WO2024062228A1 - Composition comprising abutilon fruticosum, acacia nubica, acacia bussei and myrsine africana, methods of preparation and therapeutic uses - Google Patents
Composition comprising abutilon fruticosum, acacia nubica, acacia bussei and myrsine africana, methods of preparation and therapeutic uses Download PDFInfo
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- WO2024062228A1 WO2024062228A1 PCT/GB2023/052420 GB2023052420W WO2024062228A1 WO 2024062228 A1 WO2024062228 A1 WO 2024062228A1 GB 2023052420 W GB2023052420 W GB 2023052420W WO 2024062228 A1 WO2024062228 A1 WO 2024062228A1
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Classifications
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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)
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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)
- A61K36/32—Burseraceae (Frankincense family)
- A61K36/324—Boswellia, e.g. frankincense
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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)
- A61K36/48—Fabaceae or Leguminosae (Pea or Legume family); Caesalpiniaceae; Mimosaceae; Papilionaceae
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/0012—Galenical forms characterised by the site of application
- A61K9/0043—Nose
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/08—Solutions
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- 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
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- 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
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61P31/12—Antivirals
- A61P31/14—Antivirals for RNA viruses
- A61P31/16—Antivirals for RNA viruses for influenza or rhinoviruses
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- A—HUMAN NECESSITIES
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- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2236/00—Isolation or extraction methods of medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicine
- A61K2236/10—Preparation or pretreatment of starting material
- A61K2236/15—Preparation or pretreatment of starting material involving mechanical treatment, e.g. chopping up, cutting or grinding
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A61K2236/10—Preparation or pretreatment of starting material
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- A—HUMAN NECESSITIES
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- A61K2236/30—Extraction of the material
- A61K2236/37—Extraction at elevated pressure or temperature, e.g. pressurized solvent extraction [PSE], supercritical carbon dioxide extraction or subcritical water extraction
Definitions
- the present invention relates to a composition which can be used in the treatment and prophylaxis of an infection of the human body.
- the composition can be used as an anti-viral treatment.
- the innate immune system of the human body is the first line of defence against invading viruses.
- the innate immune system relies on the invading virus having a conserved molecular pattern, which is used by the host body to trigger the immune response.
- Innate immune mechanisms such as granulocytes, monocytes and macrophages can inhibit pathogen entry and/or prevent the establishment of infection.
- the innate immune response either succeeds in eliminating the virus or helps keep the infection at bay until an adaptive immune response, which is generally virus specific, can be developed.
- vaccines and other medicines are used to boost the adaptative immune system of a host, such as a human, in order to lessen the effects of a viral infections on a host.
- a host such as a human
- the innate immune response initially strengthens with ages but declines sharply on average around the age of 70 yrs old.
- viruses such as SARS-CoV-2
- viruses have developed strategies to evade innate immune detection, for example by masking viral RNA and generating viral proteins that actively block anti-viral responses. Accordingly, the use of vaccination and other medicines are important to boost the adaptive response mechanism of the host.
- anti- viral drugs which inhibit some function of the specific virus can be given to help treat some viral infections.
- An object of the present invention may be to provide a composition which aids the host body in the prevention and/or treatment of a viral infection.
- a composition comprising at least one of an extract of Abutilon fruticosum, an extract of Acacia nubica and/or an extract of Acacia bussei and, optionally, an extract from Boswellia frereana and/or, optionally, an extract of Qorsheen, which is also known as Myrsine Africana or Qurjeen.
- the composition comprises at least two extracts selected from an extract of Abutilon fruticosum, an extract of Acacia nubica and an extract of Acacia bussei and optionally, an extract of Boswellia frereana, and/or optionally an extract of Qorsheen.
- the composition comprises an extract of Abutilon fruticosum, an extract of Acacia nubica and an extract of Acacia bussei and optionally an extract of Boswellia frereana, and/or optionally an extract of Qorsheen.
- the composition comprises an extract of Boswellia frereana, an extract of Abutilon fruticosum, an extract of Acacia nubica, an extract of Acacia bussei and an extract of Qorsheen.
- extract used herein is intended to include raw material of the plant along with solutions of essential constituents taken from the plant material.
- the raw material can be in the form of the bark, the root the fruit or the gum of the tree.
- an antiviral composition comprising the above-mentioned compositions.
- Boswellia frereana is a species of plant native to Somalia where the locals call it "Maydi” (other spellings include: “Meydi”, “Meyti”, “Maidi”, “Maieti”, and “Mayeti”) (which is the frankincense) or the “king of all frankincense”. It is also known as the “Yigaar” (or “Yegaar”) tree and by the common name for all frankincense, “Luban”.
- Maydi or frankincense extract is used in the present invention.
- Boswellia frereana and “Maydi” are used interchangeably herein.
- the composition could comprise an extract of Boswellia sacra and/or Boswellia carterii.
- the proportion of the composition given for Boswellia frereana would be the total proportion of the Boswellia extract in the composition whether it is Boswellia frereana, Boswellia sacra and/or Boswellia carterii.
- Abutilon fruticosum, Acacia nubica, Acacia bussei and Qorsheen are plants are native to Somalia.
- Abutilon fruticosum is known in Somalia as “Wancad” and the terms are used interchangeably herein.
- Acacia nubica is known in Somalia as “Gumar” and the terms are used interchangeably herein.
- Acacia bussei is known in Somalia as “Galool” and the terms are used interchangeably herein.
- Qorsheen also known as Myrsine Africana or Qurjeen
- the Golis mountains are also known as Qar Golis. Pictures of the plant are shown in Figures 16A to 16D and were taken in the Sanaag region of Somalia. Surprisingly, it has been found that the above-mentioned compositions can be used as a prophylactic treatment for viral infections as well as a treatment therefor.
- the extract of Boswellia frereana is the resin from the Boswellia frereana tree. The resin is produced in trees generally above 8 to 10 years old. The trees are tapped 2 to 3 times per year. Conveniently, the extract of Boswellia frereana is the resin from the final tap of the tree.
- This resin can have higher levels of aromatic terpene, sesquiterpene and diterpene content. Generally, a more opaque resin is a better quality.
- the extract of Abutilon fruticosum is a powder of the root of the shrub. The Abutilon fruticosum root is harvested, dried and then ground.
- the extract of Acacia nubica is a powder of the root of the shrub. The Acacia nubica root is harvested, dried and then ground.
- the extract of Acacia bussei is a powder of the bark. The Acacia bussei bark is harvested, dried and then ground.
- the extract of Qorsheen is a powder of the fruit. The fruit is toxic unless specially prepared.
- the Qorsheen fruit is dried in the sun for around 15 days and then ground into a powder.
- the powder can then be enclosed in a cloth and placed in water to dissolve.
- the composition comprises at least four extracts, for example, an extract of Abutilon fruticosum, an extract of Acacia nubica, an extract of Acacia bussei and an extract of Qorsheen.
- the composition comprises an extract of Boswellia frereana, an extract of Abutilon fruticosum, an extract of Acacia nubica, an extract of Acacia bussei and an extract of Qorsheen.
- the composition is an aqueous solution.
- the composition is in liquid and/or tablet form.
- the liquid form can be used for intranasal administration in the form of a nasal drop or spray or for oral administration in the form of mouth spray or gargle.
- the use of the composition of the present invention at the nose and/or mouth means that the innate immune system is being triggered at the entry points of the virus. Accordingly, it is convenient to have the composition in a form which can be administered to the mouth and nose. If a spray form is used, this may aid the coating for the nasal passages which have a complex structure and, in the context of the mouth, aid covering of the buccal membrane and the back of the throat.
- the tablets according to the present invention are prepared by grinding the extracts and compacting them into a suitable form which would can be prepared by any suitable method known to persons skilled in the art.
- the tablet form can be used when the viral infection has progressed and the patient is, for example suffering from symptoms in the lungs. Accordingly, ingestion of the herbs will lead to a systemic increase of the innate immune system.
- the tablet form can be used when suitable cooling facilities are not available to store the liquid form of the composition, if cooling facilities are required for such storage.
- the liquid form comprises 300 to 500g of Maydi extract, 4 to 10 g of Wancad extract, 4 to 10g of Gumar extract, 1 to 3g Galool extract and, optionally, 1 to 3g Qorsheen extract in 750ml to 1250ml of liquid.
- the liquid form comprises 350 to 450g of Maydi extract, 5 to 7g of Wancad extract, 5 to 7g of Gumar extract, 1 to 3g Galool extract and, optionally, 1 to 3g Qorsheen extract in 750ml to 1250ml of liquid.
- the liquid form comprises 400g of Maydi extract, 6g of Wancad extract, 6g of Gumar extract, 2g Galool extract and, optionally, 2g Qorsheen extract dissolved in 750ml to 1250ml of liquid.
- the tablet form comprises 40 to 59% Galool extract, 40 to 59% Gumar extract and 0.5 to 3% Wancad extract, and optionally 0.5 to 3% Qorsheen extract.
- the tablet form comprises 45 to 55% Galool extract, 45 to 55% Gumar extract and 0.5 to 3% Wancad extract, and optionally 0.5 to 3% Qorsheen extract.
- the tablet form comprises 49% Galool extract, 49% Gumar extract and 1% Wancad extract, and optionally 1% Qorsheen extract.
- the tablet form may be administered according to any regimen known to those skilled in the art, for example 2 tablets, 3 times per day for 7 days.
- the liquid and tablet form may comprise any suitable excipient known and/or used by those skilled in the art.
- the liquid form may be any suitable form including but not limited to aqueous solutions, suspensions and emulsions. Liquid formulation may be prepared by dissolving or suspending the active compounds in water or other suitable vehicles.
- compositions according to the present invention are effective in the treatment of viral infections and prophylactic treatment thereof, especially in RNA viral infections, such as SARS-Covid 19.
- the virus is selected from influenza virus, respiratory syncytial virus, human metapneumovirus, parainfluenza virus, coronaviruses, and rhinoviruses. These viruses are among the most common viruses causing respiratory diseases such as mild seasonal colds and influenza. These RNA viruses can also cause lower respiratory tract infections leading to bronchiolitis and pneumonia. Young children, the elderly, and patients with compromised cardiac, pulmonary, or immune systems are at greatest risk for serious disease associated with these RNA virus respiratory infections. In addition, swine and avian influenza viruses, together with severe acute respiratory syndrome-associated and Middle Eastern respiratory syndrome coronaviruses, represent significant pandemic threats to the general population.
- the virus is a coronavirus.
- the virus is SARS-CoV-2.
- a composition according to the present invention for use in the treatment of an infection caused by a virus wherein administration comprises 3 to 5 drops/spray per nostril every 3 to 5 hrs for 1 to 3 days and then 3 to 5 drops/spray per nostril every 7 to 9 hrs for 4 to 6 days.
- a composition according to the present invention wherein administration comprises 2 to 4 sprays into mouth every 3 to 5 hrs for 1 to 3 days and then 2 to 4 sprays into mouth every 7 to 9 hrs for 4 to 6 days.
- Conveniently administration comprises 4 drops/spray per nostril every 4hrs for 2 days and then 4 drops/spray per nostril every 8hrs for 5 days.
- Conveniently administration comprises 3 sprays into mouth every 4 hrs for 2 days and then 3 sprays into mouth every 8hrs for 5 days.
- Conveniently administration comprises 4 drops/spray per nostril at least 6 times per day for 2 days and then 4 drops/spray per nostril at least 8 times per day for 5 days.
- Conveniently administration comprises 3 sprays into mouth at least 6 times per day for 2 days and then 3 sprays into mouth at least 8 times per day for 5 days.
- a composition according to the present invention wherein administration comprises gargling with 5 to 15 ml every 3 to 5 hours for 1 to 3 days and then gargling with 2 to 10 ml every 7 to 9hrs for 4 to 6 days.
- Conveniently administration comprises gargling with 10ml every 4 hours for 2 days and then gargling with 5ml every 8hrs for 5 days.
- a composition according to the present invention wherein administration comprises gargling with 5 to 15 ml at least 6 times per day for 1 to 3 days and then gargling with 2 to 10 ml at least 3 times per day for 4 to 6 days.
- Conveniently administration comprises gargling with 10ml at least 6 times per day for 2 days and then gargling with 5ml at least 3 times per day for 5 days.
- Conveniently gargling takes place between 5 and 30 minutes post administration of nasal drops/spray and/or mouth spray.
- Conveniently gargling takes place between 10 and 20 minutes post administration of nasal drops/spray and/or mouth spray.
- Conveniently gargling takes place 15 minutes post administration of nasal drops/spray and/or mouth spray.
- the above mentioned dosage regimens and method of treatment have been found to be effective in children over 12 and adults.
- the timings of the doses may be at the same intervals, such that if the nostril drop/spray and mouth spray are both to be administered, they are administered at the same time.
- the nasal and buccal administration may be given at different intervals such that there is a space therebetween.
- the form of administration of the dosage regimens may be decided by the patient or a person skilled in the art such that a single form of administration may be chosen or two or more forms of administration, such as nasal spray/drops and gargle or nasal spray/drops, mouth spray and gargle, may be chosen.
- a patient may be treated using nasal drops/spray, a mouth spray and/or the gargle. If the compositions is administered by mouth spray and gargling then the gargling may take place at a spaced interval after the mouth spray.
- Dosage regimens and methods of treatment using oral administration can be effective for children under 5yrs old.
- the oral administration can be via drinking the composition or by mouth spray. In babies, only 1 spray per administration may be necessary.
- Such an oral preparation can be effective for children between around 5yrs old and 12yrs old.
- the oral preparation may be given in a usual drink for the children, for example, water, juice or milk. The latter choices will provide flavour for the children to try to make drinking the composition more palatable.
- An oral preparation which is drunk can be easier to administer to younger patients than a mouth spray or gargle.
- the prophylactic treatment according to the present invention has been found to be effective for patients of all ages.
- a method for preparation of the composition of the present invention comprising: a) mixing extracts of Wancad, Gumar and Galool, and optionally, Qorsheen together; b) adding mixture of extracts to water; c) optionally adding extract of Maydi; d) bring to the boil; e) take off heat; f) filter; and g) place in sealed container.
- Steps b) and c) can be performed in either order.
- the filtering step can be performed by any suitable means such as a mesh or metal sieve. Conveniently, the filter is chosen to remove greater than 10mm particles within the solution. Conveniently, the filter is chosen to remove greater than 5mm particles within the solution.
- the filter is chosen to remove greater than 1mm particles within the solution.
- 750ml to 1250ml of water is used per 350 to 450g of Maydi, 5 to 7g of Wancad, 5 to 7g of Gumar, 0.5 to 2.5g of Galool and optionally 0.5 to 2.5g of Qorsheen.
- 1000ml of water is used per 400g of Maydi, 6g of Wancad, 6g of Gumar, 2g of Galool and optionally 2g of Qorsheen.
- the liquid form of the composition of the present invention is stored at 1 ⁇ C to 10 ⁇ C.
- Figure 1 shows results of the review into the cell toxicity of the composition of the present invention
- Figures 2A, 2B and 2C show the impact of the composition of the present invention on the SARS- CoV-2 replication (A- Treatment, B- Prophylactic, C- Mix)
- Figures 3A, 3B, 3C, 3D and 3E show the composition of the present invention reduced SARS-CoV-2 replication in plaque assays (A- Treatment, B- Prophylactic, C- Mix, D- Negative Control, E- SARS- CoV control)
- Figure 4 shows the composition of the present invention reduced SARS-CoV-2 replication in plaque assays shown in Figures 3A to 3E in graphical format
- Figure 5 shows treatment of VeroE6 cells with the composition of the present invention inhibited the SARS-CoV-2 in situ (A – Positive Control, B - Treatment
- FIG. 9 shows treatment of cells infected with SARS-CoV-2 with 1:250 dilution of composition of the present invention and immune staining using anti-S antibodies in three replicates; 1, 2 and 3;
- Figure 10 shows treatment of cells infected with SARS-CoV-2 with 1:500 dilution of composition of the present invention and immune staining using anti-S antibodies in three replicates; 1, 2 and 3;
- Figure 11 shows treatment of untreated cells infected with SARS-Cov-2 and immune stained using anti-S antibodies in three replicates; 1, 2 and 3.
- Figure 12 shows an assessment of the quality of RNA used in the RT-PCR using Nano-drop TM spectrophotometer at 10mm Absorbance;
- Figures 13A, 13B and 13 C show expression of interferon-stimulated genes (ISGs) in cells treated with the composition of the present invention.
- y Sample ID;
- Figure 14 shows the ability of the composition of the present invention in reducing the virus infectivity.
- y entry of pseudoparticles (%)
- Figures 16A to 16D show pictures of the Qorsheen plant in situ in Sangaa region of Somalia.
- Example 1 The following composition was prepared for use in all the following Examples: GALOOL, GUMAR, WANCAD, QORSHEEN were ground together. Then put in water with MAYDI and brought to the boiling point. The boiled solution was filtered through a 1mm metal sieve and sealed to ensure no vapour escapes and then stored until needed.
- QURE20-IM3 400 Grams Maydi 16 Grams of herbs • 6 Grams WANCAD • 6 Grams GUMAR • 2 Grams GALOOL • 2 Grams QORSHEEN This formulation is herein referred to as “QURE20-IM3”.
- Example 2 In order to demonstrate the level of toxicity for VeroE6 cells, QURE20-IM3 was diluted in PBS into 1:1, 1:10, 1:50, and 1:100 and cells were incubated for 2 hours. Thereafter, live cell images were captured at 6 hours, 12 hours, and 24 hours after the treatment. The analysis of the healthy and stressed cells indicated that dilutions until 1:10 were toxic whereas 1:50 to 1:100 dilutions appeared to be safe (see Figure 1).
- VeroE6 cells were treated with QURE20-IM3 using three different combinations: 1. Cells were first treated with QURE20-IM3 and then infected with the virus (Prophylactic); 2. Cells were first infected with the SARS-CoV-2 and then treated with QURE20-IM3 (Treatment); 3. Both QURE20-IM3 and SARS-CoV-2 were mixed and then VeroE6 cells were infected (mix). Under all three conditions, cells were used to extract total RNA and quantitative real time PCR was applied to assess the replication of SARS-CoV-2.
- Both QURE20-IM3 and SARS-CoV-2 were mixed and then VeroE6 cells were infected and incubated for 6 hours before analysis (Mix). Thereafter, a plaque assay was performed in serial dilution. The analysis indicates that treatment and prophylactic application of the composition of the present invention significantly inhibited the virus replication (see Figures 3A to 3E and Figure 4).
- cells were infected with virus before and after the treatment with the composition of the present invention. Additionally, a mixture of QURE20-IM3 and virus was also incubated before infecting VeroE6 cells.
- QURE20-IM3 induced substantially some of the innate immune genes including IFN-beta, IFI35, IFIT5 and Mx genes (see Figure 6).
- Example 3 The following patients were given QURE20-IM3 in 2020 and 2021 under a non-disclosure agreement. No patient other than the inventors were given access to the exact composition of the treatment given nor the method of preparation.: Patient A Age: +70, Female, Medical history Do you suffer from symptoms of Covid-19?
- Patient B After approximately 15 mins after administration of the nasal drops and mouth spray, Patient B gargled with 10ml of composition on days 1 and 2 and 5ml of composition on the remaining days. After 3 days of taking the above composition, Patient B seemed to be improving and recovering from all her Covid-19 symptoms.
- Patient C received 3 sprays of the same composition into the mouth every 4 hours for two days and then for the next five days received 3 sprays into the mouth every 8 hours. After approximately 15 mins after administration of the nasal drops and mouth spray, Patient C gargled with 10ml of composition on days 1 and 2 and 5ml of composition on the remaining days. Patient C was a difficult case. She had symptoms cough, shortness of breath, and difficulty breathing and acute pneumonia. She took her first dose on the same day she knew she was positive with covid-19. After 48 hours, her major symptoms disappeared, and she stopped taking the treatment on the fifth day because she felt she was back to normal.
- Patient D After approximately 15 mins after administration of the nasal drops and mouth spray, Patient D gargled with 10ml of composition on days 1 and 2 and 5ml of composition on the remaining days. Patient D’s loss of smell and taste came back within 2 days.
- Patient E After approximately 15 mins after administration of the nasal drops and mouth spray, Patient E gargled with 10ml of composition on days 1 and 2 and 5ml of composition on the remaining days. After 24 hours of taking the above-mentioned treatment, Patient E had his sense of taste back, and on his second day, his sense of smell was back. Patient stopped taking the treatment after seven days.
- Patient H received 3 sprays of the same composition into the mouth every 4 hours for two days and then for the next five days received 3 sprays into the mouth every 8 hours. After approximately 15 mins after administration of the nasal drops and mouth spray Patient H gargled with 10ml of composition on days 1 and 2 and 5ml of composition on the remaining days. After 24 hours of taking the aforementioned treatment, the symptoms (fever - loss of appetite - body aches - sore throat - chills - nausea - diarrhea) disappeared. After 48 hours, Patient H recover his sense of smell and taste slightly and the cough subsided. Within 72 hours Patient H was feeling better, his coughing was less as he can talk without coughing a lot. In addition, his sense of smell and taste was much better, and his fatigue gone.
- Patient J received 3 sprays of the same composition into the mouth every 4 hours for two days and then for the next five days received 3 sprays into the mouth every 8 hours. After approximately 15 mins after administration of the nasal drops and mouth spray, Patient J gargled with 10ml of composition on days 1 and 2 and 5ml of composition on the remaining days. After approximately 72 hours the patient started to improve, and all symptoms had disappeared within 7 days when treatment stopped.
- Patient K received 3 sprays of the same composition into the mouth every 4 hours for two days and then for the next five days received 3 sprays into the mouth every 8 hours. After approximately 15 mins after administration of the nasal drops and mouth spray, Patient K gargled with 10ml of composition on days 1 and 2 and 5ml of composition on the remaining days. After approximately 72 hours the patient started to improve, and all symptoms had disappeared within 7 days when treatment stopped.
- Patient L received 3 sprays of the same composition into the mouth every 4 hours for two days and then for the next five days received 3 sprays into the mouth every 8 hours. After approximately 15 mins after administration of the nasal drops and mouth spray, Patient L gargled with 10ml of composition on days 1 and 2 and 5ml of composition on the remaining days. After approximately 72 hours the patient started to improve, and all symptoms had disappeared within 7 days when treatment stopped. Patient M Male Medical history Do you suffer from symptoms of Covid-19?
- Patient M received 3 sprays of the same composition into the mouth every 4 hours for two days and then for the next five days received 3 sprays into the mouth every 8 hours. After approximately 15 mins after administration of the nasal drops and mouth spray, Patient M gargled with 10ml of composition on days 1 and 2 and 5ml of composition on the remaining days. After approximately 72 hours the patient started to improve, and all symptoms had disappeared within 7 days when treatment stopped. Patient N Female Medical history Do you suffer from symptoms of Covid-19?
- Patient N received 3 sprays of the same composition into the mouth every 4 hours for two days and then for the next five days received 3 sprays into the mouth every 8 hours. After approximately 15 mins after administration of the nasal drops and mouth spray, Patient N gargled with 10ml of composition on days 1 and 2 and 5ml of composition on the remaining days. After approximately 72 hours the patient started to improve, and all symptoms had disappeared within 7 days when treatment stopped.
- Example 4 15 subjects were given 1 spray of composition in their mouth and 1 drop of the composition into each nostril every day as a prophylactic treatment. To date, despite going to their place of work and continuing daily life none of the subject contracted Covid-19. The findings of these in vivo studies are concurrent with the in vitro studies given in Example 2.
- Example 5 Toxicity: Required material: VeroE6 cells 96-wells plate (Nuc 136101 Thermofisher) Medium (DMEM, Thermofisher, 11965084) Crystal Violet (0.2% crystal violet in distal water for staining) Procedure: 1. Seed 60,000 VeroE6 cells in 96-well plates. 2. Expect 100% confluency in 24 hours 3. Teat VeroE6 cells with treatment with different dilutions. 4. After 16 hours, stain the cells with crystal violet and calculate the healthy cells compared to untreated cells In summary, a total of 60,000 VeroE6 cells were seeded in 96-well plates. Once fully confluent, cells were exposed to increasing doses of QURE20-IM3 (1:1 to 1:1000).
- VeroE6 cells 96-wells plate (Nuc 136101 Thermofisher) Medium (DMEM, Thermofisher, 11965084) Crystal Violet (0.2% crystal violet in distal water for staining) SARS-COV-2 Procedure: • VeroE6 cells were seeded in 96-well plates with a density of 60,000. • Treat cells with increasing doses of treatment (1:1 to 1:1000).
- QURE20-IM3- treated and untreated cells were infected with SARSCoV-2 (UK/Victoria/2020 strain) with MOI (multiplicity of infection) of 1.0. Twenty-four hours post-infection, supernatant was collected and seeded into VeroE6 cells for 24 hours. Cells were fixed with 5% paraformaldehyde and stained with crystal violet and the TCID 50 values were estimated in QURE20-IM3-treated and non-treated cells. Based on the analysis of the TCID50/ml, both 1:250 and 1:500 dilution treated cells showed inhibition of SARS-COV-2 (see Figure 8).
- Example 7 This Method covers fixation, fluorescent labelling and mounting of cells grown on glass coverslips for imaging in the confocal microscope.
- the aim of this procedure is to fluorescently label specific proteins in cells in order to see where those proteins originate/move to or to see if they interact with other proteins or cell components to understand the level of expression under specific treatments.
- the protocol should always be followed as described in this method.
- VeroE6 cells were subjected to treatment with either 1:250 or 1:500 dilutions of QUER20-IM3 for 16 hours and then infected with SARS-CoV-2 for 24 hours.
- the cells were fixed post-infection with 5% paraformaldehyde for 4 h and permeabilized with 0.5% Triton X-100 for 5 min. After blocking with 3% bovine serum albumin (BSA) for 30 min, the cells were incubated for 1 h at room temperature with rabbit anti-SARS-CoV Spike antibodies, which exhibits strong cross- reactivity with SARS-CoV-2.
- BSA bovine serum albumin
- RNA was assessed with the NanoDrop TM spectrophotometer at 10mm Absorbance (see Figure 12 and Table 1).
- Real-time PCR was performed to determine the gene expression of IFIT5, IFIT35, and Mx and were compared with housekeeping gene.
- the chosen genes can be considered are key host genes involved in the innate immunity, cytokine and antiviral actions against SARS-CoV-2.
- Treatment of QURE20-IM3 induced more ISGs expression compared to gene transcription induced by the virus (see Figures 13A, 13B and 13C).
- Example 10 Material Required • HEK293T cells • VeroE6 cells • Medium (DMEM, Thermofisher, 11965084) • Viafect (Promega) • Luciferase Cell Culture Lysis 5X reagent (REF E1531, Promega) • Corning® 96-well Black Flat Bottom Polystyrene High Bind Microplate (REF 3925, Corning) • Promega Nano-Glo Luciferase Assay System (REF N1150, Promega) Part 1: Virus pseudotype production Protocol • Seed 5 million 293T cells per 10 cm dish in 10ml DMEM++ (10% FSC, antibiotics) • Transfection per 10cm dish with 3-plasmid NanoLuc2AEGFP system (VSV-G pseudotyped ⁇ G- luciferase VSV) along with SARS-CoV-2 S.
- VSV vesicular stomatitis virus
- BHK-21/WI-2 cells were transfected to express the S proteins were inoculated with VSV-G pseudotyped ⁇ G-luciferase VSV (Kerafast). After a 2-h incubation at 37 °C, the inoculum was removed and DMEM supplemented with 5% FBS, 50 units/ml penicillin and 50 ⁇ g/ml streptomycin were added back to cells. Pseudotyped particles were collected 24 h post-inoculation, then centrifuged at 1,320g to remove cell debris and stored at ⁇ 80 °C unYl use.
- VeroE6 cells were treated with QURE20-IM3 for 1 h before inoculation with respective pseudotyped VSV. After 2 h inoculation in the presence of the QURE20-IM3, the inoculum was removed, and fresh medium were added to cells for further culture. The activity of firefly luciferase as a readout of infected cells were measured using the bright-Glo luciferase assay (Promega) for quantitative determination at 16 hpi. The data analysis indicated a reduced virus entry into the cells treated with the QURE20- IM3 compared to untreated and pseudoparticle infected cells (see Figure 15).
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Abstract
The present invention provides a composition which can be used in the treatment and prophylaxis of an infection of the human body. For example, the composition can be used as an anti-viral treatment.
Description
Composition The present invention relates to a composition which can be used in the treatment and prophylaxis of an infection of the human body. In particular, the composition can be used as an anti-viral treatment. The innate immune system of the human body is the first line of defence against invading viruses. The innate immune system relies on the invading virus having a conserved molecular pattern, which is used by the host body to trigger the immune response. Innate immune mechanisms such as granulocytes, monocytes and macrophages can inhibit pathogen entry and/or prevent the establishment of infection. The innate immune response either succeeds in eliminating the virus or helps keep the infection at bay until an adaptive immune response, which is generally virus specific, can be developed. Traditionally, vaccines and other medicines are used to boost the adaptative immune system of a host, such as a human, in order to lessen the effects of a viral infections on a host. In humans, the innate immune response initially strengthens with ages but declines sharply on average around the age of 70 yrs old. Further, viruses, such as SARS-CoV-2, have developed strategies to evade innate immune detection, for example by masking viral RNA and generating viral proteins that actively block anti-viral responses. Accordingly, the use of vaccination and other medicines are important to boost the adaptive response mechanism of the host. In addition, anti- viral drugs, which inhibit some function of the specific virus can be given to help treat some viral infections. However, generally anti-viral treatments must be given during the initial stages of viral infection otherwise the host can become overwhelmed by virus numbers. The problem with vaccinations and anti-viral treatments are that they are often virus specific. Accordingly, although a vaccination may have been given for a specific virus or variant of a virus, if the host is infected with a different virus or variant, then the vaccination may have no effect. Further, anti-viral treatments work in relation to very specific viral replication pathways and, therefore, if the virus infecting the host does not use such a specific replication pathway the anti- viral treatment may have no effect. An object of the present invention may be to provide a composition which aids the host body in the
prevention and/or treatment of a viral infection. According to a first aspect of the invention there is provided a composition comprising at least one of an extract of Abutilon fruticosum, an extract of Acacia nubica and/or an extract of Acacia bussei and, optionally, an extract from Boswellia frereana and/or, optionally, an extract of Qorsheen, which is also known as Myrsine Africana or Qurjeen. Conveniently, the composition comprises at least two extracts selected from an extract of Abutilon fruticosum, an extract of Acacia nubica and an extract of Acacia bussei and optionally, an extract of Boswellia frereana, and/or optionally an extract of Qorsheen. Conveniently, the composition comprises an extract of Abutilon fruticosum, an extract of Acacia nubica and an extract of Acacia bussei and optionally an extract of Boswellia frereana, and/or optionally an extract of Qorsheen. Conveniently, the composition comprises an extract of Boswellia frereana, an extract of Abutilon fruticosum, an extract of Acacia nubica, an extract of Acacia bussei and an extract of Qorsheen. As is demonstrated in the present application, the term “extract” used herein is intended to include raw material of the plant along with solutions of essential constituents taken from the plant material. The raw material can be in the form of the bark, the root the fruit or the gum of the tree. According to a further aspect of the invention there is provided an antiviral composition comprising the above-mentioned compositions. Boswellia frereana is a species of plant native to Somalia where the locals call it "Maydi" (other spellings include: “Meydi”, “Meyti”, “Maidi”, “Maieti”, and “Mayeti”) (which is the frankincense) or the “king of all frankincense”. It is also known as the “Yigaar” (or “Yegaar”) tree and by the common name for all frankincense, “Luban”. Maydi or frankincense extract is used in the present invention. The terms “Boswellia frereana” and “Maydi” are used interchangeably herein. In addition or as an alternative to Boswellia frereana, the composition could comprise an extract of Boswellia sacra and/or Boswellia carterii. The proportion of the composition given for Boswellia frereana would be the total proportion of the Boswellia extract in the composition whether it is Boswellia frereana, Boswellia sacra and/or Boswellia carterii. Abutilon fruticosum, Acacia nubica, Acacia bussei and Qorsheen are plants are native to Somalia. Abutilon fruticosum is known in Somalia as “Wancad” and the terms are used interchangeably herein. Acacia nubica is known in Somalia as “Gumar” and the terms are used interchangeably
herein. Acacia bussei is known in Somalia as “Galool” and the terms are used interchangeably herein. Qorsheen (also known as Myrsine Africana or Qurjeen) is a plant found in the Golis mountains and on the slopes of the Dallo Mountain, which is part of the Ogo Mountain range in Somalia. The Golis mountains are also known as Qar Golis. Pictures of the plant are shown in Figures 16A to 16D and were taken in the Sanaag region of Somalia. Surprisingly, it has been found that the above-mentioned compositions can be used as a prophylactic treatment for viral infections as well as a treatment therefor. Conveniently, the extract of Boswellia frereana is the resin from the Boswellia frereana tree. The resin is produced in trees generally above 8 to 10 years old. The trees are tapped 2 to 3 times per year. Conveniently, the extract of Boswellia frereana is the resin from the final tap of the tree. This resin can have higher levels of aromatic terpene, sesquiterpene and diterpene content. Generally, a more opaque resin is a better quality. Conveniently the extract of Abutilon fruticosum is a powder of the root of the shrub. The Abutilon fruticosum root is harvested, dried and then ground. Conveniently the extract of Acacia nubica is a powder of the root of the shrub. The Acacia nubica root is harvested, dried and then ground. Conveniently the extract of Acacia bussei is a powder of the bark. The Acacia bussei bark is harvested, dried and then ground. Conveniently the extract of Qorsheen is a powder of the fruit. The fruit is toxic unless specially prepared. Accordingly, the Qorsheen fruit is dried in the sun for around 15 days and then ground into a powder. The powder can then be enclosed in a cloth and placed in water to dissolve. Conveniently the composition comprises at least four extracts, for example, an extract of Abutilon fruticosum, an extract of Acacia nubica, an extract of Acacia bussei and an extract of Qorsheen.
Conveniently, the composition comprises an extract of Boswellia frereana, an extract of Abutilon fruticosum, an extract of Acacia nubica, an extract of Acacia bussei and an extract of Qorsheen. Conveniently, the composition is an aqueous solution. Conveniently, the composition is in liquid and/or tablet form. Conveniently the liquid form can be used for intranasal administration in the form of a nasal drop or spray or for oral administration in the form of mouth spray or gargle. The use of the composition of the present invention at the nose and/or mouth means that the innate immune system is being triggered at the entry points of the virus. Accordingly, it is convenient to have the composition in a form which can be administered to the mouth and nose. If a spray form is used, this may aid the coating for the nasal passages which have a complex structure and, in the context of the mouth, aid covering of the buccal membrane and the back of the throat. The tablets according to the present invention are prepared by grinding the extracts and compacting them into a suitable form which would can be prepared by any suitable method known to persons skilled in the art. Conveniently the tablet form can be used when the viral infection has progressed and the patient is, for example suffering from symptoms in the lungs. Accordingly, ingestion of the herbs will lead to a systemic increase of the innate immune system. Further, the tablet form can be used when suitable cooling facilities are not available to store the liquid form of the composition, if cooling facilities are required for such storage. Conveniently the liquid form comprises 300 to 500g of Maydi extract, 4 to 10 g of Wancad extract, 4 to 10g of Gumar extract, 1 to 3g Galool extract and, optionally, 1 to 3g Qorsheen extract in 750ml to 1250ml of liquid. Conveniently the liquid form comprises 350 to 450g of Maydi extract, 5 to 7g of Wancad extract, 5 to 7g of Gumar extract, 1 to 3g Galool extract and, optionally, 1 to 3g Qorsheen extract in 750ml to 1250ml of liquid.
Conveniently the liquid form comprises 400g of Maydi extract, 6g of Wancad extract, 6g of Gumar extract, 2g Galool extract and, optionally, 2g Qorsheen extract dissolved in 750ml to 1250ml of liquid. Conveniently the tablet form comprises 40 to 59% Galool extract, 40 to 59% Gumar extract and 0.5 to 3% Wancad extract, and optionally 0.5 to 3% Qorsheen extract. Conveniently the tablet form comprises 45 to 55% Galool extract, 45 to 55% Gumar extract and 0.5 to 3% Wancad extract, and optionally 0.5 to 3% Qorsheen extract. Conveniently the tablet form comprises 49% Galool extract, 49% Gumar extract and 1% Wancad extract, and optionally 1% Qorsheen extract. The tablet form may be administered according to any regimen known to those skilled in the art, for example 2 tablets, 3 times per day for 7 days. The liquid and tablet form may comprise any suitable excipient known and/or used by those skilled in the art. The liquid form may be any suitable form including but not limited to aqueous solutions, suspensions and emulsions. Liquid formulation may be prepared by dissolving or suspending the active compounds in water or other suitable vehicles. The excipient may, for example, be selected from carriers, support materials, lubricants, fillers, solvents, diluents, colourants, flavours, antioxidants and/or agglutinating agent. Tablets and solid forms of the compositions may be coated in any conventional and convenient manner. Conveniently, the liquid is water. Surprisingly, it has been found that compositions according to the present invention are effective in the treatment of viral infections and prophylactic treatment thereof, especially in RNA viral infections, such as SARS-Covid 19. A composition according to the present invention for use in the treatment of an infection caused by a virus.
A composition according to the present invention for use as a prophylactic against any infection caused by a virus. Conveniently, the virus is selected from influenza virus, respiratory syncytial virus, human metapneumovirus, parainfluenza virus, coronaviruses, and rhinoviruses. These viruses are among the most common viruses causing respiratory diseases such as mild seasonal colds and influenza. These RNA viruses can also cause lower respiratory tract infections leading to bronchiolitis and pneumonia. Young children, the elderly, and patients with compromised cardiac, pulmonary, or immune systems are at greatest risk for serious disease associated with these RNA virus respiratory infections. In addition, swine and avian influenza viruses, together with severe acute respiratory syndrome-associated and Middle Eastern respiratory syndrome coronaviruses, represent significant pandemic threats to the general population. After our clinical trials on covid-19 patients, we had two patients who had bronchiolitis and pneumonia with covid-19, and after they were administered the composition of the present invention, they were clear from it. Conveniently, the virus is a coronavirus. Conveniently, the virus is SARS-CoV-2. A composition according to the present invention for use in the treatment of an infection caused by a virus wherein administration comprises 3 to 5 drops/spray per nostril every 3 to 5 hrs for 1 to 3 days and then 3 to 5 drops/spray per nostril every 7 to 9 hrs for 4 to 6 days. A composition according to the present invention wherein administration comprises 2 to 4 sprays into mouth every 3 to 5 hrs for 1 to 3 days and then 2 to 4 sprays into mouth every 7 to 9 hrs for 4 to 6 days. Conveniently administration comprises 4 drops/spray per nostril every 4hrs for 2 days and then 4 drops/spray per nostril every 8hrs for 5 days. Conveniently administration comprises 3 sprays into mouth every 4 hrs for 2 days and then 3 sprays into mouth every 8hrs for 5 days. A composition according to the present invention for use in the treatment of an infection caused
by a virus wherein administration comprises 3 to 5 drops/spray per nostril at least 5 to 7 times per day for 1 to 3 days and then 3 to 5 drops/spray per nostril at least 7 to 9 times per day for 4 to 6 days. A composition according to the present invention for use in the treatment of an infection caused by a virus wherein administration comprises 2 to 4 sprays into mouth at least 5 to 7 times per day for 1 to 3 days and then 2 to 4 sprays into mouth at least 7 to 9 times per day for 4 to 6 days. Conveniently administration comprises 4 drops/spray per nostril at least 6 times per day for 2 days and then 4 drops/spray per nostril at least 8 times per day for 5 days. Conveniently administration comprises 3 sprays into mouth at least 6 times per day for 2 days and then 3 sprays into mouth at least 8 times per day for 5 days. A composition according to the present invention wherein administration comprises gargling with 5 to 15 ml every 3 to 5 hours for 1 to 3 days and then gargling with 2 to 10 ml every 7 to 9hrs for 4 to 6 days. Conveniently administration comprises gargling with 10ml every 4 hours for 2 days and then gargling with 5ml every 8hrs for 5 days. A composition according to the present invention wherein administration comprises gargling with 5 to 15 ml at least 6 times per day for 1 to 3 days and then gargling with 2 to 10 ml at least 3 times per day for 4 to 6 days. Conveniently administration comprises gargling with 10ml at least 6 times per day for 2 days and then gargling with 5ml at least 3 times per day for 5 days. Conveniently gargling takes place between 5 and 30 minutes post administration of nasal drops/spray and/or mouth spray. Conveniently gargling takes place between 10 and 20 minutes post administration of nasal drops/spray and/or mouth spray.
Conveniently gargling takes place 15 minutes post administration of nasal drops/spray and/or mouth spray. The above mentioned dosage regimens and method of treatment have been found to be effective in children over 12 and adults. The timings of the doses may be at the same intervals, such that if the nostril drop/spray and mouth spray are both to be administered, they are administered at the same time. Alternatively, the nasal and buccal administration may be given at different intervals such that there is a space therebetween. The form of administration of the dosage regimens may be decided by the patient or a person skilled in the art such that a single form of administration may be chosen or two or more forms of administration, such as nasal spray/drops and gargle or nasal spray/drops, mouth spray and gargle, may be chosen. A patient may be treated using nasal drops/spray, a mouth spray and/or the gargle. If the compositions is administered by mouth spray and gargling then the gargling may take place at a spaced interval after the mouth spray. A composition according to the present invention for use in the treatment of an infection caused by a virus wherein administration comprises 1 to 3 drops/spray per nostril every 7 to 9 hrs for 6 to 8 days. Conveniently administration comprises 1 drop per nostril every 8 hrs for 7 days. Dosage regimens and methods of treatment using nasal administration can be effective for children under 12yrs old. A composition according to the present invention for use in the treatment of an infection caused by a virus wherein administration comprises taking orally 3 to 15ml every 7 to 9 hrs for 6 to 8 days. A composition according to the present invention for use in the treatment of an infection caused by a virus wherein administration comprises taking orally 5ml every 8 hrs for 7 days. Dosage regimens and methods of treatment using oral administration can be effective for children
under 5yrs old. The oral administration can be via drinking the composition or by mouth spray. In babies, only 1 spray per administration may be necessary. A composition according to the present invention for use in the treatment of an infection caused by a virus wherein administration comprises taking orally 10ml every 8 hrs for 7 days. Such an oral preparation can be effective for children between around 5yrs old and 12yrs old. The oral preparation may be given in a usual drink for the children, for example, water, juice or milk. The latter choices will provide flavour for the children to try to make drinking the composition more palatable. An oral preparation which is drunk can be easier to administer to younger patients than a mouth spray or gargle. A composition according to the present invention for use as a prophylactic wherein administration comprises 1 to 3 drops/spray per nostril per day. A composition according to the present invention for use as a prophylactic wherein administration comprises 1 to 3 sprays into the mouth per day. A composition according to the present invention for use as a prophylactic wherein administration comprises 1 drop per nostril per day. A composition according to the present invention for use as a prophylactic wherein administration comprises 1 spray into the mouth per day. The prophylactic treatment according to the present invention has been found to be effective for patients of all ages. A method for preparation of the composition of the present invention, the method comprising: a) mixing extracts of Wancad, Gumar and Galool, and optionally, Qorsheen together; b) adding mixture of extracts to water; c) optionally adding extract of Maydi; d) bring to the boil;
e) take off heat; f) filter; and g) place in sealed container. Steps b) and c) can be performed in either order. The filtering step can be performed by any suitable means such as a mesh or metal sieve. Conveniently, the filter is chosen to remove greater than 10mm particles within the solution. Conveniently, the filter is chosen to remove greater than 5mm particles within the solution. Conveniently, the filter is chosen to remove greater than 1mm particles within the solution. Conveniently, 750ml to 1250ml of water is used per 350 to 450g of Maydi, 5 to 7g of Wancad, 5 to 7g of Gumar, 0.5 to 2.5g of Galool and optionally 0.5 to 2.5g of Qorsheen. Conveniently, 1000ml of water is used per 400g of Maydi, 6g of Wancad, 6g of Gumar, 2g of Galool and optionally 2g of Qorsheen. Conveniently the liquid form of the composition of the present invention is stored at 1◦C to 10◦C. Conveniently the liquid form of the composition of the present invention is stored at 2◦C to 8◦C. The invention will now be particularly described by way of example with reference to the accompanying Figures in which: Figure 1 shows results of the review into the cell toxicity of the composition of the present invention; Figures 2A, 2B and 2C show the impact of the composition of the present invention on the SARS- CoV-2 replication (A- Treatment, B- Prophylactic, C- Mix); Figures 3A, 3B, 3C, 3D and 3E show the composition of the present invention reduced SARS-CoV-2 replication in plaque assays (A- Treatment, B- Prophylactic, C- Mix, D- Negative Control, E- SARS- CoV control); Figure 4 shows the composition of the present invention reduced SARS-CoV-2 replication in plaque assays shown in Figures 3A to 3E in graphical format; Figure 5 shows treatment of VeroE6 cells with the composition of the present invention inhibited the SARS-CoV-2 in situ (A – Positive Control, B - Treatment with 1/100 of the composition of the present invention for 6 hours then infection for 2 hours then left for 16 hours, C - 100ul virus + 100ul 1/100 of the composition of the present invention, mixing and incubate over cells for 2 hours
then remove and add fresh media for 22 hours, D- Infection with SARS-CoV-2 for 2 hours then treatment with 1/100 of the composition of the present invention for 22 hours); and Figure 6 shows the composition of the present invention activates innate immune genes; Figure 7 shows further results of investigation into the toxicity of the composition of the present invention in cell culture system (VeroE6 cells); Figure 8 shows antiviral activity of the composition of the present invention in cell cultures using TCID50. y=1E+10x2-5E+10x+5E+10; R2=1; A - virus only – positive control, B - 1:250 dilution, C – 1:500 dilution; Figure 9 shows treatment of cells infected with SARS-CoV-2 with 1:250 dilution of composition of the present invention and immune staining using anti-S antibodies in three replicates; 1, 2 and 3; Figure 10 shows treatment of cells infected with SARS-CoV-2 with 1:500 dilution of composition of the present invention and immune staining using anti-S antibodies in three replicates; 1, 2 and 3; Figure 11 shows treatment of untreated cells infected with SARS-Cov-2 and immune stained using anti-S antibodies in three replicates; 1, 2 and 3. (i.e. positive control); Figure 12 shows an assessment of the quality of RNA used in the RT-PCR using Nano-drop TM spectrophotometer at 10mm Absorbance; Figures 13A, 13B and 13 C show expression of interferon-stimulated genes (ISGs) in cells treated with the composition of the present invention. Figure 13A – Interferon-induced protein 35 (IFI35), Figure 13B – Interferon-induced proteins with tetratricopeptide repeats (IFITS) and Figure 13C – MxA. y = Sample ID; Figure 14 shows the ability of the composition of the present invention in reducing the virus infectivity. y = percentage virus recovery, x = samples; and Figure 15 shows the percentage entry inhibition of pseudotyped viruses in cells treated with the composition of the present invention. y = entry of pseudoparticles (%) A: Virus-like particles only (VLPs), B: 1:250 treatment, C: 1:500 treatment, D: Cells only. Figures 16A to 16D show pictures of the Qorsheen plant in situ in Sangaa region of Somalia. Example 1 The following composition was prepared for use in all the following Examples: GALOOL, GUMAR, WANCAD, QORSHEEN were ground together. Then put in water with MAYDI and brought to the boiling point. The boiled solution was filtered through a 1mm metal sieve and sealed to ensure no vapour escapes and then stored until needed. The solution was stored between 2◦C and 8◦C.
400 Grams Maydi 16 Grams of herbs • 6 Grams WANCAD • 6 Grams GUMAR • 2 Grams GALOOL • 2 Grams QORSHEEN This formulation is herein referred to as “QURE20-IM3”. Example 2 In order to demonstrate the level of toxicity for VeroE6 cells, QURE20-IM3 was diluted in PBS into 1:1, 1:10, 1:50, and 1:100 and cells were incubated for 2 hours. Thereafter, live cell images were captured at 6 hours, 12 hours, and 24 hours after the treatment. The analysis of the healthy and stressed cells indicated that dilutions until 1:10 were toxic whereas 1:50 to 1:100 dilutions appeared to be safe (see Figure 1). Therefore, 1:100 dilution was used to understand the antiviral potential of the QURE20-IM3. In order to assess the impact of the composition of the present invention on the replication kinetics of SARSCoV-2, VeroE6 cells were treated with QURE20-IM3 using three different combinations: 1. Cells were first treated with QURE20-IM3 and then infected with the virus (Prophylactic); 2. Cells were first infected with the SARS-CoV-2 and then treated with QURE20-IM3 (Treatment); 3. Both QURE20-IM3 and SARS-CoV-2 were mixed and then VeroE6 cells were infected (mix). Under all three conditions, cells were used to extract total RNA and quantitative real time PCR was applied to assess the replication of SARS-CoV-2. As shown in Figures 2A to 2C the analysis indicated that in all three conditions, the replication of SARS-CoV-2 was significantly reduced, however, the most reduced replication was observed in treatment regimen (see Fig 2A). In order to assess the impact of QURE20-IM3 on the infectivity of the SARS-CoV2, the following three combinations were made: 1. Cells were first treated with QURE20-IM3 (1:100 dilution) and then infected with the virus before incubation for 6 hours before analysis (Prophylactic); 2. Cells were first infected with the SARS-CoV-2 and then treated with QURE20-IM3 (1:100 dilution) for 2 hours before analysis (Treatment);
3. Both QURE20-IM3 and SARS-CoV-2 were mixed and then VeroE6 cells were infected and incubated for 6 hours before analysis (Mix). Thereafter, a plaque assay was performed in serial dilution. The analysis indicates that treatment and prophylactic application of the composition of the present invention significantly inhibited the virus replication (see Figures 3A to 3E and Figure 4). In order to visually inspect the replication of SARS-CoV-2 in VeroE6 cells treated with QURE20-IM3, cells were infected with virus before and after the treatment with the composition of the present invention. Additionally, a mixture of QURE20-IM3 and virus was also incubated before infecting VeroE6 cells. Thereafter, immunofluorescence assay using DAPI staining was applied to understand the replication of SARS-CoV-2 in the cell culture treated with or without QURE20-IM3. Analysis indicated a profound reduction in the replication of SARS-CoV-2 particularly when QURE20-IM3 was applied before the infection with SARS-CoV-2 or after the infection with SARS-CoV-2 (see Figure 5). In order to understand the nature of the innate immunity induced by the composition of the present invention, VeroE6 cells were infected with SARS-CoV-2 or left untreated. Additionally, cells were stimulated with QURE20-IM3. Thereafter, RNA was extracted, and multiple genes of innate immunity were measured quantitatively to ensure the gene expression using qRT-PCR. Quantitative analysis indicated that QURE20-IM3 induced substantially some of the innate immune genes including IFN-beta, IFI35, IFIT5 and Mx genes (see Figure 6). Example 3 The following patients were given QURE20-IM3 in 2020 and 2021 under a non-disclosure agreement. No patient other than the inventors were given access to the exact composition of the treatment given nor the method of preparation.: Patient A Age: +70, Female, Medical history Do you suffer from symptoms of Covid-19? Yes Fever (√) fatigue (√), dry cough (√), loss of appetite, () body pain (√), shortness of breath () Mucus or phlegm () sore throat () headache (√), Loss of smell or taste (√) Nasal congestion or runny nose (), Nausea or vomiting (√), chills () diarrhoea (√) difficulty breathing (). Other diseases: diabetes, heart disease
Patient A received 4 nasal drops per nostril approximately every 4 hours for two days. Then for the next five days received 4 drops per nostril approximately every 8 hours. At the same time. Patient A received 3 sprays of the same composition into the mouth every 4 hours for two days and then for the next five days received 3 sprays into the mouth every 8 hours. After approximately 15 mins after administration of the nasal drops and mouth spray, Patient A gargled with 10ml of composition on days 1 and 2 and 5ml of composition on the remaining days. After 8 Hours from treatment, Patient A started to feel the benefit of the treatment. She had no more diarrhoea, nausea, high temperature, body pain and headache, and her sense of smell returned slightly. On her second day after treatment, she felt much better, her sense of smell and taste were back within less than 48 hours. Patient B Age: 15, Female Medical history Do you suffer from symptoms of Covid-19? Yes Fever () fatigue (√) dry cough (√) loss of appetite (√) body pain (√) shortness of breath () Mucus or phlegm () sore throat () headache () Loss of smell or taste (√) Nasal congestion or runny nose () Nausea or vomiting () chills () diarrhea (√) Difficulty breathing (√) Constant pain or pressure in your chest () Patient B received 4 nasal drops per nostril approximately every 4 hours for two days. Then for the next five days received 4 drops per nostril approximately every 8 hours. At the same time. Patient B received 3 sprays of the same composition into the mouth every 4 hours for two days and then for the next five days received 3 sprays into the mouth every 8 hours. After approximately 15 mins after administration of the nasal drops and mouth spray, Patient B gargled with 10ml of composition on days 1 and 2 and 5ml of composition on the remaining days. After 3 days of taking the above composition, Patient B seemed to be improving and recovering from all her Covid-19 symptoms.
Patient C Age: 46, Female Medical history • Do you suffer from symptoms of Covid-19? Yes Fever () fatigue (√) dry cough (√) loss of appetite (√) body pain (√) shortness of breath (√) Mucus or phlegm () sore throat () headache () Loss of smell or taste () Congestion or runny nose () Nausea or vomiting () chills () diarrhea (√) Difficulty breathing () Constant pain or pressure in your chest (√) Other diseases: Diabetes - Endocrinology - Hysterectomy 2019 Endorsement Patient C received 4 nasal drops per nostril approximately every 4 hours for two days. Then for the next five days received 4 drops per nostril approximately every 8 hours. At the same time. Patient C received 3 sprays of the same composition into the mouth every 4 hours for two days and then for the next five days received 3 sprays into the mouth every 8 hours. After approximately 15 mins after administration of the nasal drops and mouth spray, Patient C gargled with 10ml of composition on days 1 and 2 and 5ml of composition on the remaining days. Patient C was a difficult case. She had symptoms cough, shortness of breath, and difficulty breathing and acute pneumonia. She took her first dose on the same day she knew she was positive with covid-19. After 48 hours, her major symptoms disappeared, and she stopped taking the treatment on the fifth day because she felt she was back to normal. Patient D Age: 42, Male Medical history () Fever () Fatigue () Dry cough () Loss of appetite () Body pain () Shortness of breath () Mucus or phlegm () sore throat () headache () Loss of smell or taste (√) Nasal congestion or runny nose () Nausea or vomiting () chills () diarrhea () Difficulty breathing () constant pain or pressure in your chest () Patient D received 4 nasal drops per nostril approximately every 4 hours for two days. Then for the
next five days received 4 drops per nostril approximately every 8 hours. At the same time. Patient D received 3 sprays of the same composition into the mouth every 4 hours for two days and then for the next five days received 3 sprays into the mouth every 8 hours. After approximately 15 mins after administration of the nasal drops and mouth spray, Patient D gargled with 10ml of composition on days 1 and 2 and 5ml of composition on the remaining days. Patient D’s loss of smell and taste came back within 2 days. Patient E Age: 46, Male Medical history Do you suffer from symptoms of Covid-19? Yes Fever (√) fatigue (√) dry cough (√) loss of appetite () body aches () shortness of breath (√) Mucus or phlegm (√) sore throat () headache () Loss of smell or taste (√) Nasal congestion or runny nose (√) Nausea or vomiting () chills () diarrhea () Difficulty breathing () constant pain or pressure in your chest () Patient E received 4 nasal drops per nostril approximately every 4 hours for two days. Then for the next five days received 4 drops per nostril approximately every 8 hours. At the same time. Patient E received 3 sprays of the same composition into the mouth every 4 hours for two days and then for the next five days received 3 sprays into the mouth every 8 hours. After approximately 15 mins after administration of the nasal drops and mouth spray, Patient E gargled with 10ml of composition on days 1 and 2 and 5ml of composition on the remaining days. After 24 hours of taking the above-mentioned treatment, Patient E had his sense of taste back, and on his second day, his sense of smell was back. Patient stopped taking the treatment after seven days. Patient F Age: 14, Male Medical history Do you suffer from symptoms of Covid-19? Yes
(√) Fever () Fatigue (√) Dry cough (√) Loss of appetite () Body pain () Shortness of breath () Mucus or phlegm () sore throat () headache () Loss of smell or taste (√) Nasal congestion or runny nose () Nausea or vomiting () chills () diarrhea () Difficulty breathing () constant pain or pressure in your chest () Patient F received 4 nasal drops per nostril approximately every 4 hours for two days. Then for the next five days received 4 drops per nostril approximately every 8 hours. At the same time. Patient F received 3 sprays of the same composition into the mouth every 4 hours for two days and then for the next five days received 3 sprays into the mouth every 8 hours. After approximately 15 mins after administration of the nasal drops and mouth spray, Patient F gargled with 10ml of composition on days 1 and 2 and 5ml of composition on the remaining days. After approximately 72 hours the patient started to improve, and all symptoms had disappeared within 7 days when treatment stopped. Patient G Male Medical history (√) Fever (√) Fatigue () Dry cough (√) Loss of appetite (√) Body pain () Shortness of breath () Mucus or phlegm () sore throat () headache () Loss of smell or taste (√) Nasal congestion or runny nose () Nausea or vomiting () chills () diarrhea () • Are you taking medications before the QURE20-IM3 treatment? Yes (√) No () Name: Pandol Patient G received 4 nasal drops per nostril approximately every 4 hours for two days. Then for the next five days received 4 drops per nostril approximately every 8 hours. At the same time. Patient G received 3 sprays of the same composition into the mouth every 4 hours for two days and then for the next five days received 3 sprays into the mouth every 8 hours. After approximately 15 mins after administration of the nasal drops and mouth spray, Patient G gargled with 10ml of composition on days 1 and 2 and 5ml of composition on the remaining days.
After approximately 72 hours the patient started to improve, and all symptoms had disappeared within 7 days when treatment stopped. Patient H Age: 34, Male Medical history Do you suffer from symptoms of Covid-19? Yes Fever () fatigue (√) dry cough (√) loss of appetite (√) body pain (√) shortness of breath () Mucus or phlegm () sore throat () headache () Loss of smell or taste (√) Nasal congestion or runny nose () Nausea or vomiting () chills () diarrhea () Difficulty breathing () constant pain or pressure in your chest () • Are you taking medications before the Cure-20 treatment? Yes (√) No () Name: Pandol Patient H received 4 nasal drops per nostril approximately every 4 hours for two days. Then for the next five days received 4 drops per nostril approximately every 8 hours. At the same time. Patient H received 3 sprays of the same composition into the mouth every 4 hours for two days and then for the next five days received 3 sprays into the mouth every 8 hours. After approximately 15 mins after administration of the nasal drops and mouth spray Patient H gargled with 10ml of composition on days 1 and 2 and 5ml of composition on the remaining days. After 24 hours of taking the aforementioned treatment, the symptoms (fever - loss of appetite - body aches - sore throat - chills - nausea - diarrhea) disappeared. After 48 hours, Patient H recover his sense of smell and taste slightly and the cough subsided. Within 72 hours Patient H was feeling better, his coughing was less as he can talk without coughing a lot. In addition, his sense of smell and taste was much better, and his fatigue gone. And he continued taking the rest of the treatment for seven days. Patient J Age: 30, Male Medical history Do you suffer from symptoms of Covid-19? Yes Fever (√) fatigue (√) dry cough (√) loss of appetite () body pain () shortness of breath ()
Mucus or phlegm () sore throat () headache () Loss of smell or taste () Nasal congestion or runny nose (√) Nausea or vomiting () chills () diarrhea () Difficulty breathing () constant pain or pressure in your chest () Are you taking medicines with Cure-20? Yes (√) No () Name: Paracetamol Patient J received 4 nasal drops per nostril approximately every 4 hours for two days. Then for the next five days received 4 drops per nostril approximately every 8 hours. At the same time. Patient J received 3 sprays of the same composition into the mouth every 4 hours for two days and then for the next five days received 3 sprays into the mouth every 8 hours. After approximately 15 mins after administration of the nasal drops and mouth spray, Patient J gargled with 10ml of composition on days 1 and 2 and 5ml of composition on the remaining days. After approximately 72 hours the patient started to improve, and all symptoms had disappeared within 7 days when treatment stopped. Patient K Age: 29, Female Medical history Do you suffer from symptoms of Covid-19? Yes Fever (√) fatigue (√) dry cough (√) loss of appetite (√) body pain (√) shortness of breath () Mucus or phlegm () sore throat () headache () Loss of smell or taste () Nasal congestion or runny nose (√) Nausea or vomiting () chills () diarrhea () Difficulty breathing () constant pain or pressure in your chest () • Are you taking medications before the Cure-20 treatment? Yes (√) No () Name: Pandol Patient K received 4 nasal drops per nostril approximately every 4 hours for two days. Then for the next five days received 4 drops per nostril approximately every 8 hours. At the same time. Patient K received 3 sprays of the same composition into the mouth every 4 hours for two days and then for the next five days received 3 sprays into the mouth every 8 hours. After approximately 15 mins after administration of the nasal drops and mouth spray, Patient K
gargled with 10ml of composition on days 1 and 2 and 5ml of composition on the remaining days. After approximately 72 hours the patient started to improve, and all symptoms had disappeared within 7 days when treatment stopped. Patient L Age: 46, Male Medical history Do you suffer from symptoms of Covid-19? Yes Fever (√) fatigue (√) dry cough (√) loss of appetite (√) body pain (√) shortness of breath () Mucus or phlegm (√) sore throat () headache (√) Loss of smell or taste (√) Nasal congestion or runny nose (√) Nausea or vomiting () chills () diarrhea () Difficulty breathing () constant pain or pressure in your chest () Are you taking medications before the QURE20-IM3 treatment? Yes () No (√) Patient L received 4 nasal drops per nostril approximately every 4 hours for two days. Then for the next five days received 4 drops per nostril approximately every 8 hours. At the same time. Patient L received 3 sprays of the same composition into the mouth every 4 hours for two days and then for the next five days received 3 sprays into the mouth every 8 hours. After approximately 15 mins after administration of the nasal drops and mouth spray, Patient L gargled with 10ml of composition on days 1 and 2 and 5ml of composition on the remaining days. After approximately 72 hours the patient started to improve, and all symptoms had disappeared within 7 days when treatment stopped. Patient M Male Medical history Do you suffer from symptoms of Covid-19? Yes Fever (√) fatigue (√) dry cough (√) loss of appetite () body pain (√) shortness of breath (√) Mucus or phlegm () sore throat (√) headache (√) Loss of smell or taste (√) Nasal congestion or runny nose (√) Nausea or vomiting () chills () diarrhea () Difficulty breathing (√) constant pain or pressure in your chest (√)
Are you taking medications before the QURE20-IM3 treatment? Yes () No (√) Patient M received 4 nasal drops per nostril approximately every 4 hours for two days. Then for the next five days received 4 drops per nostril approximately every 8 hours. At the same time. Patient M received 3 sprays of the same composition into the mouth every 4 hours for two days and then for the next five days received 3 sprays into the mouth every 8 hours. After approximately 15 mins after administration of the nasal drops and mouth spray, Patient M gargled with 10ml of composition on days 1 and 2 and 5ml of composition on the remaining days. After approximately 72 hours the patient started to improve, and all symptoms had disappeared within 7 days when treatment stopped. Patient N Female Medical history Do you suffer from symptoms of Covid-19? Yes Fever (√) fatigue (√) dry cough (√) loss of appetite () body pain (√) shortness of breath (√) Mucus or phlegm () sore throat (√) headache (√) Loss of smell or taste () Nasal congestion or runny nose () Nausea or vomiting () chills () diarrhea () Difficulty breathing () constant pain or pressure in your chest (√) Are you taking medications before the QURE20-IM3 treatment? Yes () No (√) Patient N received 4 nasal drops per nostril approximately every 4 hours for two days. Then for the next five days received 4 drops per nostril approximately every 8 hours. At the same time. Patient N received 3 sprays of the same composition into the mouth every 4 hours for two days and then for the next five days received 3 sprays into the mouth every 8 hours. After approximately 15 mins after administration of the nasal drops and mouth spray, Patient N gargled with 10ml of composition on days 1 and 2 and 5ml of composition on the remaining days. After approximately 72 hours the patient started to improve, and all symptoms had disappeared within 7 days when treatment stopped.
Patient P Age: 19 Months, Male Medical history Date of the first treatment dose: 11/06/2020 Do you suffer from symptoms of Covid-19? Yes Fever (√) fatigue (√) dry cough () loss of appetite () body pain () shortness of breath () Mucus or phlegm () sore throat () headache () Loss of smell or taste () Nasal congestion or runny nose () Nausea or vomiting () chills () diarrhea () Difficulty breathing () constant pain or pressure in your chest () Patient P received 1 spray into the mouth approximately every 6 hours for seven days. After approximately 72 hours the patient started to improve, and all symptoms had disappeared within 7 days when treatment stopped. Patient Q Age: 37, Male Medical history Do you suffer from symptoms of Covid-19? Yes Fever (√) fatigue (√) dry cough () loss of appetite () body pain () shortness of breath () Mucus or phlegm (√) sore throat () headache () Loss of smell or taste () Nasal congestion or runny nose () Nausea or vomiting () chills () diarrhea () Difficulty breathing () constant pain or pressure in your chest () Patient Q received 4 nasal drops per nostril approximately every 4 hours for two days. Then for the next five days received 4 drops per nostril approximately every 8 hours. At the same time. Patient Q received 3 sprays of the same composition into the mouth every 4 hours for two days and then for the next five days received 3 sprays into the mouth every 8 hours. After approximately 15 mins after administration of the nasal drops and mouth spray, Patient Q gargled with 10ml of composition on days 1 and 2 and 5ml of composition on the remaining days. After approximately 72 hours the patient started to improve, and all symptoms had disappeared within 7 days when treatment stopped.
Patient R Age: 35, Female Medical history Do you suffer from symptoms of Covid-19? Yes Fever () fatigue (√) dry cough (√) loss of appetite (√) body pain (√) shortness of breath () Mucus or phlegm (√) sore throat (√) headache (√) Loss of smell or taste (√) Nasal congestion or runny nose (√) Nausea or vomiting () chills (√) diarrhea () Difficulty breathing () constant pain or pressure in your chest () Patient R received 4 nasal drops per nostril approximately every 4 hours for two days. Then for the next five days received 4 drops per nostril approximately every 8 hours. At the same time. Patient R received 3 sprays of the same composition into the mouth every 4 hours for two days and then for the next five days received 3 sprays into the mouth every 8 hours. After approximately 15 mins after administration of the nasal drops and mouth spray, Patient R gargled with 10ml of composition on days 1 and 2 and 5ml of composition on the remaining days. After approximately 72 hours the patient started to improve, and all symptoms had disappeared within 7 days when treatment stopped. Example 4 15 subjects were given 1 spray of composition in their mouth and 1 drop of the composition into each nostril every day as a prophylactic treatment. To date, despite going to their place of work and continuing daily life none of the subject contracted Covid-19. The findings of these in vivo studies are concurrent with the in vitro studies given in Example 2. Example 5 Toxicity: Required material: VeroE6 cells 96-wells plate (Nuc 136101 Thermofisher) Medium (DMEM, Thermofisher, 11965084)
Crystal Violet (0.2% crystal violet in distal water for staining) Procedure: 1. Seed 60,000 VeroE6 cells in 96-well plates. 2. Expect 100% confluency in 24 hours 3. Teat VeroE6 cells with treatment with different dilutions. 4. After 16 hours, stain the cells with crystal violet and calculate the healthy cells compared to untreated cells In summary, a total of 60,000 VeroE6 cells were seeded in 96-well plates. Once fully confluent, cells were exposed to increasing doses of QURE20-IM3 (1:1 to 1:1000). After 16 hours, cells were stained, and safe doses were estimated. Based on cell toxicity, 1:500 was the safest dose determined (see Figure 7). For comparison purposes, both 1:250 and 1:500 dilutions were used in Examples 6 to 10 to provide a breadth of results on both doses. Example 6 Antiviral Activity Required material VeroE6 cells 96-wells plate (Nuc 136101 Thermofisher) Medium (DMEM, Thermofisher, 11965084) Crystal Violet (0.2% crystal violet in distal water for staining) SARS-COV-2 Procedure: • VeroE6 cells were seeded in 96-well plates with a density of 60,000. • Treat cells with increasing doses of treatment (1:1 to 1:1000). • Infect cells with SARSCoV-2 (UK/Victoria/2020 strain) with moi of 1.0. • 24 hours post-infection, supernatant was collected and seeded into VeroE6 cells for 24 hours. • Fix cells with 5% paraformaldehyde • Stained cells with crystal violet • Determine TCID50 values in treated versus non-treated cells.
In summary, to determine the antiviral actions of QURE20-IM3, VeroE6 cells were exposed in 96 cells to either 1:250 or 1:500 dilutions of QURE20-IM3 for 16 hours. Thereafter, QURE20-IM3- treated and untreated cells were infected with SARSCoV-2 (UK/Victoria/2020 strain) with MOI (multiplicity of infection) of 1.0. Twenty-four hours post-infection, supernatant was collected and seeded into VeroE6 cells for 24 hours. Cells were fixed with 5% paraformaldehyde and stained with crystal violet and the TCID50 values were estimated in QURE20-IM3-treated and non-treated cells. Based on the analysis of the TCID50/ml, both 1:250 and 1:500 dilution treated cells showed inhibition of SARS-COV-2 (see Figure 8). Example 7 This Method covers fixation, fluorescent labelling and mounting of cells grown on glass coverslips for imaging in the confocal microscope. The aim of this procedure is to fluorescently label specific proteins in cells in order to see where those proteins originate/move to or to see if they interact with other proteins or cell components to understand the level of expression under specific treatments. To produce reproducible and comparable results, the protocol should always be followed as described in this method. Materials • Fine forceps • Glass coverslips covered with cells that have been treated appropriately • Appropriate container for staining, eg.24 well plate for 13mm coverslips • Set of calibrated Gilson pipettes and tips • Ca/Mg free phosphate buffered saline (PBS) (from CSU) • 4% paraformaldehyde solution in PBS • 0.1% Triton X-100 in de-ionised water • Blocking buffer (0.5% bovine serum albumin in PBS) • Primary antibody diluted in PBS/BSA • Species specific secondary antibody diluted in PBS/BSA (AlexaFluor, Molecular Probes) • DAPI in de-ionised water (1:10,000 solution) • Glass microscope slides • Vectashield mounting medium (Vector Labs, UK) • Nail varnish Procedure
• All steps carried out at room temperature. • Do not allow the cells to dry at any stage in the procedure. • Ensure cells are uppermost in wells throughout procedure. • Grow VeroE6 cells in 24 well plate with cover slips • Treat cells with treatment with 1:250 and 1:500 dilutions • Infect with SARS-COV-2 for 24 hours 1. Remove medium and fix for 60 min in 4% paraformaldehyde in PBS. Transfer to PBS. CELLS ARE STABLE IN PBS AND CAN BE LEFT FOR A FEW WEEKS AT THIS STAGE @ 2 - 8°C 2. Treat with 0.1% Triton X100 in PBS for approx 10 mins and then wash with PBS. 3. Block non-specific binding with 0.5% bovine serum albumin in PBS (PBS/BSA) for approx 60 min. 4. Remove PBS and add primary antibody (Mouse monoclonal to SARS Rabbit monoclonal to SARS Spike Protein) diluted in PBS/BSA for 60 - 90 min. 5. Wash 3 x approx 5 min in PBS 6. Incubate with Alexa Fluor 488-conjugated goat-anti-rabbit IgG secondary antibodies in PBS (PBS/BSA) for 60 - 90 min at RT 7. Wash 3 x approx 5 min in PBS then 1x with distal water. 8. Add 1:10,000 DAPI (made up in de-ionised water) for approx 30 mins. 9. Whilst waiting, label a glass slide with appropriate details and place a small drop of an aqueous mounting medium e.g.Vectashield on a glass slide. 10. Dip coverslips in de-ionised water using fine forceps to hold coverslips. 11. Hold vertically against tissue to pull as much liquid off cells as possible and dry bottom of coverslip on tissue. 12. Place coverslip, cells down, on this drop. 13. Seal coverslips with nail varnish. Allow to dry. 14. Samples are ready for viewing under the confocal microscope. 15. Images captured under confocal microscope are edited in PowerPoint and presented. In summary, VeroE6 cells were subjected to treatment with either 1:250 or 1:500 dilutions of QUER20-IM3 for 16 hours and then infected with SARS-CoV-2 for 24 hours. The cells were fixed post-infection with 5% paraformaldehyde for 4 h and permeabilized with 0.5% Triton X-100 for 5 min. After blocking with 3% bovine serum albumin (BSA) for 30 min, the cells were incubated for 1 h at room temperature with rabbit anti-SARS-CoV Spike antibodies, which exhibits strong cross-
reactivity with SARS-CoV-2. After two washes with phosphate buffered saline (PBS), the cells were incubated with Alexa Fluor 488-conjugated goat-anti-rabbit IgG secondary antibodies for 1 h at room temperature. After two additional washes, PBS supplemented with 0.1 μg/ml DAPI was added to the cells for 30 min before imaging. Images were acquired using the confocal microscope. Analysis indicated a profound reduction in the replication of SARS-CoV-2 particularly when QURE20- IM3 was applied with lower dilution of 1:250 (see Figure 8) compared to 1:500 dilution (see Figure 10). The untreated and virus infected cells showed increased replication of SARS-COV-2 (see Figure 11) compared to the treated cells. The results and data analysis of this experiment confirm reduced infectivity and replication of SARS-CoV-2 in the presence of QURE20-IM3. Example 8 Material Required: VeroE6 cells 24 wells plate SARS-COV-2 RNA Qiagen RNeasy Plus Mini Kit NanoDrop SuperScript® III Platinum® SYBR® Green One-Step qRT-PCR Kit Protocol: • Seed Vero cells in 24 wells plate • Treat VeroE6 cells with safe dose of treatment • Infect or mock infect cells with SARS-CoV-2 (MOI = 1.0) • 24 hours-post infection, collect cells and extract total RNA Qiagen RNeasy Plus Mini Kit (standard Qiagen RNeasy Plus Mini Kit protocol was used). • Determine the quality of RNA using NanoDrop • Perform real-time PCR using SuperScript® III Platinum® SYBR® Green One-Step qRT-PCR Kit standard protocol. • Determine the gene expression of innate immune genes compared to housekeeping gene. In summary, the VeroE6 cells were treated with either 1:250 or 1:500 dilutions of QURE20-IM3 and were either mock-infected or infected with SARS-CoV-2 (MOI = 1.0). Twenty-four hours after infection, cells were collected, and total RNA was extracted using the Qiagen RNeasy Plus Mini Kit (see Figure 11). The quality of the extracted RNA was assessed with the NanoDrop TM spectrophotometer at 10mm Absorbance (see Figure 12 and Table 1). Real-time PCR was performed to determine the gene expression of IFIT5, IFIT35, and Mx and were compared with housekeeping
gene. The chosen genes can be considered are key host genes involved in the innate immunity, cytokine and antiviral actions against SARS-CoV-2. Treatment of QURE20-IM3 induced more ISGs expression compared to gene transcription induced by the virus (see Figures 13A, 13B and 13C). Table 1
Example 9 Material required VeroE6 cells 96-wells plate (Nuc 136101 Thermofisher) SARS-COV-2 Medium (DMEM, Thermofisher, 11965084) Crystal Violet (0.2% crystal violet in distal water for staining) Procedure: • Seed VeroE6 cells in 96-well plate • Once confluent, infect cells with SARS-CoV-2 (MOI = 1.0). • After 1 h, remove the viral inoculum
• Wash cells with ice-cold PBS, before addition of fresh medium. • Treat cells for 1, 10, and 16 hours. • After these hours, cells were fixed and stained with crystal violet • Determine TCID50 and data is presented on the inhibition of virus compared to untreated cells In summary, twenty thousand VeroE6 cells were seeded in 96-well plate and cells were infected with SARS-CoV-2 (MOI = 1.0). After 1 h, the viral inoculum was removed and cells were washed with ice-cold PBS, before addition of fresh medium. The QURE20-IM3 were added onto cells for 1, 10, and 16 hours. After these hours, cells were fixed and stained to determine TCID50. Data analysis indicated that the inhibitory effect of QURE20-IM3 was profoundly observed compared to untreated virus infected cells (see Figure 14). Example 10 Material Required • HEK293T cells • VeroE6 cells • Medium (DMEM, Thermofisher, 11965084) • Viafect (Promega) • Luciferase Cell Culture Lysis 5X reagent (REF E1531, Promega) • Corning® 96-well Black Flat Bottom Polystyrene High Bind Microplate (REF 3925, Corning) • Promega Nano-Glo Luciferase Assay System (REF N1150, Promega) Part 1: Virus pseudotype production Protocol • Seed 5 million 293T cells per 10 cm dish in 10ml DMEM++ (10% FSC, antibiotics) • Transfection per 10cm dish with 3-plasmid NanoLuc2AEGFP system (VSV-G pseudotyped ΔG- luciferase VSV) along with SARS-CoV-2 S. • Add 7µg NanoLuc2AEGFP, 7 µg NLGagPol, and 2.5 µg SARS-CoV-2-Strunc to 500 µl DMEM w/o serum, mix thoroughly • Dilute 30 µl Viafect (Promega) in 500 µl DMEM w/o serum, mix thoroughly • Mix diluted DNA and Viafect thoroughly by pipetting or vortexing, incubate 20 min at RT • Add dropwise to 293T cells in DMEM++ Day 2 or 3 • At least 8 hours after transfection (usually overnight): carefully wash cells 2x with PBS and add 10
ml DMEM++ Day 4 (48 hours after transfection). Take up all 10ml supernatant, clarify by centrifugation at 300g, 5 minutes and syringe filter through a 0.22 µm pore size PVDF filter (e.g., SLGVR33RS, Millipore) • aliquot and freeze at -80°C or use directly for transduction of final cells Part 2: Inhibition of Viral Entry • Plate target cells (e.g. VeroE6) in 100 µl DMEM++ in 96 well plates • Treat cells with the treatments for 1 hour. • Dilute virus as prepared in Part 1 • add 100 µl virus dilution to cells plated • Remove inoculum, and wash carefully with 200 µl PBS and add 200 µl fresh DMEM++ • To take NanoLuc Readout, wash cells once with 100 µl PBS (aspirate and add PBS very carefully when working with cells), remove all PBS. • Add 50 µl 1x Lysis buffer (prepared with H2O from 5x Cell lysis reagent) • Incubate with shaking, 15 min RT • Distribute 25 µl substrate per well in a 96-well black plate • Add 25 µl lysate per well, mix by moving the plate, incubate 5 min at RT in the dark • Read in Luminometer (0.1 seconds integration time) • Plot the data for the intensity of luciferase activity (representing %age entry of the virus) In summary, vesicular stomatitis virus (VSV) pseudotyped with S proteins of SARS-CoV-2 were generated in mammalian cells. In brief, BHK-21/WI-2 cells (Kerafast) were transfected to express the S proteins were inoculated with VSV-G pseudotyped ΔG-luciferase VSV (Kerafast). After a 2-h incubation at 37 °C, the inoculum was removed and DMEM supplemented with 5% FBS, 50 units/ml penicillin and 50 μg/ml streptomycin were added back to cells. Pseudotyped particles were collected 24 h post-inoculation, then centrifuged at 1,320g to remove cell debris and stored at −80 °C unYl use. To determine the effect of the QURE20-IM3 on viral entry, VeroE6 cells were treated with QURE20-IM3 for 1 h before inoculation with respective pseudotyped VSV. After 2 h inoculation in the presence of the QURE20-IM3, the inoculum was removed, and fresh medium were added to cells for further culture. The activity of firefly luciferase as a readout of infected cells were measured using the bright-Glo luciferase assay (Promega) for quantitative determination at 16 hpi. The data analysis indicated a reduced virus entry into the cells treated with the QURE20- IM3 compared to untreated and pseudoparticle infected cells (see Figure 15).
Claims
Claims: 1. A composition comprising an extract of Abutilon fruticosum, an extract of Acacia nubica, an extract of Acacia bussei and an extract of Myrsine Africana (Qorsheen). 2. A composition according to claim 1 further comprising an extract of Boswellia frereana. 3. A composition according to any preceding claim wherein the extract of Abutilon fruticosum is in the form of a powder of root of shrub. 4. A composition according to any preceding claim wherein the extract of Acacia nubica is in the form of a powder of root of shrub. 5. A composition according to any preceding claim wherein the extract of Acacia bussei is in the form of a powder of bark of tree. 6. A composition according to any one or more of claims 2 to 6 wherein the extract of Boswellia frereana is in the form of the resin from Boswellia frereana tree. 7. A composition according to any preceding claims wherein the extract of Qorsheen is in the form of a powder of fruit. 8. A composition according to any preceding claim which is in liquid and/or tablet form. 9. A composition according to claim 8 wherein the liquid form comprises 300-500g of Maydi extract, 4-10 g of Wancad extract, 4-10g of Gumar extract, 1-3g Galool extract and 1-3g Qorsheen extract and 750ml to 1250ml of a suitable liquid. 10. A composition according to any preceding claim further comprising water. 11. An antiviral composition comprising the composition of any preceding claims. 12. A composition according to claim 11 wherein administration comprises 3 to 5 nasal drops/sprays per nostril every 3 to 5 hrs for 1 to 3 days and then 3 to 5 nasal drops/spray per nostril
every 7 to 9 hrs for 4 to 6 days. 13. A composition according to claim 11 wherein administration comprises 3 to 5 drops/spray per nostril at least 5 to 7 times per day for 1 to 3 days and then 3 to 5 drops/spray per nostril at least 7 to 9 times per day for 4 to 6 days. 14. A composition according to any one or more of claims 11 to 13 wherein administration comprises 2 to 4 mouth sprays into mouth every 3 to 5 hrs for 1 to 3 days and then 2 to 4 mouth sprays into mouth every 7 to 9 hrs for 4 to 6 days. 15. A composition according to any one or more of claims 11 to wherein administration comprises 2 to 4 sprays into mouth at least 5 to 7 times per day for 1 to 3 days and then 2 to 4 sprays into mouth at least 7 to 9 times per day for 4 to 6 days. 16. A composition according to any one or more of claims 11 to 15 wherein administration comprises gargling with 5 to 15 ml every 3 to 5 hours for 1 to 3 days and then gargling with 2 to 10 ml every 7 to 9hrs for 4 to 6 days. 17. A composition any one or more of claims 11 to 15 wherein administration comprises gargling with 5 to 15 ml at least 6 times per day for 1 to 3 days and then gargling with 2 to 10 ml at least 3 times per day for 4 to 6 days. 18. A composition according to either claim 16 or 17 wherein gargling takes place between 5 and 30 minutes post administration of nasal drops/spray and/or mouth spray. 19. A composition according to claim 11 wherein administration comprises 1 to 3 drops/spray per nostril every 7 to 9 hrs for 6 to 8 days. 20. A composition according to either claim 11 or 19 wherein administration comprises taking orally 3 to 15ml every 7 to 9 hrs for 6 to 8 days. 21. A viral prophylactic composition according to any one or more of claims 1 to 10. 22. A composition according to claim 21 wherein administration comprises 1 to 3 nasal
drops/spray per nostril per day. 23. A composition according to either claim 21 or 22 wherein administration comprises 1 to 3 sprays into the mouth per day. 24. A composition according to any one or more of claims 11 to 23 wherein the virus is selected from influenza virus, respiratory syncytial virus, human metapneumovirus, parainfluenza virus, coronaviruses, and rhinoviruses. 25. A composition according to claim 9 wherein the tablet form comprises 40-59% Galool extract by weight, 40-59% Gumar extract by weight and 0.5-3% Wancad extract by weight. 26. A composition according to claim 25 further comprising 0.5-3% Qorsheen extract by weight. 27. A method of preparation of the composition according to claim 10, the method comprising: a) mixing extracts of Wancad, Gumar and Galool, and optionally Qorsheen together; b) adding mixture of extracts to water; c) optionally adding extract of Maydi; d) bring to the boil; e) take off heat; f) filtered and f) place in sealed container. 28. A method according to claim 27, wherein 750ml to 1250ml of water is used per 300 to 500g of Maydi, 4 to 10g of Wancad, 4 to 10g of Gumar, 1 to 3g of Galool and 1 to 3g of Qorsheen.
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| Application Number | Priority Date | Filing Date | Title |
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| GBGB2213842.4A GB202213842D0 (en) | 2022-09-22 | 2022-09-22 | Composition |
| GB2213842.4 | 2022-09-22 | ||
| GB2307863.7A GB2622661A (en) | 2022-09-22 | 2023-05-25 | Composition |
| GB2307863.7 | 2023-05-25 |
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| PCT/GB2023/052420 WO2024062228A1 (en) | 2022-09-22 | 2023-09-19 | Composition comprising abutilon fruticosum, acacia nubica, acacia bussei and myrsine africana, methods of preparation and therapeutic uses |
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| KR20190122562A (en) * | 2018-04-21 | 2019-10-30 | 류형준 | Antiviral agent and use thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
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