro cellular inflammation model, culture supernatant from peripheral blood monocytes (PBMC) stimulated with LPS and then treated with an immunomodulatory composition containing some concentrations of a combination of extracts from Moringa olifera (38.46%), T. cordifolia (3.84%), Aloe vera (38.46%) and Curcuma xanthorrhiza (19.23%) contained significantly decreased TNF-o levels as compared to the culture supernatant stimulated with LPS and dexamethasone (anti-inflammatory positive control). In the sample treated with LPS and 5 pg/mL of the combination of extracts, an decrease of 25.92% in TNF level was observed as compared to the LPS+ dexamethasone control. This result indicates that the extract combination offers potential to downregulate TNF-o help combat the SARS-CoV-2 virus.
Eotaxin-1
[0183] Interestingly, increased levels of Eotaxin-1 in serum and lung tissue have been demonstrated in COVID-19 patients, despite eosinopenia (Horvath et al., 2014; Gebremeskel et al., 2021). Although some reports demonstrated the increase and activation of peripheral eosinophils in severe cases of Sars-CoV-2 infection, but the role of eosinophils in COVID-19-related inflammation still remains ambiguous (Lucas et al., 2020; Mitamura et al., 2022; Lindsley et al., 2020).
[0184] Neurologic complication associated with the SARS-CoV-2 infection in COVID-19 patients and survivors comprise symptoms including anxiety, depression, sleep impairment, headaches, dizziness, muscle pain, fatigue, myopathies and anosmia/hyposmia have been reported, this issue can prolonged for months and have been reported Goertz et al., 2020; Heneka et al., 2020; Seeano-Castro et al., 2020; Mei Junhua et al., 2020). As Eotaxin-1 plays roles in physiosomatic and neuroinflammation, analyzing the level of this chemokine in COVID-19 patients during hospitalization and to predicting post-COVID-19-related neurologic complications may be worthwhile. It is thought that, using chemokine modulators may be helpful in lessening the neurologic complications in such patients (Nazarinia et al., 2022).
[0185] As shown in Figure 10, The extract combination appears to downregulate the Eotaxin-1 level reflected by the obtained result. Eotaxin-1 levels appears to be lower when using extract combination particularly in the range of 5 - 200 pg/mL. Particularly at 5 pg/mL, the Eotaxin-1 is significantly down-regulated (-13.99% fold) than the negative control DMSO+LPS (**p<0.05). Moreover, the lowering capability of Eotaxin-1 secretion at 5 pg/mL comparable to the dexamethasone+LPS control with a small difference of -2.79% fold (* >0.05). The result demonstrated that the extract combination an act as chemokine modulators and do have potential as neuron protection as it lowers the amount of Eotaxin-1 significantly when compared towards the DMSO+LPS control.
IP-10
[0186] It has been reported that several pro-inflammatory cytokines and chemokines, including IP-10/CXCL-10, were higher in the plasma of COVID-19 patients as compared to healthy controls. More importantly, among infected patients, IP-10/CXCL-10 circulating concentration was significantly higher in patients requiring admission to intensive care units as compared to patients experiencing a less severe clinical course (Huang et al., 2020c). A study by Zhicheng et. al. corroborates this. The result revealed significant lower levels of immune cells (CD3+ T, CD4+ T, CD8+ T, B and NK cells). On the other hand, significantly higher levels of chemokines (including IP-10/CXCL-10) in severe cases compared with mild cases of COVID-19 patients were also observed (Zhicheng et al., 2020).
[0187] As shown in Figure 12, in the in vitro cellular inflammation model, culture supernatant from peripheral blood monocytes (PBMC) stimulated with LPS and then treated with an immunomodulatory composition containing some concentrations of a combination of extracts from Moringa olifera (38.46%), T. cordifolia (3.84%), Aloe vera (38.46%) and Curcuma xanthorrhiza (19.23%) contained significantly decreased IP-10 levels as compared to the culture supernatant stimulated with LPS and dexamethasone (anti-inflammatory positive control). In the sample treated with LPS within the range of 5 pg/mL to 200 pg/mL of the combination of extracts a clear downregulation of IP-10 was observed. At 5 pg/mL extract combination, the IP-10/CXCL-10 is lower (-8.29% fold) than the negative control DMSO+LPS (**p<0.05). On the other hand, although not statistically significant, at 5 pg/mL, the lowering capability of IP-10 secretion is more noticeable than the dexamethasone+LPS control with a small difference of - 6.64% fold (*p>0.05). The result demonstrated that the extract combination an act as chemokine modulators and can down-regulate
MIP-1B
[0188] MIP-ip/CCL-4 is a CC type Chemokine. The relationship between cytokines and chemokines exist to enhance/regulate innate or adaptive (or both) effector functions. In vitro and in vivo evidences, demonstrated that the chemokine milieu induced by one pathogen, by specifically recruiting T cells in the infected tissue, underpins a crucial role in determining the nature of the immune response (Sokol et al., 2015; Kaiko et al., 2008). The pro-inflammatory feedback loop resulting from SARS-COV-2 infection generating a massive production of chemokines, which orchestrate the immune cells, infiltration and the further secretion of both chemokine and cytokines resulting in the cytokine storm. The particular importance of chemokines in COVID-19 was highlighted by the analysis of BALF.
[0189] Xiong et al., found that peripheral blood mononuclear cells (PBMC) isolated from the BALF of COVID- 19 patients, over-expressed the genes encoding for several chemokines, including MIP-ip/CCL-4 (Zhou et al., 2020). Additionally, Xiong reported that the use transcriptomics data has revealed distinct host inflammatory cytokine profiles to SARS-CoV-2 infection in patients, and highlight the association between COVID-19 pathogenesis and excessive cytokine release including MIP-ip/CCL-4.
[0190] As shown in Figure 12, in the in vitro cellular inflammation model, culture supernatant from peripheral blood monocytes (PBMC) stimulated with LPS and then treated with an immunomodulatory composition containing some concentrations of a combination of extracts from Moringa olifera (38.46%), T. cordifolia (3.84%), Aloe vera (38.46%) and Curcuma xanthorrhiza (19.23%) contained significantly decreased MIP-ip levels as compared to the culture supernatant stimulated with LPS and dexamethasone (anti-inflammatory positive control). In the sample treated with LPS and 5 pg/mL of the combination of extracts, of extract combination to the LPS-stimulated cells, the level of MIP- ip/CCL-4 has been down-regulated significantly by -27.87% folds and -38.75% folds when compared to dexamethasone+LPS (*p<0.05) and DMSO+LPS (**p<0.05) controls respectively. Both MIP-lo/CCL-3 and MIP-ip/CCL-4 are chemoattractant. When dysregulated and highly secreted by the macrophages/monocytes will call upon various immune cells and may lead to CRS. Here at 5 pg/mL the extract combination significantly down-regulate the MIP- ip/CCL-4 secretion even lower than the dexamethasone+LPS control.
SUMMARY
[0191] As shown in Figure 13, culture supernatant from peripheral blood monocytes (PBMC) stimulated with LPS and then treated with an immunomodulatory composition containing 5 pg/mL of a combination of extracts from Moringa olifera (38.46%), T. cordifolia (3.84%), Aloe vera (38.46%) and Curcuma xanthorrhiza (19.23%) modulates the levels of at least IFN-y, IL-ip, IL- 2, IL-4, TNF-o, Eotaxin-l/CCL-11, IL-8/CXC-10, MCP-l/CCL-2, MIP-lo/CCL-3, and MIP-ip/CCL-4 compared to samples stimulated with either LPS alone or LPS and dexamethasone. This accordingly shows that the combination of extracts enables an immunomodulation mechanism that assists to reduce cytokines and chemokines imbalances caused by the LPS stimulation. Moreover, this result indicates that the combination of extracts may assist to modulate immune responses associated with cytokine storms, such as those seen in severe inflammatory lung disease such as COVID-19, SARS, MERS and influenza, etc.
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Claims

WHAT IS CLAIMED IS: An immunomodulatory composition comprising a combination of agents consisting of a curcuminoid, an emodin, bioavailable zinc and an at least one active agent obtained from Tinospora cordifolia. The immunomodulatory composition of claim 1 further comprising an agent selected from the list consisting of an albumin, a glutathione, ascorbic acid and bioavailable iron. The immunomodulatory composition of any one of claims 1 or 2 wherein at least one of the agents are provided as extracts derived from plants and/or animals. The immunomodulatory composition of any one of claims 1 to 3 wherein the at least one active agent obtained from T. cordifolia is provided by a Tinospora cordifolia extract. The immunomodulatory composition of any one of claims 1 to 4 wherein the curcuminoid is provided by a Curcuma spp. extract. The immunomodulatory composition of any one of claims 1 to 5 wherein the emodin is provided by an Aloe spp. extract. The immunomodulatory composition of any one of claims 1 to 6 wherein the bioavailable zinc is provided by a moringa extract. The immunomodulatory composition of any one of claims 1 to 7 wherein the albumin is provided by a Channa striata (snakehead fish) extract. An immunomodulatory composition comprising a combination of agents consisting of a Tinospora cordifolia extract, a Curcuma xanthorrhiza extract, an aloe extract and a moringa extract. The immunomodulatory composition of claim 9 further comprising a Channa striata (snakehead fish) extract. An immunomodulatory composition comprising a combination of agents consisting of a Tinospora cordifolia extract, a Curcuma xanthorrhiza extract, an aloe extract, a moringa extract and a Channa striata extract. The immunomodulatory composition of any one of claims 1 to 11, formulated for oral delivery. The immunomodulatory composition of any one of claims 1 to 11, comprising a dried powder. The immunomodulatory composition of any one of claims 1 to 13, when used to treat a viral infection. The immunomodulatory composition of claim 14, wherein the viral infection is a coronavirus infection. The immunomodulatory composition of claim 14 wherein the viral infection is a SARS-Cov-2 infection. Use of the immunomodulatory composition of any one of claims 1 to 12 to treat a viral infection. Use of the immunomodulatory composition of any one of claims 1 to 12 in the manufacture of a medicament for the treatment of a viral infection. Use of any one of claims 1 to 13 wherein the viral infection is a coronavirus infection. Use of any one of claims 1 to 13, wherein the viral infection is COVID-19. A method of treating a viral infection comprising administering to a to a subject in need thereof a therapeutically effective amount of the immunomodulatory composition of any one of claims 1 to 13. The method of claim 21 wherein the viral infection is a coronavirus infection. The method of claim 21 wherein the viral infection is COVID-19.
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ADHIKARI, B. ET AL.: "Potential roles of medicinal plants for the treatment of viral diseases focusing on COVID-19: A review", PHYTOTHERAPY RESEARCH, vol. 35, no. 3, 2021, pages 1298 - 1312, XP093000661, DOI: 10.1002/ptr.6893 *
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