Methods for preparing zingerone, compositions containing zingerone, and uses thereof

Abstract

Methods for producing zingerone from ginger, compositions obtained using these methods, and methods of using these compositions are disclosed. In particular, pharmaceutical compositions and methods of treatment utilizing the same are disclosed.

Description

[Technical field]

[0001] This application claims the benefit of New Zealand Provisional Application No. 779010, filed August 11, 2021, the entire contents of which are incorporated herein by reference.

[0002] The present disclosure relates to methods of preparing zingerone and compositions comprising zingerone. In particular, pharmaceutical compositions and uses of these compositions are described. [Background technology]

[0003] Ginger (Zingiber officinale) is a flowering plant whose rhizomes are widely used as a spice and in traditional medicine. When consumed in moderate amounts, ginger has few adverse side effects. It is listed as "generally recognized as safe" by the FDA.

[0004] The characteristic aroma and flavor of ginger is due to a volatile oil present in fresh ginger that is essentially composed of gingerone, shogaols, and gingerols, with gingerol ([6]-gingerol (1-[4'-hydroxy-3'-methoxyphenyl]-5-hydroxy-3-decanone))) as the main pungent compound, present in amounts of 1% to 3% by weight. [ka]

[0005] Zingerone (also known as gingerone) is reported to be produced from gingerol during drying or heat treatment at a temperature of about 40 degrees Celsius as reported by Li et. al., 2016, “Chemical Characterisation and antioxidant activities comparison in fresh, dried, stir frying and carbonized ginger” Journal of Chromatography B Analyt. Technol. Biomed. Life Sci. 1011: 223-232. Zingerone has a low pungency and a tangy-sweet aroma. Zingerone, also called vanillylacetone, is a crystalline solid that is reported to be sparingly soluble in water and soluble in ether. The water solubility value of zingerone is 0.57 g / L, LogP is 2.02 1.92, and logS is -2.5 as listed in the FoodB compound database. See https: / / foodb.ca / compounds / FDB010527. [ka]

[0006] Fresh ginger contains very little zingerone, and is known to be produced by cooking or drying ginger root, which is caused by the loss of water molecules and the dehydration of gingerol to produce zingerone and hexanal. See, for example, Gopi et al. 2016, “Study on temperature dependent conversion of active components of ginger” Int. J. of Pharma Sciences 6(1): 1344-1347.

[0007] Shogaol, which has a more pungent taste and high antioxidant activity, is not found in fresh ginger but is formed from gingerol by heating, storage, or acidification. [ka]

[0008] Zingerone was first isolated from ginger root by Hiroshi Nomura in 1917. Nomura identified and later patented a method for the synthesis of zingerone by reacting vanillin with acetone under basic conditions to form dehydrozingerone (US 1,263,796, issued April 23, 1918). This compound was obtained in approximately 95% yield. After this reaction, the intermediate compound was catalytically hydrogenated to produce zingerone in approximately 100% yield.

[0009] Ginger compounds have been shown to be active against enterotoxigenic Escherichia coli heat-labile enterotoxin-induced diarrhea. This type of diarrhea is a major cause of infant mortality in developing countries. It has been reported that zingerone may be the active component responsible for the antidiarrheal effect of ginger. The study concluded that the bioactive compounds in ginger significantly blocked the binding of enterotoxigenic Escherichia coli heat-labile enterotoxin to the cell surface receptor G M1, thereby inhibiting fluid retention in closed ileal loops in mice. See, for example, Chen et al., 2007, “Ginger and its bioactive component inhibit enterotoxigenic Escherichia coli heat-labile enterotoxin-induced diarrhoea in mice” Journal of Agricultural and Food Chemistry 55 (21): 8390-7.

[0010] Zingerone has been shown to have anti-inflammatory effects on liver inflammation in a mouse model of peritonitis as reported by Kumar et al., “Zingerone suppresses liver inflammation induced by antibiotic mediated endotoxemia through down regulating hepatic mRNA expression of inflammatory markers in Pseudomonas aeruginosa peritonitis mouse model” PLOS ONE 9(9): e106536.

[0011] Kumar et al. also reported that zingerone increases the susceptibility of Pseudomonas aeruginosa cells to antibiotics. See Kumar et al., 2014, Life Sciences 117: 24-32. Kumar et al. concluded that zingerone was found to cause changes in the cell surface properties of Pseudomonas aeruginosa, thereby increasing the susceptibility of Pseudomonas aeruginosa cells to antibiotics.

[0012] Limited research has been conducted on optimizing the production of zingerone from natural sources. Given the current emphasis on compositions derived from natural sources, there is a need for new compositions, including plant-based compositions, particularly those with antibacterial activity. The present application is directed to meeting these and other needs. Summary of the Invention

[0013] In one aspect, the disclosure encompasses a method of producing zingerone by (i) subjecting ginger root to alkaline treatment in an alkaline solution; or (ii) subjecting juice obtained from ginger root to alkaline treatment in an alkaline solution.

[0014] In certain embodiments

[0015] The ginger root is fresh.

[0016] The ginger root is dried.

[0017] The ginger root is dried at about 40 degrees Celsius (about 40°C) to about 70 degrees Celsius, or at about 55 degrees Celsius to about 65 degrees Celsius, or at about 60 degrees Celsius.

[0018] The juice is obtained by soaking and / or pressing ginger root.

[0019] The ginger root is cut and subjected to an alkaline treatment.

[0020] The ginger root is cut, dried and subjected to an alkaline treatment.

[0021] The alkali treatment is carried out at a temperature of about 40 degrees Celsius to about 70 degrees Celsius.

[0022] The alkali treatment is carried out at about 50 degrees Celsius to about 60 degrees Celsius.

[0023] The alkali treatment is carried out at about 55 degrees Celsius to about 65 degrees Celsius.

[0024] The alkaline treatment is carried out at about 60 degrees Celsius.

[0025] The alkali treatment is carried out for about 1 to 72 hours.

[0026] The alkali treatment is carried out for about 1 to 48 hours.

[0027] The alkali treatment is carried out for about 1 to 24 hours.

[0028] The alkali treatment is carried out for about 1 to 30 hours, or about 1 to 20 hours, or about 1 to 10 hours, or about 1 to 5 hours.

[0029] The alkali treatment is carried out for about 0.5 hours to about 3 hours, or about 0.5 hours to about 2 hours, or about 1 hour to about 2 hours.

[0030] The alkaline treatment is carried out for about 2 hours.

[0031] The alkaline treatment is carried out for about 1 hour.

[0032] Potassium hydroxide is used.

[0033] A liquid form of potassium hydroxide (KOH) is used.

[0034] About 0.1% to about 1.0% KOH (v / v) is used. About 0.5% to about 0.7% KOH (v / v) is used.

[0035] About 1% to about 3% KOH (v / v) is used. About 1.5% to about 2.5% KOH (v / v) is used. About 2% KOH (v / v) is used.

[0036] Calcium hydroxide, Ca(OH)2, is used.

[0037] About 0.5% to about 4% Ca(OH)2 (v / v) is used. About 1.5% to about 3.5% Ca(OH)2 (v / v) is used. About 2% to about 3% Ca(OH)2 (v / v) is used.

[0038] After the alkaline treatment, the alkaline solution is neutralized.

[0039] The alkaline solution is neutralized to obtain a pH of about 6.5 to about 7.5.

[0040] The neutralized alkaline solution is freeze-dried.

[0041] The neutralized alkaline solution is subjected to extraction of zingerone.

[0042] The neutralized alkaline solution is dried and then subjected to extraction of zingerone.

[0043] The drying is carried out at about 60 degrees Celsius for up to 24 hours.

[0044] The zingerone may optionally be extracted by one or more alcohol extraction steps. The zingerone is extracted by one or more ethanol extraction steps.

[0045] The ethanol extraction is carried out for at least 7 days.

[0046] The ethanol extraction is carried out for 24 hours or less.

[0047] The zingerone is extracted using supercritical fluid extraction.

[0048] The zingerone is extracted by a supercritical fluid extraction step followed by an alcohol extraction step.

[0049] The process produces a product consisting essentially of zingerone.

[0050] The process produces an aldehyde-free or substantially aldehyde-free product.

[0051] In one aspect, the present disclosure encompasses a method for producing zingerone by subjecting ginger root extract to alkaline treatment.

[0052] The ginger root extract is obtained by supercritical fluid extraction of ginger root.

[0053] The ginger root extract is obtained by alcohol extraction of ginger root.

[0054] The ginger root extract is obtained by squeezing the juice from ginger root.

[0055] The juicing involves soaking and / or pressing the ginger root.

[0056] The alkali treatment is carried out at a temperature of about 30 degrees Celsius to about 70 degrees Celsius.

[0057] The alkali treatment is carried out at about 50 degrees Celsius to about 60 degrees Celsius.

[0058] The alkali treatment is carried out at about 55 degrees Celsius to about 65 degrees Celsius.

[0059] The alkaline treatment is carried out at about 60 degrees Celsius.

[0060] The alkali treatment is carried out for about 1 to 72 hours.

[0061] The alkali treatment is carried out for about 1 to 48 hours.

[0062] The alkali treatment is carried out for about 1 to 24 hours.

[0063] The alkali treatment is carried out for about 1 to 30 hours, or about 1 to 20 hours, or about 1 to 10 hours, or about 1 to 5 hours.

[0064] The alkali treatment is carried out for about 0.5 hours to about 3 hours, or about 0.5 hours to about 2 hours, or about 1 hour to about 2 hours.

[0065] The alkaline treatment is carried out for about 2 hours.

[0066] The alkaline treatment is carried out for about 1 hour.

[0067] Potassium hydroxide (KOH) is used.

[0068] A liquid form of potassium hydroxide (KOH) is used.

[0069] About 0.1% to about 5.0% KOH (v / v) is used. About 0.5% to about 0.7% KOH (v / v) is used.

[0070] About 1% to about 3% KOH (v / v) is used. About 1.5% to about 2.5% KOH (v / v) is used. About 2% KOH (v / v) is used.

[0071] Calcium hydroxide, Ca(OH)2, is used.

[0072] About 0.5% to about 4% Ca(OH)2 (v / v) is used. About 1.5% to about 3.5% Ca(OH)2 (v / v) is used. About 2% to about 3% Ca(OH)2 (v / v) is used.

[0073] After the alkaline treatment, the alkaline solution is neutralized.

[0074] The alkaline solution is neutralized to obtain a pH of about 6.5 to about 7.5.

[0075] After neutralization of the alkaline solution, zingerone is further extracted.

[0076] The zingerone may optionally be further extracted by one or more alcohol extraction steps.

[0077] The zingerone may optionally be further extracted by one or more ethanol extraction steps.

[0078] The ethanol extraction is carried out for at least 7 days.

[0079] The ethanol extraction is carried out for 24 hours or less.

[0080] The zingerone may optionally be further extracted using supercritical fluid extraction.

[0081] The zingerone may optionally be further extracted by a supercritical fluid extraction step followed by an alcohol extraction step.

[0082] The process produces a product consisting essentially of zingerone.

[0083] The process produces an aldehyde-free or substantially aldehyde-free product.

[0084] The method includes: (i) subjecting ginger root to an alkaline treatment in an alkaline solution; or (ii) subjecting juice obtained from ginger root to an alkaline treatment in an alkaline solution, wherein the alkaline solution contains about 1.5% to about 2.5% KOH (v / v), the alkaline treatment is carried out for about 1 hour to about 2 hours, and after the alkaline treatment, neutralizing the alkaline solution to a pH of about 6.5 to about 7.5.

[0085] The present disclosure also encompasses a composition comprising zingerone, wherein the zingerone is prepared by the method of any one of the preceding aspects.

[0086] The present disclosure further encompasses a composition consisting essentially of zingerone, wherein the zingerone is prepared by the method of any one of the preceding aspects.

[0087] In one embodiment, the disclosure encompasses a method of treating or preventing a microbial infection comprising administering to a subject a composition described in any one of the preceding embodiments, thereby treating or preventing said infection.

[0088] In another embodiment, the disclosure encompasses the use of a composition according to any one of the preceding embodiments for the preparation of a medicament for treating or preventing a microbial infection.

[0089] The present disclosure also includes compositions comprising zingerone for treating or preventing a microbial infection.

[0090] In various aspects:

[0091] The zingerone-containing composition is obtainable by the method according to any one of the preceding aspects.

[0092] The zingerone-containing composition is obtained from ginger root.

[0093] The zingerone-containing composition is obtained from fresh ginger root.

[0094] The zingerone-containing composition is obtained from dried ginger root.

[0095] The zingerone-containing composition is obtained from juice prepared from ginger root.

[0096] The juice is prepared by soaking and / or pressing ginger root.

[0097] The zingerone-containing composition is obtained using an alkaline conversion process to convert gingerol in ginger root or in juice from ginger root into zingerone.

[0098] The zingerone-containing composition is free or substantially free of aldehydes.

[0099] The zingerone-containing composition is formulated as a powder.

[0100] The zingerone-containing composition is formulated as a tincture.

[0101] The composition further comprises one or more antimicrobial agents.

[0102] The composition further comprises one or more aminoglycoside antibiotics and / or one or more glycopeptide antibiotics.

[0103] The composition further comprises vancomycin.

[0104] The composition further comprises gentamicin.

[0105] The microorganism is selected from the group consisting of bacteria and fungi.

[0106] The bacteria may be a gram-positive or gram-negative bacterium.

[0107] The bacteria is selected from the group consisting of Bacillus bacteria, Clostridium bacteria, Escherichia bacteria, Mycoplasma bacteria, Neissaria bacteria, Pseudomonas bacteria, Salmonella bacteria, Shigella bacteria, Streptococcus bacteria, Staphylococcus bacteria, and Vibrio bacteria.

[0108] The probacterium is selected from the group consisting of Escherichia coli (Escherichia coli), Staphylococcus aureus (Staphylococcus aureus), Streptococcus pneumoniae (Streptococcus pneumoniae), Pseudomonas aeruginosa (Pseudomonas aeruginosa) and Klebsiella pneumoniae (Klebsiella pneumoniae).

[0109] The fungus is selected from the group consisting of Aspergillus fungi, Candida fungi, Coccidioides fungi, Cryptococcus fungi, Histoplasma fungi, Pneumocystis fungi and Stachybotrys fungi.

[0110] The fungus is selected from the group consisting of Candida albicans, Aspergillus species, Histoplasma capsulatum, Coccidioides immitis, and Pneumocystis carinii and tinea.

[0111] In various embodiments of the composition:

[0112] The compositions are formulated for topical or oral administration.

[0113] The compositions are formulated as liquids, powders, tablets or capsules.

[0114] The composition comprises a dosage of zingerone from about 1 mg to about 5000 mg.

[0115] The composition comprises a dosage of zingerone from about 1 mg to about 1500 mg.

[0116] The composition comprises a dosage of zingerone from about 5 mg to about 500 mg.

[0117] The composition comprises a dosage of about 1 mg to about 150 mg of zingerone.

[0118] The composition comprises a dosage of about 5 mg to about 50 mg of zingerone.

[0119] The composition comprises a dosage of about 1 mg to about 15 mg of zingerone.

[0120] The composition comprises a dosage of about 1 mg to about 10 mg of zingerone.

[0121] The composition is provided in a sachet.

[0122] The compositions are formulated for co-administration with one or more antimicrobial agents.

[0123] The compositions are formulated for co-administration with one or more aminoglycoside antibiotics and / or one or more glycopeptide antibiotics.

[0124] The composition is formulated for co-administration with gentamicin or vancomycin.

[0125] The microbial infection is an infection affecting one or more of the skin, eyes, ears, nose, mouth, throat, esophagus, lungs, circulatory system, digestive system or genitourinary system.

[0126] The foregoing brief summary broadly describes the features and technical advantages of certain embodiments of the present disclosure. Further technical advantages are described in the detailed description and examples that follow.

[0127] The novel features believed to be characteristic will be better understood from the detailed description when considered in conjunction with the accompanying figures and examples. However, the figures and examples provided herein are intended to serve to explain the disclosed matter or to aid in the understanding of the disclosed matter, and are not intended to limit the scope of the present disclosure. [Brief description of the drawings]

[0128] [Figure 1] A photo showing fresh ginger root.

[0129] [Diagram 2] HPLC UV chromatogram trace (280 nm) of alkali-treated ginger.

[0130] [Diagram 3] Schematic diagram showing process comparison

[0131] [Figure 4A] Photograph showing the juicing equipment and raw ginger before juicing.

[0132] [Figure 4B] A photo showing the process of extracting juice from fresh ginger.

[0133] [Figure 5A] Ginger juice was treated with KOH (0.5%) and analyzed by HPLC. The peak areas of zingerone (Z) and gingerol (G) are shown.

[0134] [Figure 5B] Ginger juice was treated with KOH (1%) and analyzed by HPLC. The peak areas of zingerone (Z) and gingerol (G) are shown.

[0135] [Figure 5C] Ginger juice was treated with KOH (2%) and analyzed by HPLC. The peak areas of zingerone (Z) and gingerol (G) are shown.

[0136] [Figure 6A] Ginger pomace produced by squeezing

[0137] [Figure 6B] Ginger juice produced by squeezing

[0138] [Figure 7] Schematic showing the ethanol extraction process and evaporation.

[0139] [Figure 8A] GCMS TIC analysis of ethanol extracts

[0140] [Figure 8B] Comparison of the ethanol extract with a hexanal standard. The chromatogram shows the 2-7 min region.

[0141] [Figure 9A] Checkerboard assay of zingerone and gentamicin combinations.

[0142] [Figure 9B] Checkerboard assay of zingerone and vancomycin combinations.

[0143] [Figure 9C] Checkerboard assay of zingerone and cefotaxime combination.

[0144] Detailed Description In the following description, numerous example configurations, parameters, etc., are set forth. It should be recognized, however, that such description is not intended to limit the scope of the present disclosure, but is instead provided as a description of example embodiments.

[0145] All documents cited in this specification, including patents and patent applications, are hereby incorporated by reference. No admission is made that any document is prior art, and the discussion of any reference is not an admission that such reference forms part of the general knowledge in the art in New Zealand or any other country.

[0146] definition In each instance herein, in the descriptions, embodiments, and examples of the present disclosure, the terms "comprising," "including," and the like are to be read expansively, without limitation. Thus, unless the context clearly requires otherwise, throughout this specification and the claims, the words "comprise," "comprising," and the like are to be interpreted in an inclusive sense, i.e., "including, but not limited to," as opposed to an exclusive sense.

[0147] As used herein, the articles "a" and "an" are used to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" may mean one or more elements.

[0148] Throughout this specification, the term "about" is used to indicate that a value includes the standard deviation of error for the method employed to determine, for example, compound levels or dosage levels, as described in detail herein. In particular, the term "about" encompasses deviations (positive and negative) of up to 10% in the stated value or range.

[0149] The term "comprising" as used herein may refer to the presence of zingerone or zingerone extract in the composition. By way of example, zingerone or zingerone extract may be at least 0.1% by weight, at least 0.2% by weight, at least 0.3% by weight, at least 0.4% by weight, at least 0.5% by weight, at least 0.6% by weight, at least 0.7% by weight, at least 0.8% by weight, at least 0.9% by weight, at least 1% by weight, at least 2% by weight, at least 5% by weight, at least 10% by weight, at least 20% by weight, at least 30% by weight, at least 40% by weight, at least 50% by weight, at least 60% by weight, at least 70% by weight, at least 80% by weight, or at least 90% by weight (% w / w) of the composition. If liquid, zingerone or zingerone extract can be at least 0.1%, at least 0.2%, at least 0.3%, at least 0.4%, at least 0.5%, at least 0.6%, at least 0.7%, at least 0.8%, at least 0.9%, at least 1%, at least 2%, at least 5%, at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% by volume (% v / v) of the composition.

[0150] The term "consisting essentially of" as used herein may refer to the presence of zingerone in a product. The product may be, for example, a composition described herein, or may be, for example, a product produced by a method described herein. By way of illustration, zingerone may be at least 90% by weight of the product, or at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% by weight (% w / w) of the product. In the case of a liquid, zingerone may be at least 90% by volume of the product, or at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or at least 99.9% by volume (% v / v) of the product.

[0151] The term "substantially free" in relation to aldehydes refers to a product having negligible levels of aldehydes. The product may be, for example, a composition described herein, or may be, for example, a product produced by a method described herein. By way of example, the level of aldehydes may be less than 20 ppm, less than 15 ppm, less than 10 ppm, less than 7.5 ppm, less than 5 ppm, less than 2 ppm, less than 1.5 ppm, less than 1 ppm, less than 0.75 ppm, less than 0.5 ppm, less than 0.2 ppm, less than 0.1 ppm, less than 0.05 ppm, less than 0.005 ppm, or less than 0.0005 ppm.

[0152] The term "alkaline treatment" as used herein means exposing a sample of ginger or ginger extract to an aqueous solution containing an alkali having a pH greater than 7. Solutions containing, but not limited to, sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide, and any combination thereof are included. It is understood that the alkaline treatment, as described herein, can occur at various temperatures, and that the alkaline solution can be heated before or during exposure to the sample containing ginger or ginger extract. The alkaline solution is a chemically effective amount of alkali present that converts at least some of the gingerol present in the sample to zingerone. Specific methodologies are described in detail herein.

[0153] By "extract" prepared from ginger root is meant a composition in which one or more liquid, solid, or chemical components of the root are isolated or concentrated. For example, a liquid, solid, or semi-solid extract can be obtained from ginger root. The extract can be obtained by one or more of juicing, squeezing, soaking, mashing, grinding, or other known processes. Extraction with a solvent is also included. Solid extracts include, for example, powders obtained by drying or evaporation. As a specific example, the extract can be prepared in a dry form or in the form of a solution. By "zingerone extract" is meant an extract prepared / produced from ginger root, comprising zingerone or consisting essentially of zingerone. The specific extracts and the method of their preparation are described in detail herein.

[0154] By "pharmaceutical composition" is meant a composition administered to a subject to treat or prevent, for example, an infection.

[0155] As used herein, "microbes" or "microbial organisms" includes pathogenic organisms such as bacteria, fungi, protozoa, and viruses. This includes all harmful microorganisms, including organisms associated with a variety of infections and other health conditions, including human and non-human diseases.

[0156] As used herein, "bacteria" includes, but is not limited to, Bacillus bacteria, Bartonella bacteria, Bordetella bacteria, Borrelia bacteria, Brucella bacteria, Campylobacter bacteria, Chlamydia bacteria, Chlamydophila bacteria, Clostridium bacteria, Corynebacterium bacteria, Enterococcus bacteria, Escherichia bacteria, Francisella bacteria, Haemophilus bacteria, Helicobacter bacteria, Legionella Examples of suitable bacterial organisms include, but are not limited to, Legionella bacteria, Leptospira bacteria, Listeria bacteria, Mycobacterium bacteria, Mycoplasma bacteria, Neisseria bacteria, Pseudomonas bacteria, Rickettsia bacteria, Salmonella bacteria, Shigella bacteria, Staphylococcus bacteria, Streptococcus bacteria, Treponema bacteria, Ureaplasma bacteria, Vibrio bacteria, and Yersinia bacteria. Gram-positive and Gram-negative bacteria are included. Specific bacterial organisms are described in detail herein.

[0157] As used herein, "fungi" includes, but is not limited to, Aspergillus fungi, Candida fungi, Cryptococcus fungi, Histoplasma fungi, Pneumocystis fungi, and Stachybotrys fungi. In particular, tinea fungi are included. Specific fungal organisms are described in more detail herein.

[0158] As used herein, "protozoa" includes, but is not limited to, Acanthamoeba, Cryptosporidium, Entamoeba, Giardia, Leishmania, Plasmodium, Trypanosoma, and Toxoplasma. Specific protozoan organisms are described in more detail herein.

[0159] "Antimicrobial agents" refer to ingredients that inhibit the growth or infection of microorganisms, including, for example, medicines, herbal compositions, essential oils, and a variety of other ingredients that aid in the suppression or elimination of fungi, which may be utilized in combination with the compounds and extracts of the present disclosure.

[0160] As used herein, a "subject" may be a human or a non-human animal, particularly a mammal, including cows, sheep, goats, pigs, horses, and other livestock (as well as dogs, cats, and other domesticated pets). In certain embodiments, the subject is a human.

[0161] As used herein, "treatment" means reducing or resolving an infection, or ameliorating a disease or other condition caused by an infection. Treatment is expected to reduce and / or eliminate the infection. Treatment may stop, reduce, or slow the progression of an infection.

[0162] As used herein, "prevention" means preventing the onset of an infectious disease or the onset of a disease or other condition resulting from an infection. A prophylactic measure is expected to stop or delay the onset of an infectious disease or to reduce the severity of an infectious disease if it does occur. It should be understood that the term "treatment or prevention" does not exclude the possibility of obtaining both treatment and prevention (e.g., simultaneously or at different times) of a disorder in any given subject.

[0163] As used herein, "inhibiting" or "arresting" growth means slowing or stopping the growth of one or more microorganisms. This can be accomplished by killing one or more microorganisms, e.g., eradicating or reducing the number of microorganisms present, or eradicating or reducing the reproductive units of the microorganisms, e.g., spores. Alternatively, or in addition, growth inhibition or arrest can be accomplished by partially or completely preventing the division or replication of the microorganisms.

[0164] Methods for preparing the compositions The inventors have found that zingerone compositions prepared from ginger root according to the disclosed methods have significant antimicrobial activity. Accordingly, the present disclosure generally relates to zingerone compositions prepared from ginger root, and methods of preparing such compositions.

[0165] In one aspect, the present disclosure provides a method for producing zingerone from ginger root by subjecting the ginger root to alkaline treatment. The alkaline treatment may include incubation in an alkaline solution as described herein. As a starting material, the ginger root may be fresh ginger root. For example, to aid in preparation, it may be useful to optimize the period that the ginger root is kept in soil before harvesting. In this way, the ginger root utilized is fresh and retains the advantageous properties of fresh ginger root.

[0166] By way of example, to optimize freshness, ginger root can be harvested less than 48 hours prior to processing, less than 24 hours prior to processing, or less than 12 hours prior to processing, or less than 6 hours prior to processing, or less than 3 hours prior to processing. For example, fresh ginger can have a moisture content of about 80% to about 95%, about 81% to about 95%, about 82% to about 95%, about 83% to about 95%, or about 85% to about 95% on a wet basis.

[0167] Alternatively, the ginger root may be dried prior to treatment. For example, the ginger root may be dried at about 40° C. to about 70° C., or about 55° C. to about 65° C., or about 60° C. Drying may be performed for about 1 to 72 hours, or about 1 to 48 hours, or about 1 to 24 hours, or about 1 to 20 hours, or about 1 to 18 hours, or about 1 to 10 hours, or about 1 to 5 hours.

[0168] In certain embodiments, the ginger root selected for use in the disclosed methods may have a minimum level of gingerols, such as 6-gingerol. For example, ginger root (e.g., fresh ginger root) may have about 0.3 mg / g to about 10 mg / g, or about 0.3 mg / g to about 9 mg / g, or about 0.3 mg / g to about 8 mg / g, or about 0.3 mg / g to about 7 mg / g, or about 0.3 mg / g to about 6 mg / g, or about 0.4 mg / g to about 5 mg / g of 6-gingerol. By way of further illustration, ginger root may have at least 1 mg / g, at least 2 mg / g, at least 3 mg / g, at least 4 mg / g, or at least 5 mg / g of 6-gingerol. Thus, in certain circumstances, it may be advantageous to test the level of gingerols, such as 6-gingerol, in the starting material before commencing the methods disclosed herein.

[0169] In one embodiment, the method includes subjecting juice from ginger root to an alkaline treatment. Ginger juice can be obtained by steeping and / or pressing. Steeping can include homogenization using a blender, food processor, or similar device. Pressing can utilize a machine or a hand press, with screw presses being particularly popular. The solids remaining after juicing (ginger pomace) can be squeezed again to obtain ginger juice. This can be repeated as necessary. The various juice samples can be combined prior to alkaline treatment.

[0170] Optionally, diluted juice can be obtained by subjecting the ginger pomace to a hot water treatment. For example, water may be added to the pomace in a weight ratio of about 6 to about 1 (about 6:1), or about 5 to about 1 (about 5:1), or about 4 to about 1 (about 4:1), or about 3 to about 1 (about 3:1). The water can be, for example, about 40°C to about 80°C, or about 50°C to about 70°C, or about 55°C to about 65°C, or about 60°C. The incubation time in water can be about 5 minutes to about 60 minutes, or about 10 minutes to about 30 minutes, or about 15 minutes to about 20 minutes, or about 15 minutes. The diluted juice sample can then be subjected to an alkali treatment. The diluted juice sample can be combined with other juice samples before the alkali treatment.

[0171] In one embodiment, potassium hydroxide (KOH) may be used for the alkaline treatment. For example, KOH in liquid form may be used in the alkaline solution. The concentration of KOH used in the treatment mixture may be, for example, about 0.1% to about 1.0%, or about 0.5% to about 0.7%, or about 1% to about 3%, or about 1.5% to about 2.5%, or about 2% (v / v). Alternatively, calcium hydroxide Ca(OH)2 may be used for the alkaline treatment. For example, Ca(OH)2 in liquid form may be used in the alkaline solution. The concentration of Ca(OH)2 used in the treatment mixture may be, for example, about 0.5% to about 4%, about 1.5% to about 3.5%, about 1% to about 2%, or about 3.0% (v / v). Examples of liquid forms include about 25% to about 65%, or about 30% to about 60%, or about 35% to about 55%, or about 45% to about 55%, or about 50% concentrate.

[0172] In certain embodiments, the alkali treatment can achieve a pH level of the treatment solution of about pH 9 to about pH 14, or about pH 9.5 to about pH 13.5, or about pH 10 to about pH 13, or about pH 10.5 to about pH 12.5, or about pH 11 to about pH 12. The alkali treatment can be carried out for a sufficient time and at a sufficiently elevated temperature to obtain the desired level of zingerone. For example, the alkali treatment can be carried out for about 1 to about 72 hours, or about 1 to about 48 hours, or about 1 to about 24 hours. Further examples include treatment for about 1 to about 30 hours, or about 1 to about 20 hours, or about 1 to about 10 hours, or about 1 to about 15 hours, or about 1 to about 7 hours, or about 1 to about 6 hours, or about 1 to about 5 hours, or about 1 to about 4 hours, or about 0.5 to about 3 hours, or about 0.5 to about 2 hours, or about 1 to about 2 hours, or about 2 hours, or about 1 hour. As a specific example, the alkali treatment may be carried out at about 40° C. to about 70° C., or about 50° C. to about 60° C., or about 55° C. to about 65° C., or about 60° C. It is understood that lower temperatures may allow for longer treatment periods. For example, an alkali treatment carried out at room temperature may be carried out for about 3 days to about 9 days, or about 5 days to about 9 days, or about 5 days to about 7 days.

[0173] After alkaline treatment, the treated mixture may be further processed, for example, by one or more of neutralization, extraction, and drying. Neutralization may utilize citric acid or other acid compositions. Illustratively, neutralization may achieve a pH of about 6.4 to about 7.4, or about 6.5 to about 7.5, or about 6.6 to about 7.6, or about 6.9 to about 7.4, or about 6.6, about 6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, or about 7.5. For example, about 10 to about 700 g / L of citric acid may be used, or about 10 to about 600 g / L, or about 10 to about 500 g / L, or about 10 to about 400 g / L, or about 10 to about 300 g / L, or about 10 to about 200 g / L, or about 10 to about 100 g / L, or about 10 to about 50 g / L, or about 10 to about 40 g / L, or about 10 to about 30 g / L, or about 10 to about 20 g / L, or about 15 to about 16 g / L of citric acid may be used.

[0174] For extraction, zingerone extraction may be accomplished by one or more alcohol extractions, for example, one or more ethanol extractions. Illustratively, the alcohol extraction, for example, ethanol extraction, may be performed for about 1-72 hours, or about 1-48 hours, or about 1-24 hours, or about 6-24 hours, or about 8-24 hours, or about 12-24 hours, or about 18-24 hours. In a particular embodiment, ethanol extraction at room temperature may be utilized for 24 hours or less. One or more drying steps may be utilized before and / or after extraction. For example, freeze-drying may be utilized.

[0175] The composition may be prepared as a disinfecting composition or a pharmaceutical composition. The composition may also be prepared as a functional food or beverage, a natural ingredient (e.g., a natural additive), or a natural supplement (e.g., a dietary supplement). In various embodiments, the composition may be prepared in a liquid or solid form, or in a semi-solid form. Various formulations are encompassed in the present disclosure. In certain embodiments, it may be desirable to formulate the composition into a powder. The powder may be provided in a free-flowing form or a solid cake. The composition may be provided as a powder for forming a suspension, a powder for forming a solution, bulk granules, or bulk powder. The powder may be prepared as a tablet or capsule, or other pharmaceutical formulation, as described in detail herein.

[0176] As part of the initial process, the ginger root may be washed or sterilized. The plant components (e.g., fruit or seeds) may be passed through an assembly having one or more roller brushes to remove any attached foreign material. Next, conventional washing techniques may be utilized. For example, the components may be washed using a series of spray nozzles. Washing additives that aid in washing or reduce the bacterial count of the plant components may be utilized in accordance with local regulations and requirements. For example, the plant components may be washed by a chlorine wash and / or an ozone impregnated water wash, then rinsed with fresh water.

[0177] As mentioned above, it may be desirable to prepare a liquid or semi-solid zingerone composition from ginger root. As described herein, zingerone components may be extracted by chemical means (e.g., solvent-based extraction). Solvent-based extraction may utilize one or more of water extraction, methanol extraction, ethanol extraction, or 2-propanol extraction. Supercritical fluid extraction, e.g., CO2 extraction, may also be used to extract zingerone. Emulsions, pastes, suspensions, and syrups are also suitable. For example, in certain embodiments, it may be desirable to use a paste from ginger root or from components of ginger root (e.g., zingerone or zingerone extract). As an example, ginger root may be heated for several hours and strained to produce a thick, concentrated form. The thickened paste may be spread on a flat sheet or transferred to packaging, e.g., bags, tubes, jars, bottles, or other containers. The paste may be transferred aseptically. It may also be desirable to prepare a paste from mature plant components. The paste may be a smooth preparation.

[0178] In certain embodiments, the present disclosure encompasses mechanical means (e.g., juicing means such as soaking and / or squeezing) for extracting zingerone from ginger root. In one embodiment, the squeezing assembly can be adapted to perform a pulping or comminution process. Such a process is relatively mild and gentle (soft pulping) compared to conventional fruit pulping techniques. Soft pulping does not utilize significant cell disruption or lysis. The press belts may be multiple loops rotating around a series of pulleys. The distance separating the press belts may be shorter toward the direction of travel of the plant components. In this way, more force may be exerted on the plant components as they move along the length of the squeezing assembly. In certain embodiments, a squeezing assembly or mechanical press may be used to obtain juice from ginger root as described herein. Alternatively, or in addition, mechanical soaking may be used to obtain juice. For example, commercially available juicing equipment may be utilized.

[0179] The ingredients of ginger root (e.g., zingerone or zingerone extract) may be processed by a freezing step. This step may be followed or combined with a drying or evaporation step. In an alternative embodiment, the ingredients are dried or evaporated and then processed to a powder without a freezing step. Methods including air drying or heat-assisted drying (e.g., oven drying) may be used. Drying may be obtained, for example, by one or more of sun or solar drying, solar drying, hot air drying, batch drying, rotary drying, tunnel drying, belt drying, fluidized bed drying, impingement drying, puff drying, cylinder drying, spray drying, vacuum drying, freeze drying, or osmotic drying. Exemplary temperatures for drying include about 50°C to 70°C, about 55°C to 65°C, or at least 50°C, at least 55°C, at least 60°C, or at least 65°C. Evaporation can be achieved, for example, by one or more of pan evaporation, batch evaporation, tube evaporation, rising film evaporation, falling film evaporation, rising-falling film evaporation, or agitated film evaporation. Various drying and evaporation techniques and combinations can be used. For example, filtration followed by freeze drying can be used.

[0180] If freezing is used, it may be desirable to freeze the ginger root ingredients (e.g., zingerone or zingerone extract) as soon as possible after production to maintain freshness. However, freezing can occur within 24 or 48 hours, if desired. Freezing methodologies are well known to those skilled in the art. Blast freezing is particularly desirable for use with the present disclosure. The ingredients can be frozen in standard size pails used to collect the frozen product after processing. The ingredients can be stored frozen (e.g., at -18°C), for example, until needed. Optionally, the ingredients can then be freeze dried, i.e., lyophilised. Freeze drying techniques are widely known and commonly used. The freeze drying cycle can be up to 48 hours. In certain embodiments, the process can be carried out such that water mass formation is avoided and moisture content during the process is minimized. It is understood that freeze drying / lyophilising does not preclude the use of higher temperatures (i.e., higher than the freezing temperature). For example, higher temperatures can be used to remove residual moisture in the secondary drying step of a lyophilisation / freeze drying procedure.

[0181] The dried or evaporated ingredients obtained from ginger root (e.g., zingerone or zingerone extract) can then be ground into powder and then used as appropriate. Grinding methods are well known and widely used by those skilled in the art. Standard mesh sizes can be used to produce the powder, for example, US 20, US 23, US 30, US 35, US 40, US 45, or US 50 mesh sizes can be used. The sieve size of the powder can be in the range of 1.0-0.3 mm; or 0.84-0.4 mm; or 0.71-0.5 mm; or about 1.0 mm, about 0.84 mm, about 0.71 mm, about 0.59 mm, about 0.5 mm, about 0.47 mm, about 0.465 mm, about 0.437 mm, about 0.4 mm, about 0.355 mm, or about 0.3 mm.

[0182] For any liquid or semi-solid product obtained from ginger root, it will be understood that the liquid / semi-solid may be used in this form or may be dried or evaporated to obtain a powder form for use as a disinfecting or pharmaceutical composition as described herein.Similarly, for any solid product obtained from ginger root, it will be understood that the solid may be used as such (e.g., through a grinding process, sieving process, or other process) or may be resuspended to obtain a liquid or semi-solid form for use as a disinfecting or pharmaceutical composition as described herein.

[0183] composition The present inventors have discovered that zingerone compositions prepared from ginger root according to the methods disclosed herein have significant antimicrobial properties useful in inhibiting or stopping the growth of microorganisms and in treating or preventing infections by these organisms.

[0184] The compositions of the present disclosure may be prepared as one or more disinfecting compositions or pharmaceutical compositions. As non-limiting examples, the percentage of zingerone or zingerone extract in the composition may be about 0.01% to about 0.5%, or about 0.02% to about 0.2%, or about 0.03% to about 0.1%, or about 0.04% to about 0.09%, or about 0.05% to about 0.08%, or about 0.06% to about 0.07%, or at least about 1%, at least about 5%, at least about 6%, at least about 10%, at least about 12%, at least about 15%, at least about 20%, at least about 23%, at least about 25%, or at least about 30%, or about 6.25%, about 12.5%, or about 25%, with these percentages being expressed as v / v values ​​for liquid compositions or w / w values ​​for solid or semi-solid compositions.

[0185] For example, the solid or semi-solid composition may contain about 1 mg / g to about 100 mg / g zingerone, or about 5 mg / g to about 50 mg / g zingerone, or about 5 mg / g to about 25 mg / g zingerone, or about 5 mg / g to about 20 mg / g zingerone, or about 5 mg / g to about 15 mg / g zingerone, or about 10 mg / g to about 15 mg / g zingerone, or about 10 mg / g to about 13 mg / g zingerone, or about 12 mg / g zingerone (w / w). Similarly, the liquid composition may contain from about 1 mg / ml to about 100 mg / ml of zingerone, or from about 5 mg / ml to about 50 mg / ml of zingerone, or from about 5 mg / ml to about 25 mg / ml of zingerone, or from about 5 mg / ml to about 20 mg / ml of zingerone, or from about 5 mg / ml to about 15 mg / ml of zingerone, or from about 10 mg / ml to about 15 mg / ml of zingerone, or from about 10 mg / ml to about 13 mg / ml of zingerone, or about 12 mg / ml of zingerone (w / v).

[0186] In various embodiments, disinfectant compositions can be prepared for use on hands (e.g., hand sanitizers), preoperative tissues (e.g., skin surgical preparations), mucous membranes (e.g., for treating bladder, urethra or vaginal infections, or for cleaning these cavities before medical procedures), wounds or burns (e.g., disinfectant ointments, bandages, or dressings), or mouth or throat (e.g., mouthwashes or disinfectant lozenges). Disinfectant formulations and their preparations are well known in the art. See, for example, Antiseptic Prophylaxis and Therapy in Ocular Infections: Principles, Clinical Practice and Infection Control, 2002, Karger, Basel.

[0187] As non-limiting examples, disinfecting compositions may include one or more diluents (e.g., ethanol or other alcohols), emollients (e.g., PEG-45, palm kernel glyceride, or isopropyl myristate), humectants (e.g., glycerin or methylpropanediol), carriers (e.g., one or more oils), occlusive agents (e.g., mineral oil or dimethicone), other conditioning agents (e.g., behentrimonium methosulfate or polyquaternium-7), and surfactants (e.g., mild surfactants (e.g., amphoacetates, isethionates, sulfosuccinates, etc.). The carrier may include, in particular, sodium lauroamphoacetate, sodium cocoyl isethionate, disodium oleamidosulfosuccinate, sodium lauryl sulfate, sodium C14-16 olefin sulfonate. Exemplary oils include olive oil, coconut oil (e.g., coconut-derived MCT oil), palm oil (e.g., palm kernel-derived MCT oil), any other MCT oil (medium chain triglyceride oil), and combinations thereof. Other possible carriers include lecithin (e.g., in liquid form) and propylene glycol. Also, combinations of any of the carriers described herein are permitted.

[0188] In yet another embodiment, the pharmaceutical composition may be prepared for various routes of administration, including topical or oral formulations. Also included are compositions prepared for other routes of enteral or parenteral administration. Enteral preparations include, but are not limited to, oral, rectal, sublingual, sublabial, and buccal preparations. Parenteral preparations include, but are not limited to, nasal, ocular, vaginal, intralesional, transdermal, and transmucosal preparations. Methods for formulating pharmaceutical compositions are well known in the art. See, for example, Remington: Essentials of Pharmaceutics, 2012, Pharmaceutical Press, London.

[0189] In certain embodiments, the composition of the present disclosure can be prepared as powder or any other suitable form.Topical formulations can be prepared as, for example, aerosol, balm, cream, dressing, drops, emulsion, film, foam, gel, liquid, lotion, mask, oil, ointment, paste, powder, ointment, soap, spray, suspension, solution, tincture and vapor.Additional topical formulations include bandage, dressing, patch, pad, sponge, strip, tape and others as indicated herein.

[0190] As described herein, the compositions may be formulated, for example, as semi-solid or liquid compositions for oral administration, or as solutions for enteral or parenteral administration. Alternatively, the compositions may be formulated as powders for encapsulation, tableting, or addition or incorporation into other products.

[0191] Oral formulations can be prepared, for example, as draughts, drops, elixirs, emulsions, liquids, linctuses, solutions, sprays, suspensions, syrups, tonics, or as thin films, gels, gummies, jellies, troches, nuggets, pastes, purees, pressed cakes, powders, pills, or strips. In other embodiments, oral formulations can be prepared as tablets or as capsules, for example, with liquid, semi-solid, or solid contents. Oral formulations can be provided in sachet form, for example, powder sachets, or gel or jelly sachets. Also included are oral formulations that include thin strips, or solids in capsules to be mixed with food or beverages. Oral formulations can be provided as shooters or shots (to be taken by mouth), for example, liquid shots, gel or jelly shots, paste shots, or powder shots.

[0192] Particularly included are delayed release formulations, sustained release formulations, and fast disintegration formulations.Particularly included are capsules, such as gel capsules, and also sachets and chewable tablets.Furthermore, also included are combination formulations in which the powder of the present disclosure is mixed with other beneficial agents, such as one or more antimicrobial agents.Other formulations are possible as described herein.

[0193] The dissolution time of oral formulations can be modified for rapid effect or sustained release. Oral formulations can also contain a mixture of slow and fast release particles to provide rapid and sustained absorption at the same dose. Special coatings can be used on oral formulations such as tablets and capsules to make them resistant to stomach acid. Oral formulations can also be coated with sugars, varnishes, or waxes to improve taste.

[0194] Thus, tablets can be prepared as fast dissolving tablets and capsules can be prepared as sustained release capsules. Tablets can be split, chewable, effervescent, orally disintegrating, or tablets for forming suspensions. Capsules can be gel capsules, for example, containing powdered contents. This includes gel capsules made by one-piece gel encapsulation and two-piece gel encapsulation. Capsules are also included, as well as non-gelatin capsules.

[0195] It will be understood that certain formulations are suitable for either disinfecting or medicinal use. Particular formulations of interest are ophthalmic formulations (e.g., drops, ointments), otic formulations (e.g., drops, ointments), nasal or respiratory tract formulations (e.g., drops, sprays, insufflation compositions, inhalation compositions, nebulisation compositions), skin formulations (e.g., soaps, sprays, aerosols, gels, pastes, lotions, creams, ointments, pads, patches, tapes, bandages, dressings, sponges, vapours), throat or mouth formulations (e.g., drops, lozenges, mouthwashes, toothpastes, sprays, pastes, gels), mucosal formulations (e.g., sprays, aerosols, gels, pastes, lotions, creams, ointments, pads, dressings, sponges).

[0196] Solid and liquid compositions can combine zingerone or zingerone extract with one or more compounds to ensure a stable and active composition. For example, an oral formulation such as a tablet or capsule can contain about 5 to about 50% w / w zingerone or zingerone extract; up to about 80% w / w of one or more fillers, lubricants, glidants or binders; and up to about 10% w / w of a compound to ensure easy disintegration, disintegration and dissolution of the tablet in the stomach or intestines.

[0197] Thus, the compositions may include various excipients, such as one or more solubilizers, stabilizers, buffers, tonicity modifiers, bulking agents, viscosity enhancing / reducing agents, surfactants, chelating agents, adjuvants, anti-adherents, anti-caking agents, binders, coating agents, disintegrants, lubricants, glidants, flow agents, adsorbents, flavors, colours, sweetening agents, or preservatives. The composition may contain less than 1% preservative, for example, about 0.005% to about 0.5%, or about 0.05% to about 0.15%, or about 0.04%, about 0.06%, about 0.08%, about 0.1%, about 0.12%, about 0.14%, about 0.16%, about 0.18%, or about 0.2% preservative, these percentages being representative of w / v or w / w values. Useful preservatives include, but are not limited to, sorbic acid, sodium sorbate, potassium sorbate, citric acid, ascorbic acid, malic acid, tartaric acid, propionic acid, and benzoic acid, including the sodium salts thereof, such as sodium benzoate.

[0198] Other useful excipients include, but are not limited to, stearin, magnesium stearate, and stearic acid; saccharides and derivatives thereof, such as disaccharides: sucrose, lactose; polysaccharides and derivatives thereof, such as starch, cellulose or modified cellulose, such as microcrystalline cellulose and cellulose ethers, such as hydroxypropylcellulose; sugar alcohols, such as isomalt, xylitol, sorbitol, and maltitol; proteins, such as gelatin; synthetic polymers, such as polyvinylpyrrolidone, polyethylene glycol; fatty acids, vegetable-based surfactants, such as sunflower lecithin, waxes, shellac, plastics, vegetable fibers, such as corn protein zein; hydroxypropylmethylcellulose; crosslinked polymers, such as crosslinked polyvinylpyrrolidone (crospovidone) and crosslinked sodium carboxymethylcellulose (croscarmellose sodium); sodium starch glycolate; silicon dioxide, fumed silica, talc, and magnesium carbonate.

[0199] Liquid compositions can be stored as tinctures in vials, bags, ampoules, cartridges, or pre-filled syringes. The compositions can be transferred from the vials to larger containers and mixed with other materials. Dry or evaporated compositions can be stored in vials, cartridges, dual-chamber syringes, or pre-filled mixing systems. Also, before administration, dry forms of the compositions can be reconstituted as liquids and then administered.

[0200] Exemplary unit doses of the composition include about 0.1 mg to about 1000 mg of zingerone or zingerone extract, about 1 mg to about 500 mg of zingerone or zingerone extract, about 1 mg to about 200 mg of zingerone or zingerone extract, and about 1 mg to about 100 mg of zingerone or zingerone extract. Doses may be formulated to be administered once a week, twice a week, three times a week, every other day, once a day, twice a day, or three times a day, or more, as needed. Doses may be adjusted for pediatric, geriatric, overweight, underweight, and other patients, as needed. Dose modifications may be made according to known methods. The European Food Safety Authority (EFSA) has classified zingerone as safe for human consumption with no observed adverse effects (NOAEL) based on a dose of 128 mg / kg / day (EFSA 2016; 14(8):4557). It should therefore be understood that a wide range of unit dosage forms are envisioned.

[0201] Methods of using the composition As discussed above, the disclosed compositions can be used to reduce the prevalence of microbial organisms and / or to treat or prevent various health conditions caused by such organisms.

[0202] In certain embodiments, the composition may comprise (or consist essentially of) zingerone or zingerone extract produced by the method defined herein.The composition of the present disclosure may also be formulated for one or more of disinfectant, sanitizing, or therapeutic uses.In addition, or alternatively, the composition may be utilized as a functional food or beverage, a natural ingredient (e.g., natural additive), or a natural supplement (e.g., dietary supplement).

[0203] In various embodiments, the disclosed compositions can be used to target one or more bacterial, fungal, or protozoan organisms. Specific fungi include, but are not limited to, Aspergillus fumigatus, Aspergillus flavus, Candida albicans, Cryptococcus neoformans, Cryptococcus gattii, Histoplasma capsulatum, Pneumocystis jirovecii, Pneumocystis carinii, and Stachybotrys chartarum. Of particular interest are drug-resistant strains.

[0204] Specific protozoa include Acanthamoeba culbertsoni, Acanthamoeba polyphaga, Acanthamoeba castellanii, Acanthamoeba astronyxis, Acanthamoeba hatchetti, Acanthamoeba griffini, Acanthamoeba lugdenensis, Acanthamoeba polyphaga, Acanthamoeba rhysodes, Balamuthia mandrillaris, Acanthamoeba spp. ... mandrillaris, Cryptosporidium parvum, Entamoeba histolytica, Giardia lamblia, Leishmania donovani, Leishmania infantum, Leishmania major, Naegleria fowleri, Plasmodium vivax, Plasmodium malariae, Plasmodium falciparum, Plasmodium ovale, Plasmodium kunowresii knowlesi, Trypanosoma cruzi, Trypanosoma brucei gambiense, Trypanosoma brucei rhodesiense, and Toxoplasma gondii.gondii). Of particular interest are drug-resistant strains.

[0205] Specific bacteria include Bacillus anthracis, Bacillus cereus, Bartonella henselae, Bartonella quintana, Bordetella pertussis, Borrelia burgdorferi, Borrelia garinii, Borrelia afzelii, Borrelia recurrentis, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Campylobacter jejuni, and jejuni, Chlamydia pneumoniae, Chlamydia trachomatis, Chlamydophila psittaci, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium diphtheriae, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, coli, Francisella tularensis, Haemophilus influenzae, Helicobacter pylori, Legionella pneumophilapneumophila, Leptospira interrogans, Leptospira santarosai, Leptospira weilii, Leptospira noguchii, Listeria monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis, Mycobacterium ulcerans, Mycoplasma pneumoniae, Neisseria gonorrhoeae, Neisseria meningitidis, Pseudomonas aeruginosa aeruginosa, Rickettsia, Salmonella typhi, Salmonella typhimurium, Shigella sonnei, Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Treponema pallidum, pallidum, Ureaplasmaurealyticum, Vibrio cholerae, Yersinia pestis, Yersinia enterocoliticaOf particular interest are drug resistant strains, e.g., antibiotic resistant microorganisms.

[0206] In certain embodiments, the disclosed compositions can be used with one or more antimicrobial agents. For example, the compositions can be prepared as a combined formulation with one or more antimicrobial agents. Alternatively, the compositions can be utilized as separate formulations with one or more antimicrobial agents. When separate formulations are used (e.g., zingerone compositions and antimicrobial agents), it is possible to coordinate use by simultaneous or sequential application / administration of the separate formulations. Furthermore, the compositions described herein can be used in combination with various medical or non-medical treatments. Use of the compositions can be performed before, during, or after the treatment, or any combination thereof.

[0207] Exemplary antimicrobial agents include, but are not limited to, ethanol, isopropanol, glutaraldehyde, formaldehyde, triclocarbon, phenol, o-phenol, chlorophenol, amylmetacresol, thymol, cresol, resorcinol, chloroxylenol, triclosan, hexachlorophene, chlorhexidine, propamidine, hypochlorous acid, chloramine, iodophor, iodine, povidone-iodine, dibromine, mercuric chloride, thiomersal, silver nitrate, silver sulfadiazine, zinc sulfate, zinc oxide, hydrogen peroxide, ozone, peracetic acid, cetrimide, benzalkonium chloride, gentian violet, basic fuchsine, methylene blue, acriflavine, salacrin, mercurochrome, boric acid, acetic acid, azelaic acid, nitrofuran, and ethylene oxide.

[0208] Other antimicrobial agents include natural products (essential oils or plant extracts, e.g., from witch hazel, hops, thyme, oregano, calendula, tea tree, lavender, and anise, in particular lemon oil, orange oil, grapefruit oil, lime oil, neroli oil, mandarin oil, citronella oil, petitgrain oil, marjolan oil, rosemary oil, thyme oil, thymol, oregano oil, basil oil, clove oil, tea tree oil, juniper oil, myrrh oil, patchouli oil, pepper oil, black pepper oil, rose oil (e.g., rose otto oil), spikenard oil, vetiver oil, vervain oil, among others. oil, fennel oil, lemongrass oil, cinnamon oil, lavender oil, geranium oil, sandalwood oil, eucalyptus oil, pine oil, fir oil, balsam oil, cedar leaf oil, cedarwood oil, spearmint oil, wintergreen oil, peppermint oil, and menthol. Also included are honey (e.g., manuka honey), activated charcoal, yarrow (e.g., for various skin preparations), and comfrey (e.g., for salves or creams).

[0209] Antimicrobial agents further include various antibiotics, such as bacitracin, ceftriaxone, ciprofloxacin, clarithromycin, clindamycin, chloramphenicol, dapsone, dexamethasone, flucloxacillin, framycetin, fusidic acid, gentamicin, gramicidin, lincomycin, macrolides, mupirocin, nadifloxacin, neomycin, nitrofurazone, polymyxin B, retapamulin, soframycin, and sulfadiazine. Further antibiotics include, for example, ampicillin, ampicillin, e.g., ampicillin and clavulanate, amoxicillin clavulanate, azithromycin, cefotaxime, cephalexin, ciprofloxacin, clioquinol, dicloxacillin, doxycycline, erythromycin, flumethasone, metronidazole, nafcillin, nitrofurantoin, ornidazole, oxacillin, penicillins, e.g., benzathine penicillin, phenoxymethylpenicillin, penicillin G sodium, penicillin V potassium, roxithromycin, sulfamethoxazole, trimethoprim, and vancomycin. Notable among these are glycopeptide antibiotics, e.g., dalbavancin, oritavancin, ramoplanin, teicoplanin, telavancin, and vancomycin. Of particular note are aminoglycoside antibiotics such as gentamicin (e.g., Cidomycin®, Garamycin®, G-Myticin®, Pred-G®, Gentak®, Genoptic®), and amikacin, amikacin liposomal, dibekacin, kanamycin, neomycin, netilmicin, paromomycin, plazomicin, sisomicin, streptomycin, and tobramycin. It will be understood that any combination of antimicrobial agents may be utilized in the methods and compositions of the present disclosure.

[0210] As described herein, the composition of the present disclosure is useful as an antibacterial preparation.In certain aspects, the disinfectant composition can be used in a method to inhibit or stop the growth of bacteria on or in certain tissues.These tissues include skin, nails, ears, eyes, nose, mouth, gums, throat, vagina, and urinary tract tissues, as well as other tissues as indicated herein.The disinfectant composition can be applied, for example, to burns to reduce the possibility of infection, or to skin before surgery to combat microorganisms on the skin around the surgical site. The disinfecting compositions may be used as hand cleansers (e.g., soaps or hand sanitisers) and may be applied with or without water. The disinfecting compositions may be used for minor skin infections, cuts, or scrapes. The disinfecting compositions may be used as mouthwashes or gargles, for example, to combat microorganisms in the mouth or on the gums. The disinfecting compositions may also be utilized as lozenges or throat sprays, for example, to soothe a sore throat. Disinfecting eye drops or ointments may be used to combat microorganisms in or on the eye.

[0211] The compositions of the present disclosure also find use as formulations that can be used in methods for treating or preventing microbial infection or other conditions resulting from infection, as described herein.Infectious diseases can affect one or more physiological components, including one or more parts of the circulatory system, respiratory system, digestive system, renal system, excretory system, reproductive system, integumentary system, nervous system, lymphatic system, endocrine system, muscular system, skeletal system, and sensory system.

[0212] As described herein, various routes of administration can be used for the compositions, including parenteral administration (e.g., topical administration) and enteral administration (e.g., oral administration). Enteral administration can be by duodenal tubing or gastric tubing, including nasogastric tubing, or other known means. Oral administration can be by tablets, capsules, sachets, drops, elixirs, linctus, solutions, emulsions, suspensions, draughts, purees, pastes, presses, syrups, gels, jellies, tonics, or other known means. Topical administration can be by drops, sprays, ointments, soaps, pads, sponges, dressings, bandages, or other various means. Different modes of administration are known in the art and can be utilized by those skilled in the art. The compositions disclosed herein are not limited to a particular form for administration.

[0213] As exemplary doses, the compositions are administered to obtain a dose of about 1 mg to 5000 mg of zingerone or zingerone extract for a human subject of average body weight of 70 kg, or a dose of about 1 mg to 1500 mg of zingerone or zingerone extract, or a dose of about 5 mg to about 500 mg of zingerone or zingerone extract, or a dose of about 1 mg to about 500 mg of zingerone or zingerone extract, or a dose of about 1 mg to about 150 mg of zingerone or zingerone extract for a human subject of average body weight of 70 kg. The ginger extract may be administered to obtain a dose of about 1 mg to about 100 mg of gingerone or zingerone extract, or a dose of about 1 mg to about 50 mg of gingerone or zingerone extract, or a dose of about 1 mg to about 30 mg of gingerone or zingerone extract, or a dose of about 1 mg to about 20 mg of gingerone or zingerone extract, or a dose of about 1 mg to about 15 mg of gingerone or zingerone extract, or a dose of about 1 mg to about 10 mg of gingerone or zingerone extract. Doses in this range are particularly useful for ginger ingredients (e.g., gingerone or zingerone extract) that have been dried and ground into a powder. The above doses may be administered once a day, twice a day, three times a day, or less or more times, as needed. Administration may be with a meal or before a meal. Appropriate doses and dosage forms will be readily determined by those skilled in the art. EXAMPLES

[0214] The examples described herein are provided for the purpose of illustrating particular embodiments and are not intended to limit the disclosure in any way.

[0215] Example 1: Preparation of Zingerone An initial sample of fresh ginger (400 gm) as shown in Figure 1 was sourced from New Zealand and washed of dirt and soil. The washed ginger was then diced or finely chopped and subjected to an alkaline treatment (800 g of 0.5% potassium hydroxide in distilled water). The resulting mixture was stirred and placed in an oven at 60°C for 22 hours. It is understood that the resulting mixture can also be placed in a water bath and maintained at about 60°C for a desired period of time.

[0216] The pH of the mixture was then adjusted to pH 7 by the addition of concentrated citric acid and the treated plant material was spread on a metal tray and dried in an oven at 60°C for 20 hours. The resulting dried material weighed 35 grams, giving a dry yield of 8.75%. This dried material was extracted by placing it in a flask and covering it with 95% ethanol (210 ml). The flask was shaken and placed in an oven at 40°C for 16 hours. The extract (Extract 1) was filtered using a glass funnel with a glass wool plug. The remaining plant was extracted again with 95% ethanol (Extract 2) and then extracted twice more with 50% ethanol (Extracts 3 and 4).

[0217] The extracts were analyzed directly for zingerone content. A sample of 294 mg of freshly chopped ginger was also extracted with 2 ml of ethanol and this extract was also analyzed. The results are shown in Table 1. [Table 1]

[0218] result The 400 g fresh ginger root supplied was shown to contain 260 mg of 6-gingerol (i.e. 0.65 mg / g). This means that the theoretical maximum yield of zingerone would be around 171 mg from the processed material (weight loss due to the lower molecular weight of zingerone compared to 6-gingerol). Approximately 50% of the 6-gingerol was not converted. It is believed that further studies could increase the alkaline conversion of 6-gingerol to zingerone.

[0219] Ethanol extractions were performed in the minimum volume necessary to cover the treated plant material. Thus, the most concentrated extract 1 had a zingerone content of 0.47 mg / mL. It is envisaged that this concentration could, in principle, be increased by a multi-step process or by evaporating part of the ethanol. Reducing the ethanol by 80% would yield 2.35 mg of zingerone / mL of solution.

[0220] It is expected that a concentration of approximately 16 mg / g of solid extract will be obtained if the extract is completely dried. Higher concentrations are expected if one starts with a higher initial content of 6-gingerol and / or a more complete conversion. For example, the theoretical 25 mg dose could be achieved by formulating the dried extract directly into an oil or glycerol carrier to provide the required dose of zingerone in one or two 500 mg capsules.

[0221] The majority of the zingerone was extracted in the first extraction. Double extraction may be effective in some cases. The third and fourth extractions produced slightly increased totals. Overall, the ethanol extraction was found to be very effective and a very efficient and inexpensive preparation method.

[0222] Example 2: Preparation of Zingerone Summary: These studies show that freeze-drying fresh ginger after aqueous alkali treatment significantly improved the conversion, leading to a zingerone content of about 1% in dried ginger. The treated ginger was then extracted with supercritical CO2 and CO2 + ethanol co-solvent, with a combined extraction yield of 3% and an average concentration of zingerone in the extracted oleoresin of about 12%. Furthermore, drying fresh ginger at medium temperature (60°C) and then extracting with supercritical CO2 resulted in an extraction yield of 4.6%. The extracted oleoresin was then alkali-treated, and the final product contained about 15% zingerone.

[0223] Drying: Fresh ginger material samples imported from Fiji were sliced ​​into 2-5 mm pieces and placed in a single layer on perforated oven trays. Drying was carried out by forced convection. Drying was carried out at moderate temperature (60°C) with the goal of removing moisture without conversion of gingerols. The process was terminated when the moisture content of the ginger reached 7%. The resulting dried ginger was kept refrigerated until use in extraction studies.

[0224] Catalytic Conversion: A small scale preliminary test was carried out by treating approximately 1.6 g of chopped fresh Fiji ginger with an aqueous solution of 0.5% KOH (pH 14), 1% Ca(OH)2 (pH 11.6) and 1% sodium carbonate (pH 10.5). The volume:weight ratio of added reagents:ginger was approximately 3:1. The samples were then shaken and placed at room temperature or in an oven at 37°C or 60°C overnight before analysis. It should be understood that a suitable water bath can be used to keep the samples at the desired temperature for the desired period of time.

[0225] After the alkaline treatment was selected, 5.2 kg of fresh ginger was chopped using a vertical cutter mixer (RobotCoupe R 45). The ginger was then mixed with KOH 0.5% (about 0.1N) in a 3:1 liquid:solid ratio (volume:weight) to a pH of about 12.5. The mixture was manually stirred and placed in a 60°C oven for 24 hours. After this time, the mixture was neutralized to a pH of about 7.2 by adding concentrated citric acid (625 g / L). The neutralized mixture was then freeze-dried and the ginger obtained from this process was stored refrigerated until it was used in the extraction test.

[0226] Catalytic conversion results: As mentioned before, different alkalis were tested: 0.5% potassium hydroxide or KOH (pH 14), 1% calcium hydroxide or Ca(OH)2 (pH 11.6), and 1% sodium carbonate or Na2CO3 (pH 10.5). For these experiments, the contents of zingerone and 6-gingerol were quantified (Table 2), and it was concluded that the treatment with KOH at 60°C was the most efficient, resulting in the highest zingerone concentration. Table 2 shows the amount of 6-gingerol (mg / g) and zingerone (mg / g) obtained under each condition, and the results are expressed on a wet basis. The HPLC traces are shown in Figure 2. From the HPLC traces, the 1% Ca(OH)2 treatment showed the highest zingerone concentration, while the 1% Ca(OH)2 treatment showed the highest zingerone concentration. )2 It can be seen that 0.5% KOH performs similarly. For 0.5% KOH at 60°C, the ratio of zingerone:6-gingerol is 6.5. For 1.0% Ca(OH) at 60°C, )2 In the case of gingerone:6-gingerol ratio is 4.0. [Table 2]

[0227] This treatment was applied to a larger fresh ginger sample (5.2 kg) and the resulting treated ginger was then neutralized and freeze-dried. The yield of freeze-dried ginger was 17%, i.e. 17 g of treated freeze-dried ginger per 100 g of chopped fresh ginger. The zingerone and 6-gingerol contents of the freeze-dried ginger were determined to be 10.2 mg / g and 3.3 mg / g, respectively (on a dry basis). See Table 2-1. The zingerone content was at least 10 times that of oven-dried ginger. [Table 3]

[0228] Extraction: Supercritical extraction tests were performed with alkali-treated ginger. Alkaline-treated freeze-dried ginger was lightly crushed by hand and placed into a 2 L extraction vessel fitted with sintered filter disks on both ends, filling the vessel completely. Extraction was performed as above until a sudden drop in extraction rate was observed, corresponding to a CO2:feed ratio of 13:1. At this point, the ethanol co-solvent pump was started and ethanol was added to the CO2 stream at a rate of approximately 10 wt% (i.e., 10 g ethanol per 100 g CO2). The ethanol pump was stopped after 2:1 ethanol:feed (2 g ethanol per 1 g feed) was introduced. The CO 2を The bed was circulated to wash away any remaining ethanol. Once the extraction was complete, the plant was depressurized and the remaining pomace was allowed to vent overnight before being unloaded. The ethanol present in the extract was removed by rotary evaporation under vacuum. The extraction parameters are given in Table 3. [Table 4]

[0229] Analysis: Samples were prepared for analysis by adding methanol after neutralization if necessary. Extracts were dissolved directly in methanol. Analysis was performed by HPLC with an acetonitrile / 0.1% formic acid gradient. Detection was at 280 nm and the column used was a Phenomenex Kinetex C18 (150 x 2.1 mm). Zingerone eluted at approximately 2 minutes and 6-gingerol at 5.2 minutes. Quantitation of zingerone and 6-gingerol was obtained from standard curves generated using analytical standards of these compounds.

[0230] Extraction results: Ginger, freeze-dried after alkali treatment as described above, was extracted with CO2 followed by CO2 + ethanol co-solvent. No free water was observed in the CO2 extract and the ethanol in the CO2 + ethanol extract was removed by rotary evaporation under vacuum. The extract had a sweet caramel-like aroma. The CO2 yield was 1.5%. An additional 1.5% could be extracted by the addition of 10% ethanol co-solvent. The composition of the different fractions is shown in Tables 4 and 5. See also Figure 3. [Table 5] [Table 6]

[0231] As can be seen from the results, in the alkali treated samples, the CO2 extract contained 153 mg / g zingerone (15.3%) and 103 mg / g 6-gingerol (10.3%) with a zingerone / gingerol ratio of about 1.5. The CO2 + ethanol extract contained 91 mg / g zingerone and 52 mg / g gingerol (zingerone / gingerol ratio of about 1.75). The ginger pomace or residue after extraction was also analyzed and found to contain 5.4 mg / g zingerone. Considering the mass of the feed, extract and pomace (Table 4), the mass balance of zingerone can be calculated to be 90.6%. This indicates that 90.6% of the zingerone initially present in the feed is present in the extract and pomace. This difference may be due to degradation during extraction or in the ethanol removal step. The extraction yield of zingerone (i.e. grams of zingerone extracted per 100 g of zingerone in the feed) was only 37% when calculated based on the extract. However, 54% of the zingerone initially present in the feed remained unextracted in the pomace, so the zingerone extraction yield calculated based on the pomace results is as high as 46%. This takes into account the "missing" zingerone. The proportion of unextracted zingerone is large, and the extraction process can be further improved to reduce it. The extraction yield of gingerol is higher than zingerone (82% for untreated samples and 71% for treated samples) because it is more soluble in CO2.

[0232] In another experiment, the conversion of gingerol to zingerone was tested by subjecting a CO2 extract obtained from untreated ginger root to an alkali treatment. Overnight treatment with both 0.1N and 1N KOH at 60°C worked well, resulting in a zingerone concentration of about 15% in the treated ginger. The resulting material appeared to be much cleaner than the alkali-treated crude ginger, and is therefore an interesting alternative process that could provide an overall more cost-effective extraction process, since the conversion process is carried out on a smaller amount of material. In fact, starting with the same amount of fresh ginger (100 kg), and based on the results obtained in this work, extracting untreated ginger and then treating the extract with alkali would yield almost twice as much zingerone in the final product than the alternative process (see comparison in the table below). However, even in this case, the total zingerone yield of the process is about 0.1% (0.1 kg zingerone per 100 kg fresh ginger). It is expected that further optimization is possible.

[0233] In another experiment, a sample of the CO2 extract of raw ginger was mixed with KOH and converted to a zingerone-rich extract. A neutralization step after processing involves the separation of zingerone from the aqueous reaction mixture as a zingerone-rich resin. 3 g of oleoresin was taken twice and 9 ml of 1N KOH was added to the sample in a plastic vial. The sample was then shaken and left overnight in a 60°C oven. The treated sample was then neutralized by the addition of 10 ml of 1N HCl. There was a slight excess of acid to ensure that all the KOH was neutralized. The addition was done in two stages with mixing after each stage. The sample was centrifuged at 2000 rpm to separate the water from the oleoresin. After centrifugation, most of the water was removed with a pipette. The resulting resin was then removed. A tincture was made by adding absolute alcohol to one of the resin samples (5 ml of ethanol in about 3 g of resin) (sample 1). The other sample was kept in resin form (sample 2). Samples 1 and 2 were analyzed for zingerone content. The concentration of zingerone in the tincture (sample 1) was calculated to be 23 mg / g tincture, and the concentration of zingerone in the treated resin (sample 2) was 52 mg / g.

[0234] For both samples 1 and 2, these values ​​were lower than those obtained in previous treatments, where the zingerone content was estimated to be 150 mg / g. In this work, resin sample 2 was separated from the water and analyzed. The separated water was then also analyzed and estimated to contain an additional 50-55 mg (about 25%) of zingerone for each 3 g batch. This result was rather unexpected, since zingerone has been reported to have a fairly limited water solubility. This method contrasts with the method of adding alcohol directly to the crude neutralized product, since a significant portion of the zingerone remained in the water and was not separated from the resin.

[0235] Since water can extract some of the zingerone from the resin, it may be possible to produce zingerone in high yield after treatment by drying the entire neutralized alkali-treated product. Considering that zingerone after alkali treatment was found to be more soluble in water than previously reported, further process optimization will be performed to improve the yield.

[0236] Example 3: Further reaction methods and comparisons Abstract: Zingerone does not occur naturally in ginger, but is a conversion product of gingerol through processing processes. In these studies, samples of alkali-treated and dried ginger were obtained from Samoa and extracted with ethanol using two different extraction conditions: 20 hours at 40°C (extract A) and 7 days at room temperature (extract B). The zingerone concentrations of the resulting extracts were 41.4 mg / g for extract A and 43.8 mg / g for extract B.

[0237] Extraction A: Processed ginger produced by SROS (Scientific Research Organisation of Samoa) was supplied in two separate plastic bags. The contents of both bags were combined and frozen at -80°C, then ground using a knife mill (Wiley) fitted with a 2 mm mesh. The ground material (782.3 g) was then placed in a round-bottom flask and food-grade ethanol was added at a weight ratio of approximately 5:1. The flask was then placed in a water bath at 40°C, 5 rpm and stirred overnight (total extraction time 20 h). After this, the mixture was filtered under vacuum and the ethanol removed by rotary evaporation under vacuum, producing 43.8 g of a highly viscous dark brown resin with a characteristic ginger aroma. The extraction yield was 5.6%. 10 g of this resin (Extract A) was removed and stored refrigerated under nitrogen flush for future bioassays.

[0238] Extraction B: Ginger was frozen and ground as above. The ground material (785.9 g) was placed in a bucket with food-grade ethanol in a weight ratio of approximately 5:1. Ginger was soaked in ethanol for 7 days at room temperature (22-29 °C). Samples were taken on days 1, 3, and 7. After 7 days, the mixture was filtered under vacuum and the ethanol was removed by rotary evaporation under vacuum to produce 49.3 g of a highly viscous dark brown resin with a characteristic ginger aroma, very similar to that obtained in extraction A (Figure 1). The extraction yield was 6.3%.

[0239] Zingerone and aldehyde analysis: Quantification of zingerone was performed by HPLC in the starting material (i.e., treated ginger) and the final two resins, as well as in the 1st, 3rd and 7th day samples of Extraction B (note that the 7th day liquid sample corresponds to the final resin sample). For the HPLC quantification method, methanol was added and the samples were ground before analysis. The zingerone content of all fractions is shown in Table 6. The mass balance and yield of zingerone are shown in Table 7. [Table 7] [Table 8]

[0240] As mentioned above, extract A was obtained by treatment at 40° C. for 20 hours and extract B was obtained by treatment at room temperature for 7 days. The results in Tables 6 and 7 show that although increasing the temperature to 40° C. also gave high yields, longer treatment at room temperature gave higher levels of zingerone.

[0241] To determine the zingerone content in the starting material, it was first extracted in a suitable solvent. In one process, extraction was performed with ethanol, resulting in a low zingerone content (1.44 mg / g). In the second process, extraction was performed with methanol, and the ginger along with the solvent was ground in a mortar and pestle, resulting in a high zingerone content (3.1 mg / g). For reference, the zingerone content of this material reported by a Samoan laboratory was 1.86 mg / g.

[0242] On this basis, the zingerone yield (i.e., the amount of zingerone in the extract relative to the zingerone in the feed) was estimated to be 75% for Extract A and 89% for Extract B. The 6-gingerol peak seen in the HPLC analysis in determining the zingerone content was consistently observed at approximately 1 / 8 the area of ​​the zingerone peak, suggesting that the extraction has little effect on the zingerone to gingerol ratio.

[0243] Samples of both final resins were taken in ethanol and tested by GCMS for aldehyde analysis. Very low levels of hexanal were found (too low to quantify). Hexanal is a by-product of the reaction that forms zingerone, but is somewhat volatile. The identity of the hexanal peak was confirmed by library matching of the MS data and by ordering a hexanal standard.

[0244] Each of the tests described herein was effective in producing zingerone. A comparison of the results of Examples 2 and 3 is shown in Table 8. [Table 9]

[0245] For fresh and dried ginger, similar values ​​were obtained by Li et al. See Li et al., 2016, "Chemical characterization and antioxidant activities comparison in fresh, dried, stir-frying and carbonized ginger" J Chromatogr B Analyt. Technol. Biomed. Life Sci. 1011: 223-32. Example 2 utilizes a retroaldol reaction as described above. Example 3 utilizes temperature and pH adjustment and extraction as described above.

[0246] For Samoan ginger, it has been noted that the ginger was not harvested at the required time (9 months in the soil), which affected the level of gingerols present in the ginger and therefore the zingerone content in the final product. Further benefits are therefore expected to be obtained. The Fijian ginger had a significantly higher zingerone content (10.2:1.44 = 7.08 times) than the Samoan ginger. This means that if the experimental conditions of Example 3 are applied, the total yield of Fijian ginger can be estimated to be 310 mg / g, i.e. 43.8 mg / g (amount obtained from Samoan ginger in Example 3) x 7.08 (higher starting content in Fijian ginger) = 310 mg / g.

[0247] Table 5 in Example 2 shows the results of pH treated ginger and CO2 extraction. It was found that 322 grams of fresh ginger provided 153 mg / g zingerone. In comparison, Example 3 utilized 785.9 grams (2.4 times the amount of product used in Example 2) providing 43.8 mg / g zingerone. However, this lower yield can be explained by the lower level of Samoa starting material (1.44 mg / g zingerone).

[0248] Example 4: Processing method using juicing and alkaline treatment Summary: Ginger root was mechanically saped and the levels of 6-gingerol were determined in the sap and residual solids. The majority of 6-gingerol was present in the juice. Treatment of the sap with alkali demonstrated that virtually all of the 6-gingerol was converted to zingerone at 60°C for 5-6 hours.

[0249] Juice extraction overview: Fresh ginger (500-1000g) was pre-treated by blending / steeping and squeezing. The liquid fraction was kept and the pomace was further washed in warm water (4 parts water to 1 part ginger) for 10-15 minutes at a temperature of 55-60°C. This was done in a covered container. It was then squeezed again. Each fraction was analyzed for 6-gingerol content (5 analyses in total): 1) fresh ginger sample just before the process; 2) liquid fraction after the first blending / steeping; 3) ginger pomace after the first blending / steeping; 4) second liquid fraction taken after further washing of the first pomace; 5) final ginger pomace after washing and squeezing as above. Moisture content readings were taken on the ginger pomace of each lot after the final squeezing (samples 3) and 5) above).

[0250] Alkaline treatment summary: The liquid fraction was subjected to an alkali treatment to establish the conversion to zingerone. Samples were taken at various time points and analyzed for zingerone content. Samples were subjected to small scale treatment with KOH (conventional 5%). Treatments were performed at room temperature, 30°C and 60°C for 1, 2, 3 and 5 hours using 1 ml samples. Additionally, one sample was treated at 60°C for 24 hours. Up to 15 samples were analyzed.

[0251] Juicing method: Two samples of fresh ginger root were obtained, one locally (organic from Ebisu, Pitone) and the other received from Phil Rasmussen in Auckland. Both samples appeared plumper and juicier than regular supermarket ginger root. Moisture content was measured by slicing approximately 10g of each root, freezing in liquid air and then freeze-drying. The roots were extracted with methanol and the 6-gingerol content was measured. For this, approximately 5g of each sample was cut into 4-5 pieces and crushed using a small kitchen garlic press. The crushed roots and juice were extracted with methanol (2 x 15ml) for 20 minutes at 60°C. HPLC analysis with detection at 280nm was then performed. The results are shown in Table 9 below. [Table 10]

[0252] A sample from Auckland was selected and subjected to juicing. For this, 635 g of ginger root was subjected to the process using a domestic juice extractor. This included a rotating screw drive with a mesh juice filter and an adjustable solids nozzle (see Figures 4A-4B). The extractor removed 516.3 g (81%) of liquid (juice 1, J1) and collected 101.5 g of solids (pomace 1, M1). A portion of the dried solids was removed for analysis and 90.5 g was extracted with 360 ml of hot tap water. This was left for 15 minutes before the material was passed through the juicer. From this process, 350 g of juice (juice 2, J2) was removed together with 70.2 g of solids (pomace 1, M2).

[0253] The two liquids were refrigerated overnight. Both were cloudy with precipitated solids. The samples were shaken before analysis or processing. The juice samples were analyzed for 6-gingerol content by mixing the samples with ethanol (1:1), centrifuging, and injecting the supernatant directly. The solid contents of the two pressed solids, M1 and M2, were 37% and 26.3%, respectively.

[0254] The results are shown in Table 10 below. The gingerol values ​​were multiplied by weight to obtain the total amount of gingerol in each material. The juice was found to contain 81.6% of the measured gingerols. The total gingerols calculated for the feed were lower than the recovered amount, suggesting partial extraction of gingerols from the root. The gingerol percentages were based on the total measured gingerols in the pomace and juice (not the feed measurement). [Table 11]

[0255] Alkaline treatment: This was done on the first juice (J1) collected from the juice extraction process (above). Juice samples (after shaking to suspend all solids) were reacted with KOH at RT, 30°C or 60°C for 1, 2, 3, 5 hours and at 60°C for 24 hours. Three concentrations of KOH were also tested (0.5%, 1.0% and 2.0%). A 2N KOH solution was prepared (5.6g KOH in 50ml water). To produce 0.5%, 1% and 2% KOH concentrations in each sample, 0.25ml, 0.5ml or 1ml of 2N KOH was added to 5.5ml of juice and shaken. The samples were then placed at RT (laboratory), 30°C (water bath) or 60°C (drying oven). Sampling for HPLC analysis was performed by taking 200 μl from each sample and adding 200 μl of 1N HCl, followed by the addition of 500 μl of ethanol. After centrifugation, the samples were directly injected into the HPLC. The peak areas of zingerone and gingerol were compared (see below).

[0256] These results demonstrated that treatment with 2% KOH could achieve complete conversion to zingerone within 5 hours; see Figure 5C. Incubation at 60°C was particularly successful; see Figure 5C. Results were presented as peak areas of zingerone (Z) and gingerol (G). It was noted that the KOH treated samples had solids that precipitated in the tube. For analysis, the tubes were shaken and samples were taken at the wide bore tip to avoid clogging.

[0257] Example 5: Additional Processing Methods Using Juice Extraction and Alkaline Treatment Summary: The aim is to produce an extract from ginger with 6-gingerol converted to zingerone by an alkali catalyzed retro-aldol reaction. The current process aims to reduce processing time and water usage. The process was tested at approximately 40 kg scale before further production at 200 kg scale was undertaken.

[0258] Briefly, fresh ginger was received and processed by treating with alkali and then freeze-drying to produce treated ginger powder. This powder was extracted with ethanol at room temperature for 3 days, with samples taken at 24, 48, and 72 hours to assess the progress of the extraction. The ethanol extraction is described in detail in Example 6.

[0259] Methodology: Imported fresh ginger was subjected to alkaline pretreatment and drying. For this, fresh ginger (36.67 kg) was pressed in a Vincent Corporation CP-4 screw press to produce two streams: ginger juice and pomace. The screw press settings were 50% VSD speed and 2 bar cone air pressure. The pomace from the first press was pressed again to remove the remaining juice.

[0260] The two juices were combined. The juice was heated to 60°C and KOH was added to a final concentration of 2% w / w. The alkalized juice was kept at 60°C for 5 hours to allow for the conversion of gingerol to zingerone. The pH was neutralized to 7.2 by adding anhydrous citric acid. The juice containing zingerone was freeze-dried and milled.

[0261] Samples were taken: fresh ginger (ZINGO); two pomaces (GMARC and GMARC2); juice before KOH addition (GKOH0); juice after 2, 3 and 5 hours of treatment (GKOH2, GKOH3, GKOH5); and the final dry extract (GPE).

[0262] Additionally, a sample of GMARC2 was extracted with hot water as follows: Water was added to GMARC2 in a 5:1 w:w ratio. The mixture was heated to 60°C and held at this temperature for 15 minutes. The extract was separated from the solids by a screw press with the same settings as above. Samples were taken: Extract (GMARC2 HWEX); Pomace (HW MARC). Each sample was analyzed by HPLC for total solids (LOD, 16h 100°C) and gingerol or zingerone content.

[0263] Sample analysis: Analysis was performed using HPLC with UV detection at 280 nm. Sample preparation was as follows: (1) Liquid samples such as juice were diluted 1:1 with ethanol and centrifuged. In-process liquid samples with alkali were diluted 1:1:1 with 1N HCl and ethanol. (2) Solid samples such as fresh ginger or ginger pomace were double extracted with ethanol (sonication, heating at 60°C for 20 min, vortexing, centrifugation) and the supernatants were combined. Solid extracts (approximately 5 g) were typically prepared to 50 mL for analysis. Raw fresh ginger was coarsely chopped and then blended with ethanol using an ULTRA-TURRAX® type mixer. Quantitation was performed by comparison to a standard curve generated using zingerone. Molecular weight correction was performed for gingerols.

[0264] Pressing: 36.67 kg of Fijian fresh ginger was received and pressed. Pressing was effective, producing a large quantity of pale green juice and fibrous pomace. 28.92 kg of juice was recovered in the first press. 6.6 kg of pomace was then pressed a second time, recovering a further 2.18 kg of juice. The total juice yield was 31.1 kg, which represented 86% of the incoming fresh ginger mass. The final recovered pomace mass was 4.05 kg. Generally, there was a hold-up of 1-2 kg in the screw press at the end of the run. This resulted in a small difference between the feed mass and the combined mass of pomace and juice.

[0265] Hot water extraction: 3.62 kg of GMARC2 and 18.1 kg of water were heated to 60°C and then extracted at 60°C for 15 minutes before being separated by a screw press. 21.18 kg of the mixture was then pressed. It should be noted that approximately 500 g of water was lost as evaporation during the extraction. From this, 17.62 kg of extract was collected and subsampled for analysis. Additionally, 2.69 kg of pomace was collected.

[0266] Alkaline treatment: For this, 1.236 kg of 50% KOH solution was added to 31.1 kg of ginger juice to reach a target KOH concentration of 2%. The pH after KOH addition was 12.18. Upon addition of KOH, the color of the juice changed from light green to reddish brown. The juice was kept at 60°C for 5 hours and then neutralized by adding 500 g of anhydrous citric acid. The pH after addition of citric acid was 7.23.

[0267] Freeze Drying: The processed juice was transferred to freeze dryer trays and frozen overnight before being transferred to a Cuddon FD 80 freeze dryer. A total of 28.51 kg of juice was loaded onto the trays and dried. Approximately 3 kg of juice was lost prior to freeze drying as a result of manual handling. After drying, 2.86 kg of dried extract (total 10% of the dried juice mass) was collected. This was ground and subsampled. After grinding, subsampling and loss handling, a total of 2.19 kg was packaged in foil bags and stored until further processing. Of this, approximately 1.6 kg was sent for ethanol extraction (see Example 6).

[0268] A summary of the mass balance is shown below. [Table 12] [Table 13] [Table 14]

[0269] Gingerol and zingerone levels were measured as described above, and the results are shown in Table 11 below. [Table 15]

[0270] This table shows the gingerol content of the various fractions in the pretreatment and treatment process. From these measurements, there was a total of 19.1 g of 6-gingerol in the 36.67 kg raw ginger feed. The zingerone content of the dried ginger extract (GPE) was 5.7 mg / g. Thus, the total zingerone in the 2.86 kg dried powder (before losses and grinding losses) was 16.30 g. The gingerol concentration in the juice before conversion (GKOH-0) was 6.9 mg / g on a dry basis. Converted to a wet basis using a solids concentration of 6.67% (before the total solids content was increased to about 10% by the addition of KOH and anhydrous citric acid), the total gingerol in the juice was 14.32 g. The GKOH-2, -3, and -5 values ​​of 6-gingerol were confirmed.

[0271] The mass balance between gingerols in the juice and zingerone in the final powder extract does not match exactly. This is likely due to measurement variability. The pomace count appears to be somewhat inflated when the total mass balance calculation is taken into account. When the pomace was extracted with hot water, the 6-gingerol content in the extract was 0.22 mg / g, which corresponds to a total of 5.7 g of 6-gingerol in this extract, approximately 25% of the 6-gingerol in the feed. A total of 18 kg of water was required for the water extraction. This leads to an increase in the mass of KOH and citric acid, which further increases the drying load by 58%. Therefore, in certain situations it may be desirable to omit the water extraction.

[0272] In particular, these experiments showed that nearly complete recovery of 6-gingerol (in the form of zingerone) in the final product was achieved. Samples taken during the conversion reaction (GKOH2, GKOH3, and GKOH5) showed that the conversion of 6-gingerol to zingerone occurred in the first 2 hours of treatment, and there was no significant increase in the level of zingerone in samples taken after 2 hours of treatment. If the conversion were complete after 2 hours, this incubation time (or even shorter incubation times) would be sufficient.

[0273] It was concluded that the process involving KOH treatment of the juice phase after expression was a valid manufacturing method. Further experiments will utilize over 200 kg of ginger as starting material. The 6-gingerol level of this batch was 1.1 mg / g, which was expected to be reflected in the corresponding zingerone level.

[0274] Example 6: Ethanol Extraction Process and Analysis Summary: Fresh ginger was received and treated with alkali and then freeze-dried to produce treated ginger powder (see Example 5 above). This powder was extracted with ethanol at room temperature for 3 days, with samples taken at 24, 48, and 72 hours to assess the progress of the extraction.

[0275] Methodology: Treated ginger produced as above (Example 5) was kept refrigerated until use. Approximately half of the ginger received was extracted with XNS food grade ethanol at room temperature. The treated ginger was placed in a 10 L glass vessel equipped with an overhead stainless steel stirrer along with ethanol (using a 1:5 weight ratio of ginger:ethanol). The mixture was stirred for 72 hours at a stirring speed sufficient to prevent solids from accumulating on the bottom.

[0276] After 24 hours, the stirrer was stopped and the solids were allowed to settle for 10 minutes before a 50 mL sample was taken from the top. After another 24 hours, a second sample was taken using the same procedure. After a total of 72 hours, the stirrer was stopped and the mixture was filtered under vacuum using filter paper. A sample was taken from the cake and the final tincture sample was taken from the 72 hour filtrate. The remaining filtrate was labelled ZINGOEE. Approximately 300 mL of ZINGOEE sample was taken and stored refrigerated in a glass bottle. In a further step, all the ZINGOEE was evaporated, making a total resin weight of 43.5 g with 88.9 mg / g zingerone.

[0277] The remaining ethanolic tincture was evaporated under vacuum using a Buchi R220SE rotary evaporator operating at 50 mbar and 40°C until approximately a 31-fold volume reduction was achieved. The resulting concentrated extract (ZINGOCE) was then analyzed and once the zingerone content was confirmed, a small sample of this concentrated extract was used to produce a standard grade tincture containing approximately 12 mg / g zingerone by dilution with food grade ethanol. Two separate samples of this standardized tincture were sent to SCU (Australia) for analysis and a third sample was kept on-site for zingerone analysis.

[0278] Results: The process and results are shown in Figure 7. In these experiments, 801.5 g of received treated ginger was used for ethanol extraction along with 4007.3 g of food-grade ethanol. After 72 h at room temperature (16-20 °C) under stirring, the mixture was filtered to obtain 3556.7 g of clear brown aromatic ethanol tincture [ZINGOEE], and 1033.4 g of cake (i.e., spent ginger solids). Approximately 140 g of ethanol was lost due to evaporation during the extraction. The total weight of the produced ethanolic tincture was 3631.7 g, including samples taken at 24 and 48 h.

[0279] Quantification of zingerone was performed by HPLC on the starting material (i.e., treated ginger, GPE) and samples after ethanol extraction for 24, 48, and 72 hours at room temperature. The spent ginger solid (i.e., the cake from the filtration process) was also analyzed. The zingerone content of all fractions is shown in Table 12. The HPLC results showed little difference among the three extraction times, indicating that 24 hours was sufficient for extraction. [Table 16]

[0280] The zingerone concentration in the filtrate [ZINGOEE] was 1.1mg / g, i.e., 3.88g of zingerone in the liquid, or 85% of the starting zingerone, indicating a reasonable recovery. The zingerone concentration in the cake was 0.61mg / g, i.e., 0.63g of zingerone in the cake. However, it was noted that there was still some ethanol solution present in the cake. In future processing, the cake could be washed with clean ethanol to wash off as much of the extractables as possible.

[0281] The zingerone mass balance in the ethanol extraction process is 98.5%. This is 3.88g in the extract and 0.63g in the cake divided by 4.58g in the feed. Room temperature extraction was found to be favorable for zingerone recovery. Although it is possible that higher extraction temperatures could result in higher zingerone recovery, room temperature extraction is clearly effective.

[0282] After taking approximately 300 mL of ZINGOEE sample, the remaining extract (3249 g) was evaporated under vacuum to obtain 104.9 g of concentrated extract [ZINGOCE] with zingerone content of 33 mg / g (3.46 g zingerone). The total solids content of this concentrated extract was 37.1% (measured as loss on drying at 110°C), indicating a final oleoresin weight of 38.9 g if all ethanol is removed. Extrapolating this figure to the total production of ZINGOEE, the extraction yield of this process is approximately 5.4%. A standardized tincture containing 12 mg / g zingerone was prepared by mixing 25.5 g ZINGOCE with 44.5 g food grade ethanol. A sample of this tincture was sent for further testing.

[0283] Additionally, further analysis showed that no aldehydes were present in the final product. See Figures 8A-8B. For these evaluations, samples of the treated ethanolic ginger extracts were analyzed using GCMS for the presence of aldehydes. If present, this is expected to be (mainly) hexanal derived from 6-gingerol. Powder preparations (e.g., pre-extraction powders as in Examples 4 and 5 above) are also expected to be free of aldehydes. As a next step, the ethanolic tincture is evaporated to produce a thick ethanolic paste.

[0284] Conclusion: The proposed method, which involves screw pressing followed by KOH treatment at the juice stage, neutralization with citric acid, and then freeze-drying, proved to be very effective, as it could recover almost all of the 6-gingerol at the juice stage and convert it all into zingerone after treatment with 2% KOH at 60°C for 5 hours. The dried powder before the process now contains 5.32 mg / g zingerone, which is at least twice as efficient as the previous production technique.

[0285] Ethanol extraction at room temperature for 24 hours is an optional step that achieves at least 85% recovery of zingerone. Recovery can be further increased by washing the solids with fresh ethanol after extraction. In these methods, the ethanol extract [ZINGOEE] was evaporated to significantly reduce the volume, and the concentrated extract [ZINGOCE] was then reconstituted with fresh ethanol to produce a standardized tincture containing a target dose of 12 mg / g zingerone.

[0286] For the extraction process, the overall Z mass balance (out / in) was 98.5%. The zingerone content in the filtrate (ZINGOEE) was 1.1 mg / g, and the zingerone content in the concentrated extract (ZINGOCE) was 33 mg / g. The method produced 104.9 g of ZINGOCE at 33 mg / g, leaving 3.46 g of zingerone. This figure is lower than the 3.88 g of ZINGOCE because 400 g of ZINGOCE was removed before evaporation (plus a retentate sample of approximately 300 mL for testing). Taking this into account, the calculation fits very well. The overall zingerone mass balance before evaporation was calculated to be 98.5% (=(0.63+3.88) / 4.58).

[0287] The results obtained in this study show that from 100 kg of fresh ginger with a 6-gingerol content of 0.5 mg / g, 3.6 kg of standardized tincture with a zingerone content of 12 mg / g can be obtained.

[0288] Example 7: Combination of Zingerone with Antimicrobial Agents Gentamicin is an antibiotic in the aminoglycoside class used to treat severe gram-negative cell infections. It is available in a variety of dosage forms, including injectable, as well as intravenous and oral formulations. Invasive Staphylococcus aureus bacteria frequently develop resistance to antibiotic monotherapy with gentamicin. Some strains of Staphylococcus aureus develop resistance to gentamicin and other aminoglycosides through expression of antibiotic-modifying enzymes that mediate bacterial resistance to gentamicin.

[0289] Vancomycin is a glycopeptide antibiotic that is most widely used to treat gram-positive infections in adults, children, and neonates. It is available in a variety of formulations, including oral (e.g., capsules or solutions) and intravenous injections. However, vancomycin-resistant enterococci have emerged, making treatment more difficult. E. faecium is the most common strain to acquire vancomycin resistance. The primary mechanism of glycopeptide resistance (e.g., vancomycin) in enterococci involves alterations in the peptidoglycan synthesis pathway, specifically the substitution of D-alanine-D-alanine (D-Ala-D-Ala) with D-alanine-lactate (D-Ala-D-Lac) or D-alanine-D-serine (D-Ala-D-Ser).

[0290] Checkerboard analysis was used to determine whether combinations of compounds could reduce the MIC of Staphylococcus aureus compared to individual compounds. In this experiment, combinations of zingerone and gentamicin, zingerone and vancomycin, and zingerone and cefotaxime were tested. For this test, 96-well plates were inoculated with Staphylococcus aureus and then incubated with the test compounds for 24 hours at 37°C. Zingerone was added at 0 mg / ml, 0.78 mg / ml, 1.56 mg / ml, 3.125 mg / ml, 6.26 mg / ml, 12.5 mg / ml, and 25 mg / ml. Gentamicin was added at 0, 4, 8, 16, 32, 64, 128, and 256 μg / ml. Vancomycin was added at 0, 4, 8, 16, 32, 64, 128, and 256 μg / ml. Cefotaxime was added at 0, 8, 16, 32, 64, 128, 256, and 512 μg / ml. MRSA cells (Methicillin-resistant Staphylococcus aureus) were grown in Mueller-Hinton medium supplemented with 2% NaCl. 10 per ml 8 cells were used. The FICI index was used to quantify compound interactions using the following formula: FICI = (MIC A 組み合わせ / MIC A 単独 )+(MIC B 組み合わせ / MIC B 嘆息 ). An FICI of 0.5 or less was defined as synergy, and an FICI of greater than 4.0 was defined as antagonism. An FICI between 0.5 and 1.0 was considered a non-synergistic but significant improvement. The results are shown in Tables 13-16 below. [Table 17] [Table 18] [Table 19] [Table 20]

[0291] These results established that gentamicin in combination with zingerone and vancomycin in combination with zingerone reduced the MIC of Staphylococcus aureus compared to the individual addition of gentamicin, vancomycin, and zingerone (see Figures 9A-9B). In combination with zingerone and gentamicin, this activity was synergistic: FCI = 0.5 = (MIC A 組み合わせ 4 μg / mL / MIC A 単独 16μg / mL)+(MIC B 組み合わせ 6.25mg / g / MIC B 単独 25 mg / g). The combination of zingerone with vancomycin significantly increased the inhibitory activity, approaching synergistic activity: FIC1 = 0.1 = (MIC A 組み合わせ 4 μg / mL / MIC A 単独 8μg / mL)+(MIC B 組み合わせ 12.5mg / g / MIC B 単独In comparison, the combination of zingerone with cefotaxime (a member of the cephalosporin family of antibiotics) did not result in any significant change in activity compared to each compound applied individually.

[0292] Gentamicin and vancomycin are representatives of aminoglycoside and glycopeptide antibiotics, respectively. The favorable increase in inhibitory activity of zingerone and gentamicin combinations (synergistic activity) and zingerone and vancomycin combinations (significant increase / close to synergy) provide valuable tools for use in combating infections, especially antibiotic-resistant strains (including the antibiotic-resistant strain Staphylococcus aureus). Such combination treatments increase efficacy and decrease the chance of resistance, making them highly beneficial as therapeutic approaches.

[0293] Those of ordinary skill in the art will be able to make other embodiments and modifications using the disclosure and teachings herein without undue experimentation, and all such embodiments and modifications are considered to be part of this disclosure.

[0294] Thus, one skilled in the art will readily appreciate from this disclosure that subsequent modifications, substitutions, and / or variations that perform substantially the same function or achieve substantially the same results as the subject matter described herein may be utilized in accordance with such related embodiments. Accordingly, the present disclosure is intended to encompass within its scope modifications, substitutions, and variations of the processes, manufacture, compositions, compounds, means, methods, and / or steps disclosed herein.

[0295] This specification may include subject matter that is outside the scope of the claimed invention. This subject matter is included to aid in the understanding of the present invention.

[0296] References herein to external sources, including patents and other documents, are generally for the purpose of providing a context for discussing features of the present disclosure, and unless otherwise stated, reference to such sources shall not be construed as an admission that such sources are prior art or form part of the general knowledge in the art in any jurisdiction.

Claims

1. 1. A method for producing zingerone, comprising: (i) subjecting ginger root to an alkaline treatment in an alkaline solution; or (ii) subjecting the juice obtained from the ginger root to alkaline treatment in an alkaline solution; thereby producing zingerone.

2. 10. The method of claim 1, comprising one or more of the following: (a) The ginger root is fresh; (b) chopping the ginger root in (i); (c) chopping and drying the ginger root of (i); (d) obtaining the juice of (ii) by steeping and / or pressing; (e) potassium hydroxide (KOH) is used in the alkaline solution; (f) the alkaline solution uses potassium hydroxide (KOH) in liquid form; (g) Add calcium hydroxide (Ca(OH) 2 ) is used; (h) the method further comprises the step of neutralizing the alkaline solution; (i) the method further comprises the step of extracting zingerone after alkaline treatment.

3. The alkaline solution (a) about 0.1% to about 1.0% KOH (v / v); (b) about 0.5% to about 0.7% KOH(v / v); (c) about 1% to about 3% KOH (v / v); (d) about 1.5% to about 2.5% KOH (v / v); (e) approximately 2% KOH (v / v); (f) About 0.5% to about 4% Ca(OH) 2 (v / v); (g) Approximately 1.5% to approximately 3.5% Ca(OH) 2 (v / v); or (h) About 2.0% to 3.0% Ca(OH) 2 (v / v), 3. The method of claim 2, comprising:

4. 3. The method of claim 2, comprising one or more of the following: (a) the alkaline treatment is carried out at a temperature of about 40 degrees Celsius to about 70 degrees Celsius; (b) the alkaline treatment is carried out at about 50 degrees Celsius to about 60 degrees Celsius; (c) the alkaline treatment is carried out at about 55 degrees Celsius to about 65 degrees Celsius; (d) the alkaline treatment is carried out at about 60 degrees Celsius; (e) the alkaline treatment is carried out for about 1 to 30 hours, about 1 to 20 hours, about 1 to 10 hours, or about 1 to 5 hours; (f) the alkaline treatment is carried out for about 0.5 hours to about 3 hours, or about 0.75 hours to about 2.5 hours, or about 1 hour to about 2 hours; (g) The alkaline treatment is carried out for about 1 hour or about 2 hours.

5. 3. The method of claim 2, comprising one or more of the following: (a) the zingerone is extracted by one or more ethanol extraction steps; (b) the zingerone is extracted by a supercritical fluid extraction process; (c) the zingerone is extracted by a supercritical fluid extraction step followed by an ethanol extraction step; (d) producing a product consisting essentially of zingerone by said process; (e) producing an aldehyde-free or substantially aldehyde-free product by said process.

6. A composition comprising zingerone, wherein the zingerone is obtainable by the method according to any one of claims 1 to 5.

7. 7. The composition of claim 6, comprising one or more of the following: (a) the composition is formulated as a pharmaceutical composition; (b) the composition is formulated as a liquid or a powder; (c) the composition is formulated for topical or oral administration; (d) the composition is formulated to contain a dose of zingerone from about 1 mg to about 5000 mg; (e) the composition is formulated to contain a dose of zingerone from about 1 mg to about 1500 mg; (f) the composition is formulated to contain a dose of zingerone from about 5 mg to about 500 mg; (g) the composition is formulated to contain a dose of zingerone from about 1 mg to about 15 mg; (h) the composition is formulated to contain a dose of zingerone from about 1 mg to about 10 mg; (i) the composition is formulated for co-administration with an additional antimicrobial agent; (j) the composition is formulated for co-administration with one or more aminoglycoside antibiotics or one or more glycopeptide antibiotics; (k) the composition is formulated for co-administration with gentamicin or vancomycin; (l) the composition is formulated to contain gentamicin or vancomycin; (m) The composition is formulated for use in treating or preventing a microbial infection.

8. 8. The composition of claim 7, comprising one or more of the following: (a) the microorganism is selected from the group consisting of bacteria and fungi; (b) the microorganism is selected from the group consisting of gram-positive and gram-negative bacteria; (c) the microorganism is selected from the group consisting of Bacillus bacteria, Clostridium bacteria, Escherichia bacteria, Mycoplasma bacteria, Neissaria bacteria, Pseudomonas bacteria, Salmonella bacteria, Shigella bacteria, Streptococcus bacteria, Staphylococcus bacteria, and Vibrio bacteria; (d) the microorganism is selected from the group consisting of Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), Streptococcus pneumoniae (S. pneumoniae), Pseudomonas aeruginosa (P. aeruginosa), and Klebsiella pneumoniae (K. pneumoniae); (e) the microorganism is selected from the group consisting of Aspergillus fungi, Candida fungi, Coccidioides fungi, Cryptococcus fungi, Histoplasma fungi, Pneumocystis fungi, and Stachybotrys fungi; (f) the microorganism is selected from the group consisting of Candida albicans fungi, Aspergillus species fungi, Histoplasma capsulatum fungi, Coccidioides immitis fungi, and Pneumocystis carinii fungi, and tinea fungi; (g) The infection affects one or more of the skin, eyes, ears, nose, mouth, throat, esophagus, lungs, circulatory system, digestive system, or genitourinary system.

9. A medicament for treating or preventing infections caused by microbial agents, comprising zingerone, wherein the zingerone is obtained by the method according to any one of claims 1 to 5.

10. 10. The medicament of claim 9, comprising one or more of the following: (a) the medicament reduces or slows the progression of the infection; (b) the medicament is formulated as a liquid or powder; (c) the medicament is formulated for topical or oral administration; (d) the medicament is formulated as a liquid, tablet, or capsule; (e) the medicament is formulated to contain zingerone in a dose of about 1 mg to about 5000 mg; (f) the medicament is formulated to contain zingerone in a dose of about 1 mg to about 1500 mg; (g) the medicament is formulated to contain zingerone in a dose of about 5 mg to about 500 mg; (h) the medicament is formulated to contain zingerone in a dose of about 1 mg to about 15 mg; (i) the medicament is formulated to contain a dose of zingerone from about 1 mg to about 10 mg; (j) the medicament is adapted for co-administration with one or more antimicrobial agents; (k) the medicament is formulated for co-administration with one or more aminoglycoside antibiotics or one or more glycopeptide antibiotics; (l) the medication is formulated for co-administration with gentamicin or vancomycin; (m) The medicine contains gentamicin or vancomycin.

11. 10. The medicament of claim 9, comprising one or more of the following: (a) the microorganism is selected from the group consisting of bacteria and fungi; (b) the microorganism is selected from the group consisting of gram-positive and gram-negative bacteria; (c) the microorganism is selected from the group consisting of Bacillus bacteria, Clostridium bacteria, Escherichia bacteria, Mycoplasma bacteria, Neissaria bacteria, Pseudomonas bacteria, Salmonella bacteria, Shigella bacteria, Streptococcus bacteria, Staphylococcus bacteria, and Vibrio bacteria; (d) the microorganism is selected from the group consisting of Escherichia coli (E. coli), Staphylococcus aureus (S. aureus), Streptococcus pneumoniae (S. pneumoniae), Pseudomonas aeruginosa (P. aeruginosa), and Klebsiella pneumoniae (K. pneumoniae); (e) the microorganism is selected from the group consisting of Aspergillus fungi, Candida fungi, Coccidioides fungi, Cryptococcus fungi, Histoplasma fungi, Pneumocystis fungi, and Stachybotrys fungi; (f) the microorganism is selected from the group consisting of Candida albicans fungi, Aspergillus species fungi, Histoplasma capsulatum fungi, Coccidioides immitis fungi, and Pneumocystis carinii fungi, and tinea fungi; (g) The infection is an infection affecting one or more of the skin, eyes, ears, nose, mouth, throat, esophagus, lungs, circulatory system, digestive system, or genitourinary system.

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