Production method

Abstract

The present invention relates, inter alia, to a process for the preparation of garlic oil, to garlic oil produced using this process, to pharmaceutical compositions comprising this garlic oil and to the use of this garlic oil in the treatment of viral infections such as SARS-COV-2 and influenza A. A method of making a garlic oil comprises the steps of: a) processing a portion of a garlic plant to increase its surface area; b) aging the portion of the garlic plant for at least 18 months to provide aged garlic, the step not comprising the addition of ethanol; and c) separating garlic oil from the aged garlic. Step c) may be performed using rotating cone distillation or supercritical fluid extraction at a temperature of less than 110 DEG C or such that the pressure (atmospheric pressure) divided by the temperature (Kelvin temperature) is equal to or less than about 0.0026.

Description

Technical Field

[0001] In one embodiment, the present invention relates to a method for producing garlic oil. In other embodiments, the present invention relates to garlic oil produced by this method, as well as methods and uses relating to garlic oil. Background Technology

[0002] It will be clearly understood that if prior art publications are cited in this document, such citation does not constitute an acknowledgment that such publications constitute common general knowledge in the field in Australia or any other country.

[0003] Garlic is known to contain volatile organic compounds (VOCs) that exhibit a wide range of biological effects, such as antioxidant, immunomodulatory, antifungal, anti-inflammatory, antibacterial, and antiviral activities. Organic sulfates are a type of VOC found in garlic, and they are generally believed to possess these beneficial effects. Due to their broad activity, garlic products are frequently used as dietary supplements and herbs, marketed as effective against a variety of ailments.

[0004] Fresh garlic can be used, but it is limited by its short shelf life and must be eaten raw to obtain the maximum benefits, as cooking garlic usually causes the degradation of its bioactive compounds. Additionally, fresh garlic contains only about 2% to 4% by weight of organic sulfates and can have a strong taste and odor, which may be unpalatable to consumers. To overcome some of these difficulties, a range of techniques have been used to extract bioactive compounds from garlic.

[0005] One method of producing garlic preparations is by maceration. In this method, chopped garlic is soaked in a liquid such as water, ethanol, or a carrier oil such as soybean oil or vegetable oil for up to several days to several weeks. This allows the active compounds in the raw garlic to be extracted into the liquid, which is then filtered to remove any remaining raw garlic. While maceration does avoid the use of high temperatures that could degrade the bioactive compounds, the resulting concentration of the active components is inevitably very low due to the large excess of liquid, thus significantly reducing bioactivity. Furthermore, such preparations typically produce emulsions or liquids with high water content, which may be microbially unstable and therefore have a shortened shelf life.

[0006] To produce products with higher microbial stability, some methods form garlic oil that is essentially anhydrous. One such method is through chemical extraction. In this method, the garlic is washed with a solvent to extract the active components into the solvent. The solvent is then separated from the garlic, filtered, and concentrated to remove the solvent and any excess water. This typically produces an oily composition containing compounds obtained from garlic. While these compositions have higher microbial stability compared to aqueous preparations, many of the bioactive compounds present in garlic are volatile, and therefore some are lost when the solution is concentrated.

[0007] Commercial garlic oil is typically produced using distillation techniques such as steam distillation. During steam distillation, fresh garlic is mixed with water, and the mixture is heated until the water boils. The steam generated by the boiling water then carries volatile compounds to a cooling system, which condenses the volatile-rich steam into a liquid. This liquid then spontaneously separates into an aqueous phase and an oil phase, allowing pure garlic oil to be collected by decantation. While steam distillation allows for the production of garlic oil containing volatile compounds, the solution must be boiled for an extended period to achieve a sufficient yield. This prolonged exposure to heat can cause degradation of the bioactive compounds within the garlic, resulting in a significant loss of the bioactivity of the resulting oil.

[0008] Therefore, the concentration of bioactive compounds in garlic preparations can vary widely, and many methods produce compositions that are either structurally unstable with low bioactive concentrations or lead to the degradation of key bioactive compounds present in garlic. Summary of the Invention

[0009] In view of the above, the present invention relates in one aspect to a method for producing garlic oil that maintains or improves the biological activity of garlic.

[0010] In a first aspect, the present invention provides a method for preparing garlic oil, the method comprising the following steps: a) Process a portion of the garlic plant to increase its surface area; b) Aging this part of the garlic plant for at least 18 months to provide aged garlic; and c) Separating garlic oil from aged garlic, wherein the separation is carried out using rotary cone distillation or supercritical fluid extraction.

[0011] In one embodiment of the first aspect, a method for preparing garlic oil is provided, the method comprising the following steps: a) Process a portion of the garlic plant to increase its surface area; b) Aging this part of the garlic plant for at least 18 months to provide aged garlic; and c) Separating garlic oil from aged garlic, wherein the separation is performed using rotary cone distillation or supercritical fluid extraction. Step b) does not include the addition of ethanol.

[0012] In a second aspect of the present invention, a method for preparing garlic oil is provided, the method comprising the following steps: a) Process a portion of the garlic plant to increase its surface area; b) Aging this part of the garlic plant for at least 18 months to provide aged garlic; and c) Separating garlic oil from aged garlic, wherein the separation is carried out at a temperature below 110°C.

[0013] In one embodiment of the second aspect, a method for preparing garlic oil is provided, the method comprising the following steps: a) Process a portion of the garlic plant to increase its surface area; b) Aging this part of the garlic plant for at least 18 months to provide aged garlic; and c) Separating garlic oil from aged garlic, wherein the separation is carried out at a temperature below 110°C. Step b) does not include the addition of ethanol.

[0014] In a third aspect of the present invention, a method for preparing garlic oil is provided, the method comprising the following steps: a) Process a portion of the garlic plant to increase its surface area; b) Aging this part of the garlic plant for at least 18 months to provide aged garlic; and c) Separating garlic oil from aged garlic, wherein during the separation process, the pressure (atmospheric pressure) divided by the temperature (Kelvin temperature) is equal to or less than about 0.0026.

[0015] In one embodiment of the third aspect, a method for preparing garlic oil is provided, the method comprising the following steps: a) Process a portion of the garlic plant to increase its surface area; b) Aging this part of the garlic plant for at least 18 months to provide aged garlic; and c) Separating garlic oil from aged garlic, wherein during the separation process, the pressure (atmospheric pressure) divided by the temperature (Kelvin temperature) is equal to or less than approximately 0.0026. Step b) does not include the addition of ethanol.

[0016] The features of the first to third aspects (and the implementation schemes of the first to third aspects described above) can be described as follows.

[0017] Advantageously, the method of the present invention provides a garlic oil that substantially maintains or improves the biological activity of garlic.

[0018] Unwilling to be bound by any theory, the inventors believe that the aging process advantageously disrupts the cellular structure of garlic, thereby releasing oils and active compounds contained within the cellular environment. Furthermore, the aging process transforms unstable reactive compounds into more stable analogues.

[0019] As used herein, the term "garlic oil" refers to a hydrophobic aqueous solution derived from a portion of the garlic plant. While garlic oil may include other types of oil (e.g., mixtures with oils derived from another plant), in some embodiments, garlic oil consists of (or is substantially composed of) oil derived solely from the garlic plant. The term "garlic oil" as used herein may also contain a significantly low water content, but the oil is hydrophobic overall and may not be an emulsion.

[0020] The part of the garlic plant in step a) can be a whole garlic bulb, an unpeeled garlic clove, a peeled garlic clove, or a combination thereof.

[0021] In some implementations, in step a), this part of the garlic plant is a peeled garlic clove.

[0022] In some implementations, the portion of the garlic plant in step a) is derived from or originates from Australian garlic varieties, particularly Australian Red (MOF), Australian White (WHT), or Australian Purple (AUP).

[0023] As used in this article, the term "Australian Red (MOF)" refers to a hard-necked garlic variety with 8 to 12 pink, red, or purple cloves and a white outer skin. This variety is often called Morado in Europe, Creole and Rojo in the United States, and Colorado in Argentina. In Australia, this garlic variety is typically harvested around December.

[0024] As used in this article, the term "Australian White (WHT)" refers to a soft-necked garlic variety with 10 to 16 white, yellow, or light brown cloves and a white outer skin. This variety is commonly known as Spanish White, Italian White, Messiderome, or Thermidrome in Europe, artichoke or early-maturing garlic in the United States, and Blanco in Argentina. In Australia, this garlic variety is typically harvested around November.

[0025] As used in this article, the term "Australian Purple (AUP)" refers to a hard-necked garlic variety with 8 to 14 brown, pink, red, or purple cloves and a light purple outer skin. This variety is often called Spring Purple in Europe, Turban or Chinese Purple in the United States, and Chino / Asiatico Morado in Argentina. In Australia, this variety is typically harvested around October.

[0026] In some implementations, this part of the garlic plant does not come from or is not derived from the Griffith White garlic variety.

[0027] As used in this article, the term "Griffith White (AUW)" refers to a hard-necked garlic variety with 8 to 14 yellow, brown, or light pink cloves and a thin white or very pale purple outer skin. This variety is often called Sprint, Primor, or Spring White in Europe, Turban or Chinese White in the United States, and Chino Morado Blanco in Argentina. In Australia, this variety is typically harvested around October.

[0028] In some implementations, step a) includes chopping, cutting, mincing, slicing, grinding, crushing, pulverizing, milling, blending, threshing, grinding, crushing, extruding, cutting into pieces, dividing, grinding, pulverizing, granulating, breaking, rubbing, crushing, pressing, pounding, or any combination thereof.

[0029] Step a) may include cutting, grinding, crushing, or any combination thereof. In one embodiment, cutting includes chopping, slicing, dicing, splitting, blending, pulverizing, grinding, or any combination thereof. In another embodiment, crushing includes grinding, pounding, threshing, milling, pounding, extruding, crushing, breaking, pressing, or any combination thereof. In yet another embodiment, grinding includes milling, crushing, pulverizing, granulating, or any combination thereof.

[0030] In some implementations, step a) includes chopping.

[0031] In some implementations, step a) reduces the size of this portion of the garlic plant to approximately 0.1 mm, 0.2 mm, 0.3 mm, 0.4 mm, 0.5 mm, 0.6 mm, 0.7 mm, 0.8 mm, 0.9 mm, 1.0 mm, 1.25 mm, 1.5 mm, 1.75 mm, 2.0 mm, 2.25 mm, 2.5 mm, 2.75 mm, 3.0 mm, 3.25 mm, 3.5 mm, 3.75 mm, 4.0 mm, 4.25 mm, 4.5 mm, 4.75 mm, 5.0 mm, 5.5 mm, 6.0 mm, 6.5 mm, or approximately 7.0 mm.

[0032] In other embodiments, step a) reduces the size of this portion of the garlic plant to less than 0.2 mm, less than 0.3 mm, less than 0.4 mm, less than 0.5 mm, less than 0.6 mm, less than 0.7 mm, less than 0.8 mm, less than 0.9 mm, less than 1.0 mm, less than 1.25 mm, less than 1.5 mm, less than 1.75 mm, less than 2.0 mm, less than 2.25 mm, less than 2.5 mm, less than 2.75 mm, less than 3.0 mm, less than 3.25 mm, less than 3.5 mm, less than 3.75 mm, less than 4.0 mm, less than 4.25 mm, less than 4.5 mm, less than 4.75 mm, less than 5.0 mm, less than 5.5 mm, less than 6.0 mm, less than 6.5 mm, or less than 7.0 mm.

[0033] In another embodiment, after step a), the size of this portion of the garlic plant is greater than 0.1 mm, greater than 0.2 mm, greater than 0.3 mm, greater than 0.4 mm, greater than 0.5 mm, greater than 0.6 mm, greater than 0.7 mm, greater than 0.8 mm, greater than 0.9 mm, greater than 1.0 mm, greater than 1.25 mm, greater than 1.5 mm, greater than 1.75 mm, greater than 2.0 mm, greater than 2.25 mm, greater than 2.5 mm, greater than 2.75 mm, greater than 3.0 mm, greater than 3.25 mm, greater than 3.5 mm, greater than 3.75 mm, greater than 4.0 mm, greater than 4.25 mm, greater than 4.5 mm, greater than 4.75 mm, greater than 5.0 mm, greater than 5.5 mm, greater than 6.0 mm, greater than 6.5 mm, or about 7.0 mm.

[0034] In one implementation, step a) is performed without adding a solvent.

[0035] Step a) can be performed with the addition of a solvent. In some embodiments, the solvent is a polar solvent, such as water, methanol, ethanol, isopropanol, ethyl acetate, or a combination thereof. In other embodiments, the solvent is a nonpolar solvent such as hexane, or a vegetable-based oil such as olive oil, rapeseed oil, vegetable oil, sunflower seed oil, and grape seed oil, or a combination thereof. In one embodiment, the polar solvent is not (or does not contain) ethanol. In another embodiment, the polar solvent is not (or does not contain) an alcohol.

[0036] In one implementation, step a) is performed using only that part of the garlic plant.

[0037] Step a) can be performed at any suitable pressure and temperature. In one embodiment, step a) is performed at atmospheric pressure. In another embodiment, step a) is performed below atmospheric pressure. In some embodiments, step a) is performed above atmospheric pressure. In another embodiment, step a) is performed at room temperature. In one embodiment, step a) is performed above room temperature. In another embodiment, step a) is performed below room temperature. In one embodiment, step a) is performed at -10°C, -5°C, 0°C, 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, or 50°C, or any temperature in between. In one embodiment, step a) is performed above 50°C, above 40°C, above 30°C, above 20°C, above 10°C, above 0°C, or above -10°C. In another embodiment, step a) is performed below 50°C, below 40°C, below 30°C, below 20°C, below 10°C, below 0°C, or below -10°C. In another embodiment, step a) is carried out at a temperature of 0°C to 50°C, particularly 10°C to 40°C or 15°C to 35°C.

[0038] In some embodiments, prior to step a), the method for producing garlic oil includes harvesting garlic. The method of the first aspect may also include steps prior to step a) of separating the green parts from the garlic bulb, splitting the garlic bulb, and / or peeling the garlic cloves.

[0039] In some embodiments, step b) does not include adding a solvent. In other embodiments, step b) does not include adding an aqueous solution, such as water. In still other embodiments, step b) does not include adding an alcohol, such as ethanol.

[0040] Not wishing to be bound by any theory, the inventors believe that adding ethanol during the aging process may have a detrimental effect on the aged garlic, and therefore on the resulting garlic oil. This is because ethanol may destroy or damage enzymes active during the aging process, harm plant cells, and / or react with the active components in the garlic, causing unwanted chemical transformations or potentially inhibiting the aging process. Consumers also have some aversion to using solvents such as ethanol to extract plant-based health products. For these reasons, adding ethanol in step b) ultimately results in a different compound distribution, which may have a very significant impact on the garlic oil separated in step c), as it may not possess the surprising activity exhibited by the garlic oil of this invention. Furthermore, adding ethanol during step b) may hinder step c), as any ethanol present in the solution may separate along with the garlic oil, which could subsequently affect the recovery of volatile compounds. This could significantly dilute the concentration of garlic oil in the final product and further necessitate a more laborious purification step to separate the garlic oil from the ethanol.

[0041] Step b) can be performed at any temperature between -20°C, -19°C, -18°C, -17°C, -16°C, -15°C, -14°C, -13°C, -12°C, -11°C, -10°C, -9°C, -8°C, -7°C, -6°C, -5°C, -4°C, -3°C, -2°C, -1°C, 0°C, 1°C, 2°C, 3°C, 4°C, or 5°C. In one implementation, step b) is performed at temperatures above -20°C, above -19°C, above -18°C, above -17°C, above -16°C, above -15°C, above -14°C, above -13°C, above -12°C, above -11°C, above -10°C, above -9°C, above -8°C, above -7°C, above -6°C, above -5°C, above -4°C, above -3°C, above -2°C, above -1°C, above 0°C, above 1°C, above 2°C, above 3°C, or above 4°C. In another embodiment, step b) is performed at a temperature below -19°C, below -18°C, below -17°C, below -16°C, below -15°C, below -14°C, below -13°C, below -12°C, below -11°C, below -10°C, below -9°C, below -8°C, below -7°C, below -6°C, below -5°C, below -4°C, below -3°C, below -2°C, below -1°C, below 0°C, below 1°C, below 2°C, below 3°C, or below 4°C, or below 5°C.In another embodiment, step b) is performed at approximately -20°C to approximately 5°C, approximately -20°C to approximately 4°C, approximately -20°C to approximately 3°C, approximately -20°C to approximately 2°C, approximately -20°C to approximately 1°C, approximately -20°C to approximately 0°C, approximately -20°C to approximately -1°C, approximately -20°C to approximately -2°C, approximately -20°C to approximately -3°C, approximately -20°C to approximately -4°C, approximately -20°C to approximately -5°C, approximately -20°C to approximately -6 ... ℃ to -7℃, about -20℃ to -8℃, about -20℃ to -9℃, about -20℃ to -10℃, about -20℃ to -11℃, about -20℃ to -12℃, about -20℃ to -13℃, about -20℃ to -14℃, about -20℃ to -15℃, about -20℃ to -16℃, about -20℃ to -17℃, about -20℃ to -18℃, about -20℃ to - 19°C, approximately -20°C to approximately 5°C, approximately -19°C to approximately 5°C, approximately -18°C to approximately 5°C, approximately -17°C to approximately 5°C, approximately -16°C to approximately 5°C, approximately -15°C to approximately 5°C, approximately -14°C to approximately 5°C, approximately -13°C to approximately 5°C, approximately -12°C to approximately 5°C, approximately -11°C to approximately 5°C, approximately -10°C to approximately 5°C, approximately -9°C to approximately 5°C, approximately -8°C to approximately 5°C, approximately -7°C to approximately 5°C, approximately -6°C The temperature shall be carried out at approximately 5°C, approximately -5°C to approximately 5°C, approximately -4°C to approximately 4°C, approximately -3°C to approximately 3°C, approximately -2°C to approximately 2°C, approximately -1°C to approximately 1°C, approximately -5°C to approximately 4°C, approximately -5°C to approximately 3°C, approximately -5°C to approximately 2°C, approximately -5°C to approximately 1°C, or approximately -5°C to approximately 0°C, 0°C to approximately 1°C, 0°C to approximately 2°C, 0°C to approximately 3°C, 0°C to approximately 4°C, 0°C to approximately 5°C, or approximately -20°C to approximately 0°C.

[0042] Step b) can be performed at a temperature of about -3°C to about 3°C.

[0043] In some embodiments, step b) does not include freezing. In other embodiments, step b) includes freezing. In one embodiment, freezing is freeze-drying or lyophilization. Step b) may include thermally cycling the portion of the garlic plant, wherein one cycle includes cooling the portion of the garlic plant until it is frozen, and then warming the portion of the garlic plant until it is no longer frozen. In some embodiments, step b) includes thermally cycling the portion of the garlic plant 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times. In some embodiments, thermally cycling the portion of the garlic plant more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times. In other embodiments, the portion of the garlic plant is thermally cycled less than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 times.

[0044] In some embodiments, step b) is performed for approximately 1.5 years, 2 years, 3 years, 4 years, 5 years, 6 years, 7 years, 8 years, 9 years, or approximately 10 years, or any amount of time in between. In some embodiments, step b) is performed for at least 1.5 years, at least 2 years, at least 3 years, at least 4 years, at least 5 years, at least 6 years, at least 7 years, at least 8 years, at least 9 years, or at least 10 years. In other embodiments, step b) is performed for less than 10 years, less than 9 years, less than 8 years, less than 7 years, less than 6 years, less than 5 years, less than 4 years, less than 3 years, or less than 2 years. In yet another embodiment, step b) is performed for 1.5 to 10 years, 1.5 to 9 years, 1.5 to 8 years, 2 to 7 years, 2 to 6 years, 2 to 5 years, 2 to 4 years, 2 to 3 years, 3 to 10 years, 3 to 9 years, 3 to 8 years, 3 to 7 years, 3 to 6 years, 3 to 5 years, or 3 to 4 years. In some implementations, step b) is performed for 18, 19, 20, 21, 22, 23, or 24 months.

[0045] Step b) can take approximately 3 to 5 years.

[0046] In some implementations, step b) is performed at atmospheric pressure. Step b) may be performed at a pressure below atmospheric pressure. Step b) may be performed at a pressure above atmospheric pressure.

[0047] In one embodiment, step b) is performed under an inert atmosphere. The inert atmosphere may be nitrogen (e.g.).

[0048] Step c) can be carried out by rotating cone distillation.

[0049] In some embodiments, step c) includes adding an aqueous solution to aged garlic to form a slurry, and then subjecting the slurry to rotary conical distillation. In one embodiment, the aqueous solution is water. In another embodiment, the aqueous solution is a mixture of water and a solvent. In yet another embodiment, the aqueous solution is a mixture of water and an alcohol (such as ethanol). In some embodiments, the aqueous solution does not contain alcohol or ethanol.

[0050] Before performing rotary conical distillation on the slurry, the slurry can be heated to approximately 50°C.

[0051] Before rotary conical distillation, the slurry can be heated to approximately 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, 95°C, or approximately 99°C, or any temperature between these values. In some embodiments, the slurry is heated to at least 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, or 95°C before rotary conical distillation. In other embodiments, the slurry is heated to no more than 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, 35°C, 40°C, 45°C, 50°C, 55°C, 60°C, 65°C, 70°C, 75°C, 80°C, 85°C, 90°C, or 95°C before being distilled in a rotary conical column. In some embodiments, the slurry is heated to 5°C to 95°C, 10°C to 90°C, 15°C to 85°C, 20°C to 80°C, 25°C to 75°C, 30°C to 70°C, 35°C to 65°C, 40°C to 60°C, or 45°C to 55°C before being distilled in a rotary conical column.

[0052] In some implementations, the slurry is not heated before being distilled in a rotating conical column.

[0053] In some embodiments, step c) can be performed at a temperature below 110°C. In some embodiments, step c) is performed at a temperature below 110°C, 109°C, 108°C, 107°C, 106°C, 105°C, 104°C, 103°C, 102°C, or 101°C. In some embodiments, step c) is performed at a temperature above 109°C, 108°C, 107°C, 106°C, 105°C, 104°C, 103°C, 102°C, 101°C, or 100°C. In one embodiment, step c) is performed at a temperature between 110°C and 100°C, between 109°C and 100°C, between 108°C and 100°C, between 107°C and 100°C, between 106°C and 100°C, between 105°C and 100°C, between 104°C and 100°C, between 103°C and 100°C, between 102°C and 100°C, or between 101°C and 100°C.

[0054] In some embodiments, the temperature of the slurry during step c) is above room temperature for approximately 30 to 300 seconds. The temperature of the slurry during step c) may be above room temperature for approximately 30, 45, 60, 75, 90, 105, 120, 135, 150, 165, 180, 195, 210, 225, 240, 255, 270, 285, or 300 seconds. In one embodiment, the temperature of the slurry during step c) is above room temperature for less than 45, 60, 75, 90, 105, 120, 135, 150, 165, 180, 195, 210, 225, 240, 255, 270, 285, or 300 seconds. In another embodiment, during step c), the temperature of the slurry is above room temperature for at least 45 seconds, 60 seconds, 75 seconds, 90 seconds, 105 seconds, 120 seconds, 135 seconds, 150 seconds, 165 seconds, 180 seconds, 195 seconds, 210 seconds, 225 seconds, 240 seconds, 255 seconds, 270 seconds, 285 seconds, or 300 seconds. In another embodiment, during step c), the temperature of the slurry is above room temperature for a duration between 30 and 300 seconds, between 30 and 300 seconds, between 30 and 270 seconds, between 30 and 240 seconds, between 30 and 210 seconds, between 30 and 180 seconds, between 30 and 150 seconds, between 30 and 120 seconds, between 30 and 90 seconds, or between 30 and 60 seconds.

[0055] Without being bound by any theory, the inventors believe that rotating cone distillation (SCC) uses steam to separate volatile compounds from a liquid or slurry under reduced pressure. This is achieved by allowing the garlic slurry to flow downwards as a thin film along a vertical column of rotating and fixed cones within the SCC unit. Simultaneously, steam is introduced to the bottom of the column, flows upwards, and passes through the surface of the volatile-rich film of the garlic slurry, thereby separating the volatile compounds from the slurry. This steam flow then passes through a condensation system, which concentrates the volatile compounds separated from the garlic slurry into a garlic oil extract and a distillate. The concentrated garlic oil and distillate can then be collected and bottled separately. When the garlic slurry is pumped into the SCC unit, it can optionally be heated to increase the volatility of the compounds therein. Both the oil and the distillate may contain volatile compounds extracted from garlic.

[0056] Advantageously, the conditions in rotating cone distillation (SCC) are very mild. Because SCC is a flow-based method, the garlic slurry is at elevated temperatures for only a relatively short period (30 seconds to a maximum of 1 minute) as it flows through the SCC unit. In contrast, conventional methods must heat the entire garlic clove at once, resulting in a longer period at elevated temperatures and thus increased degradation of volatile compounds. Furthermore, the internal environment of the SCC unit is oxygen-deficient because it is under reduced pressure (and subsequently filled with steam). This further reduces the rate of degradation of bioactive compounds (especially organosulfates) within the garlic, as any possibility of oxidation reactions is minimized.

[0057] Another advantage of this method is that the resulting garlic oil has a low water content and is not an emulsion. Emulsions and compositions with high water content inevitably lead to a lower concentration of active ingredients and are often microbially unstable. Other methods involve soaking garlic in oils from other plants (i.e., soybean oil or vegetable oil) to extract hydrophobic compounds and produce garlic-flavored oils. However, these methods inevitably produce very dilute products, resulting in reduced or even complete loss of biological activity.

[0058] In one implementation, the garlic oil produced in step c) has a water content of less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.5%, or less than 0.1% v / v.

[0059] In some implementations, step c) is performed at a temperature below 105°C.

[0060] In some embodiments, step c) is performed by supercritical fluid extraction. This technique uses high pressure to generate a supercritical solvent (e.g., supercritical CO2) at a relatively low temperature, and then uses this supercritical solvent to separate volatile compounds from the garlic. Supercritical fluid extraction can be supercritical carbon dioxide extraction. Supercritical fluid extraction can also be supercritical air, ammonia, nitrogen, or water extraction. In some embodiments, aged garlic is freeze-dried prior to supercritical fluid extraction.

[0061] In a fourth aspect of the invention, garlic oil is provided by means of the method described in any one of the first to third aspects.

[0062] In a fifth aspect of the invention, a pharmaceutical composition comprising garlic oil according to the fourth aspect is provided. The composition may further comprise a pharmaceutically acceptable carrier, diluent, and / or excipient.

[0063] Although the garlic oil described in the fourth aspect can be applied as a pure chemical, it can also be applied as part of a pharmaceutical composition containing at least one carrier or excipient.

[0064] The type of pharmaceutical composition may depend on the absorption, distribution, metabolism, and excretion (ADME) characteristics of the pharmaceutical composition. The pharmaceutical composition may comprise a composition suitable for oral or rectal administration or suitable for administration via a non-intravenous route. Oral compositions for oral administration are preferred.

[0065] Topical application includes buccal, sublingual, epidermal, ocular, rectal, and nasal administration, as well as inhalation or aerosol administration. Those skilled in the art will be able to prepare suitable formulations.

[0066] The properties of the pharmaceutical composition and carrier or excipient will depend on the route of administration, the nature of the condition, and the patient being treated. It is believed that those skilled in the art can readily determine the choice of a particular carrier, excipient, or delivery system, and the route of administration. In some cases, it may be necessary to protect the pharmaceutical composition by means known in the art, such as microencapsulation. The route of administration should also be selected to ensure that the active agent reaches its site of action. The pharmaceutical composition may contain any suitable effective amount of active agent commensurate with the intended dose range to be employed.

[0067] The pharmaceutical composition may be in liquid form (including solutions, suspensions, syrups, emulsions, colloids, elixirs, creams, gels, and foams).

[0068] Pharmaceutically acceptable carriers or excipients must be acceptable, meaning they are compatible with other components in the composition and harmless to the patient. Pharmaceutically acceptable carriers or excipients can be solid or liquid. They can function as diluents, buffers, stabilizers, isotonic agents, flavoring agents, antioxidants, solubilizers, lubricants, suspending agents, binders, preservatives, tablet disintegrants, or encapsulating materials. Suitable carriers and excipients are known to those skilled in the art. Regarding buffers, aqueous compositions may contain buffers for maintaining the composition at near-physiological pH or at least in the range of about pH 6.0 to 9.0.

[0069] Preparations in liquid form may include, for example, solutions of water, saline, water-glucose, water-propylene glycol, petroleum, or oils (including animal, plant, mineral, or synthetic oils). For example, a parenteral injection liquid preparation may be formulated as a solution in an aqueous polyethylene glycol solution. Such a liquid preparation may contain at least 0.1% by weight of garlic oil.

[0070] Liquid pharmaceutical compositions may be formulated into unit dosage forms and may contain preservatives. These compositions may also contain formulations such as suspending agents, stabilizers, and / or dispersants. Liquid carriers and excipients may include colorants, flavoring agents, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizers, suspending agents, etc.

[0071] For topical application to the epidermis, the composition can be formulated as an ointment, cream, or lotion, or as a transdermal patch.

[0072] In a sixth aspect of the invention, a method for treating or preventing viral infection is provided, the method comprising administering to a patient in need garlic oil according to the fourth aspect or a pharmaceutical composition according to the fifth aspect.

[0073] In a seventh aspect of the invention, the use of garlic oil according to the fourth aspect is provided in the manufacture of a medicament for treating or preventing viral infections.

[0074] In an eighth aspect of the invention, garlic oil according to the fourth aspect is provided for treating or preventing viral infections.

[0075] According to aspects six through eight, the viral infection may be selected from at least one of the following groups: adenovirus-3 (AdV-3), adenovirus-41 (AdV-41), coronavirus (CoV), severe acute respiratory syndrome coronavirus (SARS-CoV), dengue virus (DENV), herpes simplex virus-1 (HSV-1), herpes simplex virus-2 (HSV-2), human cytomegalovirus (HCMV), influenza A virus subtype H9N2 (IAV-H9N2), influenza B virus (IBV), influenza A virus-H1N1 (I... AV-H1N1), Coxsackievirus B-3 (CBV-3), Echovirus-11 (ECHO), Enterovirus-71 (EV-71), Human Rhinovirus-2 (HRV-2), Hepatitis A Virus (HAV), Measles Virus (MeV), Newcastle Disease Virus (NDV), Variant Influenza Virus-3 (PIV-3), Vaccinium Virus (VV), Herpetic Stomatitis Virus (VSV), Human Immunodeficiency Virus-1 (HIV-1), Reticuloendotheliosis Virus (REV), Porcine Rotavirus (PRV), and Rotavirus SA-11 (RV-SA-11).

[0076] In some embodiments, the viral infection described in aspects six through eight is selected from the group consisting of SARS-CoV-2 and influenza A. In one embodiment, the viral infection described in aspect six or seven is SARS-CoV-2 infection. In another embodiment, the viral infection described in aspect six or seven is influenza A.

[0077] In a ninth aspect of the invention, the use of garlic oil according to the fourth aspect is provided in the manufacture of a medicament for treating or preventing fungal infections.

[0078] In a tenth aspect of the invention, garlic oil according to the fourth aspect is provided for treating or preventing fungal infections.

[0079] In an eleventh aspect of the present invention, a pharmaceutical composition comprising garlic oil is provided.

[0080] In a twelfth aspect of the invention, a method for treating or preventing viral infection is provided, the method comprising administering garlic oil, fresh garlic, or a pharmaceutical composition according to an eleventh aspect to a patient in need.

[0081] For aspect 12, viral infection can be selected from the group consisting of the following: adenovirus-3 (AdV-3), adenovirus-41 (AdV-41), coronavirus (CoV), severe acute respiratory syndrome coronavirus (SARS-CoV), dengue virus (DENV), herpes simplex virus-1 (HSV-1), herpes simplex virus-2 (HSV-2), human cytomegalovirus (HCMV), influenza A virus subtype H9N2 (IAV-H9N2), influenza B virus (IBV), and influenza A virus-H1N1 (IAV-H1N1). 1) Coxsackievirus B-3 (CBV-3), Echovirus-11 (ECHO), Enterovirus-71 (EV-71), Human Rhinovirus-2 (HRV-2), Hepatitis A Virus (HAV), Measles Virus (MeV), Newcastle Disease Virus (NDV), Variant Influenza Virus-3 (PIV-3), Vaccinium Virus (VV), Herpetic Stomatitis Virus (VSV), Human Immunodeficiency Virus-1 (HIV-1), Reticuloendotheliosis Virus (REV), Porcine Rotavirus (PRV), and Rotavirus SA-11 (RV-SA-11).

[0082] In some embodiments, the viral infection described in aspect 12 is selected from the group consisting of SARS-CoV-2 and influenza A. In some embodiments, the viral infection described in aspect 12 is SARS-CoV-2. In other embodiments, the viral infection described in aspects 13 and 14 is influenza A.

[0083] In some embodiments described in aspects eleven and twelfth, the garlic oil is the garlic oil described in aspect four.

[0084] In a thirteenth aspect of the invention, a method for treating or preventing viral infections in plants is provided, the method comprising applying to the plant an effective amount of garlic oil according to the fourth aspect. In some embodiments, the garlic oil is applied to the leaves, stems, flowers, roots, or the soil surrounding the roots of the plant, or a combination thereof. In one embodiment, the viral infection is at least one selected from the group consisting of: potato virus Y; leaf spot virus, grape leafroll-associated virus 2, tomato mosaic virus, and potato virus Y.

[0085] In a fourteenth aspect of the invention, a method for treating or preventing fungal infections in plants is provided, the method comprising applying to the plant an effective amount of garlic oil according to a fourth aspect. In some embodiments, the garlic oil is applied to the leaves, stems, flowers, roots, or soil surrounding the roots of the plant, or a combination thereof. In one embodiment, the fungal infection is at least one of the following genera: *Verticillium*, *Alternaria* (such as *Alternaria brassicicola*), *Botrytis cinerea*, *Magnaporthe* (such as *Magnaporthe grisea*), and *Plectospherella* (such as *Plectospherella cucumerina*).

[0086] As used herein, the terms “treatment” and “prevention” should be considered in their broadest context. For example, the term “treatment” does not necessarily mean that a patient receives treatment until fully recovered. The term “treatment” includes improving the symptoms of a disease, disorder, or condition, or reducing the severity of a disease, disorder, or condition. Similarly, “prevention” does not necessarily mean that a subject will never develop a disease, disorder, or condition. “Prevention” can be considered as reducing the likelihood of the onset of a disease, disorder, or condition, or preventing the development of a disease, disorder, or condition, or otherwise reducing its risk.

[0087] As used herein, the terms “subject” or “individual” or “patient” can refer to any subject in need of therapy, particularly vertebrate subjects, and even more particularly mammal subjects. Suitable vertebrates include, but are not limited to, primates, birds, livestock (e.g., sheep, cattle, horses, donkeys, pigs), laboratory test animals (e.g., rabbits, mice, rats, guinea pigs, hamsters), companion animals (e.g., cats, dogs), and captive wild animals (e.g., foxes, deer, dingoes). Humans are the preferred subject.

[0088] As used herein, "effective amount" means an amount of garlic oil applied that is sufficient to at least partially achieve the desired response, or prevent the onset of symptoms of the treated virus, or cause symptoms to worsen or cease, or treat and alleviate or at least reduce the severity of symptoms. This amount can vary depending on factors such as the health and physical condition of the individual applying the garlic oil, the taxonomic group of the individual applying the garlic oil, the degree of treatment / prevention required, the formulation of the composition, and an assessment of the medical condition. The "effective amount" is expected to fall within a wide range that can be determined through routine testing. Dosing regimens can be adjusted to provide the optimal therapeutic response. For example, doses can be administered daily, every two weeks, or weekly, or at other suitable intervals, or the dose can be reduced proportionally as indicated. Decisions regarding dosage, etc., will be made within the skill of the practicing physician or veterinarian responsible for the patient's care.

[0089] In this specification and claims, the word "comprising" and its derivatives (including "including" and "contains") include each of the integers, but do not exclude the inclusion of one or more other integers.

[0090] The transitional phrase "composed of..." excludes any unspecified elements, steps, or ingredients. If in a claim, this limits the claim to exclude materials other than those listed, except for impurities typically associated with the listed materials. When the phrase "composed of..." appears in a clause of the body of a claim rather than immediately following the preamble, it only limits the elements set forth in that clause; other elements are not excluded by the entire claim.

[0091] The transitional phrase "consistently composed of..." is used to define a composition, process, or method as including materials, steps, features, components, or elements other than those literally disclosed, provided that such additional materials, steps, features, components, or elements do not materially affect the essential and novel characteristics of the invention protected by the claims. The term "consistently composed of..." falls between "comprising" and "composed of...".

[0092] Furthermore, the indefinite articles “a” and “an” preceding the elements or components of the invention are intended to be non-limiting in terms of the number of instances (i.e., occurrences) of the elements or components. Thus, “a” or “an” should be interpreted as including one (a) or at least one (a), and the singular form of the elements or components also includes the plural, unless the numerical value obviously indicates the singular.

[0093] As used herein, when referring to values ​​within a range of numerical values, the terms "about," "approximately," and "substantially" should be understood to mean -10% to +10% of the reference value, preferably -5% to +5%, more preferably -1% to +1%, and most preferably -0.1% to +0.1% of the reference value. Furthermore, when referring to a range of numerical values, these terms should be understood to support claims relating to any value or subset of values ​​within that range. For example, disclosures of 1 to 10 should be understood to support ranges of 1 to 8, 3 to 7, 1 to 9, 3.6 to 4.6, 3.5 to 9.9, 8 to 10, etc.

[0094] Unless explicitly stated otherwise, "or" refers to inclusive or rather than exclusive or. For example, condition A or B is satisfied by either of the following: A is true (or exists) and B is false (or does not exist); A is false (or does not exist) and B is true (or exists); and both A and B are true (or exist).

[0095] Throughout this specification, references to "one embodiment" or "implementation" mean that a particular feature, structure, or characteristic described in connection with that embodiment is included in at least one embodiment of the invention. Therefore, the phrases "in one embodiment" or "in an embodiment" appearing throughout this specification do not necessarily refer to the same embodiment. Furthermore, specific features, structures, or characteristics may be combined in one or more suitable combinations.

[0096] Within the scope of this invention, any feature described herein may be combined with any one or more of the other features described herein in any combination.

[0097] Any reference to prior art in this specification is not and should not be construed as an admission or any form of implication that the prior art is part of common general knowledge. Attached Figure Description

[0098] Preferred features, embodiments, and variations of the present invention can be discerned from the following detailed description, which provides sufficient information for those skilled in the art to carry out the invention. The detailed description should not be construed as limiting the scope of the foregoing invention in any way. The detailed description will be referenced to the following figures:

[0099] Figure 1 A flowchart is shown for producing garlic oil using a rotating cone distillation column.

[0100] Figure 2 The TCID50 of the garlic juice samples is shown, where ** indicates p < 0.01 compared to inoculum only, using one-way ANOVA and Dunningt's multiple comparison test.

[0101] Figure 3 The TCID50 of garlic juice samples compared to the control is shown, where *** indicates p < 0.001 compared to inoculum only, using one-way ANOVA and Dunningt's multiple comparison test.

[0102] Figure 4 The TCID50 of garlic juice samples 2 and 3, for which measures were taken to lower the detection limit, are shown, where *** indicates that a one-way ANOVA was used, with p < 0.001 compared to the control without garlic juice.

[0103] Figure 5 The infectious viral titer of oil 1 is shown using ELISA-based SARS-CoV-2 viral genome detection, where *** indicates p<0.001, two-way ANOVA and Dunningt multiple comparison test.

[0104] Figure 6 The infectious virus titers of garlic oil 1 to 5 are shown using ELISA-based SARS-CoV-2 virus genome detection, where *** indicates p < 0.001 compared to the untreated control. One-way ANOVA and Dunningt multiple comparison test are used.

[0105] Figure 7 The infectious viral titer of oil 10 is shown using ELISA-based SARS-CoV-2 viral genome detection.

[0106] Figure 8 The assay of infectious viral titers against influenza virus (PR8) in garlic juice samples is shown, where *** indicates the use of one-way ANOVA, p<0.001 compared with the untreated control.

[0107] Figure 9 The assay shows the infectious viral titer of an oil sample against influenza virus (PR8). Detailed Implementation

[0108] Figure 1 An exemplary method 1 is shown. The method begins, for example, by harvesting garlic plants at a farm. The garlic is then transported to a facility for further processing 2 and may be stored 4. The garlic is then processed to increase its surface area 6, which may include cutting off the top of the garlic and peeling it (although these steps can also be performed before step 2), and then chopping the garlic to produce raw garlic mince. The garlic mince is then aged for 18 months to 5 years, particularly at a temperature of about -5°C to 5°C. Aging is carried out under sterile, food-grade conditions.

[0109] After aging, the garlic is pumped into batch tank 8, where water is added to provide a slurry of appropriate consistency. The slurry can be preheated (e.g., to about 50°C-55°C) before being used in the rotating cone tower 10. The garlic oil 12 is then decanted, labeled, stored, and dispensed 14.

[0110] Example 1: Preparation of Garlic Oil

[0111] Processing of raw garlic

[0112] To prepare garlic oil, garlic bulbs are first harvested and separated into cloves. The cloves are then peeled to remove the outer skin and chopped to produce raw garlic paste. Before being used in garlic oil production, the raw garlic paste is aged in a chamber for 18 months to 5 years at a temperature between -3°C and 3°C. The garlic paste is aged under sterile, food-grade conditions to avoid the growth of harmful microorganisms.

[0113] The garlic varieties used for garlic oil production and their respective aging times are summarized in Table 1 below.

[0114]

[0115] Garlic oil is produced from aged garlic powder.

[0116] Garlic oil is extracted from aged minced garlic using a rotating cone distillation (SCC) system. To extract garlic oil using SCC distillation, water is added to the raw minced garlic to provide a garlic slurry. The amount of water added is adjusted as needed to provide the appropriate consistency. The garlic slurry is then preheated to approximately 50°C-55°C and pumped into the SCC unit. The SCC unit is operated using the following parameters:

[0117]

[0118] The product flow rate can vary, but is preferably between about 425 L / hr and 475 L / hr. The product heater temperature is between about 96°C and about 100°C. Preferably, the product heater temperature is greater than about 95°C and less than about 101°C. The top steam temperature of the SCC is between about 98°C and 101°C. The bottom product temperature of the SCC is between about 98°C and 101°C. The top steam temperature and the bottom product temperature are preferably similar. The difference between the top steam temperature and the bottom product temperature is typically less than about 1.5°C to 2°C. The steam valve position is preferably less than 100%.

[0119] Chemical analysis of oil

[0120] Using the methods described above, oil samples 1-4 were prepared from different garlic varieties, as shown in Table 3. Sample 5 was a commercially available garlic oil prepared by conventional distillation from an unknown secondary (mixed) Spanish garlic variety. Sample 6 was pure olive oil and represents a control containing no garlic oil.

[0121]

[0122] Each of the oils was analyzed by gas chromatography-mass spectrometry (GCMS) using an Agilent 6890 gas chromatograph coupled with an Agilent 5973 mass spectrometer. The results are detailed in Table 4.

[0123]

[0124] As shown in Table 5 below, organosulfur compounds present in the oil account for a high percentage of the separated compounds. This data indicates that the above method can essentially maintain the high levels of organosulfur compounds present in raw garlic.

[0125]

[0126] Example 2: Preparation of Garlic Juice

[0127] To prepare garlic juice, freshly harvested garlic bulbs are broken open and peeled to produce peeled garlic cloves. The peeled garlic cloves are then juiced using a household juicer to produce a mixture of garlic juice and garlic pulp. The garlic pulp is then filtered using filter paper to separate the garlic juice. Garlic juice is prepared using this method from the garlic variety range specified in Table 6.

[0128]

[0129] Example 3: Antiviral activity of garlic juice against SARS-CoV-2 by infectivity titer assay

[0130] The virucidal activity of garlic juices 2 and 3 against SARS-CoV-2 (human isolate - δ variant, isolate Victoria / 18440 / 2021) at the highest non-cytotoxic concentration was then tested by infectious titer assay.

[0131] method

[0132] To conduct the experiment, a known amount of SARS-CoV-2 was added to fresh garlic juice and incubated at room temperature for 30 minutes. Changes in the infection titer were then determined by performing TCID50. The experiment was repeated to ensure the scientific rigor of the findings.

[0133] First, 96-well plates were seeded with Vero cells and incubated overnight at 37°C and 5% CO2. Then, 900 μL of each garlic juice sample was added to a separate Eppendorf tube. Next, 100 μL of SARS-CoV-2 stock solution (δ variant (isositor Victoria / 18440 / 2021)) was added to each sample and the tube was sealed. The samples were manually shaken every 5 minutes to ensure as much mixing as possible. At 30 minutes, the samples were serially diluted (1 / 5 s) and TCID50 was performed to determine if there were any changes in the infectious virus titer. The supernatant obtained from the initial TCID50 was passaged a second time to determine the presence of any remaining infectious virus.

[0134] result

[0135] Microscopic observation did not reveal the presence of the undiluted wells, 5. -1 Hole and 5 -2 Whether virus-induced cytopathic effect (CPE) exists in the wells. From these wells and 5 -3 Hole 5 -4 Hole and 5 -5 Samples were taken from the wells and added to a new Vero cell monolayer to assess the presence of infectious viruses.

[0136] Undiluted pores, 5 -1 Diluted pores and 5 -2 Cells in the diluted wells died due to monolayer blockage. Therefore, the limit of detection (LOD) for this assay was 10. 3.5 TCID50 / mL. Virus-induced CPE was recorded when each well was examined under a microscope. Most samples appeared to have a higher incidence of CPE in the wells than inoculum alone. This could be due to the direct effect of garlic juice or its influence on the odor intensity of the culture.

[0137] like Figure 2 As best seen in Table 7, garlic juices 2 and 3 significantly reduced infectious titers compared to the inoculum-only control. Taking into account the detection limit, this resulted in reductions in infectious titers of up to 55% and 80.67%, respectively, compared to the inoculum-only control.

[0138]

[0139] Example 4: Antiviral activity of garlic juice against SARS-CoV-2 by infectivity titer assay

[0140] The above assays were repeated using uninfected controls containing the same garlic juice dilutions obtained from samples produced by exposing garlic juice to the virus. This experiment investigated whether the cytotoxicity was caused by the garlic juice or by the presence of the infectious virus, thus ultimately determining the effect of garlic juice on the viral infectivity titer.

[0141] method

[0142] First, 96-well plates were seeded with Vero cells and incubated overnight at 37°C and 5% CO2. Then, 450 μL of each garlic juice sample was added in duplicate to separate Eppendorf tubes and transferred to the PC3 laboratory. Next, 50 μL of SARS-CoV-2 stock solution (δ variant (isolate Victoria / 18440 / 2021)) was added to each sample tube and sealed. The second tube contained only culture medium (virus-free). The samples were shaken by hand every 5 minutes to ensure as much mixing as possible. At 30 minutes, the samples were serially diluted (1 / 5 s) and TCID50 was performed to determine if there were any changes in the infectious virus titer. If a toxic odor affected the wells when the sample was inoculated undiluted, the TCID50 was increased from 5... -1 Dilution begins. The supernatant obtained from the initial TCID50 will be passaged a second time to determine the presence of any remaining infectious virus.

[0143] result

[0144] It is impossible to distinguish between uninfected control samples and samples exposed to the virus through microscopic observation. -1 Hole and 5 -2 Does the pore contain virus-induced CPE caused by the presence of garlic juice? In 5 -3 At the dilution, significant cell death was observed in wells containing uninfected sample controls. Therefore, from 5 -3 Starting with dilution, samples were taken a second time from both infected and uninfected control wells to determine whether persistent cell death due to the presence of garlic juice was present, or whether virus-induced CPE could be identified.

[0145] For samples from the second passage of uninfected control wells, all monolayers appeared healthy, and no cell death was observed. Virus-induced CPE was recorded when each well was examined under a microscope for detection.

[0146] like Figure 3As best seen in Table 8, garlic juices 2 and 3 reduced the infectious titer of SARS-CoV-2 to an undetectable (ND) level. Taking into account the detection limit, this is equivalent to an 82.7% and 73.8% reduction in infectious titer, respectively, compared to the inoculum-only control. One-way ANOVA (Dunningter's multiple comparison test) for garlic juices 2 and 3 showed significance of p < 0.001 compared to the inoculum-only control. Surprisingly, garlic juice 2 produced a TCID50 below the detection limit of this assay.

[0147]

[0148] Example 5: Antiviral activity of garlic juice against SARS-CoV-2 by infectivity titer assay

[0149] Because the experiment in Example 4 surprisingly produced TCID50 values ​​below the detection limit of that determination, the experiment was repeated and modified to lower the detection limit. This experiment used garlic juices No. 2 and No. 3, as well as a control without garlic juice.

[0150] method

[0151] The method is as outlined in Example 4, with the following modifications: (a) at sampling, 200 μL of elution buffer was taken from the samples exposed to 2 and 3 and the control without garlic juice and added directly to the RNA extraction buffer; and (b) the supernatant sample was also taken and passaged a second time in Vero cells, thus lowering the detection limit of the assay.

[0152] result

[0153] As shown in Table 9 and Figure 4 In best-of-the-line observations, garlic juice 2 and garlic juice 3 reduced the infectious titer of SARS-CoV-2 to an almost undetectable (ND) level, which is equivalent to an average reduction of 99.9% and 99.3% of the infectious titer (respectively) compared to the control without garlic juice.

[0154]

[0155] Example 6: Antiviral activity of garlic oil against SARS-CoV-2

[0156] Garlic oil 1 was tested against SARS-CoV-2 human isolate -δ variant (isolated Victoria / 18440 / 2021) by infectivity titer assay.

[0157] method

[0158] The method is as follows:

[0159] 1. Heat the oil in a water bath (37°C) until completely dissolved, then let it cool to room temperature.

[0160] 2. The oil test solution is prepared in the following manner:

[0161] a. Produce a 1 / 50 dilution (100 μL oil to 4.9 mL infection culture medium).

[0162] b. Prepare a 1 / 500 dilution (0.5 mL of 1 / 50 dilution plus 4.5 mL of infection culture medium).

[0163] 3. Vortex the sample for 30 seconds, then add it to the following DMSO or isopropanol:

[0164] a. DMSO = 10 μL DMSO + 990 μL of any diluted oil sample; final DMSO concentration = 1% v / v

[0165] b. Isopropanol = 100 μL isopropanol + 900 μL of any diluted oil sample; final concentration of isopropanol = 10% v / v

[0166] 4. Vortex the sample for at least 30 seconds and monitor the separation. The solution should appear cloudy / emulsified and stable. Produce two samples for each test condition.

[0167] 5. Add 100 μL of SARS-CoV-2 stock solution (δ variant (isolated Victoria / 18440 / 2021)) to the test solution and seal.

[0168] 6. Shake the sample by hand every 5 minutes to ensure it is mixed as much as possible. The solution will appear cloudy / emulsified throughout the incubation period (30 minutes at room temperature).

[0169] 7. At 30 minutes, serially dilute the sample and perform TCID50 to determine if there are any changes in the infectious viral titer.

[0170] 8. To detect the presence of infectious viruses, enzyme-linked immunosorbent assay (ELISA) is used.

[0171] result

[0172] As shown in Table 10 and Figure 5Best observed, solutions prepared using garlic oil 1 (MOF) exhibited significant antiviral activity against SARS-CoV-2 when dissolved at a 1 / 50 dilution in both DMSO and isopropanol (***p<0.001, 2-factor ANOVA, Dunningt multiple comparison test). When dissolved in DMSO, garlic oil 1 reduced the infectious SARS-CoV-2 titer by at least 98.6% on average, and when dissolved in isopropanol, reduced the infectious titer by at least 97.71% on average. Since the DMSO and isopropanol controls did not produce any antiviral activity against SARS-CoV-2 (titers were not different from the solvent-free control), these results are attributable to the antiviral effect of garlic oil itself.

[0173]

[0174] It has been confirmed that garlic oil 1 can cause a significant reduction in infection titers. The above method was used to analyze the antiviral activity of oils 1-3, 5, and 6 against SARS-CoV-2. The TCID50 value of oil 6 can be obtained without dissolution.

[0175] As shown in Table 11 and Figure 6 and Figure 7 Of the best observed values, garlic oils 1–3 showed a significant reduction in viral titer (99.90%, 82.29%, and 78.07%, respectively). Specifically, garlic oil 1 showed a significant reduction in viral titer (p<0.001, one-way ANOVA, Dunningt multiple comparison test). In contrast, samples 5 and 6 did not result in any reduction in viral titer.

[0176]

[0177] Example 7: Garlic juice as an antiviral agent against influenza A virus by measuring infectivity titer

[0178] This experiment demonstrated the antiviral activity of fresh garlic juice against influenza A (A / Puerto Rico / 8 / 1934; PR8) as determined by infection titer.

[0179] method

[0180] First, 96-well plates were seeded with Vero cells and incubated overnight at 37°C and 5% CO2. Then, in the PC2 laboratory, 450 μL of each garlic juice sample was added in duplicate to separate Eppendorf tubes. Next, 50 μL of influenza A virus stock solution (isolated strain A / Puerto Rico / 8 / 1934; also known as PR8) was added to each sample and the tubes were sealed. For the second aliquot, 50 μL of infection medium (uninfected control) was added. The samples were shaken by hand every 5 minutes to ensure as much mixing as possible. At 30 minutes, the samples were serially diluted (1 / 5 s) and TCID50 was performed to determine if there were any changes in the infectious virus titer. After 3 days, the supernatant was added to chicken erythrocytes for erythrocyte agglutination (HA) titer determination. For wells containing infectious virus, erythrocytes agglutinated, while for samples without infectious virus, erythrocytes precipitated. This provided a clear and definitive answer regarding the presence of infectious virus.

[0181] Previous experiments have shown that undiluted garlic juice is cytotoxic; therefore, the TCID50 was started at a 1 / 5 dilution. Consequently, the detection limit for infectious viruses was 10-1. 1.85 TCID50 / mL. For all wells inoculated with serially diluted, virus-free garlic juice, hemagglutination assays showed no agglutination; therefore, garlic juice does not interfere with the ability of this method to detect infectious influenza virus.

[0182] result

[0183] like Figure 8 As best seen in Table 12, garlic juices 1, 2 and 3 reduced the infectious titer of influenza A by an average of 54.91%, 99.95% and 100%, respectively.

[0184]

[0185] Example 8: Antiviral activity of garlic oil against influenza A virus

[0186] To determine the antiviral activity of garlic oil, the TCID50 assay was used to measure the infectious titer of influenza A virus (A / PuertoRico / 8 / 1934; PR8). The presence of the virus in the sample was confirmed by microscopic examination using a hemagglutination assay with chicken erythrocytes.

[0187] method

[0188] The oil was heated in a water bath (37°C) until completely dissolved, then cooled to room temperature. The test solution was prepared by first producing a 1 / 50 dilution (100 μL oil to 4.9 mL saline). The diluted sample was then vortexed for 30 seconds, and isopropanol was added to each sample at a ratio of 100 μL isopropanol:900 μL garlic oil sample (final isopropanol concentration = 10% v / v). The sample was vortexed for at least 30 seconds, and separation was monitored. The solution appeared turbid / emulsified and stable. 100 μL of PR8 stock solution was added to the test solution and the container was sealed. The sample was vortexed every 5 minutes to ensure as much mixing as possible. The solution remained turbid / emulsified throughout the incubation period (30 minutes at room temperature). At 30 minutes, the sample was serially diluted and TCID50 was performed to determine if there were any changes in the infectious virus titer. The HA assay was performed on the TCID50 supernatant to confirm the presence of infectious virus in each well. Given that the results of the hemagglutination assay are clear, a second passage of TCID50 is not required to confirm the presence (or absence) of the infectious virus.

[0189] result

[0190] As shown in Table 13 and Figure 9 Best observed was a 1-log decrease in influenza infectivity titer when the virus was exposed to garlic oil samples 1–4. Cell monolayers appeared intact and similar to uninfected control wells, indicating that the oil did not interfere with MDCK cell viability at the tested dilutions.

[0191]

[0192] According to regulations, inventions have been described to some extent in language specific to their structural or methodological features. It should be understood that the invention is not limited to the specific features shown or described, as the manner described herein includes preferred forms that enable the invention to take effect. Therefore, the invention is protected by the claims in any form or modification thereof within the appropriate scope of the appended claims as properly interpreted by those skilled in the art.

Claims

1. A method for preparing garlic oil, the method comprising the following steps: a) Process a portion of the garlic plant to increase its surface area; b) Aging the said portion of the garlic plant for at least 18 months to provide aged garlic; and c) Separating the garlic oil from the aged garlic, wherein the separation is performed using rotary cone distillation or supercritical fluid extraction. Step b) does not include the addition of ethanol.

2. A method for preparing garlic oil, the method comprising the following steps: a) Process a portion of the garlic plant to increase its surface area; b) Aging the said portion of the garlic plant for at least 18 months to provide aged garlic; and c) Separating the garlic oil from the aged garlic, wherein the separation is carried out at a temperature below 110°C. Step b) does not include the addition of ethanol.

3. A method for preparing garlic oil, the method comprising the following steps: a) Process a portion of the garlic plant to increase its surface area; b) Aged the garlic plant for at least 18 months to provide aged garlic; as well as c) Separating the garlic oil from the aged garlic, wherein during the separation, the pressure (atmospheric pressure) divided by the temperature (Kelvin temperature) is equal to or less than about 0.0026. Step b) does not include the addition of ethanol.

4. The method according to any one of claims 1 to 3, wherein step a) comprises cutting, grinding, crushing, or any combination thereof.

5. The method according to any one of the preceding claims, wherein step a) comprises chopping.

6. The method according to any one of the preceding claims, wherein step b) does not include adding a solvent.

7. The method according to any one of the preceding claims, wherein step b) is carried out at a temperature of about -3°C to about 3°C.

8. The method according to any one of the preceding claims, wherein step b) does not include freezing.

9. The method according to any one of the preceding claims, wherein step b) is carried out for about 3 to about 5 years.

10. The method according to any one of the preceding claims, wherein step b) is carried out at atmospheric pressure.

11. The method according to any one of the preceding claims, wherein the portion of the garlic plant in step a) is a peeled garlic clove.

12. The method according to any one of the preceding claims, wherein step c) is carried out by rotating conical distillation.

13. The method of claim 12, wherein step c) comprises adding an aqueous solution to the aged garlic to form a slurry, and then subjecting the slurry to the rotary cone distillation.

14. The method of claim 13, wherein the slurry is heated to about 50°C before being subjected to the rotary conical distillation.

15. The method according to any one of claims 1 to 11, wherein step c) is performed by supercritical fluid extraction.

16. The method of claim 15, wherein the supercritical fluid extraction is supercritical carbon dioxide extraction.

17. The method according to any one of the preceding claims, wherein the portion of said garlic plant is obtained from garlic varieties selected from the group consisting of: Australian White, Australian Red and Australian Purple.

18. The method according to any one of the preceding claims, wherein the portion of the garlic plant is not obtained from the Griffith white garlic variety.

19. A garlic oil, said garlic oil being produced by the method according to any one of claims 1 to 18.

20. A pharmaceutical composition comprising the garlic oil according to claim 19.

21. A method of treating or preventing viral infection, the method comprising administering to a patient in need the garlic oil of claim 19 or the pharmaceutical composition of claim 20.

22. The method of claim 21, wherein the viral infection is selected from the group consisting of SARS-CoV-2 and influenza A.

23. Use of the garlic oil according to claim 19 in the manufacture of a medicament for treating or preventing viral infections.

24. The use according to claim 23, wherein the viral infection is selected from the group consisting of SARS-CoV-2 and influenza A.

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