WO2010046572A2 - Anti-influenza device and composition containing extracts from olive tree leaves, grapefruit pips, rosemary, green tea and curcumin - Google Patents

Anti-influenza device and composition containing extracts from olive tree leaves, grapefruit pips, rosemary, green tea and curcumin Download PDF

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Publication number
WO2010046572A2
WO2010046572A2 PCT/FR2009/001247 FR2009001247W WO2010046572A2 WO 2010046572 A2 WO2010046572 A2 WO 2010046572A2 FR 2009001247 W FR2009001247 W FR 2009001247W WO 2010046572 A2 WO2010046572 A2 WO 2010046572A2
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Prior art keywords
virus
extract
composition
composition according
viral
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PCT/FR2009/001247
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French (fr)
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WO2010046572A8 (en
WO2010046572A3 (en
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De Mareuil Jean Boula
Roger Treger
Liia Mokrani
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Treger Corinne
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Priority to EP09753141.2A priority Critical patent/EP3035948A2/en
Publication of WO2010046572A2 publication Critical patent/WO2010046572A2/en
Publication of WO2010046572A8 publication Critical patent/WO2010046572A8/en
Publication of WO2010046572A3 publication Critical patent/WO2010046572A3/en

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    • 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)
    • A61K36/82—Theaceae (Tea family), e.g. camellia
    • 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)
    • A61K36/53—Lamiaceae or Labiatae (Mint family), e.g. thyme, rosemary or lavender
    • 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)
    • A61K36/63—Oleaceae (Olive family), e.g. jasmine, lilac or ash tree
    • 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)
    • A61K36/75—Rutaceae (Rue family)
    • A61K36/752—Citrus, e.g. lime, orange or lemon
    • 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
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/14—Antivirals for RNA viruses
    • A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L9/00—Disinfection, sterilisation or deodorisation of air
    • A61L9/015—Disinfection, sterilisation or deodorisation of air using gaseous or vaporous substances, e.g. ozone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L9/00—Disinfection, sterilisation or deodorisation of air
    • A61L9/14—Disinfection, sterilisation or deodorisation of air using sprayed or atomised substances including air-liquid contact processes
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the field of antiviral compositions, in particular antiviral compositions related to the influenza virus. Faced with the problem of the risk of pandemic avian flu, existing therapeutic solutions are solely of synthetic and expensive.
  • the H5N1 subtype has a great ability to mutate over time, as well as to exchange its genes with influenza viruses belonging to other types of infecting other species.
  • a subtype of virus may stop circulating for several years in the human population but remain present in an animal population. If the animal population is in direct contact with a human being, they can transmit the virus again.
  • a virus subtype may also be newly created by a genetic reassortment that occurs when a host is co-infected with two different viruses.
  • this new virus has H5 and Nl segments specific to the avian virus, it will completely escape the recognition of the human immune system. If he also possesses genes that allow him to multiply effectively in mammals, he will then be able to transmit himself from man to man as effectively as classical flu.
  • influenza virus belonging to a viral subtype totally unknown to the human population renders the immune memory of humans totally ineffective. This makes plausible a rapid and worldwide spread of the virus.
  • the idea of a vaccine is not a satisfactory solution in view of the possible evolution of the virus during the passage to humans. What is known is that the production of a vaccine will take at least six months once the circulating strain is isolated and that, given the global production capacity of vaccine manufacturers, these six months will not allow to produce more than one billion monovalent doses. With two doses needed to gain protection, fewer than 500 million people, or 14 percent of the world's population, will be vaccinated. This places a special emphasis on antiviral treatments, which will therefore be at the forefront of fighting a possible pandemic in the first place.
  • the protection of populations against infection with the influenza virus has become, in the face of this pandemic, a public health problem.
  • the inventors have therefore been interested in compounds that protect animal organisms, including organisms humans, against influenza infection.
  • the present invention relates to a composition, preferably pharmaceutical, for the prevention and treatment of viral infections induced by viruses of the family Orthomyxoviridae characterized in that it comprises at least four different plant extracts selected from at least one olive leaf extract, at least one grapefruit seed extract, at least one rosemary extract, at least one green tea extract and / or curcumin in combination with any suitable pharmaceutical, if appropriate, excipient.
  • the composition according to the invention comprises extracts of olive leaves, extracts of grapefruit seeds, rosemary extracts, green tea extracts and curcumin.
  • the composition of the invention comprises 0.01 to 100 mg / kg, preferably 0.5 to 10 mg / kg of olive leaf extracts.
  • composition of the invention comprises 0.01 to 100 mg / kg, preferably 0.5 to 10 mg / kg of grapefruit seed extracts.
  • the composition of the invention comprises 0.01 to 100 mg / kg, preferably 0.5 to 10 mg / kg of rosemary extracts.
  • the composition of the invention comprises 0.01 to 100 mg / kg, preferably 0.5 to 10 mg / kg of green tea extracts. According to one embodiment, the composition of the invention comprises 0.01 to 100 mg / kg, preferably 0.5 to 10 mg / kg of curcumin. According to a second embodiment of the invention, the composition does not comprise curcumin.
  • a preferred composition of the invention comprises an olive leaf extract, a grapefruit seed extract, a rosemary extract, and a green tea extract.
  • the composition according to the invention is in the form of compacted powders or not, in liquid form, in the form of a solution or dispersion, in semi-solid or gelled or emulsified or dispersed form, or in aerosol form.
  • composition according to the invention may be administered orally, by intravenous injection, by inhalation, or by means of a spray, a diffuser, or by prior impregnation in a tissue.
  • the invention also relates to a use of the composition according to the invention for the prevention and / or treatment of diseases associated with infection with a virus, preferably an influenza virus.
  • the virus is a seasonal influenza virus.
  • the composition according to the invention is particularly useful for the prevention and / or the treatment of diseases associated with an influenza virus infection of the type H1N1 and / or H5N1 and / or H3N2.
  • the invention also relates to a device comprising a composition of the invention.
  • this device comprises a woven or non-woven fabric on which is affixed or which is impregnated with a composition according to the invention.
  • the invention also for the use of the composition according to the invention for cleaning the air in public or private places, especially by diffusion of said composition in the air ducts. More generally, an object of the invention relates to any device for the diffusion of the composition according to the invention.
  • the present invention relates to compositions comprising antioxidants, and more particularly comprising plant extracts having antioxidant properties having the property of protecting animal organisms against viral attacks or treating animal organisms with viral disease.
  • the antioxidants used in the compositions according to the invention by virtue of their antibacterial activity, constitute a barrier helping in the prevention of new infections favored by the attack of viruses.
  • the present invention resides in the combination of said substances which makes it possible to potentiate their effectiveness.
  • the present invention relates to the viruses of the family Orthomyxoviridae including the influenza virus and more particularly that of the bird flu.
  • the present invention also provides pharmaceutical or therapeutic compositions for the improvement, treatment and prevention of diseases associated with influenza viruses.
  • H5N1 bird flu virus
  • the attack of the body by H5N1, bird flu virus is characterized by the triggering of an exaggerated inflammatory reaction, which results in a secretion of mucositis, type pneumonia which causes respiratory distress in the affected subject. It is this symptom that is responsible for the exceptional severity of the disease.
  • compositions according to the invention make it possible to reduce this inflammatory response. Indeed, the various components of the composition according to the invention interfere with the general mechanisms of the inflammatory response of the body to the bird flu virus.
  • the compositions according to the invention make it possible to reduce the phenomenon of respiratory distress, which often has a lethal effect on the affected subject, enabling him to pass the critical stage of the disease.
  • compositions according to the invention make it possible to design a prophylactic barrier for contamination between humans, in the case of a pandemic, by example by impregnating masks of protections assuring them a more sure and more durable efficiency.
  • compositions according to the invention may serve as air freshener of public places by their diffusion, in particular by aerosol in the air ducts, which would reduce the financial consequences of a pandemic for society.
  • the present invention is a methodical combination of different actives each of which is described in the scientific literature as acting on several pathogenic bacteria and viruses.
  • the concept of the invention is based on the synergy of the different active ingredients in the compositions of the invention.
  • the multiplication of the influenza virus is a complex mechanism described in the form of several stages.
  • the combination of these different assets makes it possible to act on most of these stages instead of focusing on a particular stage; it is this approach that makes it possible to gain in efficiency.
  • compositions of the invention comprise non-toxic natural products and can therefore be used more easily at the various factors of propagation of the virus without toxicity to the environment and its inhabitants.
  • the extracts used are of the type marketed by the company Naturex, for example grapefruit seed extract marketed under the reference
  • the compositions according to the invention have a concentration of extracts, all extracts combined, of 0.04 to 15 g / l, preferably 0.04 to 10 g / l.
  • the compositions comprise 0.01 to 10 g / l, preferably 0.4 to 3 g / 1 of olive leaf extract of the type marketed by Naturex "extracted from 10% olive leaves”.
  • the compositions comprise 0.01 to 10 g / l, preferably 0.3 to 3 g / l of grapefruit seed extract of the type marketed by Naturex "grapefruit seed pe ws”.
  • the compositions comprise 0.01 to 10 g / l, preferably 0.3 to 3 g / l of green tea extract of the type marketed by Naturex with 4 to 6% caffeine.
  • compositions comprise 0.01 to 10 g / 1, preferably 0.3 to 3 g / 1 of rosemary extract of the type marketed by Naturex under the name Rosemary PE 2%.
  • compositions comprise 0 to 10 g / 1, preferably 0.3 to 3 g / 1 of turmeric extract type sold by Naturex.
  • compositions according to the invention comprise only natural plant extracts and water.
  • a preferred composition of the invention comprises 0.01 to 10 g / 1 of olive leaf extract, 0.01 to 10 g / 1 of extract extracted from grapefruit seed, 0.01 to 10 g / 1 of rosemary extract, and 0.01 to 10 g / i of green tea extract.
  • the composition of the invention consists or consists essentially of water and from 0.01 to 10 g / 1 of olive leaf extract, 0.01 to 10 g / 1 of extracted extract. grapefruit seed, 0.01 to 10 g / 1 rosemary extract, and 0.01 to 10 g / 1 green tea extract.
  • concentrations described in the composition of the invention are based on the implementation of the extracts mentioned above. It is within the skill of those skilled in the art implementing different concentrations of extracts, to adapt the formulations of the compositions of the invention.
  • Figure 1 shows the effect of ND235 on infection of MDCK cells by Influenza A Virus. On the abscissa are given increasing concentrations of virus and on the ordinate the absorption values proportional to the number of cells. The measurements are carried out in the absence (Control) and in the presence of ND235 formulations more or less concentrated in extracts (0.04%, 0.4% and 4%).
  • Example 1 Examples of compositions of the invention, efficacy, viral titre
  • the ND235 composition is tested in the present example 2.
  • Table 2 Dose - response effect of ND235 on the viability of cultured human lymphocytes.
  • ND235 is not toxic because, on the contrary, it stimulates cell growth when its concentrations are greater than or equal to 2 ⁇ 10 -3 g / l.
  • Toxicity was evaluated as soon as the product was added (acute toxicity) and 24h after the addition of the product (possible cytostatic effect).
  • Table 3 Direct dose - response effect of ND235 and various combinations of its active ingredients on the viability of cultured MDCKs.
  • Table 4 Effect at 24 hours in dose - response of ND235 and various combinations of its active ingredients on the viability of cultured MDCKs.
  • ND235 is not toxic even at the highest concentrations of 10g / l (first line). 2) In concentrations above 3 10 "g / 1 (lines 4-1), the ND235 and different combinations of active ingredients stimulate cell growth compared to cell controls.
  • ND235 is not toxic because on the contrary, it stimulates cell growth of MDCK when its concentrations are greater than or equal to 0.3 g / 1.
  • ND235 acts at the level of the infected cell as follows: 4.1 ND235 action on virus attachment to the cell
  • the docking of the influenza virus to the target cell is enabled by the interaction between a viral hemagglutinin and sialic acid receptors on the cell surface (Lamb, 1989).
  • This operation can be compared to a key and lock system.
  • Part of the subunit HA1 (the key), facing outward, has a very particular form and recognizes a specific molecule: sialic acid (the lock), which is present on the surface of some cells. Both forms are perfectly recognizable.
  • This recognition between the HA and the sialic acid causes the attachment of the viral particle to the target cell (the key is inserted into the lock).
  • ND235 blocks the multiplication of the virus and thus decreases the cellular interactions due to infection, this concept has a direct destructive effect of the virus on the affected cell, which reduces the excess of immune response which results in the case of bird flu respiratory distress often responsible for the death of the infected subject.
  • NA proteins break this link, allowing new viruses to break off and infect new target cells.
  • One of the actions of the active ingredient contained in the ND235 will prevent the action of neuraminidase (NA) which is responsible for the detachment of the virus from the host cell and the release of new infectious viruses.
  • ND235 Blocking this release of infected particles in the cell will thereby decrease the number of viruses circulating. ND235 will moderate the inflammatory response of different cells affected by viral attack. 4.3 Conclusion on cellular action modes of ND235
  • ND235 Several active ingredients contained in ND235 have a recognized action against the bird flu virus.
  • the antiviral actions described demonstrate their effectiveness by blocking the production of the virus in the cell, particularly in steps 6 and 7 for regulating the production of viral proteins and for synthesizing effective viral proteins.
  • MDCK cells are the reference cells for the study of influenza A virus multiplication.
  • H1N1 the strain A / PR / 8/34. This viral strain was obtained from a Puerto Rican patient and this reference virus is accessible at the American Tissue and Collection Bank (American Type Culture Collection).
  • CPE cytopathic effect
  • TCID50 is the viral concentration that will give a CPE effect on 50% of cells in culture.
  • MDCK cells are derived from cells isolated by S.H. Madin and N.B. Darby in 1958 from a kidney of an adult cocker spaniel puppy. These cells are accessible at the American bank of ATCC tissues and viruses.
  • the MDCK cells are cultured at 37 ° C. in a humid atmosphere containing
  • influenza A virus strain used is the A / PR / 8/34 type strain
  • H1N1 (ATCC VR-1249).
  • the MDCK cells are cultured at 33 ° C. in a humid atmosphere containing 5% CO 2 and in MEM medium with 0.125% BSA and in the presence of 1 ⁇ g / ml trypsin treated with TPCK ( Perbio SA, Be).
  • 96-well culture plates are seeded at 5 MDCK cells per well. These cells are cultured at 37 ° C. in a humid atmosphere containing 5% CO 2 in DMEM medium with 10% fetal calf serum in the presence of Penicillin Streptomycin antibiotics. When they form a confluent monolayer, they are infected with decreasing amounts of virus resulting from one-third dilutions of a stock solution of virus. The title of the starting virus stock solution established in 96-well culture plates is 81 TCID50. These infections are carried out in the absence or presence of ND235 at different concentrations. The cells are then kept in culture at 33 ° C. in a humid atmosphere containing 5% CO 2 for four days and then stained with Crystal Violet. The cytopathogenic effects are then recorded in order to establish the percentage inhibition of viral infection obtained by different concentrations of ND235.
  • the remaining cell quantities are estimated by measuring the monolayer uptake at 595 nm performed in a microplate reader.
  • the absorption values at 595 nm are corrected by those of the non-specific absorptions measured at 405 nm.
  • Table 5 Raw data of absorbances as a function of ND235 concentration and viral quantities.
  • Viral Input amounts of virus in TCID50 used for infection at time 0.
  • Table 6 Inhibition of infection with Influenza A virus by different concentrations of ND235 viral titre: in TCID50. Effect: +: destruction> 60% of the cells; + -: destruction of 30% to 60% of the cells; -: presence of 70% to 100% of the cells.
  • Percent viral inhibition can be calculated by comparing wells infected with the lowest effective viral dilutions in the presence and absence of the product.
  • Table 7 Mean values and standard deviation of absorbances as a function of ND235 concentration and viral input.
  • the absorption values in the control wells without virus are of the order of 0.75 and 0.45 in the presence respectively of 4% ND235 and 0% (control) of ND235.
  • the ND235 at the highest concentrations (ND235 4%) will promote the development of cells compared to the control cells not having ND235 (Control).
  • concentrations 10 and 100 times lower in ND235 this cell growth stimulating effect is less pronounced but seems nevertheless present.
  • ND235 is an effective antiviral against in vitro infection with influenza A virus. We have demonstrated that its efficacy is dose - dependent.
  • the concentrations of curcumin (Cu) products, extracts of grapefruit seed (PP), rosemary (Ro), olive leaf (01) and green tea (Tv) are the same as those contained in ND235.
  • the last column (ND235 - Cu) corresponds to the formulation containing ND235 components at concentrations of ND 235 without Curcumin.
  • the control (control) corresponds to the same viral load as the other columns without product. Viral Title: in TCID50.
  • ND235 The different products contained in ND235 are effective against the virus. Percent viral inhibition can be calculated by comparing wells infected with the lowest effective viral dilutions in the presence and absence of the product. Thus it follows that: - ND235 0.4%: 86% viral inhibition Curcumin: 7% viral inhibition
  • ND 235 is a blend of active flu-based products that work synergistically, resulting in increased efficacy.
  • ND235 is an effective antiviral against in vitro infection with mfluenza type A virus. We have shown that its efficacy is dose-dependent.

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Abstract

The present invention relates to a composition for preventing and treating viral infections induced by viruses of the Orthomyxoviridae family, characterised in that the composition comprises at least four different plant extracts selected from an olive tree leaf extract, at least one grapefruit pip extract, at least one rosemary extract and/or at least one green tea extract in combination with any adequate carrier. The invention also relates to the use of such a composition for preventing and/or treating diseases associated with a virus infection, in particular the H1N1 flu virus. The invention further relates to a device comprising a woven or unwoven textile on or with which the composition of the present invention is applied or impregnated. The invention also relates to the use of the composition of the invention for purifying air in public or private places, in particular by diffusing said composition in ventilation ducts.

Description

Composition antivirale Antiviral composition
La présente invention concerne le domaine des compositions antivirales, en particulier des compositions antivirales liées au virus de la grippe. Devant la problématique du risque de pandémie de la grippe aviaire, les solutions thérapeutiques existantes sont uniquement d'origines synthétiques et coûteuses.The present invention relates to the field of antiviral compositions, in particular antiviral compositions related to the influenza virus. Faced with the problem of the risk of pandemic avian flu, existing therapeutic solutions are solely of synthetic and expensive.
En effet, comme tous les virus de type A, le sous-type H5N1 a une grande capacité à muter au cours du temps, ainsi qu'à échanger ses gènes avec des virus grippaux appartenant à d'autres types infectants d'autres espèces. Un sous-type de virus peut cesser de circuler pendant plusieurs années dans la population humaine mais rester présent dans une population animale. Si la population animale est alors en contact direct avec un être humain, elle pourra lui transmettre à nouveau le virus.Indeed, like all type A viruses, the H5N1 subtype has a great ability to mutate over time, as well as to exchange its genes with influenza viruses belonging to other types of infecting other species. A subtype of virus may stop circulating for several years in the human population but remain present in an animal population. If the animal population is in direct contact with a human being, they can transmit the virus again.
Un sous-type de virus peut également être nouvellement créé par un réassortiment génétique qui se produit lors d'une co-infection d'un hôte par deux virus différents.A virus subtype may also be newly created by a genetic reassortment that occurs when a host is co-infected with two different viruses.
Si ce nouveau virus possède des segments H5 et Nl propres au virus aviaire, il échappera complètement à la reconnaissance du système immunitaire humain. S'il possède également des gènes lui permettant de se multiplier efficacement chez les mammifères, il aura alors la faculté de se transmettre d'homme à homme aussi efficacement que la grippe classique.If this new virus has H5 and Nl segments specific to the avian virus, it will completely escape the recognition of the human immune system. If he also possesses genes that allow him to multiply effectively in mammals, he will then be able to transmit himself from man to man as effectively as classical flu.
L'apparition d'un virus grippal appartenant à un sous-type viral totalement inconnu de la population humaine, comme le H5N1, rend la mémoire immunitaire de l'homme totalement inefficace. C'est ce qui rend plausible une dissémination rapide et mondiale du virus.The appearance of an influenza virus belonging to a viral subtype totally unknown to the human population, such as H5N1, renders the immune memory of humans totally ineffective. This makes plausible a rapid and worldwide spread of the virus.
Les experts de la grippe reconnaissent aujourd'hui qu'une nouvelle pandémie semble inévitable. Elle sera probablement causée par un virus H5N1 dérivé de la souche grippale aviaire qui circule actuellement en Asie et arrive aux portes de l'Europe. L'idée d'un vaccin n'est pas une solution satisfaisante au regard de l'évolution possible du virus lors du passage à l'homme. Ce que l'on sait, c'est que la production d'un vaccin demandera au moins six mois une fois la souche circulante isolée et que, compte tenu de la capacité de production mondiale des fabricants de vaccins, ces six mois ne permettront pas de produire plus d'un milliard de doses monovalentes. Deux doses étant nécessaires pour acquérir une protection, moins de 500 millions de personnes, soit 14 % de la population mondiale, pourront être vaccinées. Cela fait une place particulièrement importante aux traitements antiviraux qui seront donc en première ligne pour combattre une éventuelle pandémie dans un premier temps.Influenza experts now admit that a new pandemic seems inevitable. It will probably be caused by an H5N1 virus derived from the avian influenza strain currently circulating in Asia and reaching Europe's doorstep. The idea of a vaccine is not a satisfactory solution in view of the possible evolution of the virus during the passage to humans. What is known is that the production of a vaccine will take at least six months once the circulating strain is isolated and that, given the global production capacity of vaccine manufacturers, these six months will not allow to produce more than one billion monovalent doses. With two doses needed to gain protection, fewer than 500 million people, or 14 percent of the world's population, will be vaccinated. This places a special emphasis on antiviral treatments, which will therefore be at the forefront of fighting a possible pandemic in the first place.
La protection des populations contre l'infection par le virus de la grippe, est devenue, devant l'éventualité forte de cette pandémie, un problème de santé public Les inventeurs se sont donc intéressés aux composés permettant de protéger les organismes animaux, incluant les organismes humains, contre l'infection grippale.The protection of populations against infection with the influenza virus has become, in the face of this pandemic, a public health problem. The inventors have therefore been interested in compounds that protect animal organisms, including organisms humans, against influenza infection.
Ainsi, la présente invention concerne une composition, de préférence pharmaceutique, destinée à la prévention et au traitement des infections virales induites par les virus de la famille des Orthomyxoviridae caractérisée en ce qu'elle comprend au moins quatre extraits végétaux différents choisis parmi au moins un extrait de feuilles d'olivier, au moins un extrait de pépins de pamplemousse, au moins un extrait de romarin, au moins un extrait de thé vert et/ou du curcumin en combinaison avec tout excipient, le cas échéant pharmaceutique, approprié.Thus, the present invention relates to a composition, preferably pharmaceutical, for the prevention and treatment of viral infections induced by viruses of the family Orthomyxoviridae characterized in that it comprises at least four different plant extracts selected from at least one olive leaf extract, at least one grapefruit seed extract, at least one rosemary extract, at least one green tea extract and / or curcumin in combination with any suitable pharmaceutical, if appropriate, excipient.
Suivant un premier mode de réalisation, la composition selon l'invention comprend des extraits de feuilles d'olivier, des extraits de pépins de pamplemousse, des extraits de romarin, des extraits de thé vert et du curcumin. Avantageusement, la composition de l'invention comprend 0.01 à 100 mg/kg, de préférence 0.5 à 10 mg/kg d'extraits de feuilles d'olivier.According to a first embodiment, the composition according to the invention comprises extracts of olive leaves, extracts of grapefruit seeds, rosemary extracts, green tea extracts and curcumin. Advantageously, the composition of the invention comprises 0.01 to 100 mg / kg, preferably 0.5 to 10 mg / kg of olive leaf extracts.
Avantageusement, la composition de l'invention comprend 0.01 à 100 mg/kg, de préférence 0.5 à 10 mg/kg d'extraits de pépins de pamplemousse.Advantageously, the composition of the invention comprises 0.01 to 100 mg / kg, preferably 0.5 to 10 mg / kg of grapefruit seed extracts.
Avantageusement, la composition de l'invention comprend 0.01 à 100 mg/kg, de préférence 0.5 à 10 mg/kg d'extraits de romarin.Advantageously, the composition of the invention comprises 0.01 to 100 mg / kg, preferably 0.5 to 10 mg / kg of rosemary extracts.
Avantageusement, la composition de l'invention comprend 0.01 à 100 mg/kg, de préférence 0.5 à 10 mg/kg d'extraits de thé vert. Suivant un mode de réalisation, la composition de l'invention comprend 0.01 à 100 mg/kg, de préférence 0.5 à 10 mg/kg de curcumin. Suivant un second mode de réalisation de l'invention, la composition ne comprend pas de curcumin.Advantageously, the composition of the invention comprises 0.01 to 100 mg / kg, preferably 0.5 to 10 mg / kg of green tea extracts. According to one embodiment, the composition of the invention comprises 0.01 to 100 mg / kg, preferably 0.5 to 10 mg / kg of curcumin. According to a second embodiment of the invention, the composition does not comprise curcumin.
Une composition préférée de l'invention comprend un extrait de feuilles d'olivier, un extrait de pépins de pamplemousse, un extrait de romarin, et un extrait de thé vert.A preferred composition of the invention comprises an olive leaf extract, a grapefruit seed extract, a rosemary extract, and a green tea extract.
De préférence, la composition selon l'invention se présente sous forme de poudres compactées ou non, sous forme liquide, sous forme de solution ou dispersion, sous forme semi-solide ou gélifiée ou émulsionnée ou dispersée, ou sous forme d'aérosol.Preferably, the composition according to the invention is in the form of compacted powders or not, in liquid form, in the form of a solution or dispersion, in semi-solid or gelled or emulsified or dispersed form, or in aerosol form.
La composition selon l'invention peut être administrée par voie orale, par injection intraveineuse, par inhalation, soit à l'aide d'un spray, d'un diffuseur, ou par imprégnation préalable dans un tissu. L'invention a également pour objet une utilisation de la composition selon l'invention, pour la prévention et/ou le traitement de maladies associées à une infection par un virus, de préférence un virus influenza.The composition according to the invention may be administered orally, by intravenous injection, by inhalation, or by means of a spray, a diffuser, or by prior impregnation in a tissue. The invention also relates to a use of the composition according to the invention for the prevention and / or treatment of diseases associated with infection with a virus, preferably an influenza virus.
Suivant un premier mode de réalisation, le virus est un virus influenza saisonnier. Suivant un second mode de réalisation, la composition selon l'invention est particulièrement utile pour la prévention et/ou le traitement de maladies associées à une infection par le virus influenza de type HlNl et/ou H5N1 et/ou H3N2.According to a first embodiment, the virus is a seasonal influenza virus. According to a second embodiment, the composition according to the invention is particularly useful for the prevention and / or the treatment of diseases associated with an influenza virus infection of the type H1N1 and / or H5N1 and / or H3N2.
L'invention a également pour objet un dispositif comprenant une composition de l'invention. Suivant un mode de réalisation préféré, ce dispositif comprend un textile tissé ou non-tissé sur lequel est apposé ou qui est imprégné d'une composition selon l'invention.The invention also relates to a device comprising a composition of the invention. According to a preferred embodiment, this device comprises a woven or non-woven fabric on which is affixed or which is impregnated with a composition according to the invention.
L'invention a également pour l'utilisation de la composition selon l'invention pour assainir l'air dans les lieux publics ou privés, notamment par diffusion de ladite composition dans les gaines d'aération. Plus généralement, un objet de l'invention concerne tout dispositif permettant la diffusion de la composition selon l'invention. Ainsi, la présente invention porte sur des compositions comprenant des antioxydants, et plus particulièrement comprenant des extraits végétaux possédant des propriétés antioxydantes ayant la propriété de protéger les organismes animaux contre les attaques virales ou de traiter des organismes animaux atteints d'affection virale. Les antioxydants entrant dans les compositions selon l'invention, de par leur activité antibactérienne constituent une barrière aidant dans la prévention de nouvelles infections favorisées par l'attaque des virus. La présente invention réside dans l'association desdites substances qui permet d'en potentialiser l'efficacité.The invention also for the use of the composition according to the invention for cleaning the air in public or private places, especially by diffusion of said composition in the air ducts. More generally, an object of the invention relates to any device for the diffusion of the composition according to the invention. Thus, the present invention relates to compositions comprising antioxidants, and more particularly comprising plant extracts having antioxidant properties having the property of protecting animal organisms against viral attacks or treating animal organisms with viral disease. The antioxidants used in the compositions according to the invention, by virtue of their antibacterial activity, constitute a barrier helping in the prevention of new infections favored by the attack of viruses. The present invention resides in the combination of said substances which makes it possible to potentiate their effectiveness.
La présente invention porte sur les virus de la famille des Orthomyxoviridae dont le virus de la grippe et plus particulièrement celui de la Grippe aviaire. La présente invention porte également des compositions pharmaceutiques ou thérapeutiques permettant l'amélioration, le traitement et la prévention de maladies associées avec les virus grippaux.The present invention relates to the viruses of the family Orthomyxoviridae including the influenza virus and more particularly that of the bird flu. The present invention also provides pharmaceutical or therapeutic compositions for the improvement, treatment and prevention of diseases associated with influenza viruses.
L'atteinte de l'organisme par le H5N1, virus de la grippe aviaire, se caractérise par le déclenchement d'une réaction inflammatoire exagérée, qui se traduit par une sécrétion de mucosité, type pneumonie qui entraîne une détresse respiratoire chez le sujet atteint. C'est ce symptôme qui est responsable de l'exceptionnelle sévérité de la maladie.The attack of the body by H5N1, bird flu virus, is characterized by the triggering of an exaggerated inflammatory reaction, which results in a secretion of mucositis, type pneumonia which causes respiratory distress in the affected subject. It is this symptom that is responsible for the exceptional severity of the disease.
Les compositions selon l'invention permettent de diminuer cette réponse inflammatoire. En effet, les différents composants de la composition selon l'invention interfèrent avec les mécanismes généraux de la réponse inflammatoire de l'organisme au virus de la grippe aviaire. Les compositions selon l'invention permettent de diminuer le phénomène de détresse respiratoire qui a souvent un effet létal chez le sujet atteint, lui permettant de passer le stade critique de la maladie.The compositions according to the invention make it possible to reduce this inflammatory response. Indeed, the various components of the composition according to the invention interfere with the general mechanisms of the inflammatory response of the body to the bird flu virus. The compositions according to the invention make it possible to reduce the phenomenon of respiratory distress, which often has a lethal effect on the affected subject, enabling him to pass the critical stage of the disease.
La diversité des modes d'actions sur le virus, sur sa multiplication et ses effets permettent d'envisager de nombreuses applications des compositions selon l'invention, notamment grâce aux différentes formes galéniques envisageables qui représentent un avantage essentiel de lutte contre la pandémie de la grippe aviaire.The diversity of the modes of action on the virus, on its multiplication and its effects make it possible to envisage many applications of the compositions according to the invention, in particular thanks to the different conceivable galenic forms which represent an essential advantage of the fight against the pandemic of the Avian Flu.
Ainsi, les compositions selon l'invention permettent de concevoir une barrière prophylactique de contamination entre humains, dans le cas d'une pandémie, par exemple par imprégnation des masques de protections leur assurant une efficacité plus sure et plus durable.Thus, the compositions according to the invention make it possible to design a prophylactic barrier for contamination between humans, in the case of a pandemic, by example by impregnating masks of protections assuring them a more sure and more durable efficiency.
Dans un autre mode de réalisation, les compositions selon l'invention pourront servir d'assainisseur de l'air des lieux publics par leur diffusion, notamment en aérosol dans les gaines d'aération, ce qui permettrait de diminuer les conséquences financières d'une pandémie pour la société.In another embodiment, the compositions according to the invention may serve as air freshener of public places by their diffusion, in particular by aerosol in the air ducts, which would reduce the financial consequences of a pandemic for society.
La présente invention est une association méthodique de différents actifs dont chacun est décrit dans la littérature scientifique comme agissant sur plusieurs bactéries et virus pathogènes. Le concept de l'invention est basé sur la synergie des différents actifs entrant dans les compositions de l'invention.The present invention is a methodical combination of different actives each of which is described in the scientific literature as acting on several pathogenic bacteria and viruses. The concept of the invention is based on the synergy of the different active ingredients in the compositions of the invention.
En effet, la multiplication du virus de la grippe est un mécanisme complexe décrit sous la forme de plusieurs étapes. La combinaison de ces différents actifs permet d'agir sur la plupart de ces étapes au lieu de se focaliser sur une étape particulière ; c'est cette approche qui permet de gagner en efficacité.Indeed, the multiplication of the influenza virus is a complex mechanism described in the form of several stages. The combination of these different assets makes it possible to act on most of these stages instead of focusing on a particular stage; it is this approach that makes it possible to gain in efficiency.
D'autre part, les compositions de l'invention comprennent des produits naturels non toxiques et pourront donc être utilisé plus facilement au niveau des différents facteurs de propagation du virus sans toxicité pour l'environnement et ses habitants.On the other hand, the compositions of the invention comprise non-toxic natural products and can therefore be used more easily at the various factors of propagation of the virus without toxicity to the environment and its inhabitants.
Les extraits employés sont du type commercialisés par la société Naturex, par exemple l'extrait de pépins de pamplemousse commercialisé sous la référenceThe extracts used are of the type marketed by the company Naturex, for example grapefruit seed extract marketed under the reference
« grapefruit seed pe ws », l'extrait de feuilles d'olivier commercialisé sous la référence"Grapefruit seed pe ws", the olive leaf extract marketed under the reference
« extrait de feuilles d'olivier 10% », « extrait de thé vert concentré» à 4 à 6% de caféine, de l'extrait de romarin 2%."10% Olive Leaf Extract", "4% to 6% Caffeine Green Tea Extract", 2% Rosemary Extract.
Suivant un premier mode de réalisation, les compositions selon l'invention ont une concentration en extraits, tous extraits confondus de 0,04 à 15 g/1, de préférence 0,04 à 10 g/1.According to a first embodiment, the compositions according to the invention have a concentration of extracts, all extracts combined, of 0.04 to 15 g / l, preferably 0.04 to 10 g / l.
Avantageusement, les compositions comprennent 0,01 à 10 g/1, de préférence 0,4 à 3 g/1 d'extrait de feuilles d'olivier du type commercialisé par Naturex « extrait de feuilles d'olivier 10% ». Avantageusement, les compositions comprennent 0,01 à 10 g/1, de préférence 0,3 à 3 g/1 d'extrait de pépins de pamplemousse du type commercialisé par Naturex « grapefruit seed pe ws ». Avantageusement, les compositions comprennent 0,01 à 10 g/1, de préférence 0,3 à 3 g/1 d'extrait de thé vert du type commercialisé par Naturex à 4 à 6% de caféine.Advantageously, the compositions comprise 0.01 to 10 g / l, preferably 0.4 to 3 g / 1 of olive leaf extract of the type marketed by Naturex "extracted from 10% olive leaves". Advantageously, the compositions comprise 0.01 to 10 g / l, preferably 0.3 to 3 g / l of grapefruit seed extract of the type marketed by Naturex "grapefruit seed pe ws". Advantageously, the compositions comprise 0.01 to 10 g / l, preferably 0.3 to 3 g / l of green tea extract of the type marketed by Naturex with 4 to 6% caffeine.
Avantageusement, les compositions comprennent 0,01 à 10 g/1, de préférence 0,3 à 3 g/1 d'extrait de romarin du type commercialisé par Naturex sous le nom Rosemary PE 2%.Advantageously, the compositions comprise 0.01 to 10 g / 1, preferably 0.3 to 3 g / 1 of rosemary extract of the type marketed by Naturex under the name Rosemary PE 2%.
Avantageusement, les compositions comprennent 0 à 10 g/1, de préférence 0,3 à 3 g/1 d'extrait de curcuma type commercialisé par Naturex.Advantageously, the compositions comprise 0 to 10 g / 1, preferably 0.3 to 3 g / 1 of turmeric extract type sold by Naturex.
Suivant un mode de réalisation préféré, les compositions selon l'invention ne comprennent que des extraits naturels végétaux et de l'eau.According to a preferred embodiment, the compositions according to the invention comprise only natural plant extracts and water.
Une composition préférée de l'invention comprend 0,01 à 10 g/1 d'extrait de feuilles d'olivier, 0,01 à 10 g/1 d'extrait extrait de pépins de pamplemousse, 0,01 à 10 g/1 d'extrait de romarin, et 0,01 à 10 g/i d'extrait de thé vert. Très préférentiellement, la composition de l'invention est constituée, ou constituée essentiellement, d'eau et de 0,01 à 10 g/1 d'extrait de feuilles d'olivier, 0,01 à 10 g/1 d'extrait extrait de pépins de pamplemousse, 0,01 à 10 g/1 d'extrait de romarin, et 0,01 à 10 g/1 d'extrait de thé vert.A preferred composition of the invention comprises 0.01 to 10 g / 1 of olive leaf extract, 0.01 to 10 g / 1 of extract extracted from grapefruit seed, 0.01 to 10 g / 1 of rosemary extract, and 0.01 to 10 g / i of green tea extract. Very preferably, the composition of the invention consists or consists essentially of water and from 0.01 to 10 g / 1 of olive leaf extract, 0.01 to 10 g / 1 of extracted extract. grapefruit seed, 0.01 to 10 g / 1 rosemary extract, and 0.01 to 10 g / 1 green tea extract.
Les concentrations décrites dans la composition de l'invention sont basées sur la mise en œuvre des extraits ci-dessus mentionnés. Il est de la compétence de l'homme du métier mettant en œuvre différentes concentrations d'extraits, d'adapter les formulations des compositions de l'invention.The concentrations described in the composition of the invention are based on the implementation of the extracts mentioned above. It is within the skill of those skilled in the art implementing different concentrations of extracts, to adapt the formulations of the compositions of the invention.
Les exemples qui suivent illustrent non limitativement l'invention et se lisent en regard de la figure 1.The examples which follow illustrate the invention without limitation and are read in conjunction with FIG.
La figure 1 représente l'effet du ND235 sur l'infection des cellules MDCK par Influenza A Virus. En abscisse sont données des concentrations croissantes de virus et en ordonnée les valeurs d'absorption proportionnelles au nombre de cellules. Les mesures sont effectuées en absence (Control) et en présence de formulations de ND235 plus ou moins concentrées en extraits (0,04 %, 0,4 % et 4 %). Figure 1 shows the effect of ND235 on infection of MDCK cells by Influenza A Virus. On the abscissa are given increasing concentrations of virus and on the ordinate the absorption values proportional to the number of cells. The measurements are carried out in the absence (Control) and in the presence of ND235 formulations more or less concentrated in extracts (0.04%, 0.4% and 4%).
Figure imgf000008_0001
Figure imgf000008_0001
Exemple 1 : exemples de compositions de l'invention, efficacité, titre viralExample 1: Examples of compositions of the invention, efficacy, viral titre
Figure imgf000008_0002
Figure imgf000008_0002
Titre viral : en TCID50.Viral Title: in TCID50.
Effet : + : destruction > 60% des cellules ; + - : destruction de 30 % à 60 % des cellules ;Effect: +: destruction> 60% of the cells; + -: destruction of 30% to 60% of the cells;
- : présence de 70 % à 100% des cellules.-: presence of 70% to 100% of the cells.
La composition selon l'invention référencée ND235, est testée dans les exemples. Exemple 2 : Etude de toxicitéThe composition according to the invention referenced ND235, is tested in the examples. Example 2 Toxicity Study
La composition ND235 est testée dans le présent exemple 2.The ND235 composition is tested in the present example 2.
Les résultats de l'étude de toxicité sont présentés dans le tableau 2 ci-dessous. Ils montrent l'innocuité du ND235.The results of the toxicity study are shown in Table 2 below. They show the safety of ND235.
Concentration (g/1) ND235 Témoin CellulesConcentration (g / 1) ND235 Cells Control
Moyenne Ecart Type Moyenne Ecart TypeAverage Deviation Type Average Deviation Type
INDICE DE VIABILITE CELLULAIREINDEX OF CELLULAR VIABILITY
7,50E+00 0,553 0,18275393 0,126 0,052016537.50E + 00 0.553 0.18275393 0.126 0.05201653
2,50E+00 0,84733333 0,26023515 0,173 0,052016532.50E + 00 0.84733333 0.26023515 0.173 0.05201653
8,33E-Ol 1,073 0,20751626 0,273 0,052016538.33E-Ol 1.073 0.20751626 0.273 0.05201653
2,78E-Ol 1,70266667 0,07965132 0,282 0,052016532.78E-Ol 1.70266667 0.07965132 0.282 0.05201653
9,26E-02 1,44666667 0,1268122 0,266 0,052016539.26E-02 1.44666667 0.1212122 0.266 0.05201653
3,09E-02 0,70166667 0,04101626 0,302 0,05201653 l,03E-02 0,361 0,09534674 0,265 0,052016533.09E-02 0.70166667 0.04101626 0.302 0.05201653 1. 03E-02 0.361 0.09534674 0.265 0.05201653
3,43E-03 0,24666667 0,09856132 0,235 0,052016533.43E-03 0.24666667 0.09856132 0.235 0.05201653
1.14E-03 0,219 0,08758425 0,283 0,052016531.14E-03 0.219 0.08758425 0.283 0.05201653
3,81E-04 0,19466667 0,0903567 0,269 0,05201653 l,27E-04 0,206 0,10742905 0,298 0,052016533.81E-04 0.19466667 0.0903567 0.269 0.05201653 1, 27E-04 0.206 0.10742905 0.298 0.05201653
4,23E-05 0,203 0,05973274 0,259 0,052016534.23E-05 0.203 0.05973274 0.259 0.05201653
Tableau 2 : Effet dose — réponse de ND235 sur la viabilité des lymphocytes humains en Culture.Table 2: Dose - response effect of ND235 on the viability of cultured human lymphocytes.
Interprétation :Interpretation:
1) Quelle que soit la concentration de ND235, les valeurs des indices de viabilité (de 0,2 à 1,7) sont supérieures aux valeurs des témoins cellules sans produit (de 0,2 à 0,3). Ceci montre que le ND235 n'est pas toxique même aux concentrations les plus élevées de IgJl. 2) Dans les concentrations supérieures à 2 10"3 g/1 (lignes 8 à 1), le ND235 stimule la croissance cellulaire par rapport au témoin cellulaire avec un pic d'activité à la concentration de 0,3 g/1 (ligne 4)1) Whatever the concentration of ND235, the values of the viability indices (from 0.2 to 1.7) are greater than the values of the controls cells without product (from 0.2 to 0.3). This shows that ND235 is not toxic even at the highest concentrations of IgJ1. 2) In concentrations greater than 2 "-3 g / l (lanes 8-1), ND235 stimulates cell growth relative to the cell control with peak activity at 0.3 g / L (line 4)
On remarque que le ND235 n'est pas toxique puisqu'au contraire, il stimule la croissance cellulaire lorsque ses concentrations sont supérieures ou égales à 2 10"3 g/1.It is noted that ND235 is not toxic because, on the contrary, it stimulates cell growth when its concentrations are greater than or equal to 2 × 10 -3 g / l.
Après avoir vérifié la non-toxicité du ND235 sur des cellules humaines sanguines en culture, nous avons étudié l'éventuelle toxicité sur d'autres types tissulaires qui sont plus susceptibles à l'infection par le virus de la grippe, les cellules rénales MDCK.After verifying the non-toxicity of ND235 on human blood cells in culture, we investigated potential toxicity to other tissue types that are more susceptible to infection with the influenza virus, MDCK kidney cells.
Exemple 3 :Example 3
La toxicité a été évaluée dès l'ajout du produit (toxicité aiguë) et 24h après l'ajout du produit (éventuel effet cytostatique).Toxicity was evaluated as soon as the product was added (acute toxicity) and 24h after the addition of the product (possible cytostatic effect).
Concentration Témoin Association 1 Association 2 Association 3 Association 4 ND235Concentration Witness Association 1 Association 2 Association 3 Association 4 ND235
(g/1) Sans Produit(g / 1) Without Product
INDICE DE VIABILITE CELLULAIREINDEX OF CELLULAR VIABILITY
1.00E+01 0,370 ± 0,039 1,773 ± 0,022 1,896 ± 0,061 1,764 ± 0,022 1,726 ± 0,013 1,839 ± 0,0201.00E + 01 0.370 ± 0.039 1.773 ± 0.022 1.896 ± 0.061 1.764 ± 0.022 1.726 ± 0.013 1.839 ± 0.020
3,33E+00 0,353 ± 0,045 1,820 ± 0,136 1,682 ± 0,389 1,634 ± 0,099 1,749 ± 0,061 1,817 ± 0,0533.33E + 00 0.353 ± 0.045 1.820 ± 0.136 1.682 ± 0.389 1.634 ± 0.099 1.749 ± 0.061 1.817 ± 0.053
1,11E+OO 0,350 ± 0,065 1,746 + 0,122 1,617 ± 0,182 1,476 ± 0,029 1,686 + 0,105 1,531 ± 0,1001.11E + OO 0.350 ± 0.065 1.746 + 0.122 1.617 ± 0.182 1.476 ± 0.029 1.686 + 0.105 1.531 ± 0.100
3,70E-Ol 0,364 ± 0,067 1,545 ± 0,089 1,305 ± 0,051 1,443 ± 0,127 1,421 ± 0,036 1,198 ± 0,0483.70E-Ol 0.364 ± 0.067 1.545 ± 0.089 1.305 ± 0.051 1.443 ± 0.127 1.421 ± 0.036 1.198 ± 0.048
1,23E-Ol 0,391 ± 0,081 0,772 ± 0,088 0,692 ± 0,073 0,720 ± 0,082 0,775 ± 0,072 0,692 ± 0,0421.23E-Ol 0.391 ± 0.081 0.772 ± 0.088 0.692 ± 0.073 0.720 ± 0.082 0.775 ± 0.072 0.692 ± 0.042
4,12E-02 0,398 ± 0,038 0,489 ± 0,039 0,484 ± 0,058 0,484 ± 0,071 0,479 + 0,075 0,515 ± 0,055 l,37E-02 0,389 ± 0,050 0,431 ± 0,029 0,446 ± 0,035 0,440 ± 0,037 0,449 ± 0,042 0,444 ± 0,0374.12E-02 0.398 ± 0.038 0.489 ± 0.039 0.484 ± 0.058 0.484 ± 0.071 0.479 + 0.075 0.515 ± 0.055 l, 37E-02 0.389 ± 0.050 0.431 ± 0.029 0.446 ± 0.035 0.440 ± 0.037 0.449 ± 0.042 0.444 ± 0.037
4,57E-03 0,336 ± 0,061 0,412 ± 0,053 0,411 ± 0,075 0,393 ± 0,059 0,376± 0,068 0,365 ± 0,0784.57E-03 0.336 ± 0.061 0.412 ± 0.053 0.411 ± 0.075 0.393 ± 0.059 0.376 ± 0.068 0.365 ± 0.078
Tableau 3 : Effet direct en dose - réponse de ND235 et de diverses associations de ses principes actifs sur la viabilité des MDCK en Culture.Table 3: Direct dose - response effect of ND235 and various combinations of its active ingredients on the viability of cultured MDCKs.
Interprétation :Interpretation:
1) Quelle que soit la concentration de ND235 ou des différentes associations de ses principes actifs, les valeurs des indices de viabilité (de 0,3 à 1,9) sont supérieurs aux valeurs des témoins cellules sans produit (de 0,3 à 0,4, colonne 2). Ceci montre que le ND235 n'est pas toxique même aux concentrations les plus élevées de 10 g/1.1) Whatever the concentration of ND235 or the different combinations of its active ingredients, the values of viability indices (from 0.3 to 1.9) are higher than values of control cells without product (from 0.3 to 0.4, column 2). This shows that ND235 is not toxic even at the highest concentrations of 10 g / l.
2) Dans les concentrations supérieures à 1,7 10'2 g/1 (lignes 7 à 1), le ND235 et les différentes associations de ses principes actifs stimulent la croissance cellulaire par rapport aux témoins cellulaires.2) In concentrations higher than 1.7 10 -2 g / 1 (lanes 7-1), the ND235 and different combinations of active ingredients stimulate cell growth compared to cell controls.
Concentration Témoin Association Association Association Association ND235Concentration Witness Association Association Association Association ND235
(g/1) Sans Produit 1 2 3 4(g / 1) Without Product 1 2 3 4
INDICE DE VIABnLITE CEL LULAΠUEINDEX OF VIABLILITE CEL LULAΠUE
1.00E+01 0,429 + 0,010 1,856 ± 0,061 1,829 ± 0,048 1,733 ± 0,118 1,770 ± 0,076 1,819 ± 0,0781.00E + 01 0.429 + 0.010 1.856 ± 0.061 1.829 ± 0.048 1.733 ± 0.118 1.770 ± 0.076 1.819 ± 0.078
3.33E+00 0,385 ± 0,004 1,638+ 0,044 1,750 ± 0,046 1,657 ± 0,044 1,715 ± 0,080 1,839 ± 0,0793.33E + 00 0.385 ± 0.004 1.638+ 0.044 1.750 ± 0.046 1.657 ± 0.044 1.715 ± 0.080 1.839 ± 0.079
U1E+00 0,429 ± 0,011 0,962+ 0,068 0,760 ± 0,019 0,866 ± 0,043 0,897 ± 0,036 0,689 + 0,018U1E + 00 0.429 ± 0.011 0.962+ 0.068 0.760 ± 0.019 0.866 ± 0.043 0.897 ± 0.036 0.689 + 0.018
3,70E-Ol 0,448 ± 0,018 0,610 ± 0,036 0,469 ± 0,027 0,610 ± 0,066 0,601 ± 0,030 0,477 ± 0,0273.70E-Ol 0.448 ± 0.018 0.610 ± 0.036 0.469 ± 0.027 0.610 ± 0.066 0.601 ± 0.030 0.477 ± 0.027
1,23E-Ol 0,476 ± 0,001 0,464 ± 0,006 0,440 ± 0,005 0,455 + 0,006 0,448 ± 0,006 0,443 ± 0,0071.23E-Ol 0.476 ± 0.001 0.464 ± 0.006 0.440 ± 0.005 0.455 + 0.006 0.448 ± 0.006 0.443 ± 0.007
4,12E-02 0,443 ± 0,018 0,427 ± 0,023 0,436 ± 0,028 0,437 ± 0,035 0,437 ± 0,012 0,440 + 0,0264.12E-02 0.443 ± 0.018 0.427 ± 0.023 0.436 ± 0.028 0.437 ± 0.035 0.437 ± 0.012 0.440 + 0.026
1.37E-02 0,448 ± 0,024 0,431 ± 0,028 0,397 ± 0,021 0,391 ± 0,020 0,403 ± 0,004 0,401 ± 0,0301.37E-02 0.448 ± 0.024 0.431 ± 0.028 0.397 ± 0.021 0.391 ± 0.020 0.403 ± 0.004 0.401 ± 0.030
4,57E-03 0,454 ± 0,047 0,462 ± 0,053 0,422 ± 0,021 0,413 ± 0,009 0,406 ± 0,034 0,389 ± 0,009 l,52E-03 0,442 ± 0,032 0,437 ± 0,014 0,434 ± 0,001 0,397 ± 0,051 0,392 ± 0,075 0,444 ± 0,0044.57E-03 0.454 ± 0.047 0.462 ± 0.053 0.422 ± 0.021 0.413 ± 0.009 0.406 ± 0.034 0.389 ± 0.009 L, 52E-03 0.442 ± 0.032 0.437 ± 0.014 0.434 ± 0.001 0.397 ± 0.051 0.392 ± 0.075 0.444 ± 0.004
Tableau 4 : Effet à 24h en dose - réponse du ND235 et de diverses associations de ses principes actifs sur la viabilité des MDCK en Culture.Table 4: Effect at 24 hours in dose - response of ND235 and various combinations of its active ingredients on the viability of cultured MDCKs.
Interprétation :Interpretation:
1) Quelle que soit la concentration de ND235 ou des différentes associations de ses principes actifs, les valeurs des indices de viabilité (de 0,38 à 1,84) sont supérieures aux valeurs des témoins cellules sans produit (de 0,38 à 0,45). Ceci montre que le1) Whatever the concentration of ND235 or the different combinations of its active ingredients, the values of the viability indices (from 0.38 to 1.84) are greater than the values of the control cells without product (from 0.38 to 0 , 45). This shows that the
ND235 n'est pas toxique même aux concentrations les plus élevées de 10g/l (première ligne). 2) Dans les concentrations supérieures à 3 10" g/1 (lignes 4 à 1), le ND235 et les différentes associations de ses principes actifs stimulent la croissance cellulaire par rapport aux témoins cellulaire.ND235 is not toxic even at the highest concentrations of 10g / l (first line). 2) In concentrations above 3 10 "g / 1 (lines 4-1), the ND235 and different combinations of active ingredients stimulate cell growth compared to cell controls.
On remarque que le ND235 n'est pas toxique puisqu'au contraire, il stimule la croissance cellulaire des MDCK lorsque ses concentrations sont supérieures ou égales à 0,3 g/1 .It is noted that ND235 is not toxic because on the contrary, it stimulates cell growth of MDCK when its concentrations are greater than or equal to 0.3 g / 1.
Exemple 4 : Mode d'action du ND 235Example 4: Mode of Action of ND 235
Les phases de multiplication du virus de la grippe A peuvent être détaillées comme suit :The multiplication phases of influenza A virus can be detailed as follows:
I) Attachement du virus à la cellule 2) Entrée dans la cellule par endocytoseI) Attachment of the virus to the cell 2) Entry into the cell by endocytosis
3) Diminution du pH3) Decreased pH
4) Fusion, libération du contenu du virus dans la cellule4) Fusion, release of the contents of the virus into the cell
5) Entrée de TARN viral dans le noyau5) Entry of viral TARN into the nucleus
6) Fabrication de nouveaux brins d'ARN viral 7) Fabrication des protéines virales6) Manufacture of new strands of viral RNA 7) Manufacture of viral proteins
8) Sortie de TARN viral du noyau8) Release of viral TARN from the nucleus
9) Migration des éléments9) Migration of elements
10) Assemblage10) Assembly
I I) Bourgeonnement 12) Libération des nouveaux virusI I) Budding 12) Release of new viruses
Le ND235 agit au niveau de la cellule infectée de la manière suivante : 4.1 Action du ND235 sur l'attachement du virus à la celluleND235 acts at the level of the infected cell as follows: 4.1 ND235 action on virus attachment to the cell
L'amarrage du virus de la grippe à la cellule cible est permis par l'interaction entre une hémagglutinine virale et des récepteurs d'acide sialique situés à la surface des cellules (Lamb, 1989). On peut comparer ce fonctionnement à un système de clé et de serrure. Une partie de la sous unité HAl (la clé), tournée vers l'extérieur, a une forme bien particulière et reconnaît une molécule précise : l'acide sialique (la serrure), qui est présente à la surface de certaines cellules. Les deux formes se reconnaissent parfaitement. Cette reconnaissance entre l'HA et l'acide sialique entraîne l'attachement de la particule virale à la cellule cible (la clé est insérée dans la serrure).The docking of the influenza virus to the target cell is enabled by the interaction between a viral hemagglutinin and sialic acid receptors on the cell surface (Lamb, 1989). This operation can be compared to a key and lock system. Part of the subunit HA1 (the key), facing outward, has a very particular form and recognizes a specific molecule: sialic acid (the lock), which is present on the surface of some cells. Both forms are perfectly recognizable. This recognition between the HA and the sialic acid causes the attachment of the viral particle to the target cell (the key is inserted into the lock).
Le ND235 bloque la multiplication du virus et diminue donc les interactions cellulaires dues à l'infection, ce concept a un effet directement destructeur du virus au niveau de la cellule atteinte, ce qui diminue l'excès de réponse immunitaire qui entraîne dans le cas de la grippe aviaire la détresse respiratoire souvent responsable de la mort du sujet infecté.ND235 blocks the multiplication of the virus and thus decreases the cellular interactions due to infection, this concept has a direct destructive effect of the virus on the affected cell, which reduces the excess of immune response which results in the case of bird flu respiratory distress often responsible for the death of the infected subject.
4.2 Action du ND235 sur l'étape de libération des nouveaux virus4.2 Action of ND235 on the step of release of new viruses
Libération des nouveaux virus : les nouvelles particules virales restent attachées à la membrane de la cellule qui les a produits, à cause de la liaison entre l'Hémagglutinine (du virus) et l'acide sialique (de la cellule). Les protéines NA cassent cette liaison, permettant ainsi aux nouveaux virus de se détacher et d'aller infecter de nouvelles cellules cibles. Une des actions du principe actif contenu dans le ND235 va empêcher l'action de la neuraminidase (NA) qui est responsable du détachement du virus de la cellule hôte et de la libération de nouveaux virus infectieux.Release of new viruses: new viral particles remain attached to the membrane of the cell that produced them, because of the binding between hemagglutinin (of the virus) and sialic acid (of the cell). NA proteins break this link, allowing new viruses to break off and infect new target cells. One of the actions of the active ingredient contained in the ND235 will prevent the action of neuraminidase (NA) which is responsible for the detachment of the virus from the host cell and the release of new infectious viruses.
Le blocage de cette libération des particules infectées dans la cellule va de ce fait diminuer le nombre de virus mis en circulation. Le ND235 va modérer la réponse inflammatoire des différentes cellules atteintes par l'attaque virale. 4.3 Conclusion sur les modes d'actions cellulaires duND235Blocking this release of infected particles in the cell will thereby decrease the number of viruses circulating. ND235 will moderate the inflammatory response of different cells affected by viral attack. 4.3 Conclusion on cellular action modes of ND235
Plusieurs principes actifs contenus dans le ND235 présentent une action reconnue contre le virus de la grippe aviaire. Les actions antivirales décrites démontrent leur efficacité en bloquant la production du virus dans la cellule, notamment au niveau des étapes 6 et 7 de régulation de la production des protéines virales et de synthèse des protéines virales efficaces.Several active ingredients contained in ND235 have a recognized action against the bird flu virus. The antiviral actions described demonstrate their effectiveness by blocking the production of the virus in the cell, particularly in steps 6 and 7 for regulating the production of viral proteins and for synthesizing effective viral proteins.
Ainsi le nombre de virus produit sera diminué et le ND235 agira sur la cellule à l'identique des mécanismes précédents en agissant sur l'infection et ses conséquencesThus the number of viruses produced will be reduced and the ND235 will act on the cell identical to the previous mechanisms by acting on the infection and its consequences.
Une deuxième étude a été menée afin de vérifier l'effet antiviral du ND235 contre le virus de la grippe Influenza A.A second study was conducted to verify the antiviral effect of ND235 against Influenza A influenza virus.
Principe de l'étude :Principle of the study:
Les cellules MDCK sont les cellules de référence pour l'étude de la multiplication du virus de la grippe de type A. Pour notre étude d'effet antiviral, nous avons choisi d'utiliser une souche de référence du virus influenza de type HlNl, la souche A/PR/8/34. Cette souche virale a été obtenue chez un patient Portoricain et ce virus de référence est accessible au niveau de la banque Américaine de tissus et virus (American Type Culture Collection).MDCK cells are the reference cells for the study of influenza A virus multiplication. For our antiviral effect study, we chose to use a reference strain of influenza virus type H1N1, the strain A / PR / 8/34. This viral strain was obtained from a Puerto Rican patient and this reference virus is accessible at the American Tissue and Collection Bank (American Type Culture Collection).
L'infection virale est suivie par son effet cytopathogène (CPE ) sur les cellules en culture. Cet effet peut être observé par l'apparition de plages de lyse sur des monocouches cellulaires (Plaque Forming Unit ; PFU). Si l'on augmente la concentration virale on peut étendre la lyse cellulaire à tout ou partie de la monocouche cellulaire. Les expériences sont réalisées dans des plaques de culture 96 puits qui sont colorées au cristal violet. La coloration va résulter de la présence de cellules. Ainsi la disparition de la couleur attestera de la destruction viro-induite des cellules. Par ailleurs, nous vérifierons en parallèle dans des puits contenant différentes concentrations de notre produit sans virus, qu'il n'entraîne pas de lyse cellulaire confirmant son absence de toxicité dans notre système. L'activité antivirale du ND235 est établie en comparant les effets de différentes dilutions du ND235 en présence de quantités décroissantes de virus. Les quantités de virus utilisées pour l'infection sont données en TCID50. La TCID50 est la concentration virale qui va donner un effet CPE sur 50% des cellules en culture.Viral infection is followed by its cytopathic effect (CPE) on cells in culture. This effect can be observed by the appearance of lysis plaques on cell monolayers (Plaque Forming Unit, PFU). If the viral concentration is increased, cell lysis can be extended to all or part of the cell monolayer. The experiments are carried out in 96-well culture plates which are stained with crystal violet. Staining will result from the presence of cells. Thus, the disappearance of the color will attest to the viro-induced destruction of the cells. In addition, we will verify in parallel wells containing different concentrations of our product without virus, it does not cause cell lysis confirming its lack of toxicity in our system. The antiviral activity of ND235 is established by comparing the effects of different dilutions of ND235 in the presence of decreasing amounts of virus. The amounts of virus used for infection are given in TCID50. TCID50 is the viral concentration that will give a CPE effect on 50% of cells in culture.
Les expériences sont réalisées en triplicate.The experiments are performed in triplicate.
Cellules :Cells:
Les cellules MDCK dérivent de cellules isolées par S.H. Madin et N.B. Darby en 1958 à partir d'un rein d'une chienne adulte de race cocker epagneul. Ces cellules sont accessibles au niveau de la banque Américaine de tissus et virus ATCC. Les cellules MDCK sont cultivées à 37°C en atmosphère humide contenantMDCK cells are derived from cells isolated by S.H. Madin and N.B. Darby in 1958 from a kidney of an adult cocker spaniel puppy. These cells are accessible at the American bank of ATCC tissues and viruses. The MDCK cells are cultured at 37 ° C. in a humid atmosphere containing
5% de CO2 dans du milieu DMEM avec 10% de sérum de veau fœtal en présence d'antibiotiques Pénicilline Streptomycine.5% CO 2 in DMEM medium with 10% fetal calf serum in the presence of antibiotics Penicillin Streptomycin.
Virus : La souche virale d'influenza A utilisée est la souche A/PR/8/34 de typeVirus: The influenza A virus strain used is the A / PR / 8/34 type strain
HlNl (ATCC VR- 1249).H1N1 (ATCC VR-1249).
Lors de l'infection, les cellules MDCK sont cultivées à 33°C en atmosphère humide contenant 5% de CO2 et dans du milieu MEM avec de la BSA 0,125 % et en présence de 1 μg / ml de trypsine traitée à la TPCK (Perbio SA, Be).During infection, the MDCK cells are cultured at 33 ° C. in a humid atmosphere containing 5% CO 2 and in MEM medium with 0.125% BSA and in the presence of 1 μg / ml trypsin treated with TPCK ( Perbio SA, Be).
Protocole :Protocol:
Des plaques de culture de 96 puits sont ensemencées à raison de 5 10 cellules MDCK par puits. Ces cellules sont cultivées à 37°C en atmosphère humide contenant 5% de CO2 dans du milieu DMEM avec 10% de sérum de veau fœtal en présence d'antibiotiques Pénicilline Streptomycine. Lorsqu'elles forment une monocouche confluente elles sont infectées par des quantités décroissantes de virus résultant de dilutions au tiers en cascade d'une solution stock de virus. Le titre de la solution stock de départ du virus établi dans des plaques de culture 96 puits est de 81 TCID50. Ces infections sont effectuées en absence ou présence de ND235 à différentes concentrations. Les cellules sont ensuitre maintenues en culture à 33°C en atmosphère humide contenant 5% de CO2 pendant quatre jours puis sont colorées au Cristal Violet. Les effets cytopathogènes sont alors enregistrés afin d'établir le pourcentage d'inhibition de l'infection virale obtenu par les différentes concentrations de ND235.96-well culture plates are seeded at 5 MDCK cells per well. These cells are cultured at 37 ° C. in a humid atmosphere containing 5% CO 2 in DMEM medium with 10% fetal calf serum in the presence of Penicillin Streptomycin antibiotics. When they form a confluent monolayer, they are infected with decreasing amounts of virus resulting from one-third dilutions of a stock solution of virus. The title of the starting virus stock solution established in 96-well culture plates is 81 TCID50. These infections are carried out in the absence or presence of ND235 at different concentrations. The cells are then kept in culture at 33 ° C. in a humid atmosphere containing 5% CO 2 for four days and then stained with Crystal Violet. The cytopathogenic effects are then recorded in order to establish the percentage inhibition of viral infection obtained by different concentrations of ND235.
Les quantités de cellules restantes sont estimées par la mesure de l'absorption de la monocouche à 595 nm réalisée dans un lecteur de microplaque. Les valeurs d'absorption à 595 nm sont corrigées par celles des absorptions non spécifiques mesurées à 405 nm.The remaining cell quantities are estimated by measuring the monolayer uptake at 595 nm performed in a microplate reader. The absorption values at 595 nm are corrected by those of the non-specific absorptions measured at 405 nm.
Les résultats des valeurs moyennes et des écarts types sont présentés dans le tableau 3 et sous forme de diagramme.The results of the mean values and the standard deviations are presented in Table 3 and in the form of a diagram.
Résultats :Results:
Viral Input Concentration en ND 235 Viral Input Concentration in ND 23 5
0 % 0 % 0 % 4 % 4 % 4 % 0,4 % 0,4 % 0,4 % 0,04 % 0,04 % 0,04 %0% 0% 0% 4% 4% 4% 0.4% 0.4% 0.4% 0.04% 0.04% 0.04%
81 27 9 3 181 27 9 3 1
0,3 0,1 Ctrl
Figure imgf000016_0001
0.3 0.1 Ctrl
Figure imgf000016_0001
Tableau 5 : Données brutes des absorbances en fonction de la concentration en ND235 et des quantités virales.Table 5: Raw data of absorbances as a function of ND235 concentration and viral quantities.
Viral Input : quantités de virus en TCID50 utilisées pour l'infection au temps 0. Viral Input: amounts of virus in TCID50 used for infection at time 0.
Figure imgf000017_0001
Figure imgf000017_0001
Tableau 6 : Inhibition de l'infection par le virus Influenza A par différentes concentrations de ND235 Titre viral : en TCID50. Effet : + : destruction > 60% des cellules ; + - : destruction de 30 % à 60 % des cellules ; - : présence de 70 % à 100% des cellules.Table 6: Inhibition of infection with Influenza A virus by different concentrations of ND235 viral titre: in TCID50. Effect: +: destruction> 60% of the cells; + -: destruction of 30% to 60% of the cells; -: presence of 70% to 100% of the cells.
Interprétation :Interpretation:
Les différentes concentrations de produit ND235 sont efficaces contre le viras. On peut calculer le pourcentage d'inhibition virale en comparant les puits infectés par les plus faibles dilutions virales efficaces en présence et absence du produit. Ainsi il résulte que :The different concentrations of ND235 product are effective against viras. Percent viral inhibition can be calculated by comparing wells infected with the lowest effective viral dilutions in the presence and absence of the product. Thus it follows that:
ND235 4 % : 80 % d'inhibition virale ND235 0,4 % : 84 % d'inhibition viraleND235 4%: 80% viral inhibition ND235 0.4%: 84% viral inhibition
ND235 0,04 % : 73 % d'inhibition viraleND235 0.04%: 73% viral inhibition
Dans les puits témoins contenant les différentes concentrations de ND235 en absence de virus (ligne Ctrl) la coloration n'est pas diminuée en présence de ND235. La diminution de l'intensité de la coloration aurait attesté d'une toxicité. On confirme les résultats de nos études préalables de toxicité montrant que le ND235, même aux concentrations les plus élevées ne présente pas de toxicité.In the control wells containing the different concentrations of ND235 in the absence of virus (Ctrl line) the staining is not diminished in the presence of ND235. The decrease in the intensity of the coloring would have attested to a toxicity. We confirm the results of our previous toxicity studies showing that ND235, even at the highest concentrations, is not toxic.
Figure imgf000018_0001
Figure imgf000018_0001
Tableau 7 : Valeurs moyennes et déviation standard des absorbances en fonction de la concentration en ND235 et des viral input.Table 7: Mean values and standard deviation of absorbances as a function of ND235 concentration and viral input.
Viral input : en TCID50.Viral input: in TCID50.
Les valeurs d'absorptions à 595 nm ont été corrigées par les valeurs d'absorption àThe absorption values at 595 nm were corrected by the absorption values at
405nm.405nm.
Interprétation :Interpretation:
La présente interprétation se lit en regard de la figure 1.This interpretation is read in conjunction with Figure 1.
Les valeurs d'absorption dans les puits contrôle sans virus (première série, 0 TCID50) sont de l'ordre de 0,75 et 0,45 en présence respectivement de 4 % ND235 et de 0 % (control) de ND235. Ainsi, le ND235 aux concentrations les plus élevées (ND235 4 %) va favoriser le développement des cellules en comparaison des cellules témoin n'ayant pas de ND235 (Control). Pour les concentrations 10 et 100 fois inférieures en ND235, cet effet stimulant la croissance cellulaire est moins prononcé mais semble toutefois présent. Ces résultats sont en accord avec ceux que nous avons obtenus lors de l'étude de toxicité in vitro de notre produit indiquant un effet bénéfique sur la croissance cellulaire. Les différentes concentrations présentées de ND235 (série 1) bloquent efficacement des doses de virus détruisant 50 % des cellules. Pour les infections virales effectuées avec trois fois et neuf fois plus de virus (séries 3 et 9) on observe encore une inhibition du virus par le ND235 mais de façon dose-dépendante, les concentrations en ND235 les plus élevées conférant la meilleure protection.The absorption values in the control wells without virus (first series, 0 TCID50) are of the order of 0.75 and 0.45 in the presence respectively of 4% ND235 and 0% (control) of ND235. Thus, the ND235 at the highest concentrations (ND235 4%) will promote the development of cells compared to the control cells not having ND235 (Control). For concentrations 10 and 100 times lower in ND235, this cell growth stimulating effect is less pronounced but seems nevertheless present. These results are consistent with those obtained in the in vitro toxicity study of our product indicating a beneficial effect on cell growth. The different concentrations presented of ND235 (series 1) effectively block doses of virus destroying 50% of the cells. For viral infections with three and nine times more virus (series 3 and 9) there is still inhibition of the virus by ND235 but in a dose-dependent manner, the highest concentrations of ND235 conferring the best protection.
Conclusion :Conclusion:
Le ND235 est un antiviral efficace contre l'infection in vitro par le virus influenza de type A. Nous avons démontré que son efficacité est dose - dépendante.ND235 is an effective antiviral against in vitro infection with influenza A virus. We have demonstrated that its efficacy is dose - dependent.
Il ressort de cette étude que le ND235 à une concentration de 0,4 % présente une efficacité antivirale de 84 %. Il est important de noter qu'à une dose dix fois inférieure, l'efficacité du ND235 reste remarquable. En effet elle est de l'ordre de 75 %.This study shows that ND235 at a concentration of 0.4% has an antiviral efficacy of 84%. It is important to note that at a dose ten times lower, the effectiveness of ND235 remains remarkable. Indeed it is of the order of 75%.
D'autre part, il est utile de rappeler que ce produit est complètement naturel.On the other hand, it is worth remembering that this product is completely natural.
Exemple 5 : Exemple comparatifExample 5 Comparative Example
Les activités antivirales de chacun des composants aux concentrations respectives contenues dans ND235 ont été mesurées et comparées à l'activité synergique de la composition selon l'invention ND235. Les résultats sont présentés dans le tableau 4 ci- dessous.The antiviral activities of each of the components at the respective concentrations contained in ND235 were measured and compared with the synergistic activity of the composition according to the invention ND235. The results are shown in Table 4 below.
Figure imgf000019_0001
Figure imgf000020_0001
Figure imgf000019_0001
Figure imgf000020_0001
Tableau 8 : Inhibition de l'infection par le virus Influenza A par les différents composants de ND235Table 8: Inhibition of influenza A virus infection by the different components of ND235
Les concentrations des produits curcumin (Cu), extraits de pépins de pamplemousse (PP), de romarin (Ro), de feuille d'olivier (01) et de thé vert (Tv) sont les mêmes que celles contenues dans le ND235. La dernière colonne (ND235 - Cu) correspond à la formulation contenant les composants du ND235 aux concentrations du ND 235 sans Curcumin. Le contrôle (contrôle) correspond à la même charge virale que les autres colonnes sans produit. Titre viral : en TCID50.The concentrations of curcumin (Cu) products, extracts of grapefruit seed (PP), rosemary (Ro), olive leaf (01) and green tea (Tv) are the same as those contained in ND235. The last column (ND235 - Cu) corresponds to the formulation containing ND235 components at concentrations of ND 235 without Curcumin. The control (control) corresponds to the same viral load as the other columns without product. Viral Title: in TCID50.
Effet : + : destruction > 60% des cellules ; + - : destruction de 30 % à 60 % des cellules ; - : présence de 70 % à 100% des cellules.Effect: +: destruction> 60% of the cells; + -: destruction of 30% to 60% of the cells; -: presence of 70% to 100% of the cells.
Interprétation :Interpretation:
Les différents produits contenus dans ND235 sont efficaces contre le virus. On peut calculer le pourcentage d'inhibition virale en comparant les puits infectés par les plus faibles dilutions virales efficaces en présence et absence du produit. Ainsi il résulte que : - ND235 0,4 % : 86 % d'inhibition virale Curcumin : 7 % d'inhibition viraleThe different products contained in ND235 are effective against the virus. Percent viral inhibition can be calculated by comparing wells infected with the lowest effective viral dilutions in the presence and absence of the product. Thus it follows that: - ND235 0.4%: 86% viral inhibition Curcumin: 7% viral inhibition
- Pépin de pamplemousse : 21 % d'inhibition virale - Extraits de romarin : 28 % d'inhibition virale- Grapefruit seed: 21% viral inhibition - Rosemary extract: 28% viral inhibition
Extraits de feuilles d'olivier : 36 % d'inhibition viraleOlive leaf extract: 36% viral inhibition
- Extraits de thé vert : 50 % d'inhibition virale- Green tea extracts: 50% viral inhibition
- ND235 0,4 % sans curcumin : 64 % d'inhibition virale- ND235 0.4% without curcumin: 64% viral inhibition
Ceci montre que les différents composants du ND235 utilisés séparément à la même concentration que celui-ci sont bien moins efficaces que lorsqu'ils sont réunis.This shows that the different components of ND235 used separately at the same concentration as this one are much less effective than when they are combined.
Conclusion :Conclusion:
Le ND 235 est un mélange de produits actifs contre le virus de la grippe qui agissent en synergie permettant ainsi une efficacité augmentée.ND 235 is a blend of active flu-based products that work synergistically, resulting in increased efficacy.
Le ND235 est un antiviral efficace contre l'infection in vitro par le virus mfluenza de type A. Nous avons démontré que son efficacité est dose-dépendante.ND235 is an effective antiviral against in vitro infection with mfluenza type A virus. We have shown that its efficacy is dose-dependent.
Il ressort de cette étude que le ND235 à une concentration de 0,4 % présente une efficacité antivirale de 84 %. Il est important de noter qu'à une dose dix fois inférieure, l'efficacité du ND235 reste remarquable. En effet elle est de l'ordre de 75 %.This study shows that ND235 at a concentration of 0.4% has an antiviral efficacy of 84%. It is important to note that at a dose ten times lower, the effectiveness of ND235 remains remarkable. Indeed it is of the order of 75%.
D'autre part, il est utile de rappeler que ce produit est complètement naturel et que l'efficacité de ses constituants tant au niveau antiviral qu'au niveau de la diminutjpn de la réponse immunitaire a été décrite dans la littérature scientifique. Enfin, l'intérêt de ce produit réside également dans le fait qu'il va bloquer le virus de la grippe par différents mécanismes. On the other hand, it is worth remembering that this product is completely natural and that the effectiveness of its constituents both at the antiviral level and at the level of diminutomy of the immune response has been described in the scientific literature. Finally, the interest of this product lies also in the fact that it will block the flu virus by different mechanisms.

Claims

Revendications claims
1. Composition destinée à la prévention et au traitement des infections virales induites par les virus de la famille des Orthomyxoviridae caractérisée en ce qu'elle comprend au moins quatre extraits végétaux différents choisis parmi un extrait de feuilles d'olivier , au moins un extrait de pépins de pamplemousse, au moins un extrait de romarin et/ou au moins un extrait de thé vert en combinaison avec tout excipient approprié.1. Composition intended for the prevention and treatment of viral infections induced by viruses of the family Orthomyxoviridae characterized in that it comprises at least four different plant extracts selected from an extract of olive leaves, at least one extract of grapefruit seed, at least one rosemary extract and / or at least one green tea extract in combination with any suitable excipient.
2. . Composition selon la revendication 1, caractérisée en ce qu'elle comprend en outre du curcumin.2.. Composition according to claim 1, characterized in that it further comprises curcumin.
3. Composition selon la revendication 1, caractérisée en ce qu'elle comprend 0.01 à 100 mg/kg d'extrait de feuilles d'olivier.3. Composition according to claim 1, characterized in that it comprises 0.01 to 100 mg / kg of olive leaf extract.
4. Composition selon la revendication 1, caractérisée en ce qu'elle comprend 0.01 à 100 mg/kg d'extrait de pépins de pamplemousse. 4. Composition according to claim 1, characterized in that it comprises 0.01 to 100 mg / kg of grapefruit seed extract.
5. Composition selon la revendication 1, caractérisée en ce qu'elle comprend5. Composition according to claim 1, characterized in that it comprises
0.01 à 100 mg/kg d'extrait de romarin.0.01 to 100 mg / kg of rosemary extract.
6. Composition selon la revendication I5 caractérisée en ce qu'elle comprend 0.01 à 100 mg/kg d'extrait de thé vert.6. The composition of claim I 5 characterized in that it comprises 0.01 to 100 mg / kg green tea extract.
7. Composition selon la revendication 2, caractérisée en ce qu'elle comprend 0.01 à 100 mg/kg de curcuma7. Composition according to claim 2, characterized in that it comprises 0.01 to 100 mg / kg of turmeric.
8. Composition selon l'une quelconque des revendications 1 à 7 caractérisée en ce qu'elle se présente sous forme de poudres compactées ou non, sous forme liquide, sous forme de solution ou dispersion, sous forme semi-solide ou gélifiée ou émulsionnée ou dispersée, ou sous forme d'aérosol. 8. Composition according to any one of claims 1 to 7, characterized in that it is in the form of compacted powders or not, in liquid form, in the form of a solution or dispersion, in semi-solid or gelled or emulsified form or dispersed, or in aerosol form.
9. Composition selon l'une quelconque des revendications 1 à 8 caractérisée en ce qu'elle est administrée par voie orale, par injection intraveineuse, par inhalation, soit à l'aide d'un spray, d'un diffuseur, ou par imprégnation préalable dans un tissμ,9. Composition according to any one of claims 1 to 8 characterized in that it is administered orally, by intravenous injection, by inhalation, or with the aid of a spray, a diffuser, or by impregnation in a tissue,
10. Utilisation de la composition selon l'une quelconque des revendications 1 à 9, pour la prévention et/όμ |e traitement de maladies associées à une infection par un virus .10. Use of the composition according to any one of claims 1 to 9, for the prevention and / or treatment of diseases associated with infection with a virus.
Jl* Utilisation selon la revendication 10 dans laquelle le virus est un virus μifluenza. Jl * The use of claim 10 wherein the virus is a virus μifluenza.
12. Utilisation selon l'une quelconque des revendications 10 ou 11, dans laquelle le virus est le virus influenza de type HlNl et/ou H5N1 et/ou H3N2.The use according to any one of claims 10 or 11, wherein the virus is influenza virus of the type H1N1 and / or H5N1 and / or H3N2.
13. Dispositif comprenant un textile tissé ou non-tissé sur lequel est apposé ou dans lequel est imprégné une composition selon l'une quelconque des revendications 1 à 9.13. Device comprising a woven or non-woven fabric on which is affixed or in which is impregnated a composition according to any one of claims 1 to 9.
14. Utilisation de la composition selon l'une quelconque des revendications 1 à 9 pour assainir l'air dans les lieux publics ou privés, par diffusion de ladite composition dans les gaines d'aération. 14. Use of the composition according to any one of claims 1 to 9 for cleaning the air in public or private places, by diffusion of said composition in the air ducts.
PCT/FR2009/001247 2008-10-23 2009-10-23 Anti-influenza device and composition containing extracts from olive tree leaves, grapefruit pips, rosemary, green tea and curcumin WO2010046572A2 (en)

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EP09753141.2A EP3035948A2 (en) 2008-10-23 2009-10-23 ANTI-INFLUENZA DEVICE AND COMPOSITION CONTAINING EXTRACTS FROM OLIVE TREE LEAVES, GRAPEFRUIT PIPS, ROSEMARY, GREEN TEA AND CURCUMIN& xA;

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FR0805893A FR2937538B1 (en) 2008-10-23 2008-10-23 ANTIGRIPPAL PRODUCT

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Also Published As

Publication number Publication date
FR2937538B1 (en) 2010-12-31
EP3035948A2 (en) 2016-06-29
FR2937538A1 (en) 2010-04-30
WO2010046572A8 (en) 2010-06-03
WO2010046572A3 (en) 2010-07-22

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