Use of compositions comprising 3,6,7-trimethyllumazine for preventing, ameliorating or treating MMP-9-associated conditions and inflammation

Abstract

The present invention relates to compositions comprising 3,6,7-trimethyllumazine, methods and uses thereof in the prevention, amelioration or treatment of inflammation, and / or conditions associated with inflammation. More particularly, but not exclusively, the present invention relates to compositions comprising 3,6,7-trimethyllumazine and methods of use thereof in the prevention, amelioration or treatment of MMP-9-associated conditions, such as inflammation of the gastrointestinal tract and / or inflammatory conditions associated with the gastrointestinal tract.

Description

[Technical field]

[0001] Related Applications This application derives priority from New Zealand Patent Application No. 755138, which is incorporated herein by reference.

[0002] The present invention relates to compositions comprising 3,6,7-trimethyllumazine, methods and uses thereof in the prevention, amelioration or treatment of inflammation, and / or conditions associated with inflammation. More specifically, but not exclusively, the present invention relates to compositions comprising 3,6,7-trimethyllumazine, and methods of use thereof in the prevention, amelioration or treatment of MMP-9-associated conditions, such as inflammation of the gastrointestinal tract and / or inflammatory conditions associated with the gastrointestinal tract. [Background technology]

[0003] Although immune system-related inflammation can be beneficial, this is not always the case: it is often viewed as a negative reaction or one to be avoided, especially in the context of the gastrointestinal system.

[0004] Inflammation is involved in a wide range of gastrointestinal disorders. In a healthy intestine, the intestinal mucosa is in a state of controlled response, regulated by a complex balance of pro-inflammatory and anti-inflammatory cytokines and cells. Disturbance of this balance can lead to persistent activation of immune / non-immune responses, resulting in active inflammation and tissue destruction. Failure to adequately prevent or resolve inflammation is involved in the pathogenesis of several diseases of the gastrointestinal tract, including gastric ulcers, inflammatory bowel disease (IBD), Crohn's disease, and ulcerative colitis.

[0005] Depending on the severity, scope, and medical goal of treatment, conventional drug therapies for inflammatory conditions, such as sulfasalazine, mesalazine, corticosteroids, and methotrexate, are primarily used to regulate immune and inflammatory responses. Both safety and efficacy limitations encountered with current medical approaches to inflammatory conditions continue to drive the search for better and safer therapeutic alternatives. Consumers are also more generally looking for natural ways to support their health and well-being.

[0006] Although the specific cause of inflammation has yet to be identified in many diseases, cytokine activation in the intestinal mucosal system is an important target for regulating inflammation in intestinal inflammatory diseases.

[0007] Gastric ulcers are another common inflammation-related gastrointestinal disorder. Gastric ulcers are benign mucosal lesions that penetrate deep into the intestinal wall beyond the muscularis mucosae, forming craters surrounded by acute and chronic inflammatory cell infiltration. Many studies have reported that the major risk factors for gastric ulcers include Helicobacter pylori infection, smoking, aspirin / nonsteroidal anti-inflammatory drug (NSAID) use, alcohol abuse, and stress.

[0008] Matrix metalloproteinases (MMPs) are a group of enzymes that exhibit proinflammatory effects. They have been shown to have a role in inflammation and to be involved in the inflammatory response to disease (Stallmach, 2000), and MMP-1, MMP-2, and MMP-9 have been shown to be important in inflammation (Manicone, 2008).

[0009] MMP-9 is a gelatinase-type enzyme that specifically regulates acute and chronic gastric ulcers (Swarnakar et al., 2005). Several studies have reported increased MMP-9 activity (up to 10-fold) in ethanol- and indomethacin-induced gastric ulcers (Lempinen, Inkinen, Wolff, & Ahonen, 2000; Pradeepkumar Singh, Kundu, Ganguly, Mishra, & Swarnakar, 2007). MMP-9 is one of the key pro-inflammatory enzymes that can proteolytically process many cytokines and chemokines into more active forms such as pro-IL-1β and IL-8 (Van den Steen et al., 2000). Therefore, MMP-9 is of interest in the treatment of gastric ulcers to prevent excessive tissue degradation of the extracellular matrix.

[0010] Conventional treatments for gastric ulcers include medical management with medications such as omeprazole and ranitidine, which can have serious side effects such as bone marrow suppression and abnormal heart rhythms and are known to have high recurrence rates.

[0011] Thus, there is interest in identifying other anti-inflammatory agents for use in the treatment of gastrointestinal inflammation and / or conditions associated with gastrointestinal inflammation, including MMP9-associated inflammatory conditions and other MMP-9-associated conditions.

[0012] Honey is well known for its antimicrobial activity. The art also suggests that honey possesses anti-inflammatory activity, but the reasons are not fully characterized. One patent publication, WO 2015 / 030609, incorporated herein by reference, explores the anti-inflammatory activity of certain fractions of honey. This publication teaches that low molecular weight fractions from honey have a generalized anti-inflammatory effect and no immunostimulatory effect. It does not mention a specific anti-inflammatory effect.

[0013] From the above, it will be appreciated that it would be useful to provide alternative methods of treating inflammatory conditions, including those associated with the gastrointestinal tract. Summary of the Invention [Problem to be solved by the invention]

[0014] It is an object of the present invention to provide a method of treating inflammatory conditions, including inflammatory conditions associated with the gastrointestinal tract, and / or to address the problems mentioned above, or at least to provide the public with a useful choice.

[0015] Further aspects and advantages of the products, compositions, methods and uses will become apparent from the following description, which is given by way of example only. [Means for solving the problem]

[0016] Described herein are compositions comprising 3,6,7-trimethyllumazine and methods of using the compositions to prevent, ameliorate or treat MMP-9 associated conditions, inflammation of the gastrointestinal tract, and / or inflammatory conditions associated with the gastrointestinal tract. The inventors have identified 3,6,7-trimethyllumazine, a pteridine derived from honey, as having anti-inflammatory and MMP-9 inhibitory activity. The ability to isolate the compound and characterize its anti-inflammatory and MMP-9 inhibitory activity provides the ability to manufacture medicaments for a variety of uses, including the treatment, prevention, and amelioration of conditions associated with MMP-9, including inflammatory conditions of the gastrointestinal tract.

[0017] In a first particular aspect, the present invention provides a method for preventing, ameliorating, or treating an MMP-9-associated condition in a subject, comprising administering to a subject in need thereof a composition comprising 3,6,7-trimethyllumazine.

[0018] In one embodiment of the first aspect, the MMP-9 associated condition is selected from gastrointestinal inflammatory diseases, gastric ulcers (e.g. peptic ulcers), gastritis, MMP-associated inflammatory conditions, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, irritable bowel syndrome (IBS), gastrointestinal diseases, gastroesophageal reflux disease (GERD), heartburn, acid reflux, Helicobacter pylori infection, oral ulcers, stomatitis, pharyngitis, gingivitis, esophageal ulcers, neuropsychiatric diseases (such as schizophrenia, bipolar disorder, multiple sclerosis), neurodegenerative disorders (such as traumatic brain injury, multiple sclerosis, and Alzheimer's disease), cardiovascular diseases, cancer, and arthritis.

[0019] In a second particular aspect, the present invention provides a method for preventing, ameliorating, or treating an MMP-9-associated inflammatory condition in a subject, comprising administering to a subject in need thereof a composition comprising 3,6,7-trimethyllumazine.

[0020] In one embodiment of the second aspect, the MMP-9 associated inflammatory condition is associated with inflammation of the gastrointestinal tract, hi another embodiment, the MMP-9 associated inflammatory condition is selected from gastrointestinal inflammatory disease, gastric ulcer (e.g., peptic ulcer), gastritis, MMP-associated inflammatory condition, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, irritable bowel syndrome (IBS), gastrointestinal disease, gastroesophageal reflux disease (GERD), heartburn, acid reflux, Helicobacter pylori infection, oral ulcer, stomatitis, pharyngitis, gingivitis, and / or esophageal ulcer.

[0021] In one embodiment, the MMP-9 associated inflammatory condition is selected from neuropsychiatric disorders (such as schizophrenia, bipolar disorder, multiple sclerosis, etc.), neurodegenerative disorders (such as traumatic brain injury, multiple sclerosis, and Alzheimer's disease, etc.), cardiovascular disease, cancer, and arthritis.

[0022] In a third particular aspect, the present invention provides a method of preventing, ameliorating, or treating inflammation in a subject in need thereof, comprising administering to a subject a composition comprising 3,6,7-trimethyllumazine.

[0023] In one embodiment of the third particular aspect, the inflammation is associated with the gastrointestinal tract of the subject. In one embodiment, the inflammation is associated with a condition selected from gastrointestinal inflammatory disease, gastric ulcer (e.g., peptic ulcer), gastritis, MMP-associated inflammatory conditions, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, irritable bowel syndrome (IBS), gastrointestinal disease, gastroesophageal reflux disease (GERD), heartburn, acid reflux, Helicobacter pylori infection, oral ulcer, stomatitis, pharyngitis, gingivitis, esophageal ulcer, neuropsychiatric disease (such as schizophrenia, bipolar disorder, multiple sclerosis), neurodegenerative disorders (such as traumatic brain injury, multiple sclerosis, and Alzheimer's disease), cardiovascular disease, cancer, and / or arthritis.

[0024] In one embodiment of aspects 1-3, the source of 3,6,7-trimethyllumazine is Leptospermum. In one embodiment, the 3,6,7-trimethyllumazine is substantially derived from a plant selected from the group consisting of Leptospermum scoparium, Leptospermum polygalifolium, Leptospermum subtenue, and combinations thereof. In one embodiment, the 3,6,7-trimethyllumazine is derived from Leptospermum scoparium.

[0025] In one embodiment of aspects 1-3, the source of 3,6,7-trimethyllumazine is honey.

[0026] In one embodiment, the honey is of floral origin substantially derived from Leptospermum. In one embodiment, the honey is of floral origin substantially derived from Leptospermum scoparium, Leptospermum polygalifolium, Leptospermum subtenue, and / or combinations thereof. In one embodiment, the honey is of floral origin substantially derived from Leptospermum scoparium (also known as Manuka).

[0027] In one embodiment of aspects 1-3, the 3,6,7-trimethyllumazine is derived directly from a plant of the genus Leptospermum. In one embodiment, the 3,6,7-trimethyllumazine is derived directly from the flowers, nectar, roots, fruits, seeds, bark, oil, leaves, wood, stems, or other plant material of a plant of the genus Leptospermum. In one embodiment, the 3,6,7-trimethyllumazine is derived substantially from a plant selected from the group including Leptospermum scoparium, Leptospermum polygalifolium, Leptospermum subtenue, and combinations thereof.

[0028] In one embodiment of the above aspect, the composition comprising 3,6,7-trimethyllumazine comprises honey. In one embodiment, the composition comprising 3,6,7-trimethyllumazine consists of honey.

[0029] In one embodiment of the above aspect, the composition comprising 3,6,7-trimethyllumazine comprises a honey extract. In one embodiment of the above aspect, the composition comprising 3,6,7-trimethyllumazine comprises a honey extract, wherein the honey extract comprises a concentration of 3,6,7-trimethyllumazine that is greater than the concentration of 3,6,7-trimethyllumazine found to occur naturally in honey. In one embodiment, the composition comprises a honey extract, wherein the honey extract comprises a concentration of 3,6,7-trimethyllumazine that is greater than the concentration of 3,6,7-trimethyllumazine found to occur naturally in honey. In one embodiment, the honey extract comprises a concentration of 3,6,7-trimethyllumazine that is greater than the concentration of 3,6,7-trimethyllumazine found to occur naturally in the honey from which the extract is derived.

[0030] In one embodiment, the honey from which the extract is derived is of floral origin substantially from the genus Leptospermum. In one embodiment, the honey from which the extract is derived is substantially from a species selected from Leptospermum scoparium, Leptospermum polygalifolium, Leptospermum subtenue, and combinations thereof. seed In one embodiment, the composition further comprises honey.

[0031] In one embodiment of the above aspect, the honey is raw honey, heat treated honey or pasteurized honey.

[0032] In one embodiment of the above aspect, the composition comprises 3,6,7-trimethyllumazine isolated from honey. In one embodiment, the honey is of floral origin substantially from the genus Leptospermum. In one embodiment, the honey is of floral origin substantially from the genus Leptospermum scoparium, Leptospermum polygalifolium, Leptospermum subtenue, and / or combinations thereof. seed In one embodiment, 3,6,7-trimethyllumazine is isolated by subjecting honey to solid phase extraction, followed by normal phase flash chromatography and preparative thin layer chromatography.

[0033] In one embodiment of the above aspect, the 3,6,7-trimethyllumazine is synthetic. In one embodiment, the composition further comprises honey.

[0034] In one embodiment, the composition comprises about 2.5 μg / mL to about 1000 μg / mL of 3,6,7-trimethyllumazine. In one embodiment, the composition comprises about 2.5 μg / mL, about 5 μg / mL, about 10 μg / mL, about 20 μg / mL, about 40 μg / mL, about 50 μg / mL, about 60 μg / mL, about 70 μg / mL, about 80 μg / mL, about 90 μg / mL, about 100 μg / mL, 150 μg / mL, about 200 μg / mL, about 250 μg / mL, about 300 μg / mL, about Contains 350μg / mL, about 400μg / mL, about 450about 500μg / mL, about 550μg / mL, about 600μg / mL, about 650μg / mL, about 700μg / mL, about 750μg / mL, about 800μg / mL, about 850μg / mL, about 900μg / mL, about 950μg / mL to about 1000μg / mL of 3,6,7-trimethyllumazine. Or therein, the composition may be about 2.5-5 μg / mL, about 5-10 μg / mL, about 10-20 μg / mL, about 20-40 μg / mL, about 40-50 μg / mL, about 50-60 μg / mL, about 60-70 μg / mL, about 70-80 μg / mL, about 80-90 μg / mL, about 90-100 μg / mL, about 100-150 μg / mL, 150-200 μg / mL, about 200-250 μg / mL, about 250-300 μg / mL, about 300-350 μg / mL, Contains about 350-400μg / mL, about 400-450μg / mL, about 450-500μg / mL, about 500-550μg / mL, about 550-600μg / mL, about 600-650μg / mL, about 650-700μg / mL, about 700-750μg / mL, about 750-800μg / mL, about 800-850μg / mL, about 850-900μg / mL, about 900-950μg / mL, and about 950-1000μg / mL of 3,6,7-trimethyllumazine.

[0035] In one embodiment, the composition comprises about 5 mg / kg to about 3000 mg / kg of 3,6,7-trimethyllumazine. In one embodiment, the composition comprises about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 55 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, about 100 mg / kg, about 150 mg / kg, about 200 mg / kg, about 250 mg / kg, about 300 mg / kg, about 350 mg / kg, about 400 mg / kg, about 450 mg / kg, about 500 mg / kg, about 550 mg / kg, about 600 mg / kg, about 650 mg / kg, about 700 mg / kg, about 750 mg / kg, about Contains 800mg / kg, about 850mg / kg, about 900mg / kg, about 950mg / kg, about 1000mg / kg, about 1100mg / kg, about 1200mg / kg, about 1300mg / kg, about 1400mg / kg, about 1500mg / kg, about 1600mg / kg, about 1700mg / kg, about 1800mg / kg, about 1900mg / kg, about 2000mg / kg, about 2100mg / kg, about 2200mg / kg, about 2300mg / kg, about 2400mg / kg, about 2500mg / kg, about 2600mg / kg, about 2700mg / kg, about 2800mg / kg, about 2900mg / kg to about 3000mg / kg of 3,6,7-trimethyllumazine. or in which the composition is administered at a dose of 5-10 mg / kg, or 10-15 mg / kg, or 15-20 mg / kg, or 20-25 mg / kg, or 25-30 mg / kg, or 30-35 mg / kg, or 35-40 mg / kg, or is 4 0-45mg / kg, or 45-50mg / kg, or 50-55mg / kg, or 55-60mg / kg, or 60 ~70mg / kg or 70-80mg / kg, about 90-100mg / kg, about 100-150mg / kg, about 150-200mg / kg, about 200mg / kg, about 250-300mg / kg, about 300-350mg / kg, about 350-400mg / kg, about 400-450mg / kg, about 450-500mg / kg, about 500-550mg / kg, about 550~600mg / kg, approx. 600~650mg / kg, approx. 650~700mg / kg, approx. 700~750mg / kg, approx. 750~800mg / kg, approx. 800~850m g / kg, approx. 850~900mg / kg, approx. 900~950mg / kg, approx. 950~1000mg / kg, approx. 1000~1100mg / kg, approx. 1100~1200mg / k g, approx. 1200~1300mg / kg, approx. 1300~1400mg / kg, approx. 1400~1500mg / kg, approx. 1500~1600mg / kg, approx. 1600~1700mg / kg, approximately 1700~1800mg / kg, approximately 1800~1900mg / kg, approximately 1900~2000mg / kg, approximately 2000~2100mg / kg, approximately 2100~2200 200-2300 mg / kg, about 2300-2400 mg / kg, about 2400-2500 mg / kg, about 2500-2600 mg / kg, about 2600-2700 mg / kg, about 2700-2800 mg / kg, about 2800-2900 mg / kg, and about 2900-3000 mg / kg of 3,6,7-trimethyllumazine.

[0036] In one embodiment of the above aspect, the composition comprises a therapeutically effective amount of 3,6,7-trimethyllumazine.

[0037] In one embodiment, the composition comprising 3,6,7-trimethyllumazine is formulated as a drug, therapeutic product, or dietary supplement. In one embodiment, the composition comprising 3,6,7-trimethyllumazine is formulated into a range of delivery systems including, but not limited to, liquid formulations, capsules, chewable tablets, tablets, suppositories, consumer goods, intravenous formulations, intramuscular formulations, subcutaneous formulations, solutions, foods, beverages, dietary supplements, cosmetic formulations, gels, lotions, powders, or sprays.

[0038] In one embodiment of the above aspect, the method comprises administering a composition comprising 3,6,7-trimethyllumazine 1, 2, 3, 4 or 5 times daily.

[0039] In one embodiment of the above aspect, the method comprises administering a composition comprising 3,6,7-trimethyllumazine 1, 2, 3, 4, 5, 6, or 7 times weekly.

[0040] In one embodiment, the composition comprising 3,6,7-trimethyllumazine is administered as a single dose or divided doses, hi one embodiment, the composition comprising 3,6,7-trimethyllumazine is administered as 1, 2, 3 or 4 separate doses.

[0041] In one embodiment of the above aspect, the method includes administering a composition comprising 3,6,7-trimethyllumazine at a dose of about 1 mg to about 3000 mg. In one particular embodiment, the method includes administering a composition comprising 3,6,7-trimethyllumazine at a dose of about 1 mg, 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg, 450 mg, 500 mg, 550 mg, 600 mg, 650 mg, 700 mg, 750 mg, 800 mg, 850 mg, 900 mg, 95 ... The present invention includes administering a composition comprising 0 mg, 1100 mg, 1200 mg, 1300 mg, 1400 mg, 1500 mg, 1600 mg, 1700 mg, 1800 mg, 1900 mg, 2000 mg, 2100 mg, 2200 mg, 2300 mg, 2400 mg, 2500 mg, 2600 mg, 2700 mg, 2800 mg, 2900 mg, or 3000 mg of 3,6,7-trimethyllumazine.

[0042] In one embodiment of the above aspect, the method includes administering the composition at a dose of about 5 g to about 100 g of 3,6,7-trimethyllumazine.

[0043] In one embodiment of the above aspect, a composition comprising 3,6,7-trimethyllumazine having a standardized concentration of 3,6,7-trimethyllumazine comprises selecting a first composition having a known concentration of 3,6,7-trimethyllumazine; selecting at least one additional composition having a known concentration of 3,6,7-trimethyllumazine; combining the first composition with the second composition to obtain a final composition having a standardized 3,6,7-trimethyllumazine concentration of about 5 mg / kg to about 3000 mg / kg; is obtained by

[0044] In one embodiment of the above aspect, a composition comprising 3,6,7-trimethyllumazine having a standardized concentration of 3,6,7-trimethyllumazine comprises selecting a first composition having a known concentration of 3,6,7-trimethyllumazine; The selected first composition Synthetic 3,6,7-trimethyllumazine, ○ Isolated 3,6,7-trimethyllumazine, honey extract containing 3,6,7-trimethyllumazine, and / or ○ 3,6,7-trimethyllumazine derived directly from the plant Leptospermum and forming a composition having a standardized 3,6,7-trimethyllumazine concentration of about 5 mg / kg to about 3000 mg / kg; is obtained by

[0045] In one embodiment, the composition comprises honey, honey extract, isolated 3,6,7-trimethyllumazine and / or synthetic 3,6,7-trimethyllumazine.

[0046] In one embodiment, the 3,6,7-trimethyllumazine derived directly from a plant is derived directly from the flowers, nectar, roots, fruits, seeds, bark, oils, leaves, wood, stems, or other plant material of a Leptospermum plant.

[0047] In one embodiment, the normalized 3,6,7-trimethyllumazine concentration is from about 5 mg / kg to about 3000 mg / kg. In one embodiment, the normalized 3,6,7-trimethyllumazine concentration is about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 55 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, about 100 mg / kg, about 150 mg / kg, about 200 mg / kg, about 250 mg / kg, about 300 mg / kg, about 350 mg / kg, about 400 mg / kg, about 450 mg / kg, about 500 mg / kg, about 550 mg / kg, about 600 mg / kg, about 650 mg / kg, about 700 mg / kg, about 75 0 mg / kg, about 800 mg / kg, about 850 mg / kg, about 900 mg / kg, about 950 mg / kg, about 1000 mg / kg, about 1100 mg / kg, about 1200 mg / kg, about 1300 mg / kg, about 1400 mg / kg, about 1500 mg / kg, about 1600 mg / kg, about 1700 mg / kg, about 1800 mg / kg, about 1900 mg / kg, about 2000 mg / kg, about 2100 mg / kg, about 2200 mg / kg, about 2300 mg / kg, about 2400 mg / kg, about 2500 mg / kg, about 2600 mg / kg, about 2700 mg / kg, about 2800 mg / kg, about 2900 mg / kg to about 3000 mg / kg of 3,6,7-trimethyllumazine.

[0048] In one embodiment, the concentration of 3,6,7-trimethyllumazine is determined by chromatography, analytical measurement, spectrophotometry, and / or any other method known to one of skill in the art, hi one embodiment, the concentration of 3,6,7-trimethyllumazine is determined by a reverse phase HPLC instrument.

[0049] In a fourth particular aspect, the present invention provides a. testing a first composition comprising honey for a 3,6,7-trimethyllumazine concentration; b. testing at least one additional composition comprising honey for 3,6,7-trimethyllumazine concentration; c. selecting a composition comprising honey having a 3,6,7-trimethyllumazine concentration greater than about 5 mg / kg 3,6,7-trimethyllumazine; d. selecting at least one additional composition comprising honey having a 3,6,7-trimethyllumazine concentration greater than about 5 mg / kg; e. combining the selected composition with honey to form a honey composition having a 3,6,7-trimethyllumazine concentration of at least about 5 to about 80 mg / kg; The present invention provides a method for making a composition having anti-inflammatory and / or MMP-9 inhibitory activity comprising:

[0050] In one embodiment of the fourth aspect, the composition comprising honey is selected if it has a 3,6,7-trimethyllumazine concentration of greater than about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 55 mg / kg, about 60 mg / kg, about 70 mg / kg or about 80 mg / kg.

[0051] In one embodiment of the fourth aspect, the composition is honey.

[0052] In one embodiment, the concentration of 3,6,7-trimethyllumazine is determined by chromatography, analytical measurement, spectrophotometry, and / or any other method known to one of skill in the art, hi one embodiment, the concentration of 3,6,7-trimethyllumazine is determined by a reverse phase HPLC instrument.

[0053] In one embodiment of the fourth aspect, the composition having anti-inflammatory activity is suitable for use in the method of any one of aspects 1-3.

[0054] In a fifth particular aspect, the present invention relates to a method for producing a composition comprising the steps of: a. testing the composition for 3,6,7-trimethyllumazine concentration; i. identifying a composition as having anti-inflammatory and / or MMP-9 inhibitory activity if the composition contains a concentration of 3,6,7-trimethyllumazine greater than about 5 mg / kg; or ii. identifying a composition as having no anti-inflammatory and / or MMP-9 inhibitory activity if the composition contains a concentration of 3,6,7-trimethyllumazine less than about 5 mg / kg; The present invention provides a method for identifying a composition having anti-inflammatory and / or MMP-9 inhibitory activity, comprising:

[0055] In one embodiment, the composition comprises honey or a honey extract.

[0056] In one embodiment of the fifth particular aspect, a composition comprising honey is determined to have anti-inflammatory activity if it contains more than about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 55 mg / kg, about 60 mg / kg, about 70 mg / kg or about 80 mg / kg.

[0057] In one embodiment of the fifth aspect, the composition is honey or a honey extract.

[0058] In one embodiment of the fifth particular aspect, the composition having anti-inflammatory activity is suitable for use in the method of any one of aspects 1-3.

[0059] In a sixth particular aspect, the present invention relates to a method for producing a composition comprising the steps of: a. testing the composition for 3,6,7-trimethyllumazine concentration; i. identifying the composition as suitable for use in any of aspects 1-3 if the composition contains a concentration of 3,6,7-trimethyllumazine greater than about 5 to about 80 mg / kg of 3,6,7-trimethyllumazine; or ii. identifying a composition as not suitable for use in any of the embodiments 1-3 if the composition contains a concentration of 3,6,7-trimethyllumazine less than about 5 mg / kg of 3,6,7-trimethyllumazine; The present invention provides a method for identifying a composition having anti-inflammatory and / or MMP-9 inhibitory activity suitable for use in the method of any of aspects 1 to 3, comprising:

[0060] In one embodiment of the sixth specific aspect, a composition is identified as suitable for use in the method of any one of aspects 1-4 if it contains a 3,6,7-trimethyllumazine concentration of greater than about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 55 mg / kg, about 60 mg / kg, about 70 mg / kg, or about 80 mg / kg.

[0061] In one embodiment, the composition is honey or a honey extract.

[0062] In one embodiment, the concentration of 3,6,7-trimethyllumazine is determined by chromatography, analytical measurement, spectrophotometry, and / or any other method known to one of skill in the art, hi one embodiment, the concentration of 3,6,7-trimethyllumazine is determined by a reverse phase HPLC instrument.

[0063] In a seventh particular aspect, the present invention provides a composition comprising 3,6,7-trimethyllumazine suitable for use in the method of any of the above aspects.

[0064] In one embodiment of the seventh aspect, the source of 3,6,7-trimethyllumazine is Leptospermum. In one embodiment, the 3,6,7-trimethyllumazine is substantially derived from a plant selected from the group consisting of Leptospermum scoparium, Leptospermum polygalifolium, Leptospermum subtenue, and combinations thereof. In one embodiment, the 3,6,7-trimethyllumazine is derived from Leptospermum scoparium.

[0065] In one embodiment of the seventh aspect, the source of 3,6,7-trimethyllumazine is honey. In one embodiment, the honey is substantially of floral origin from Leptospermum. In one embodiment, the honey is substantially of floral origin from Leptospermum scoparium, Leptospermum polygalifolium, Leptospermum subtenue, and / or combinations thereof. In one embodiment, the honey is substantially of floral origin from Leptospermum scoparium (Manuka).

[0066] In one embodiment of the seventh aspect, the 3,6,7-trimethyllumazine is directly derived from a plant of the genus Leptospermum. In one embodiment, the 3,6,7-trimethyllumazine is directly derived from the nectar, roots, fruits, seeds, bark, oil, leaves, wood, stems, or other plant material of a plant of the genus Leptospermum. In one embodiment, the 3,6,7-trimethyllumazine is substantially derived from a plant selected from the group including Leptospermum scoparium, Leptospermum polygalifolium, Leptospermum subtenue, and combinations thereof.

[0067] In one embodiment of the seventh aspect, the composition comprising 3,6,7-trimethyllumazine comprises honey. In one embodiment, the composition comprising 3,6,7-trimethyllumazine consists of honey.

[0068] In one embodiment of the above aspect, the composition comprising 3,6,7-trimethyllumazine comprises a honey extract. In one embodiment, the composition comprising 3,6,7-trimethyllumazine comprises a honey extract, wherein the honey extract comprises a concentration of 3,6,7-trimethyllumazine that is greater than the concentration of 3,6,7-trimethyllumazine found to occur naturally in honey. In one embodiment, the composition comprises a honey extract, wherein the honey extract comprises a concentration of 3,6,7-trimethyllumazine that is greater than the concentration of 3,6,7-trimethyllumazine found to occur naturally in honey. In one embodiment, the honey extract comprises a concentration of 3,6,7-trimethyllumazine that is greater than the concentration of 3,6,7-trimethyllumazine found to occur naturally in the honey from which the extract is derived.

[0069] In one embodiment, the honey from which the extract is derived is of floral origin substantially from the genus Leptospermum. In one embodiment, the honey from which the extract is derived is substantially from a species selected from Leptospermum scoparium, Leptospermum polygalifolium, Leptospermum subtenue, and combinations thereof. seed In one embodiment, the composition further comprises honey.

[0070] In one embodiment, the honey is raw honey, heat treated honey or pasteurized honey.

[0071] In one embodiment, the composition comprises 3,6,7-trimethyllumazine isolated from honey. In one embodiment, the honey is of floral origin substantially from the genus Leptospermum. In one embodiment, the honey is of floral origin substantially from the genus Leptospermum scoparium, Leptospermum polygalifolium, Leptospermum subtenue, and / or combinations thereof. seed In one embodiment, 3,6,7-trimethyllumazine is isolated by subjecting honey to solid phase extraction, followed by normal phase flash chromatography and preparative thin layer chromatography.

[0072] In one embodiment, the composition comprises synthetic 3,6,7-trimethyllumazine.In one embodiment, the composition further comprises honey.

[0073] In one embodiment, the composition comprises about 2.5 μg / mL to about 1000 μg / mL of 3,6,7-trimethyllumazine. In one embodiment, the composition comprises about 2.5 μg / mL, about 5 μg / mL, about 10 μg / mL, about 20 μg / mL, about 40 μg / mL, about 50 μg / mL, about 60 μg / mL, about 70 μg / mL, about 80 μg / mL, about 90 μg / mL, about 100 μg / mL, 150 μg / mL, about 200 μg / mL, about 250 μg / mL, about 300 μg / mL, about 350 μg / mL, about 400 μg / mL, about 450 μg / mL, about 500 μg / mL, about 600 μg / mL, about 700 μg / mL, about 800 μg / mL, about 900 μg / mL, about 1000 μg / mL, 1500 μg / mL, about 2000 μg / mL, about 250 μg / mL, about 3000 μg / mL, about 350 μg / mL, about 400 μg / mL, about 450 μg / mL, about 500 μg / mL, about 600 μg / mL, about 700 μg / mL, about 800 μg / mL, about 900 μg / mL, about 1000 μg / mL, 1500 μg / mL, about 2000 μg / mL, about 2500 μg / mL, about 3000 μg / mL, about 3500 μg / mL, about 4000 μg / mL, about 4500 μg / mL, about 5000 μg / mL, about 5000 μg / mL, about 6 500 μg / mL, about 550 μg / mL, about 600 μg / mL, about 650 μg / mL, about 700 μg / mL, about 750 μg / mL, about 800 μg / mL, about 850 μg / mL, about 900 μg / mL, about 950 μg / mL to about 1000 μg / mL of 3,6,7-trimethyllumazine, or therein the composition may comprise about 2.5 to 5 μg / mL, about 5 to 10 μg / mL, about 10 to 20 μg / mL, about 20~40μg / mL, approx. 40~50μg / mL, approx. 50~60μg / mL, approx. 60~70μg / mL, approx. 70~80μg / mL, approx. 80~90μg / mL, approx. 90~100μg / mL, approx. 100 ~150μg / mL, 150~200μg / mL, approx. 200~250μg / mL, approx. 250~300μg / mL, approx. 300~350μg / mL, approx. 350~400μg / mL, approx. 400~450μg / mL, about 450 to 500 μg / mL, about 500 to 550 μg / mL, about 550 to 600 μg / mL, about 600 to 650 μg / mL, about 650 to 700 μg / mL, about 700 to 750 μg / mL, about 750 to 800 μg / mL, about 800 to 850 μg / mL, about 850 to 900 μg / mL, about 900 to 950 μg / mL, and about 950 to 1000 μg / mL of 3,6,7-trimethyllumazine.

[0074] In one embodiment, the composition is about 5 mg / kg g~In one embodiment, the composition comprises about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 55 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, about 100 mg / kg, about 150 mg / kg, about 200 mg / kg, about 250 mg / kg, about 300 mg / kg, about 350 ... 00mg / kg, approx. 250mg / kg, approx. 300mg / kg, approx. 350mg / kg, approx. 400mg / kg, approx. 450mg / kg, approx. 500mg / kg, approx. 550mg / kg, approx. 600mg / kg, about 650mg / kg, about 700mg / kg, about 750mg / kg, about 800mg / kg, about 850mg / kg, about 900mg / kg, about 950mg / kg, about 1000mg / kg, about 1100mg / kg, approx. 1200mg / kg, approx. 1300mg / kg, approx. 1400mg / kg, approx. 1500mg / kg, approx. 1600mg / kg, approx. 1700mg / kg, approx. 1800mg / k g, approx. 1900mg / kg, approx. 2000mg / kg, approx. 2100mg / kg, approx. 2200mg / kg, approx. 2300mg / kg, approx. 2400mg / kg, approx. 2500mg / kg, approx. 2600m g / kg, about 2700 mg / kg, about 2800 mg / kg, about 2900 mg / kg to about 3000 mg / kg of 3,6,7-trimethyllumazine, or therein the composition may comprise 5-10 mg / kg, or 10-15 mg / kg, or 15-20 mg / kg, or 20-25 mg / kg, or 25-30 mg / kg, or 30-35 mg / kg, or 35-40 mg / kg, or is 4 0-45mg / kg, or 45-50mg / kg, or 50-55mg / kg, or 55-60mg / kg, or 60 ~70mg / kg or 70-80mg / kg, about 90-100mg / kg, about 100-150mg / kg, about 150-200mg / kg, about 200mg / kg, about 250-300mg / kg, about 300-350mg / kg, about 350-400mg / kg, about 400-450mg / kg, about 450-500mg / kg, about 500-550mg / kg, about 550~600mg / kg, approx. 600~650mg / kg, approx. 650~700mg / kg, approx. 700~750mg / kg, approx. 750~800mg / kg, approx. 800~850m g / kg, approx. 850~900mg / kg, approx. 900~950mg / kg, approx. 950~1000mg / kg, approx. 1000~1100mg / kg, approx. 1100~1200mg / k g, approx. 1200~1300mg / kg, approx. 1300~1400mg / kg, approx. 1400~1500mg / kg, approx. 1500~1600mg / kg, approx. 1600~1700mg / kg, approximately 1700~1800mg / kg, approximately 1800~1900mg / kg, approximately 1900~2000mg / kg, approximately 2000~2100mg / kg, approximately 2100~2200 200-2300 mg / kg, about 2300-2400 mg / kg, about 2400-2500 mg / kg, about 2500-2600 mg / kg, about 2600-2700 mg / kg, about 2700-2800 mg / kg, about 2800-2900 mg / kg, and about 2900-3000 mg / kg of 3,6,7-trimethyllumazine.

[0075] In one embodiment, the composition comprises a therapeutically effective amount of 3,6,7-trimethyllumazine.

[0076] In one embodiment, the composition comprising 3,6,7-trimethyllumazine is formulated as a drug, therapeutic product, or dietary supplement. In one embodiment, the composition comprising 3,6,7-trimethyllumazine is formulated into a range of delivery systems, including, but not limited to, liquid formulations, consumer goods, capsules, chewable tablets, tablets, suppositories, intravenous formulations, intramuscular formulations, subcutaneous formulations, solutions, edibles, beverages, dietary supplements, cosmetic formulations, gels, lotions, powders, or sprays.

[0077] In an eighth particular aspect, the present invention provides the use of a composition comprising 3,6,7-trimethyllumazine in the manufacture of a medicament for preventing, ameliorating or treating an MMP-9 associated condition in a subject.

[0078] In one embodiment of the eighth aspect, the MMP-9 associated condition is selected from gastrointestinal inflammatory diseases, gastric ulcers (e.g., peptic ulcers), gastritis, MMP-associated inflammatory conditions, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, irritable bowel syndrome (IBS), gastrointestinal disorders, gastroesophageal reflux disease (GERD), heartburn, acid reflux, Helicobacter pylori infection, oral ulcers, stomatitis, pharyngitis, gingivitis, esophageal ulcers, neuropsychiatric disorders (such as schizophrenia, bipolar disorder, multiple sclerosis), neurodegenerative disorders (such as traumatic brain injury, multiple sclerosis, and Alzheimer's disease), cardiovascular disease, cancer, and arthritis.

[0079] In a ninth particular aspect, the present invention provides the use of a composition comprising 3,6,7-trimethyllumazine in the manufacture of a medicament for preventing, ameliorating or treating an MMP-9 associated inflammatory condition in a subject.

[0080] In one embodiment of the ninth aspect, the MMP-9 associated inflammatory condition is associated with inflammation of the gastrointestinal tract, hi another embodiment, the MMP-9 associated inflammatory condition is selected from gastrointestinal inflammatory disease, gastric ulcer (e.g., peptic ulcer), gastritis, MMP-associated inflammatory condition, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, irritable bowel syndrome (IBS), gastrointestinal disease, gastroesophageal reflux disease (GERD), heartburn, acid reflux, Helicobacter pylori infection, oral ulcer, stomatitis, pharyngitis, gingivitis, and / or esophageal ulcer.

[0081] In one embodiment, the MMP-9 associated inflammatory condition is selected from neuropsychiatric disorders (such as schizophrenia, bipolar disorder, multiple sclerosis, etc.), neurodegenerative disorders (such as traumatic brain injury, multiple sclerosis, and Alzheimer's disease, etc.), cardiovascular disease, cancer, and arthritis.

[0082] In a tenth particular aspect, the present invention provides the use of a composition comprising 3,6,7-trimethyllumazine in the manufacture of a medicament for preventing, ameliorating or treating inflammation in the gastrointestinal tract of a subject.

[0083] In one embodiment of the tenth aspect, the invention provides a use for preventing, ameliorating or treating a condition associated with inflammation of the gastrointestinal tract, in one embodiment the gastrointestinal tract condition is selected from gastrointestinal inflammatory disease, gastric ulcer (e.g. peptic ulcer), gastritis, MMP-associated inflammatory conditions, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, irritable bowel syndrome (IBS), digestive disorders, gastroesophageal reflux disease (GERD), heartburn, acid reflux, Helicobacter pylori infection, oral ulcers, stomatitis, pharyngitis, gingivitis and / or esophageal ulcers.

[0084] In an eleventh particular aspect, the present invention provides the use of a composition comprising 3,6,7-trimethyllumazine in the manufacture of a medicament for preventing, ameliorating or treating inflammation in a subject.

[0085] In one embodiment of the eleventh particular aspect, the inflammation is associated with the gastrointestinal tract of the subject. In one embodiment, the inflammation is associated with a condition selected from gastrointestinal inflammatory disease, gastric ulcer (e.g., peptic ulcer), gastritis, MMP-associated inflammatory conditions, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, irritable bowel syndrome (IBS), gastrointestinal disorders, gastroesophageal reflux disease (GERD), heartburn, acid reflux, Helicobacter pylori infection, oral ulcer, stomatitis, pharyngitis, gingivitis, and / or esophageal ulcer.

[0086] In one embodiment, inflammation is associated with a condition selected from a neuropsychiatric disorder (such as schizophrenia, bipolar disorder, multiple sclerosis), a neurodegenerative disorder (such as traumatic brain injury, multiple sclerosis, and Alzheimer's disease), cardiovascular disease, cancer, and arthritis.

[0087] In a twelfth particular aspect, there is provided a method, use, or composition of any one of the above aspects, wherein the composition further comprises a COX-2 inhibitor.

[0088] In a thirteenth particular embodiment, there is provided a method or use according to any one of the preceding embodiments, further comprising co-administration of a COX-2 inhibitor.

[0089] The advantages of the above methods and uses may vary. In some embodiments, the source of 3,6,7-trimethyllumazine is naturally occurring and can be produced on a sustainable basis. 3,6,7-trimethyllumazine is expected to have no side effects, and it may be formulated in a wide variety of ways for various administration methods.

[0090] The invention may also be generally described as consisting in the parts, components, and properties, individually or collectively, referred to or indicated in the application specification, and in any or all combinations of any two or more of said parts, components, and properties, and where a specific integer having known equivalents in the art to which the invention pertains is referred to herein, such known equivalents are deemed to be incorporated herein as if individually set forth.

[0091] Further aspects of the present invention, which should be considered in all its novel aspects, will become apparent to those skilled in the art upon reading the following description, which provides at least one example of the practice of the invention.

[0092] Embodiments of the invention will now be described, by way of example only, with reference to the accompanying drawings, in which: [Brief description of the drawings]

[0093] [Figure 1] 1 is a graph illustrating the fluorescence intensity produced by MMP-9 activity over a 10 minute period. [Diagram 2] Graph illustrating the percentage inhibition of MMP-9 activity from 3,6,7-trimethyllumazine ranging from 2.5 to 40 μg / ml. Data are shown as mean±SEM. n=4. ****p<0.0001. [Diagram 3]FIG. 1 is a graph illustrating the correlation between 3,6,7-trimethyllumazine concentration and MMP-9 inhibition. The IC50 was calculated to be 11.5 μg / ml. Data are presented as mean±SEM. n=4. [Figure 4] 1 is a graph illustrating MMP-9 activity as measured by absorbance at 412 nm over 120 minutes. [Diagram 5] 1 is a graph illustrating the percentage of inhibition of MMP-9 activity by 3,6,7-trimethyllumazine (n=5, two replicates each). [Figure 6] Graph illustrating the lack of significant interaction between 3,6,7-trimethyllumazine and the chromogenic substrate (A) or reaction product (B) over a 20 minute period. [Figure 7] A typical gelatin gel zymograph showing gels incubated in normal developing buffer (columns 3-5), 3,6,7-trimethyllumazine loading buffer (columns 6-8), and NNGH (columns 9-11). The upper clear band represents gelatinase activity from the fibronectin domain of inactive MMP-9 (~47 kDa). The lower band represents gelatinase activity of active MMP-9 (~37 kDa) in which the prodomain has been cleaved. [Figure 8] 1 is a graph illustrating the percent inhibition from 3,6,7-trimethyllumazine by gelatin zymography (n=5). The percent inhibition was calculated by comparing the optical density with the negative control (no inhibitor). [Figure 9] Illustrates the docking of 3,6,7-trimethyllumazine into the S'1 substrate-binding pocket of MMP-9. A hydrogen bond was found between the NH group of 3,6,7-trimethyllumazine and the Tyr420 residue of MMP-9. [Figure 10] The amount of 3,6,7-trimethyllumazine (ng / mL) during gastric digestion of four manuka honey samples (A, B, C, D) as a function of digestion time. Data represent the mean ± SD, n=3. [Figure 11]Amount of 3,6,7-trimethyllumazine (ng / mL) during intestinal digestion of four manuka honey samples (A, B, C, D) as a function of digestion time. Data represent mean ± SD, n=3. [Figure 12] Amount of 6,3,6,7-trimethyllumazine (ng / mL) during gastric digestion of four 50% Manuka honey samples (A, B, C, D) as a function of digestion time. Data represent mean ± SD, n=3. [Figure 13] Amount of 3,6,7-trimethyllumazine (ng / mL) during intestinal digestion of four 50% Manuka honey samples (A, B, C, D) as a function of digestion time. Data represent mean ± SD, n=3. [Figure 14] The amount of 3,6,7-trimethyllumazine (ng / mL) during gastric digestion of pure 3,6,7-trimethyllumazine as a function of digestion time. Data represent the mean ± SD, n=3. [Figure 15] The amount of 3,6,7-trimethyllumazine (ng / mL) during the intestinal digestion of pure 3,6,7-trimethyllumazine is shown as a function of digestion time. Data represent the mean ± SD, n=3. [Figure 16] Figure 1 shows the effect of 3,6,7-trimethyllumazine (2.5–40 μg / mL) on cell viability. Data are presented as mean ± SD. [Figure 17] Figure 1 illustrates the effect of 3,6,7-trimethyllumazine (based on raw values) on lipopolysaccharide (055:B5, 1 μg / mL)-induced matrix metallopeptidase 9 (MMP-9) secretion in differentiated THP-1 cells (n=2 replicates). Lowercase letters represent significant differences between treatments. a: 40 μg / mL of 3,6,7-trimethyllumazine inhibits mmp-9 secretion (P=0.02); b: 30 μg / mL of 3,6,7-trimethyllumazine inhibits mmp-9 secretion (P=0.02). [Figure 18]Illustrates the effect of 3,6,7-trimethyllumazine (based on absolute values) on lipopolysaccharide (055:B5, 1 μg / mL)-induced matrix metallopeptidase 9 (MMP-9) secretion in differentiated THP-1 cells (n=2 replicates). Lowercase letters represent significant differences between treatments. a: 40 μg / mL 3,6,7-trimethyllumazine inhibits mmp-9 secretion (P=0.00); b: 30 μg / mL 3,6,7-trimethyllumazine inhibits mmp-9 secretion (P=0.04); c: azithromycin inhibits mmp-9 secretion (P=0.00). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0094] Described herein are compositions comprising 3,6,7-trimethyllumazine, methods and uses of the compositions for preventing, ameliorating or treating inflammation and / or inflammatory conditions, particularly inflammation or inflammatory conditions associated with the gastrointestinal tract, including MMP-9 associated inflammatory conditions.

[0095] definition For the purposes of this specification, the term "comprising" as used herein means "consisting in whole or at least in part." In interpreting statements herein that include this term, all features preceded by this term in each statement must be present, although other features may also be present. Related terms such as "comprise" and "comprised" should be interpreted in the same manner.

[0096] The terms "about" or "approximately" and grammatical variations thereof mean an amount, level, degree, value, number, frequency, percentage, size, amount, weight, or length that varies by as much as 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1%.

[0097] The term "medicament" or grammatical variations thereof refers to a pharmaceutical product, including, but not limited to, liquid formulations, capsules, tablets, chewable tablets, gels, lotions, powders, consumer goods, suppositories, cosmetic formulations, spray formulations, food formulations, beverages, intravenous formulations, intramuscular formulations, subcutaneous formulations, and solutions.

[0098] The term "therapeutic product" or grammatical variations thereof refers to a product that helps support, repair, or restore health. Products include, but are not limited to, consumer goods, liquid preparations, capsules, tablets, chewable tablets, gels, lotions, powders, suppositories, spray preparations, food preparations, beverages, cosmetic formulations, intravenous preparations, intramuscular preparations, subcutaneous preparations, and solutions.

[0099] The term "inflammatory condition" refers to a condition or disorder associated with unwanted and / or abnormal inflammation.

[0100] The term "inflammation" refers to the body's response resulting in redness, warmth, swelling, and / or pain as a result of infection, irritation, injury, disease, medical condition, or other cause. Inflammation may also be characterized at the cellular level. Cellular inflammation may be characterized by the production of various inflammatory mediators, such as cytokines, chemokines, or reactive nitrogen and oxygen species.

[0101] The term "anti-inflammatory" or grammatical variations thereof refers to the prevention, mitigation, quenching, calming, suppression or reduction of inflammation-associated cytokines, chemokines, reactive nitrogen and oxygen species when compared to a period, grade or situation in which the anti-inflammatory compound is not added. It also refers to the prevention, mitigation, quenching, calming or reduction of inflammation to the extent that redness, warmth, swelling and / or pain is reduced, the amount of reduction being relative to a period, grade or situation in which the anti-inflammatory compound is not added.

[0102] The term "therapeutically effective" with respect to the amount or dosage of a composition or agent refers to an amount of the composition sufficient to effectively prevent, ameliorate, or eliminate inflammation in a subject. The term should not be considered limiting. It may also refer to a dosage of the composition or agent that optimizes the anti-inflammatory effect on a subject depending on the desired application.

[0103] The term "dietary supplement" in the context of the present invention means a product that is intended to be supplemented in a subject's diet.

[0104] The term "treatment" should be considered in its broadest context. It does not necessarily mean that the subject is treated until complete recovery. Thus, "treatment" includes alleviating or ameliorating the symptoms or severity of a particular condition, or preventing or otherwise reducing the risk of developing a particular condition. It may also include maintaining or promoting a state of complete or partial remission of a condition.

[0105] The term "raw honey" means honey that has undergone only minimal heat treatment (e.g. below 50°C) or has not undergone any heat treatment.

[0106] The term "standardized concentration" in the context of the present invention means a concentration determined to fit a given concentration range.

[0107] As used herein, the term "and / or" means "and", or "or", or both.

[0108] As used herein, "(s)" following a noun refers to the plural and / or singular form of the noun.

[0109] Reference to a range of numbers disclosed herein (e.g., 1 to 10) is intended to incorporate all rational numbers within that range (e.g., 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9, and 10) as well as any rational number range within that range (e.g., 2 to 8, 1.5 to 5.5, and 3.1 to 4.7).

[0110] A "subject" may be a human or a non-human animal. Non-limiting examples of non-human animals are companion animals (e.g., cats and dogs), horses, cows, sheep, deer, and other livestock.

[0111] As discussed above, the inventors have identified that 3,6,7-trimethyllumazine, such as 3,6,7-trimethyllumazine found in honey, has anti-inflammatory activity. In particular, the inventors have surprisingly discovered that 3,6,7-trimethyllumazine has anti-inflammatory effects. In particular, the inventors have discovered that 3,6,7-trimethyllumazine has MMP-9 inhibitory activity. The ability to characterize the activity and stability of 3,6,7-trimethyllumazine provides the ability to manufacture compositions for the prevention, amelioration, and treatment of inflammation, including the prevention, amelioration, and treatment of various MMP-9-associated conditions and inflammatory symptoms, particularly inflammatory conditions of the gastrointestinal tract.

[0112] Pteridines are a group of compounds based on a pyrimido[4,5-b]pyrazine ring structure. The bicyclic compounds are produced naturally by many organisms and are often called pterins. Pteridines and pteridine derivatives are also synthesized. Many pteridine derivatives play important metabolic roles as enzymatic cofactors, including in the synthesis of nucleic acids, amino acids, neurotransmitters, nitric oxide, and purine and aromatic amino acids.

[0113] Herein we report 3,6,7-trimethyllumazine, a pteridine derivative from Leptospermum honey, compositions containing same, and methods of use. The isolation, structural elucidation, and synthesis of 3,6,7-trimethyllumazine have been previously described in commonly-filed New Zealand Patent Application No. 722140 (NZ722140), which is incorporated herein by reference.

[0114] Inflammation is a multifactorial phenomenon that is implicated in a wide range of diseases. In a healthy intestine, the intestinal mucosa is in a state of controlled response regulated by a complex balance between pro-inflammatory cytokines (e.g., tumor necrosis factor, TNF-α, interferon, IFN-γ, IL-1, IL-6) and anti-inflammatory cytokines (e.g., IL-4, IL-10). This defect may promote a complex interplay between genetic, microbial, and environmental factors, leading to persistent activation of immune / non-immune responses, causing active inflammation and tissue destruction. Failure to resolve inflammation is involved in the pathogenesis of gastrointestinal inflammatory-associated conditions such as gastric ulcers, inflammatory bowel disease (IBD), Crohn's disease, and ulcerative colitis.

[0115] Matrix metalloproteinases (MMPs) are a group of zinc-dependent endopeptidases that are key players involved in many biological and pathological processes, including inflammation and oxidative stress.

[0116] Inflammatory and oxidative stress-related conditions associated with MMP-9 include a range of different conditions, such as gastrointestinal inflammatory diseases, gastric ulcers (e.g., peptic ulcers), gastritis, MMP-related inflammatory conditions, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, irritable bowel syndrome (IBS), gastrointestinal disorders, gastroesophageal reflux disease (GERD), heartburn, acid reflux, Helicobacter pylori infection, oral ulcers, stomatitis, pharyngitis, gingivitis, esophageal ulcers, neuropsychiatric disorders (such as schizophrenia, bipolar disorder, multiple sclerosis), neurodegenerative disorders (such as traumatic brain injury, multiple sclerosis, and Alzheimer's disease), cardiovascular diseases, cancer, and arthritis.

[0117] One of the main roles of MMPs in inflammation is the regulation of physical barriers. Migration of inflammatory cells is promoted by MMPs due to their ability to digest intercellular junctions. Several key components of endothelial adherens junctions have been identified as substrates for MMPs. Degradation of these cellular components increases vascular permeability, thus allowing the influx of inflammatory cells and plasma proteins.

[0118] MMP-9 (also known as gelatinase B) is a proinflammatory enzyme that can proteolytically process several cytokines and chemokines into more active forms, such as pro-IL-1β and IL-8 (Schonbeck et al., 1998; Van den Steen, Proost, Wuyts, Van Damme, & Opdenakker, 2000). It has also been reported that MMP-9 can regulate epithelial barrier permeability by degrading occludin in tight junctions to facilitate the influx of inflammatory cells and proteins (Caron et al., 2005; Reijerkerk et al., 2006) and is deeply involved in extracellular matrix (ECM) degradation leading to mucosal injury and cellular remodelling (Swarnakar et al., 2007). MMP-9 is associated with several conditions, including neuropsychiatric disorders (e.g., schizophrenia, bipolar disorder, multiple sclerosis), neurodegenerative disorders (e.g., traumatic brain injury, multiple sclerosis, Alzheimer's disease), cardiovascular disease, cancer, and arthritis (Rybakowski 2009, Fingleton (2007), Reinhard, 2015).

[0119] MMP-9 is also closely related to the development and severity of gastric ulcers. Many studies have reported increased expression and activity of MMP-9 in the process of gastric ulcers (Pradeepkumar Singh, Kundu, Ganguly, Mishra, & Swarnakar, 2007; Swarnakar et al., 2005, 2007). It has also been reported that ethanol-induced gastric ulcers are associated with increased pro-MMP-9 activity in a dose-, time-, and severity-dependent manner, and that MMP-9 is a risk factor for gastric ulcer recurrence (Li et al., 2013).

[0120] The expression and secretion of MMP-9 is very low in normal healthy tissues. During gastric ulcer formation, induction of oxidative stress enhances the secretion of MMP-9, leading to mucosal damage (Ganguly & Swarnakar, 2012; Li et al., 2013). Therefore, MMP-9 is a known therapeutic target in the prevention and healing of gastric ulcers.

[0121] Thus, MMP-9-associated conditions are conditions in which expression of MMP-9 is increased, including inflammatory conditions in which expression or overexpression of MMP-9 is increased. Such conditions include, but are not limited to, gastric ulcers (e.g., peptic ulcers), gastritis, other MMP-associated inflammatory conditions, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, irritable bowel syndrome (IBS), gastrointestinal disorders, gastroesophageal reflux disease (GERD), heartburn, acid reflux, Helicobacter pylori infection, oral ulcers, stomatitis, pharyngitis, gingivitis, and / or esophageal ulcers. In a particular embodiment, the MMP-9-associated inflammatory condition is a gastric ulcer or gastritis. MMP-9 associated conditions also include other conditions such as neuropsychiatric disorders (such as schizophrenia, bipolar disorder, multiple sclerosis, etc.), neurodegenerative disorders (such as traumatic brain injury, multiple sclerosis, and Alzheimer's disease), cardiovascular disease, cancer, and arthritis.

[0122] As will be appreciated from the above, MMP-9 is a desirable target for preventing, ameliorating or treating inflammation and / or for preventing, ameliorating or treating conditions associated with inflammation, particularly for preventing, ameliorating or treating conditions associated with inflammation of the gastrointestinal tract. MMP-9 is also a desirable target for treating other conditions associated with MMP-9, such as neuropsychiatric disorders (such as schizophrenia, bipolar disorder, multiple sclerosis, etc.), neurodegenerative disorders (such as traumatic brain injury, multiple sclerosis, and Alzheimer's disease), cardiovascular diseases, cancer, and arthritis.

[0123] The inventors have discovered that 3,6,7-trimethyllumazine and compositions comprising same have MMP-9 inhibitory activity and are therefore useful in methods of preventing, ameliorating or treating MMP-9-associated conditions, such as those associated with inflammation and / or oxidative stress. The inventors have surprisingly discovered that 3,6,7-trimethyllumazine inhibits both the activity and expression of MMP-9. The MMP-9 inhibitory effect is significant, suggesting good efficiency and potentially broad application and use, particularly in the prevention and / or treatment of inflammation and / or inflammatory conditions, such as gastrointestinal inflammatory conditions. In particular, gastritis and gastric ulcers.

[0124] In one aspect, the present invention provides a method of preventing, ameliorating, or treating an MMP-9 associated condition in a subject in need thereof, comprising administering to a subject a composition comprising 3,6,7-trimethyllumazine.

[0125] In one embodiment, the MMP-9 associated inflammatory condition is selected from gastrointestinal inflammatory diseases, gastric ulcers (e.g., peptic ulcers), gastritis, MMP-associated inflammatory conditions, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, irritable bowel syndrome (IBS), gastrointestinal disorders, gastroesophageal reflux disease (GERD), heartburn, acid reflux, Helicobacter pylori infection, oral ulcers, stomatitis, pharyngitis, gingivitis, esophageal ulcers, neuropsychiatric disorders (such as schizophrenia, bipolar disorder, multiple sclerosis), neurodegenerative disorders (such as traumatic brain injury, multiple sclerosis, and Alzheimer's disease), cardiovascular disease, cancer, and arthritis.

[0126] In one aspect, the present invention provides a method of preventing, ameliorating, or treating an MMP-9-associated inflammatory condition in a subject in need thereof, comprising administering to a subject a composition comprising 3,6,7-trimethyllumazine.

[0127] In one embodiment, the MMP-9 associated inflammatory condition is associated with inflammation of the gastrointestinal tract, hi one embodiment, the MMP-9 associated inflammatory condition is selected from gastrointestinal inflammatory diseases, gastric ulcers (e.g., peptic ulcers), gastritis, MMP-associated inflammatory conditions, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, irritable bowel syndrome (IBS), gastrointestinal disorders, gastroesophageal reflux disease (GERD), heartburn, acid reflux, Helicobacter pylori infection, oral ulcers, stomatitis, pharyngitis, gingivitis, and esophageal ulcers.

[0128] In one embodiment, the MMP-9 associated inflammatory condition is selected from neuropsychiatric disorders (such as schizophrenia, bipolar disorder, multiple sclerosis, etc.), neurodegenerative disorders (such as traumatic brain injury, multiple sclerosis, and Alzheimer's disease, etc.), cardiovascular disease, cancer, and arthritis.

[0129] In another aspect, the present invention provides a method of preventing, ameliorating, or treating inflammation of the gastrointestinal tract in a subject in need thereof, comprising administering to a subject a composition comprising 3,6,7-trimethyllumazine.

[0130] In another aspect, the present invention provides a method for preventing, ameliorating, or treating a condition associated with inflammation of the gastrointestinal tract.

[0131] In one aspect, the invention provides a method of preventing, ameliorating, or treating inflammation in a subject in need thereof, comprising administering to a subject a composition comprising 3,6,7-trimethyllumazine, hi one embodiment, the inflammation is inflammation of the gastrointestinal tract.

[0132] In one embodiment, the invention provides a method of preventing, ameliorating, or treating inflammation associated with conditions such as gastrointestinal inflammatory diseases, gastric ulcers (e.g., peptic ulcers), gastritis, MMP-associated inflammatory conditions, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, irritable bowel syndrome (IBS), gastrointestinal disorders, gastroesophageal reflux disease (GERD), heartburn, acid reflux, Helicobacter pylori infection, mouth ulcers, stomatitis, pharyngitis, gingivitis, esophageal ulcers, neuropsychiatric disorders (such as schizophrenia, bipolar disorder, multiple sclerosis), neurodegenerative disorders (such as traumatic brain injury, multiple sclerosis, and Alzheimer's disease), cardiovascular disease, cancer, and arthritis.

[0133] In one embodiment, the invention provides a method of preventing, ameliorating, or treating conditions such as gastrointestinal inflammatory diseases, gastric ulcers (e.g., peptic ulcers), gastritis, MMP-associated inflammatory conditions, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, irritable bowel syndrome (IBS), gastrointestinal disorders, gastroesophageal reflux disease (GERD), heartburn, acid reflux, Helicobacter pylori infection, mouth ulcers, stomatitis, pharyngitis, gingivitis, esophageal ulcers, neuropsychiatric disorders (such as schizophrenia, bipolar disorder, multiple sclerosis), neurodegenerative disorders (such as traumatic brain injury, multiple sclerosis, and Alzheimer's disease), cardiovascular disease, cancer, and arthritis.

[0134] As will be appreciated from the above, 3,6,7-trimethyllumazine, and compositions containing same, may be useful in a wide range of other uses, including supporting or maintaining normal digestion in a subject, supporting or maintaining healthy digestion in a subject, and supporting or maintaining general intestinal health and well-being in a subject.

[0135] In one embodiment, the source of 3,6,7-trimethyllumazine in the methods, uses and compositions disclosed herein of the present invention is Leptospermum. In one embodiment, the 3,6,7-trimethyllumazine is substantially derived from a plant selected from the group consisting of Leptospermum scoparium, Leptospermum polygalifolium, Leptospermum subtenue, and combinations thereof. In one embodiment, the 3,6,7-trimethyllumazine is derived from Leptospermum scoparium (Manuka).

[0136] In one embodiment of this aspect of the invention, the source of 3,6,7-trimethyllumazine is honey. In one embodiment, the honey is substantially of floral origin from Leptospermum. In one embodiment, the honey is substantially of floral origin from Leptospermum scoparium, Leptospermum polygalifolium, Leptospermum subtenue, and combinations thereof.

[0137] In one embodiment the honey is of floral origin substantially derived from Leptospermum scoparium (also known as Manuka).

[0138] In one particular embodiment, the 3,6,7-trimethyllumazine is derived directly from a plant of the genus Leptospermum. In one embodiment of the invention, the 3,6,7-trimethyllumazine is derived directly from the nectar, roots, fruits, seeds, bark, oil, leaves, wood, stems, or other plant material of a plant of the genus Leptospermum. In one embodiment of the invention, the 3,6,7-trimethyllumazine is derived directly from the nectar of a plant of the genus Leptospermum. In one aspect, the 3,6,7-trimethyllumazine is derived substantially from a plant selected from the group including Leptospermum scoparium, Leptospermum polygalifolium, Leptospermum subtenue, and combinations thereof.

[0139] In one embodiment of the present invention, the 3,6,7-trimethyllumazine is synthetic.

[0140] For example, 3,6,7-trimethyllumazine may be synthesised as described in the applicant's earlier patent published as New Zealand Patent No. 722140, which is incorporated herein by reference and shown below.

[0141] Referring to the scheme below, N-methylation of 6-aminouracil (5) at the 3-position was achieved via silylation of the exocyclic amino and carbonyl groups upon treatment with hexamethyldisilazane (HDMS) in the presence of catalytic amounts of sulfuric acid (H2SO4) following the work of Gala et al. (1997). Ammonium sulfate can also be used as a catalyst. Methylation was then effected in two steps in 71% yield using iodomethane (Mel) in the presence of dimethylformamide (DMF) as the organic solvent. Dimethyl sulfate can also be used as a methylating agent. Subsequent desilylation during aqueous workup afforded 6-amino-3-methyluracil (6) in 78% yield.

[0142] The aminouracil (6) was then treated with sodium nitrite (NaNO2) and acetic acid (AcOH) solution followed by reduction with sodium dithionite (Na2S2O4) in aqueous ammonia (NH3) at 70 °C (Chaudhari et al., 2009), affording 5,6-diamino-3-methyluracil (7) in 31% yield in two steps. Alternative acids that can be used in the first step of the nitrosation include hydrochloric acid. An alternative to the first step reduction with sodium nitrite and acetic acid is catalytic hydrogenation using catalysts such as palladium on carbon or platinum dioxide in aqueous or organic solvents.

[0143] Condensation of diaminouracil (7) with 2,3-butanedione (8) in ethanol (EtOH) and acetic acid (AcOH) solution afforded 3,6,7-trimethyllumazine (3) as a colorless solid. An alternative acid for use in the condensation step is hydrochloric acid. Spectroscopic data (UV-vis, IR, 1 H NMR, 13 1C NMR (C NMR) data were in excellent agreement with those of the isolated natural product. 1 H NMR spectra of the separate natural and synthetic products 1 The H NMR spectrum was identical to that of 3,6,7-trimethyllumazine (3). Therefore, the structure of 3,6,7-trimethyllumazine (3) was unambiguously established as 3,6,7-trimethyllumazine. [ka]

[0144] Compound (9) [ka] An alternative synthesis of [ka] or via transformation of intermediate compounds shown below into fugitive isocyanate species, including but not limited to those generated by Curtius, Hoffman, Rossen, or Schmidt rearrangements. [ka]

[0145] With reference to the above, N-deuteromethylation of 6-aminouracil (5) at the 3-position was achieved via silylation of the exocyclic amino and carbonyl groups upon treatment with hexamethyldisilazane (HDMS) in the presence of catalytic amounts of sulfuric acid (H2SO4). Methylation then took place in two steps in 71% yield using iodomethane-d3 (CD3l) in the presence of dimethylformamide (DMF) as the organic solvent. Subsequent desilylation during aqueous workup afforded 6-amino-3-( 2 H3)-methyluracil (9) was obtained.

[0146] The aminouracil (6) was then treated with sodium nitrite (NaNO2) and acetic acid (AcOH) solution, followed by reduction with sodium dithionite (Na2S2O4) in aqueous ammonia (NH3) at 70 °C (Chaudhari et al., 2009) to give 5,6-diamino-3-( 2 H3)-methyluracil (10). Alternative acids that can be used in the first nitrosation step include hydrochloric acid. An alternative to the first step reduction with sodium nitrite and acetic acid is catalytic hydrogenation using catalysts such as palladium on carbon or platinum dioxide in aqueous or organic solvents.

[0147] Condensation of diaminouracil (10) with 2,3-butanedione (8) in ethanol (EtOH) and acetic acid (AcOH) solution affords the 3,6,7-(3- 2 H3) Trimethyllumazine (11) was obtained as a colorless solid.

[0148] Materials and Methods: All reactions were carried out in flame-dried or oven-dried glassware under a dry nitrogen atmosphere. All reagents were purchased as reagent grade and used without further purification. Dimethylformamide was degassed and dried using an LC Technical SP-1 solvent purifier. Ethanol was distilled over Mg(OEt)2. Ethyl acetate, methanol, and petroleum ether were distilled prior to use. All other solvents were used as received unless otherwise stated. Solid phase extraction (SPE) was performed using StrataC 18 RP-HPLC was performed on an Agilent 1100 with a Jupiter C column. 18 Analysis was performed using a 300 Å, 5 μm, 2.0 mm×250 mm column at a flow rate of 0.2 mL / min with a DAD detector operating at 262, 280, and 320 nm. An appropriately adjusted gradient of 5% B to 100% B was used, where solvent A was 0.1% HCOOH in HO and B was 20% A in MeCN. Flash chromatography was performed using 0.063-0.1 mm silica gel and the desired solvent. Thin layer chromatography (TLC) was performed using 0.2 mm Kieselgel F254 (Merck) silica gel plates and compounds were visualized using UV irradiation at 254 or 365 nm and / or staining with potassium permanganate and potassium carbonate solutions in aqueous sodium hydroxide. Preparative TLC was performed using 500 μm, 20×20 cm Uniplate™ (Analtech) silica gel TLC plates and compounds were visualized using UV irradiation at 254 or 365 nm. Melting points were determined on a Kofler hot stage apparatus and are uncorrected. Infrared spectra were obtained using a Perkin-Elmer Spectrum 100 FTIR spectrometer on a film ATR sampling accessory. Absorption maxima are expressed in wavenumbers (cm -1 The NMR spectrum is 1 400MHz for H nuclei, 13 a Bruker Avance 400 spectrometer operating at 100 MHz for C nuclei; 1400MHz for H nuclei, 13 a Bruker DRX-400 spectrometer operating at 100 MHz for C nuclei; 1 500MHz for H nuclei, 13 A Bruker Avance AVIII-HD500 spectrometer operating at 125 MHz for C nuclei; or 1 600MHz for H nuclei, 13 For C nuclei, the data were recorded as indicated on a Bruker Avance 600 spectrometer operating at 150 MHz. 1 H and 13 The chemical shifts of C are 1 H and 13 C) or (CD3)2SO( 1 H and 13 C) are reported in parts per million (ppm). 15 The N chemical shifts are expressed as the integrated Ξ i were referenced using the scale (Harris et al., 2008). 1 H NMR data are reported as chemical shifts, relative integrals, and multiplicities (s, singlet, assignments). Assignments were made with the aid of COSY, NOESY, HSQC, and HMBC experiments as necessary. High-resolution mass spectra were recorded on a Bruker micrOTOF-Q II mass spectrometer equipped with an ESI ionization source. UV-visible spectra were run as solutions in HO on a Shimadzu UV-2101PC scanning spectrophotometer.

[0149] In one embodiment, the present invention provides a composition comprising 3,6,7-trimethyllumazine for use in the methods described above. In one embodiment, the composition comprises a therapeutically effective amount of 3,6,7-trimethyllumazine.

[0150] In one embodiment of the invention, the composition comprising 3,6,7-trimethyllumazine comprises honey. In one particular embodiment, the composition comprising 3,6,7-trimethyllumazine consists of honey.

[0151] In one embodiment, the honey is of floral origin substantially from the genus Leptospermum, hi one embodiment, the honey is substantially from a plant selected from the group including Leptospermum scoparium, Leptospermum polygalifolium, Leptospermum subtenue, and combinations thereof.

[0152] In one embodiment, the composition comprises about 2.5 μg / mL to about 80 μg / mL of 3,6,7-trimethyllumazine. In one embodiment, the composition comprises about 2.5 μg / mL, about 5 μg / mL, about 10 μg / mL, about 20 μg / mL, about 40 μg / mL, about 50 μg / mL, about 60 μg / mL, about 70 μg / mL, or about 80 μg / mL of 3,6,7-trimethyllumazine, or therein the composition comprises 3,6,7-trimethyllumazine at a concentration of 2.5 μg / mL to 5 μg / mL, or 5 μg / mL to 10 μg / mL, or 10 μg / mL to 20 μg / mL, or 20 μg / mL to 40 μg / mL, or 40 μg / mL to 50 μg / mL, or 50 μg / mL to about 60 μg / mL, or 60 μg / mL to 70 μg / mL, or 70 μg / mL to 80 μg / mL of 3,6,7-trimethyllumazine.

[0153] In one embodiment, the composition comprises about 5 to about 80 mg / kg of 3,6,7-trimethyllumazine. In one embodiment, the composition comprises about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 55 mg / kg, about 60 mg / kg, about 70 mg / kg, or about 80 mg / kg of 3,6,7-trimethyllumazine, or therein, the composition comprises 5-10 mg / kg, or 10-15 mg / kg, or 15-20 mg / kg, or 20-25 mg / kg, or 25-30 mg / kg, or 30-35 mg / kg, or 35-40 mg / kg, or is 40-45mg / kg, or 45-50mg / kg, or 50-55mg / kg, or 55-60mg / kg, or 60 ~ Contains 3,6,7-trimethyllumazine at a concentration of 70 mg / kg or 70 to 80 mg / kg.

[0154] In one embodiment, the honey is raw honey. In one embodiment, the honey is heat treated or pasteurized according to methods well known to those skilled in the art.

[0155] In one particular embodiment, the composition comprises a honey extract. In one embodiment, the composition consists of a honey extract.

[0156] In one embodiment, the honey extract comprises a concentration of 3,6,7-trimethyllumazine that is greater than the concentration of 3,6,7-trimethyllumazine found naturally occurring in honey.

[0157] In one embodiment, the honey extract comprises a concentration of 3,6,7-trimethyllumazine that is higher than the concentration of 3,6,7-trimethyllumazine found naturally occurring in the honey from which the extract is derived.

[0158] In one embodiment, the honey from which the extract is derived is of floral origin substantially from the genus Leptospermum, hi one embodiment, the honey from which the extract is derived is substantially from a plant selected from the group comprising Leptospermum scoparium, Leptospermum polygalifolium, Leptospermum subtenue, and combinations thereof.

[0159] In one embodiment, the extract contains about 2.5 μg / mL to about 1000 μg / mL of 3,6,7-trimethyllumazine. In one embodiment, the extract contains about 2.5 μg / mL, about 5 μg / mL, about 10 μg / mL, about 20 μg / mL, about 40 μg / mL, about 50 μg / mL, about 60 μg / mL, about 70 μg / mL, about 80 μg / mL, about 90 μg / mL, about 100 μg / mL, 150 μg / mL, about 200 μg / mL, about 250 μg / mL, about 300 μg / mL, about 350 μg / mL, about 400 μg / mL, about 450 μg / mL, about 500 μg / mL, about 600 μg / mL, about 700 μg / mL, about 800 μg / mL, about 900 μg / mL, about 1000 μg / mL, 1500 μg / mL, about 2000 μg / mL, about 250 μg / mL, about 3000 μg / mL, about 350 μg / mL, about 400 μg / mL, about 450 μg / mL, about 500 μg / mL, about 600 μg / mL, about 700 μg / mL, about 800 μg / mL, about 900 μg / mL, about 1000 μg / mL, 1500 μg / mL, about 2000 μg / mL, about 2500 μg / mL, about 3000 μg / mL, about 350 μg / mL, about 4000 μg / mL, about 4500 μg / mL, about 5000 μg / mL, about 6000 μg / mL, about 70 About 500 μg / mL, about 550 μg / mL, about 600 μg / mL, about 650 μg / mL, about 700 μg / mL, about 750 μg / mL, about 800 μg / mL, about 850 μg / mL, about 900 μg / mL, about 950 μg / mL, about 1000 μg / mL, or within which the composition may comprise about 2.5-5 μg / mL, about 5-10 μg / mL, about 10-20 μg / mL, about 20 ~40μg / mL, approximately 40~50μg / mL, approximately 50~60μg / mL, approximately 60~70μg / mL, approximately 70~80μg / mL, approximately 80~90μg / mL, approximately 90~100μg / mL, approximately 100~1 50μg / mL, 150~200μg / mL, approx. 200~250μg / mL, approx. 250~300μg / mL, approx. 300~350μg / mL, approx. 350~400μg / mL, approx. 400~450μg / mL mL, about 450-500 μg / mL, about 500-550 μg / mL, about 550-600 μg / mL, about 600-650 μg / mL, about 650-700 μg / mL, about 700-750 μg / mL, about 750-800 μg / mL, about 800-850 μg / mL, about 850-900 μg / mL, about 900-950 μg / mL, and about 950-1000 μg / mL of 3,6,7-trimethyllumazine.

[0160] In one embodiment, the extract contains about 5 to about 3000 mg / kg of 3,6,7-trimethyllumazine. In one embodiment, the extract contains about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 55 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, about 100 mg / kg, about 150 mg / kg, about 200 mg / kg, about 30 ...0 mg / kg, about 50 mg / kg, about 55 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, about 100 mg / kg, about 150 mg / kg, about 200 mg / kg, about 300 mg / kg, about 40 mg / kg, about 50 mg / kg, 00mg / kg, approx. 250mg / kg, approx. 300mg / kg, approx. 350mg / kg, approx. 400mg / kg, approx. 450mg / kg, approx. 500mg / kg, approx. 550mg / kg, approx. 600mg / kg, about 650mg / kg, about 700mg / kg, about 750mg / kg, about 800mg / kg, about 850mg / kg, about 900mg / kg, about 950mg / kg, about 1000mg / kg, about 1100mg / kg, approx. 1200mg / kg, approx. 1300mg / kg, approx. 1400mg / kg, approx. 1500mg / kg, approx. 1600mg / kg, approx. 1700mg / kg, approx. 1800mg / k g, approx. 1900mg / kg, approx. 2000mg / kg, approx. 2100mg / kg, approx. 2200mg / kg, approx. 2300mg / kg, approx. 2400mg / kg, approx. 2500mg / kg, approx. 2600m g / kg, about 2700 mg / kg, about 2800 mg / kg, about 2900 mg / kg to about 3000 mg / kg of 3,6,7-trimethyllumazine, or therein, the extract contains 5-10 mg / kg, or 10-15 mg / kg, or 15-20 mg / kg, or 20-25 mg / kg, or 25-30 mg / kg, or 30-35 mg / kg, or 35-40 mg / kg, or is 4 0-45mg / kg, or 45-50mg / kg, or 50-55mg / kg, or 55-60mg / kg, or 60 ~70mg / kg or 70-80mg / kg, about 90-100mg / kg, about 100-150mg / kg, about 150-200mg / kg, about 200mg / kg, about 250-300mg / kg, about 300-350mg / kg, about 350-400mg / kg, about 400-450mg / kg, about 450-500mg / kg, about 500-550mg / kg, about 550~600mg / kg, approx. 600~650mg / kg, approx. 650~700mg / kg, approx. 700~750mg / kg, approx. 750~800mg / kg, approx. 800~850m g / kg, approx. 850~900mg / kg, approx. 900~950mg / kg, approx. 950~1000mg / kg, approx. 1000~1100mg / kg, approx. 1100~1200mg / k g, approx. 1200~1300mg / kg, approx. 1300~1400mg / kg, approx. 1400~1500mg / kg, approx. 1500~1600mg / kg, approx. 1600~1700mg / kg, approximately 1700~1800mg / kg, approximately 1800~1900mg / kg, approximately 1900~2000mg / kg, approximately 2000~2100mg / kg, approximately 2100~2200 200-2300 mg / kg, about 2300-2400 mg / kg, about 2400-2500 mg / kg, about 2500-2600 mg / kg, about 2600-2700 mg / kg, about 2700-2800 mg / kg, about 2800-2900 mg / kg, and about 2900-3000 mg / kg of 3,6,7-trimethyllumazine.

[0161] In one embodiment, the composition comprises at least 0.1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% 3,6,7-trimethyllumazine.

[0162] In one embodiment, the composition comprises a honey extract and further comprises honey.

[0163] In one embodiment, the composition comprises isolated 3,6,7-trimethyllumazine isolated from honey. In one embodiment, the honey is substantially of floral origin from the genus Leptospermum. In one embodiment, the honey is substantially from a plant selected from the group including Leptospermum scoparium, Leptospermum polygalifolium, Leptospermum subtenue, and combinations thereof.

[0164] In one embodiment, 3,6,7-trimethyllumazine is isolated by any method known to one of skill in the art, hi one embodiment, 3,6,7-trimethyllumazine is isolated by subjecting honey to SPE (solid phase extraction), followed by normal phase flash chromatography and preparative TLC (thin layer chromatography).

[0165] In one embodiment, 3,6,7-trimethyllumazine is isolated by the method described in the applicant's earlier patent published as New Zealand Patent No. 722140, which is incorporated herein by reference and as set forth below. 。

[0166] Chemical isolation of 3,6,7-trimethyllumazine Raw manuka honey (51.3 g) was dissolved in HO+0.1% HCOOH (150 mL) and sonicated for 20 min. The resulting suspension was filtered through Celite and the filtrate was used in the subsequent step.

[0167] The filtrate was divided into two portions of 100 mL, and each portion was subjected to SPE using MeOH-H2O+0.1% HCOOH (1:9, 80 mL) to remove undesired materials. The desired fraction was then eluted using MeOH-H2O+0.1% HCOOH (4:1, 80 mL). The two fractions were combined and concentrated to give a crude extract (0.23 g), which was further purified by flash chromatography (1:4 petroleum ether-EtOAc) to give the purified extract (3 mg) as a brown solid.

[0168] Several purified extracts were combined (total 6 mg) and further purified by preparative TLC (1:3 petroleum ether-EtOAc, 4 times) to give 3 (4 mg) (shown below) as a colorless solid. [ka]

[0169] New Zealand and Australian honeys derived from species of the genera Leptospermum, Eucalyptus, Kunzea, and Knightia were examined using HPLC to confirm the presence of leptosperin (4), a proposed biomarker for Leptospermum honey (Kato et al. 2012 and 2014; Aitken, et al. 2013; structure shown below), which showed unexpected UV absorbance at 320 nm. [ka]

[0170] This peak was observed only in honeys from Leptospermum species (L. scoparium, L. scoparium var. eximium (exinium), L. polygalifolium, and L. subtenue), including honey from L. subtenue, where leptosperin was not detected. The use of solid phase extraction (SPE) followed by reversed-phase HPLC allowed purification of the compound, which exhibits UV absorbance at 320 nm. However, this method is time-consuming, has low yields, and is not scalable, so a more efficient isolation method was required. Subjecting manuka honey to SPE, followed by normal-phase flash chromatography and preparative TLC, allowed the isolation of 3,6,7-trimethyllumazine as a colorless solid in sufficient quantities to perform spectroscopic analysis.

[0171] In one embodiment of the invention, the composition comprises synthetic 3,6,7-trimethyllumazine or isolated 3,6,7-trimethyllumazine. In one embodiment, the composition further comprises honey. In one embodiment, the composition consists of synthetic 3,6,7-trimethyllumazine and honey. In one embodiment, the composition consists of isolated 3,6,7-trimethyllumazine and honey.

[0172] In one embodiment, the honey is of floral origin substantially from the genus Leptospermum, hi one embodiment, the honey is substantially from a plant selected from the group including Leptospermum scoparium, Leptospermum polygalifolium, Leptospermum subtenue, and combinations thereof.

[0173] In one embodiment, the composition comprises about 2.5 μg / mL to about 1000 μg / mL of 3,6,7-trimethyllumazine, synthetic 3,6,7-trimethyllumazine or isolated 3,6,7-trimethyllumazine. In one embodiment, the composition comprises about 2.5 μg / mL, about 5 μg / mL, about 10 μg / mL, about 20 μg / mL, about 40 μg / mL, about 50 μg / mL, about 60 μg / mL, about 70 μg / mL, about 80 μg / mL, about 90 μg / mL, about 100 μg / mL, 150 μg / mL, about 200 μg / mL, about 250 μg / mL, about 300 μg / mL, about 350 μg / mL, about 400 μg / mL, about 450 μg / mL, about 500 μg / mL, about 600 μg / mL, about 700 μg / mL, about 800 μg / mL, about 900 μg / mL, about 1000 μg / mL, 1500 μg / mL, about 2000 μg / mL, about 2500 μg / mL, about 3000 μg / mL, about 3500 μg / mL, about 4000 μg / mL, about 5000 μg / mL, about 5000 μg / mL, about 6000 μg / mL, about 7000 μg / mL, about 8000 μg / mL, about 9000 μg / mL, about 10000 μg / mL, about 15 ... 50 μg / mL, about 600 μg / mL, about 650 μg / mL, about 700 μg / mL, about 750 μg / mL, about 800 μg / mL, about 850 μg / mL, about 900 μg / mL, about 950 μg / mL to about 1000 μg / mL of synthetic or isolated 3,6,7-trimethyllumazine, or therein, the composition may comprise about 2.5-5 μg / mL, about 5-10 μg / mL, about 10-20 μg / mL, about 20~40μg / mL, approx. 40~50μg / mL, approx. 50~60μg / mL, approx. 60~70μg / mL, approx. 70~80μg / mL, approx. 80~90μg / mL, approx. 90~100μg / mL, approx. 100~150μg / mL, 150~200μg / mL, approx. 200~250μg / mL, approx. 250~300μg / mL, approx. 300~350μg / mL, approx. 350~400μg / mL, approx. 400~450μg / mL, approx. 450~50 The present invention includes synthetic 3,6,7-trimethyllumazine or isolated 3,6,7-trimethyllumazine of about 0 μg / mL, about 500 to 550 μg / mL, about 550 to 600 μg / mL, about 600 to 650 μg / mL, about 650 to 700 μg / mL, about 700 to 750 μg / mL, about 750 to 800 μg / mL, about 800 to 850 μg / mL, about 850 to 900 μg / mL, about 900 to 950 μg / mL, or about 950 to 1000 μg / mL.

[0174] In one embodiment, the composition comprises about 5 mg / kg to about 3000 mg / kg of synthetic or isolated 3,6,7-trimethyllumazine. In one embodiment, the composition comprises about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 55 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, about 100 mg / kg, about 150 mg / kg, about 200 mg / kg, or about 300 mg / kg. g, about 250mg / kg, about 300mg / kg, about 350mg / kg, about 400mg / kg, about 450mg / kg, about 500mg / kg, about 550mg / kg, about 600mg / kg, about 650m g / kg, about 700mg / kg, about 750mg / kg, about 800mg / kg, about 850mg / kg, about 900mg / kg, about 950mg / kg, about 1000mg / kg, about 1100mg / kg, about 1200mg / kg, about 1300mg / kg, about 1400mg / kg, about 1500mg / kg, about 1600mg / kg, about 1700mg / kg, about 1800mg / kg, about 1900mg / kg, About 2000mg / kg, about 2100mg / kg, about 2200mg / kg, about 2300mg / kg, about 2400mg / kg, about 2500mg / kg, about 2600mg / kg, about 2700mg / kg, about 2800 mg / kg, about 2900 mg / kg to about 3000 mg / kg of synthetic or isolated 3,6,7-trimethyllumazine, or therein, the composition may comprise 5-10 mg / kg, or 10-15 mg / kg, or 15-20 mg / kg, or 20-25 mg / kg, or 25-30 mg / kg, or 30-35 mg / kg, or 35-40 mg / kg, or is 4 0-45mg / kg, or 45-50mg / kg, or 50-55mg / kg, or 55-60mg / kg, or 60 ~70mg / kg or 70-80mg / kg, about 90-100mg / kg, about 100-150mg / kg, about 150-200mg / kg, about 200mg / kg, about 250-300mg / kg, about 300-350mg / kg, about 350-400mg / kg, about 400-450mg / kg, about 450-500mg / kg, about 500-550mg / kg, about 550- 600mg / kg, approx. 600~650mg / kg, approx. 650~700mg / kg, approx. 700~750mg / kg, approx. 750~800mg / kg, approx. 800~850mg / kg, approx. 850~900mg / kg, approx. 900~950mg / kg, approx. 950~1000mg / kg, approx. 1000~1100mg / kg, approx. 1100~1200mg / kg, approx. 1200~1 300mg / kg, approx. 1300~1400mg / kg, approx. 1400~1500mg / kg, approx. 1500~1600mg / kg, approx. 1600~1700mg / kg, approx. 1700~1 800mg / kg, approximately 1800~1900mg / kg, approximately 1900~2000mg / kg, approximately 2000~2100mg / kg, approximately 2100~2200mg / kg, approximately 2200~2 300 mg / kg, about 2300 to 2400 mg / kg, about 2400 to 2500 mg / kg, about 2500 to 2600 mg / kg, about 2600 to 2700 mg / kg, about 2700 to 2800 mg / kg, about 2800 to 2900 mg / kg, or about 2900 to 3000 mg / kg of synthetic 3,6,7-trimethyllumazine or isolated 3,6,7-trimethyllumazine.

[0175] In one embodiment, the composition comprises 0.1%-100% 3,6,7-trimethyllumazine.In one embodiment, the composition comprises at least 0.1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or substantially pure 3,6,7-trimethyllumazine.

[0176] Compositions containing honey-derived 3,6,7-trimethyllumazine and / or synthetic 3,6,7-trimethyllumazine are expected to be free of side effects. 3,6,7-trimethyllumazine occurs naturally in some honeys, and such honeys containing 3,6,7-trimethyllumazine have been sold and consumed for many years.

[0177] Compositions containing 3,6,7-trimethyllumazine may be formulated as a medicine, therapeutic product, or dietary supplement.

[0178] In one embodiment, the composition comprising 3,6,7-trimethyllumazine is formulated as a drug, therapeutic product, or dietary supplement. In one embodiment, the composition comprising 3,6,7-trimethyllumazine is formulated into a range of delivery systems, including, but not limited to, liquid formulations, capsules, consumer products, chewable tablets, tablets, suppositories, intravenous formulations, intramuscular formulations, subcutaneous formulations, solutions, edibles, beverages, dietary supplements, cosmetic formulations, gels, lotions, powders, or sprays.

[0179] In a specific embodiment, the method of the present invention includes administering a composition containing about 1 mg to about 3000 mg of 3,6,7-trimethyllumazine. , 1000mg, 1100mg, 1200mg, 1300mg, 1400mg, 1500mg, 1600mg, 1700mg, 1800mg, 1900mg, 2000mg, 2100mg, 2200mg, 2300mg, 2400mg, 2500mg, 2600mg, 2700mg, 2800mg, 2900mg, or 3000mg of 3,6,7-trimethyllumazine.

[0180] In one particular embodiment, the method of the invention described above includes administering a composition comprising 3,6,7-trimethyllumazine, wherein the composition is honey or a honey extract. In one embodiment, the honey in the method of the invention is administered in a dose of about 5 g to about 100 g. In one embodiment, the honey is administered in a dose of about 5 g, 10 g, 15 g, 20 g, 25 g, 30 g, 40 g, 50 g, 60 g, 70 g, 80 g, 90 g, 100 g. In one embodiment, the honey is administered in a dose equivalent to about 1 teaspoon to about 5 tablespoons of honey. In one embodiment, the honey is administered as a single dose or multiple doses.

[0181] In one embodiment, the composition comprising 3,6,7-trimethyllumazine is administered as a single dose or divided doses, hi one embodiment, the composition comprising 3,6,7-trimethyllumazine is administered as 1, 2, 3 or 4 separate doses.

[0182] In one particular embodiment, the method of the invention described above comprises administering a composition comprising 3,6,7-trimethyllumazine 1, 2, 3, or 4 times daily, hi another embodiment, the method of the invention comprises administering a composition comprising 3,6,7-trimethyllumazine 1, 2, 3, 4, 5, 6, or 7 times weekly.

[0183] The concentration of 3,6,7-trimethyllumazine can vary significantly from honey sample to honey sample, therefore, in one particular embodiment of the invention described herein, the composition comprising honey has a standardized concentration of 3,6,7-trimethyllumazine.

[0184] In one embodiment, a composition comprising 3,6,7-trimethyllumazine is - selecting a first composition having a known concentration of 3,6,7-trimethyllumazine; - selecting at least one additional composition having a known concentration of 3,6,7-trimethyllumazine; - combining the first composition with the second composition to obtain a final composition having a standardized 3,6,7-trimethyllumazine concentration of about 5 mg / kg to about 3000 mg / kg. The standardized concentration of 3,6,7-trimethyllumazine is obtained by:

[0185] In one embodiment, a composition comprising 3,6,7-trimethyllumazine is - selecting a first composition having a known concentration of 3,6,7-trimethyllumazine; - a first composition selected Synthetic 3,6,7-trimethyllumazine, ○ Isolated 3,6,7-trimethyllumazine, honey extract containing 3,6,7-trimethyllumazine, and / or ○ 3,6,7-trimethyllumazine derived directly from the plant Leptospermum to form a composition having a standardized 3,6,7-trimethyllumazine concentration of about 5 mg / kg to about 3000 mg / kg. The standardized concentration of 3,6,7-trimethyllumazine is obtained by:

[0186] In one embodiment, a composition comprising 3,6,7-trimethyllumazine is - selecting a first composition comprising honey having a known concentration of 3,6,7-trimethyllumazine; - a first composition containing honey is selected; Synthetic 3,6,7-trimethyllumazine, ○ Isolated 3,6,7-trimethyllumazine, and honey extract containing 3,6,7-trimethyllumazine, and / or ○ 3,6,7-trimethyllumazine derived directly from the plant Leptospermum to form a composition having a standardized 3,6,7-trimethyllumazine concentration of about 5 to about 3000 mg / kg. The standardized concentration of 3,6,7-trimethyllumazine is obtained by:

[0187] In one embodiment, the composition comprises honey, honey extract, isolated 3,6,7-trimethyllumazine and / or synthetic 3,6,7-trimethyllumazine.

[0188] In one embodiment, the 3,6,7-trimethyllumazine derived directly from a plant is derived directly from the flowers, nectar, roots, fruits, seeds, bark, oils, leaves, wood, stems, or other plant material of a Leptospermum plant.

[0189] In one embodiment, the standardized 3,6,7-trimethyllumazine concentration is about 2.5 μg / mL to about 1000 μg / mL of 3,6,7-trimethyllumazine. In one embodiment, the standardized 3,6,7-trimethyllumazine concentration is about 2.5 μg / mL, about 5 μg / mL, about 10 μg / mL, about 20 μg / mL, about 40 μg / mL, about 50 μg / mL, about 60 μg / mL, about 70 μg / mL, about 80 μg / mL, about 90 μg / mL, about 100 μg / mL, 150 μg / mL, about 200 μg / mL, about 250 μg / mL, About 300 μg / mL, about 350 μg / mL, about 400 μg / mL, about 450 about 500 μg / mL, about 550 μg / mL, about 600 μg / mL, about 650 μg / mL, about 700 μg / mL, about 750 μg / mL, about 800 μg / mL, about 850 μg / mL, about 900 μg / mL, about 950 μg / mL, to about 1000 3,6,7-trimethyllumazine.

[0190] In one embodiment, the normalized 3,6,7-trimethyllumazine concentration is from about 5 mg / kg to about 3000 mg / kg. In one embodiment, the normalized 3,6,7-trimethyllumazine concentration is about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 55 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, about 100 mg / kg, about 150 mg / kg, about 200 mg / kg, about 250 mg / kg, about 300 mg / kg, about 350 mg / kg, about 400 mg / kg, about 450 mg / kg, about 500 mg / kg, about 550 mg / kg, about 600 mg / kg, about 650 mg / kg, about 700 mg / kg, about 75 0 mg / kg, about 800 mg / kg, about 850 mg / kg, about 900 mg / kg, about 950 mg / kg, about 1000 mg / kg, about 1100 mg / kg, about 1200 mg / kg, about 1300 mg / kg, about 1400 mg / kg, about 1500 mg / kg, about 1600 mg / kg, about 1700 mg / kg, about 1800 mg / kg, about 1900 mg / kg, about 2000 mg / kg, about 2100 mg / kg, about 2200 mg / kg, about 2300 mg / kg, about 2400 mg / kg, about 2500 mg / kg, about 2600 mg / kg, about 2700 mg / kg, about 2800 mg / kg, about 2900 mg / kg to about 3000 mg / kg of 3,6,7-trimethyllumazine.

[0191] In one embodiment, the concentration of 3,6,7-trimethyllumazine is determined by chromatography, analytical measurement, spectrophotometry, and / or any other method known to one of skill in the art, hi one embodiment, the concentration of 3,6,7-trimethyllumazine is determined by a reverse phase HPLC instrument.

[0192] In one embodiment, the concentration of 3,6,7-trimethyllumazine in honey is determined by the method previously described in New Zealand Patent No. 722140, which is incorporated herein by reference.

[0193] In another particular aspect, the present invention provides a. testing a first composition comprising honey for a 3,6,7-trimethyllumazine concentration; b. testing at least one additional composition comprising honey for 3,6,7-trimethyllumazine concentration; c. selecting a composition comprising honey having a 3,6,7-trimethyllumazine concentration greater than about 5 mg / kg 3,6,7-trimethyllumazine; d. selecting at least one additional composition comprising honey having a 3,6,7-trimethyllumazine concentration greater than about 5 mg / kg 3,6,7-trimethyllumazine; e. combining the selected composition with honey to form a honey composition having a 3,6,7-trimethyllumazine concentration of at least about 5 to about 80 mg / kg; The present invention provides a method for making a composition having anti-inflammatory and / or MMP-9 inhibitory activity comprising:

[0194] In one embodiment, a composition comprising honey is selected if it has a concentration of 3,6,7-trimethyllumazine greater than about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 55 mg / kg, about 60 mg / kg, about 70 mg / kg or about 80 mg / kg.

[0195] In one embodiment, the method further comprises packaging the composition identified as having anti-inflammatory activity with a label identifying it as having a 3,6,7-trimethyllumazine concentration of at least about 5 to about 80 mg / kg. In one particular embodiment, the composition contains at least about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 55 mg / kg, about 60 mg / kg, about 70 mg / kg, or about 80 mg / kg of 3,6,7-trimethyllumazine. In one embodiment, the composition contains at least 5 to 10 mg / kg, or 10 to 15 mg / kg, or 15 to 20 mg / kg, or 20 to 25 mg / kg, or 25 to 30 mg / kg, or 30 to 35 mg / kg, or 35 to 40 mg / kg, or is 4 0-45mg / kg, or 45-50mg / kg, or 50-55mg / kg, or 55-60mg / kg, or 60 ~ 70 mg / kg or 70-80 mg / kg of 3,6,7-trimethyllumazine.

[0196] In one particular embodiment, the composition is honey or a honey extract.

[0197] In one embodiment, the composition having anti-inflammatory activity is suitable for use in any one of the methods described above and below.

[0198] In one embodiment, the concentration of 3,6,7-trimethyllumazine is determined by chromatography, analytical measurement, spectrophotometry, and / or any other method known to one of skill in the art, hi one embodiment, the concentration of 3,6,7-trimethyllumazine is determined by a reverse phase HPLC instrument.

[0199] In one embodiment, 3,6,7-trimethyllumazine concentration is determined by the method previously described in New Zealand Patent No. 722140, which is incorporated herein by reference.

[0200] In another particular aspect, the present invention provides a method for producing a composition comprising: a. testing the composition for 3,6,7-trimethyllumazine concentration; i. identifying the composition as having anti-inflammatory activity if it contains a concentration of 3,6,7-trimethyllumazine greater than about 5 to about 80 mg / kg of 3,6,7-trimethyllumazine; or ii. identifying a composition as having no anti-inflammatory activity if the composition contains a concentration of 3,6,7-trimethyllumazine less than about 5 mg / kg of 3,6,7-trimethyllumazine; The present invention provides a method for identifying a composition having anti-inflammatory and / or MMP-9 inhibitory activity, comprising:

[0201] In one embodiment, the composition comprises honey, honey extract, isolated 3,6,7-trimethyllumazine and / or synthetic 3,6,7-trimethyllumazine.

[0202] In one embodiment, a composition is determined to have anti-inflammatory activity if it contains greater than about 5 mg / kg, about 10 mg / kg, about 15 mg / kg, about 20 mg / kg, about 25 mg / kg, about 30 mg / kg, about 35 mg / kg, about 40 mg / kg, about 45 mg / kg, about 50 mg / kg, about 55 mg / kg, about 60 mg / kg, about 70 mg / kg, or about 80 mg / kg.

[0203] In one embodiment, the method further includes packaging the composition identified as having anti-inflammatory activity with a label that identifies it as having a 3,6,7-trimethyllumazine concentration of at least about 5 to about 80 mg / kg and as having anti-inflammatory activity.

[0204] In one embodiment, the composition having anti-inflammatory activity is suitable for use in any one of the methods described above and below.

[0205] In one particular embodiment, the composition is honey or a honey extract.

[0206] In another particular aspect, the present invention provides a method for producing a composition comprising: a. testing the composition for 3,6,7-trimethyllumazine concentration; i. identifying a composition suitable for use in a method for preventing, ameliorating, or treating a condition associated with inflammation of the gastrointestinal tract if it contains a concentration of 3,6,7-trimethyllumazine greater than about 5 to about 80 mg / kg of 3,6,7-trimethyllumazine; or ii. identifying a composition that is not suitable for use in a method for preventing, ameliorating, or treating a condition associated with inflammation of the gastrointestinal tract if it contains a concentration of 3,6,7-trimethyllumazine that is less than about 5 mg / kg of 3,6,7-trimethyllumazine; The present invention provides a method for identifying a composition having anti-inflammatory and / or MMP-9 inhibitory activity suitable for use in a method for preventing, ameliorating or treating a condition associated with inflammation of the gastrointestinal tract, comprising:

[0207] In one embodiment, the composition comprises honey, honey extract, isolated 3,6,7-trimethyllumazine and / or synthetic 3,6,7-trimethyllumazine.

[0208] In one embodiment, the condition associated with the gastrointestinal tract is selected from gastrointestinal inflammatory diseases, gastric ulcers (e.g., peptic ulcers), gastritis, MMP-associated inflammatory conditions, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, irritable bowel syndrome (IBS), digestive disorders, gastroesophageal reflux disease (GERD), heartburn, acid reflux, Helicobacter pylori infection, mouth ulcers, stomatitis, pharyngitis, gingivitis, esophageal ulcers, neuropsychiatric disorders (such as schizophrenia, bipolar disorder, multiple sclerosis), neurodegenerative disorders (such as traumatic brain injury, multiple sclerosis, and Alzheimer's disease), cardiovascular disease, cancer, and arthritis.

[0209] In one embodiment, the method further comprises packaging the composition identified by the above method with a label identifying the composition as having a 3,6,7-trimethyllumazine concentration of at least about 5 to about 80 mg / kg.

[0210] In another particular aspect, the present invention provides a method for producing a composition comprising: a. testing a batch of honey for 3,6,7-trimethyllumazine concentration; i. identifying a composition suitable for use in a method for preventing, ameliorating, or treating inflammation of the gastrointestinal tract if it contains a concentration of 3,6,7-trimethyllumazine greater than about 5 to about 80 mg / kg of 3,6,7-trimethyllumazine; or ii. identifying a composition that is not suitable for use in a method for preventing, ameliorating, or treating inflammation of the gastrointestinal tract if it contains a concentration of 3,6,7-trimethyllumazine less than about 5 mg / kg of 3,6,7-trimethyllumazine; The present invention provides a method for identifying a composition having anti-inflammatory and / or MMP-9 inhibitory activity suitable for use in a method for preventing, ameliorating or treating inflammation of the gastrointestinal tract, comprising:

[0211] In one embodiment, the composition comprises honey, honey extract, isolated 3,6,7-trimethyllumazine and / or synthetic 3,6,7-trimethyllumazine.

[0212] In one embodiment, the gastrointestinal inflammation is associated with a condition selected from gastrointestinal inflammatory diseases, gastric ulcers (e.g., peptic ulcers), gastritis, MMP-associated inflammatory conditions, inflammatory bowel disease (IBD), Crohn's disease, ulcerative colitis, irritable bowel syndrome (IBS), gastrointestinal disorders, gastroesophageal reflux disease (GERD), heartburn, acid reflux, Helicobacter pylori infection, mouth ulcers, stomatitis, pharyngitis, gingivitis, esophageal ulcers, neuropsychiatric disorders (such as schizophrenia, bipolar disorder, multiple sclerosis), neurodegenerative disorders (such as traumatic brain injury, multiple sclerosis, and Alzheimer's disease), cardiovascular disease, cancer, and arthritis.

[0213] In one embodiment, the method further comprises packaging the composition identified by the above method with a label identifying the composition as having a 3,6,7-trimethyllumazine concentration of at least about 5 to about 80 mg / kg.

[0214] In one embodiment, the concentration of 3,6,7-trimethyllumazine may be determined by chromatography, analytical measurement, spectrophotometry, and / or any other method known to one of skill in the art, hi one embodiment, the concentration of 3,6,7-trimethyllumazine is determined by a reverse phase HPLC instrument.

[0215] Quantification of 3,6,7-trimethyllumazine in Manuka honey using mass spectrometry In one embodiment of the present invention, 3,6,7-trimethyllumazine concentration is determined by the method previously described in commonly filed New Zealand Patent Application No. 722140, which is incorporated herein by reference and copied below.

[0216] A quantitative method to measure 3,6,7-trimethyllumazine concentration using tandem mass spectrometry (LC-MS / MS) is described. A heavier 3,6,7-trimethyllumazine isotope was synthesized and used as an internal standard to compensate for matrix effects from manuka honey. There is no interference from endogenous compounds in manuka honey, and the mass difference of 3 Da can be clearly distinguished on the mass spectrum. The LC-MS / MS results, described further below, correlate strongly with previous data from HPLC quantification and fluorescence spectroscopy. Thus, 3,6,7-trimethyllumazine can be accurately found using all three methods. The LC-MS / MS quantification results are relatively lower than the previous data from HPLC, which may be due to trace amounts of co-eluting compounds under the same HPLC peak. These findings support the use of quantitative mass spectrometry as an independent or alternative method for manuka honey authentication. Complementary This shows that it may be used as an approach.

[0217] To validate the LC-MS / MS method, mass spectra of a typical manuka honey were acquired before and after supplementation with the heavier 3,6,7-trimethyllumazine isotope. As shown, there were no significant interfering peaks from endogenous compounds in manuka honey at m / z 210-212. The 3 Da mass difference between the isotopes may be clearly identified on the mass spectrum. The final test concentration of manuka honey was determined to be 0.2% w / v in order to lower the sugar concentration while maintaining a relatively high mass spectral resolution.

[0218] LC-MS / MS quantification In the LC stage, endogenous 3,6,7-trimethyllumazine and the heavy isotope co-eluted at exactly the same time (12.85 min). These isomers, while showing nearly identical MS / MS spectra, were only differentiated by a mass shift of 3 Da from m / z 189 to m / z 192. The most abundant common ion was observed at 148.05 m / z. No heavy isotope was present in the portion of the structure represented by this fragment ion. This common ion was employed for quantification of 3,6,7-trimethyllumazine, reducing background interferences.

[0219] Comparison of LC-MS / MS and HPLC quantification Endogenous 3,6,7-trimethyllumazine concentrations were quantified to be 3-44 mg / kg using mass spectrometry quantification. Results demonstrated a strong linear correlation with previous data from HPLC analysis of the same set of manuka honey samples (R2=0.9517). It should be noted that the mass spectrometry results were relatively lower than the previous HPLC quantification (5-52 mg / kg). This suggests that other UV-absorbing compounds may have co-eluted with 3,6,7-trimethyllumazine in the same HPLC peak.

[0220] Results from mass spectrometry quantification also showed that ex 330nm~ em 470nm(R 2 =0.8995), which correlates well with the signature fluorescence.

[0221] Structural elucidation of 3,6,7-trimethyllumazine The chemical structure elucidation of 3,6,7-trimethyllumazine is described in New Zealand Patent Application No. 722140, which is incorporated herein by reference, and is shown below.

[0222] [Table 1]

[0223] Referring to Table 1 above, the molecular formula of the unknown compound was determined to be CH by positive ion HRESIMS. 10 The compound was determined to be N4O2. The compound was soluble in CD3OD and CDCl3, the latter of which was used to record the NMR spectrum due to the presence of a broad resonance at δ 8.55 ppm (H-1) that was not present in the spectrum recorded in CD3OD. This peak was assigned as an amide proton based on its chemical shift and the absence of characteristic hydroxyl absorption in the IR spectrum. Two singlets at δ 2.63 ppm (H-10) and δ 2.67 ppm (H-11) were assigned as heteroaryl methyl groups based on their chemical shifts, and the remaining singlet at δ 3.50 ppm (H-9) was assigned as two quaternary carbonyl 13 Due to the equal intensity of HMBC correlations with the C signals (C-2, C-4, see below) and the HSQC correlation with the carbon signal at δ 28.5 ppm (C-9), it was assigned as an N-methyl group. [ka]

[0224] From H-10 and H-11 to N-5 and N-8 at δ292.0ppm and δ329.9ppm, respectively 1 H- 15 N HMBC correlations suggested that these two methyl groups are attached to the pyrazine ring. The 2,3-dimethyl substitution pattern is from H-10 to C-7 and from H-11 to C-6. 1 H- 13Assigned based on CHMBC correlation.

[0225] Given the high degree of unsaturation and the presence of a pyrazine ring in the structure, this unknown compound was proposed to be a fused heterocyclic structure. Furthermore, similarities were noted between the chemical shifts of carbons C-2, C-4, and C-4a and those reported for analogous carbons in natural products containing the lumazine structure (Pfleiderer, 1984; Kakoi, et al. 1995; Voerman, et al., 2005; Meyer, et al. 2010; Chen, et al. 2014). This observation, coupled with HMBC correlations from H-9 to C-2 and C-4 and an additional four-bond coupling from H-10 to C-4a, led to the tentative assignment of the structure of the isolated compound as 3,6,7-trimethyllumazine (3).

[0226] 3,6,7-Trimethyllumazine (3) was first synthesized in 1958 (Curran & Angier, 1958). Since then, it has been reported in several studies on related lumazines (Pfleiderer & Fink, 1963; Pfleiderer & Hutzenlaub, 1973; Ritzmann & Pfleiderer, 1973; Ram, et al. 1977; Southon & Pfleiderer, 1978; Uhlmann & Pfleiderer, 1981; Ram, et al. 1982; Bartke & Pfleiderer, 1989; Acuna-Cueva, et al. 2000). Characterization data for lumazine 3 is limited to melting point (Curran & Angier, 1958; Pfleiderer & Hutzenlaub, 1973), elemental analysis (Curran & Angier, 1958), and UV-vis peaks (Pfleiderer & Hutzenlaub, 1973; Ritzmann & Pfleiderer, 1973; Uhlmann & Pfleiderer, 1981); so far, no NMR, MS, or IR data have been reported.

[0227] The entire disclosures of all applications, patents, and publications, cited above and below, if any, are hereby incorporated by reference.

[0228] For the avoidance of doubt, the term "composition" includes, but is not limited to, honey, honey extract, or dried honey.

[0229] The reference to any prior art in this specification is not, and should not be construed as, an admission or any form of suggestion that the prior art forms part of the common general knowledge in the field of endeavor anywhere in the world.

[0230] The invention may also be broadly described as consisting in the parts, elements, and features referred to or shown in the specification of this application, individually or collectively, in any or all combinations of two or more of the parts, elements, or features.

[0231] Where the foregoing description refers to components having integers or their known equivalents, those integers are incorporated herein as if individually set forth.

[0232] It should be noted that various changes and modifications to the presently preferred embodiments described herein will be apparent to those skilled in the art. Such changes and modifications may be made without departing from the spirit and scope of the present invention and without losing its attendant advantages. Therefore, such changes and modifications are intended to be included within the scope of the present invention. EXAMPLES

[0233] The above-identified compositions, agents and methods of use will now be described with reference to the Figures and specific examples.

[0234] Example 1 - Fluorometric assay In this example, fluorometric inhibitor screening provides a rapid, sensitive, high-throughput method for identifying potential inhibitors of MMP-9.

[0235] Methods and Materials The MMP-9 inhibitor screening assay (fluorescence measurement) kit was purchased from Abcam (Melbourne, Australia). The fluorescence measurement kit contains recombinant MMP-9 enzyme, MMP inhibitor NNGH (N-isobutyl-N-[4-methoxyphenylsulfonyl]glycylhydroxamic acid), MMP fluorogenic substrate solubilized in DMSO, fluorescence measurement assay buffer, and a 96-well clear microplate.

[0236] The inhibitory activity against MMP-9 was evaluated using a commercially available MMP-9 inhibitor screening assay kit. MMP-9 activity was expressed as the change in fluorescence intensity measured using a SpectraMax iD3 multimode microplate reader (Molecular Devices, San Jose, USA).

[0237] The site-specific assay uses a FRET-tagged (fluorescence resonance energy transfer) substrate, which can be hydrolyzed by MMP-9 at a specific site (Abcam, 2018). Cleavage of the FRET substrate releases a quenched fluorescent Mca (7-methoxycoumarin-4-yl)-acetyl group (Abcam, 2018). The kit employs the quenched fluorogenic substrate Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2, where Mca fluorescence is quenched by Dpa until cleavage by MMP. The amount of fluorescent product generated by MMP-9 can be detected fluorimetrically and is proportional to the enzyme activity. The fluorescence can be measured using a fluorometric method. ex 320nm~ em Measured at 395 nm, ex 330nm~ emFluorescence interference from 3,6,7-trimethyllumazine at 470 nm was minimized. The assay was performed in a 96-well clear microplate included in the kit, with a final reaction volume of 100 μL. The MMP-9 enzyme was incubated with test samples and inhibitor controls at 37°C for 60 minutes before adding the substrate. The fluorescent substrate was added to each well prior to the assay to initiate the reaction. The assay was run for 20 minutes, and the temperature in the reaction chamber was set to 37°C.

[0238] Test samples were prepared containing 3,6,7-trimethyllumazine prepared as outlined above.

[0239] A positive control with only MMP-9 and fluorescent substrate was used as a reference for calculating the inhibition rate. As a negative control, the broad-spectrum MMP inhibitor NNGH was included. A series of test controls was also included with test concentrations of 3,6,7-trimethyllumazine without MMP-9 and the fluorescent substrate required to measure the autofluorescence generated by 3,6,7-trimethyllumazine.

[0240] result Synthetic 3,6,7-trimethyllumazine was added to the reaction mixture at concentrations found in manuka honey ranging from 2.5 to 40 μg / ml (3 to 44 μg / ml as measured by LC-MS / MS). As shown in Figure 1, the change in fluorescence intensity was linear for all 3,6,7-trimethyllumazine samples and controls. In the NNGH positive control, there was little change in fluorescence. In contrast, a steady increase in fluorescence was observed in the negative control without inhibitor. The 3,6,7-trimethyllumazine samples exhibited higher initial fluorescence due to their autofluorescent nature. Fluorescence controls for each 3,6,7-trimethyllumazine concentration were included in the assay.

[0241] In this study, as shown in Figure 2, all concentrations of 3,6,7-trimethyllumazine tested showed inhibitory activity against MMP-9 ranging from 12% to 99%. Compared to the negative control without inhibitor, MMP-9 activity was significantly inhibited by 3,6,7-trimethyllumazine at concentrations of 5 μg / ml or higher (all p<0.05). 3,6,7-trimethyllumazine at 2.5 μg / ml inhibited MMP-9 activity by 12%, but the inhibition was not significant (p>0.05). 3,6,7-trimethyllumazine almost completely inhibited MMP-9 at 40 μg / ml, with no significant difference compared to the NNGH control (p>0.05). The inhibition of MMP-9 appeared to be dose-dependent on 3,6,7-trimethyllumazine concentration, as higher concentrations of 3,6,7-trimethyllumazine consistently showed stronger inhibition compared to lower concentrations (all p<0.05).

[0242] As shown in FIG. 3, the inhibition rate of MMP-9 was positively correlated with the concentration of 3,6,7-trimethyllumazine. The correlation was R 2 The best fit was a second-order polynomial model with a coefficient of 0.9965. Based on these data, the IC50 of 3,6,7-trimethyllumazine was calculated to be 11.5 μg / ml.

[0243] Example 2 - Colorimetric Assay In this example, the biological activity of 3,6,7-trimethyllumazine is further explored using an MMP-9 colorimetric inhibitor screening kit.

[0244] Methods and Materials The MMP-9 inhibitor screening assay (colorimetric measurement) kit was purchased from Abcam (Melbourne, Australia). The kit contains recombinant MMP-9 enzyme, MMP inhibitor NNGH, MMP chromogenic substrate, colorimetric assay buffer, and a 96-well clear microplate.

[0245] The colorimetric kit uses a thiopeptide (Ac-PLG-[2-mercapto-4-methyl-pentanoyl]-LG-OC2H5) as a chromogenic substrate, which can be hydrolyzed by MMPs to generate sulfhydryl groups. This intermediate product can further react with DTNB [5,5'-dithiobis(2-nitrobenzoic acid), Ellman's reagent] to form 2-nitro-5-thiobenzoic acid, which can be detected by absorbance at 412 nm. The change in absorbance was measured using a SpectraMax iD3 multimode microplate reader (Molecular Devices, San Jose, USA). The assay was performed on a convenient 96-well microplate with a final reaction volume of 100 μL. All test samples and inhibitor controls were incubated with MMP-9 for 60 min at 37 °C prior to the assay. The reaction was initiated by adding the chromogenic substrate to each well. The assay was run for 120 min at 37 °C. Absorbance was measured at 1 min intervals for the first 20 min, then at 10 min intervals until the assay was terminated.

[0246] Recombinant MMP-9 and a chromogenic substrate were used as a positive control corresponding to 100% enzyme activity. NNGH was used as a negative control. A series of 3,6,7-trimethyllumazine concentrations were diluted in colorimetric assay buffer, and the absorbance of the reaction product was measured.

[0247] result The underlying inhibitory bioactivity of 3,6,7-trimethyllumazine was further investigated using a colorimetric MMP-9 inhibitor screening kit. The colorimetric kit uses a thiopeptide substrate that can be hydrolyzed by MMPs to generate a sulfhydryl group intermediate, which can further react with Ellman's reagent to form 2-nitro-5-thiobenzoic acid. do. Ellman's reagent is used to detect the concentration of protein sulfhydryls, and the reaction product can be detected by absorbance at 412 nm (Riener, Kada, & Gruber, 2002).

[0248] Inhibitory bioactivity was first examined by adding 3,6,7-trimethyllumazine (40 μg / ml) to the reaction mixture (Figure 4). The rate of absorbance change was slightly smaller in the 3,6,7-trimethyllumazine-added samples compared to the negative control without inhibitor. NNGH was taken as a positive control to inhibit most MMP-9 activity. NNGH is not expected to completely inhibit MMP-9 at 1.3 μM (Abcam, 2019). Over the first 40 min, the absorbance change was linear in the 3,6,7-trimethyllumazine samples and controls. The product was unstable and appeared to start to decompose after 40 min. The first 20 min of the reaction was selected for further calculations.

[0249] 3,6,7-trimethyllumazine showed inhibitory bioactivity against MMP-9 at concentrations ranging from 2.5 to 80 μg / ml. The inhibition percentage was calculated by comparing the change in absorbance of 3,6,7-trimethyllumazine samples with the negative control (no inhibitor, 100% MMP-9 activity). As shown in Figure 5, all 3,6,7-trimethyllumazine samples inhibited MMP-9 by 3.5% to 10%. Compared to the negative control, higher concentrations (20 to 80 μg / ml) of 3,6,7-trimethyllumazine showed significant inhibition against MMP-9 (all p<0.0001). At lower 3,6,7-trimethyllumazine concentrations (2.5 to 10 μg / ml), the level of MMP-9 inhibition was not significant (all p>0.05). Increasing the concentration of 3,6,7-trimethyllumazine from 40 to 80 μg / ml did not further inhibit MMP-9 (10% inhibition for both, p>0.05), suggesting that 3,6,7-trimethyllumazine may have a relatively low binding affinity (Ki) for MMP-9 enzyme [E] or enzyme-substrate complex [ES] compared to the chromogenic substrate.

[0250] In the absence of MMP-9, 3,6,7-trimethyllumazine did not interfere with the absorbance signal generated by the chromogenic substrate and reaction product. This was examined by incubating 3,6,7-trimethyllumazine (40 μg / ml and 80 μg / ml) with the substrate (FIG. 6A) and reaction product (FIG. 6B) for 20 min. In both cases, 3,6,7-trimethyllumazine did not significantly interfere with the absorbance signal.

[0251] Example 3: Gelatin gel enzyme electrophoresis To confirm the inhibition of MMP-9 by 3,6,7-trimethyllumazine, we performed gelatin gel zymography to detect the activity of MMP-9, which is uniquely designed to detect the activity of MMP-9 (gelatinase), which has the ability to digest gelatin.

[0252] Methods and Materials Novex™ 10% Zymogram Plus (gelatin) protein gels (15 wells) were purchased from Thermo Fisher Scientific Inc. (Auckland, New Zealand). All chemicals required for zymogram analysis were also purchased from Thermo Fisher, including Novex™ Sharp pre-stained protein standards, Novex Tris-Glycine SDS sample buffer, Novex Tris-Glycine SDS running buffer, Novex Zymogram regeneration buffer, and Novex Zymogram developing buffer. Double-distilled water was purified from a Sartorius Arium® Pro (18.2 MΩ cm) water purification device. Gelatin gel zymography was performed as an independent technique to confirm the inhibition of MMP-9 from 3,6,7-trimethyllumazine. This technique uses non-reducing SDS-PAGE (sodium dodecyl sulfate polyacrylamide gel electrophoresis) gels embedded with gelatin. Proteins migrate and separate during electrophoresis. After electrophoresis, SDS is removed and the gel is then incubated with essential cofactors required for enzyme activity. Embedded gelatin can be digested with MMP-9, resulting in clear bands on a dark blue background after staining with Coomassie Blue dye. Gelatinase activity is expressed as band densitometry, which can be evaluated with image analysis software. Gelatin gel zymography is a relatively low-cost and highly sensitive technique (Leber & Balkwill, 1997). Moreover, this approach allows the simultaneous detection of both gelatinase activities, since pro-MMP and active MMP can be distinguished based on the migration distance through the gel (Rossano et al., 2014).

[0253] MMP-9 enzyme was diluted to a final test concentration of 5 μg / mL. MMP-9 enzyme was gently mixed with loading buffer and water to achieve a total loading volume of 10 μL per well. Gel electrophoresis was performed using an XCell Surelock™ Mini-Cell system (Thermo Fisher Scientific, Auckland, New Zealand). The upper chamber was filled with 200 mL of 1× Tris-Glycine SDS running buffer and the lower chamber was filled with 600 mL. The gel was run for 105 min at a constant voltage of 125 V and 30 mA (starting current). After electrophoresis, the gel was removed and incubated in 1× renaturation buffer for 30 min with gentle agitation. After incubation, the gel was carefully cut into small pieces and further incubated separately in 1× development buffer or in development buffer supplemented with 3,6,7-trimethyllumazine for 30 min with gentle agitation. The gels were further incubated with fresh developing buffer with or without 3,6,7-trimethyllumazine for 13 h overnight at 37° C. NNGH was also added to the developing buffer at 2.6 μM as a positive control.

[0254] After incubation, the gelatin gel was rinsed three times (5 min each) with water under gentle agitation. The gel was stained by adding 20 mL of SimplyBlue Safestain and incubated for 2 h at room temperature with gentle agitation. It was destained by removing the SimplyBlue Safestain and rinsed with water for 2 h at room temperature with gentle agitation. MMP-9 activity was analyzed using densitometry on ImageJ Version 1.52a.

[0255] result The bioactivity of 3,6,7-trimethyllumazine against MMP-9 was further investigated using gelatin enzyme electrophoresis by comparing gelatin gels incubated in normal developing buffer with 3,6,7-trimethyllumazine-added and NNGH-added buffers. The MMP-9 enzyme used in this study was partially activated by 4-aminophenylmercuric acetate (4-APMA) to provide more information on molecular interactions. As shown in Figure 7, the clear band on the gel represents the gelatinase activity from MMP-9. The lower band represents the gelatinase activity of active MMP-9 (approximately 37 kDa). The upper band represents the gelatinase activity from partially activated pro-MMP-9 (approximately 48 kDa). During electrophoresis, pro-MMP-9 was denatured by SDS and then regenerated by removing SDS with a detergent such as Triton X-100 (Ren, Chen, & Khalil, 2017). This refolding process autoactivates a portion of proMMP-9 without cleaving the prodomain (Woessner, 1995), but the autoactivated proMMP-9 may not represent its true activity in vivo.

[0256] 3,6,7-trimethyllumazine appeared to reduce gelatinase activity from both active and inactive MMP-9 using gelatin zymography. Compared to the negative control without inhibitor (Figure 7, rows 3-5), the area of ​​both clear bands appeared to be reduced in the 3,6,7-trimethyllumazine-treated gels (Figure 7, rows 6-8). The positive control NNGH completely inhibited gelatinase activity from active MMP-9 (Figure 7, rows 9-10). There appeared to be some gelatinase activity in the NNGH-treated gels from inactive MMP-9, but this was likely due to residues from the fibronectin domain. Remain This is likely the result of latinase activity.

[0257] 3,6,7-trimethyllumazine significantly reduced gelatinase activity from both active and inactive MMP-9 (Figure 8, both p<0.001). The inhibition rates by 3,6,7-trimethyllumazine and NNGH were analyzed by densitometry and plotted in Figure 8. As shown, 3,6,7-trimethyllumazine significantly inhibited the activity of active and inactive MMP-9 by 31% and 17%, respectively (both p<0.01). It should be noted that 3,6,7-trimethyllumazine showed significantly more potent inhibition against active MMP-9 compared to inactive MMP-9 (p<0.05). This suggests that 3,6,7-trimethyllumazine is likely to interact more with the zinc active site of MMP-9. The same pattern could also be observed with NNGH treatment, where NNGH specifically interacts with zinc ions (Bertini et al., 2005).

[0258] Example 4 - Molecular docking of 3,6,7-trimethyllumazine to MMP-9 In this example, molecular docking studies were performed to predict the non-covalent interactions between 3,6,7-trimethyllumazine and MMP-9.

[0259] principle Molecular docking is a computational procedure that attempts to predict non-covalent interactions between ligands and biopolymer targets. AutoDock and AutoDock Vina are computational tools commonly used to assist researchers in determining biomolecular complexes. The software calculates the minimum interaction energy between a target protein and a ligand while efficiently exploring all torsional degrees of freedom. AutoDock is based on an empirical free energy force field and a rapid Lamarckian genetic algorithm search method (Goodsell & Olson, 1990; Morris et al., 2009). AutoDock Vina uses a simpler scoring function and a fast gradient-optimized conformational search, greatly improving speed and accuracy (Trott & Olson, 2010).

[0260] Methods and Materials Molecular docking studies of MMP-9 and 3,6,7-trimethyllumazine were performed using AutoDock Vinav1.1.2. Docking preparation, post-docking analysis and visualization were performed on Chimera v1.13.1 (Pettersen et al., 2004). The 3D structure of 3,6,7-trimethyllumazine was drawn on Avogadro v1.2.0 (Hanwell et al., 2012). The complete 3D crystallographic structure of MMP-9 (PDB ID: 1L6J) was obtained through the RCSB Protein Data Bank (Elkins et al., 2002).

[0261] Both compounds were prepared for docking using Chimera. The 3,6,7-trimethyllumazine structure was minimized by employing the Smart Minimizer algorithm. Torsion angle detection and Gasteiger charge assignment were also performed in Chimera. The MMP-9 structure was created by adding hydrogen atoms, merging non-polar hydrogen atoms, checking for missing atoms, and assigning Gasteiger charges. On the catalytic domain of the MMP-9 enzyme, Å 3 A grid box was defined with a volume of =35, 45, 48 (x, y, z coordinates = 30, 30, 35), which defines the area of ​​the protein involved in the docking calculations.

[0262] Molecular docking was performed on AutoDock Vina, with the comprehensiveness set to 8 and the number of binding modes set to 10. The best binding conformations with the highest scores were listed on AutoDock Vina and visualized on Chimera. The potential intermolecular hydrogen bonds of each binding pose were also analyzed on Chimera.

[0263] result Molecular docking predicted significant binding affinity between 3,6,7-trimethyllumazine and MMP-9. 3,6,7-trimethyllumazine was successfully docked within the active site of MMP-9 using AutoDock Vina with a highest docking score of -7.9 (Table 2). The AutoDock Vina score represents the predicted energy required for two compounds to bind, taking into account a combination of hydrogen bonds, hydrophobic interactions, and torsional penalties (Chang, Ayeni, Breuer, & Torbett, 2010). As a result, the most favorable binding conformations are represented as negative scores. In comparison, the docking scores of the most active synthetic MMP-9 inhibitors using AutoDock Vina ranged from -7.6 to -8.9 (Rathee et al., 2018).

[0264] [Table 2]

[0265] 3,6,7-Trimethyllumazine is Tyr 420 The S'1 substrate binding site was docked into the S'1 substrate binding site by forming hydrogen bonds with the residues. The S'1 substrate binding site is enclosed in the center of the active site cleft closest to the active site zinc. Compared to other binding pockets, the S'1 pocket varies among MMPs in both amino acid structure and pocket depth (Aureli et al., 2008). As a result, the S'1 pocket determines the substrate binding specificity and targets many MMP inhibitors. In particular, for MMP-9, co-crystallization with different inhibitors has revealed that the Arg 424 revealed that the residue is highly flexible, allowing some MMP inhibitors to migrate into the S1′ pocket ( Tochowicz et al., 2007 ).

[0266] In previous docking studies, synthetic inhibitors bearing carboxylic acid and sulfonamide hydroxamic acid groups were bound to the S'1 pocket; whereas a thio-ester group interacted with both the S'1 and S1 pockets (Tandon & Sinha, 2011). The NH group of 3,6,7-trimethyllumazine and the Tyr group near the wall of the S'1 cavity 420 A potential hydrogen bond was observed between the S'1 wall residue and the Tyr 420 , Pro 421 , Tyr 423 ) (Tandon & Sinha, 2011). Zinc-binding inhibitors with carbonyl or NH groups provide opportunities for hydrogen-bonding interactions with the S1' pocket (Tandon & Sinha, 2011). Both structures are present in 3,6,7-trimethyllumazine.

[0267] These results further supported the binding of 3,6,7-trimethyllumazine at the exosite of MMPs located within the fibronectin type II domain. We further identified a high Gold Score (53.4) of 3,6,7-trimethyllumazine with MMP-9 (docking was performed with GOLD v5.7.3, a total of 10 GA runs per ligand were performed with maximum search efficiency. Docked poses were scored with GoldScore). These findings suggested that 3,6,7-trimethyllumazine may interact with the exosite of MMP-9 by disrupting the binding of gelatin. Results from molecular docking analysis further supported the binding of 3,6,7-trimethyllumazine at the exosite of MMP-9 located within the fibronectin type II domain.

[0268] Example 5 - Simulation of the gastrointestinal environment 3,6,7-Trimethyllumadil N It is unclear to what extent the anti-inflammatory activity is retained during gastrointestinal digestion: this bioactive molecule may be modified at low pH or by digestive enzymes, causing it to lose partially or completely its biological activity.

[0269] Simulated gastric digestion of 3,6,7-trimethyllumazine-containing honey samples was carried out in vitro, followed by simulated intestinal digestion, during which time points the gastric or intestinal contents were removed and analysed for residual 3,6,7-trimethyllumazine.

[0270] material Simulation of the gastrointestinal environment: Simulated gastrointestinal digestion was performed using a static model. Simulated gastric fluid (SGF) and simulated intestinal fluid (SIF) were prepared according to the global consensus protocol (Minekus et al. 2014). SGF has a pH of 3 and mimics fed conditions in the stomach. When mixed with manuka honey (or honey solution), the final mixture contains 2000 U / mL pepsin. SIF has a pH of 7, mimics fed conditions in the small intestine, and contains 2 mg / ml pancreatin (based on 8x USP, or 200 U / mL protease activity) and 20 mM porcine bile extract prior to use.

[0271] Gastric digestion: For simulated gastric digestion, 2 g of manuka honey was incubated in 2 mL of SGF at 37°C with shaking at 95 rpm for 2 h (in triplicate). At selected time points (0, 30, 60, and 120 min), a fixed amount (0.1 mL) of the mixture was withdrawn for 3,6,7-trimethyllumazine analysis. The solutions for analysis were added with 0.1 mL of SIF (pH 7) and stored on ice to stop pepsin activity. For comparison purposes, a pure 3,6,7-trimethyllumazine solution was treated similarly as a control.

[0272] Intestinal digestion: Following 2 hours of gastric digestion, the resulting solution was mixed with SIF (pH 7) in a 1:1 volume ratio to obtain a final mixture containing 1 mg / mL pancreatin and 10 mM porcine bile extract. The mixture was incubated at 37°C under shaking at 95 rpm for 4 hours (in triplicate). At selected time points (0, 60, 120, and 240 min), a fixed amount (0.1 mL) of the mixture was withdrawn for 3,6,7-trimethyllumazine concentration analysis. Pancreatin activity in the collected solution was quenched by adding 5 mmol / L Pefabloc® (Egger et al. 2019).

[0273] Analysis of 3,6,7-trimethyllumazine retention: Gastrointestinal digests for 3,6,7-trimethyllumazine analysis were processed to remove insoluble fractions (e.g., pancreatin) prior to HPLC analysis. Briefly, all samples were diluted with 0.1% formic acid and centrifuged at 14,000 rpm for 10 min. The supernatant was collected for analysis. The amount of 3,6,7-trimethyllumazine at different time points was analyzed using a reversed-phase HPLC setup previously used to analyze 3,6,7-trimethyllumazine and leptosperin as reported in the literature (BinLin et al., 2017). Briefly, samples were diluted 5-fold with 0.1% v / v formic acid. A Hypersil GOLD column (150 × 2.1 mm, 3 μM particle size) was used as the stationary phase (25 °C), and the mobile phase consisted of 0.1% formic acid (phase A), and 80:20 acetonitrile:0.1% formic acid (phase B). The injection volume was 3 μL, the flow rate was 0.200 mL, and the following gradient elution was used to separate 3,6,7-trimethyllumazine from the others: initial 2 min (5% B phase), 7 min (25% B), 14 min (50% B), 16 min (100% B), 19 min (5% B), 20 min (5% B, 10 min hold). The signal of 3,6,7-trimethyllumazine was detected at 320 nm.

[0274] Statistical analysis: The significance of differences between two means was analyzed using a two-tailed unpaired Student's t test. When more than two means were compared, significant differences were analyzed by one-way analysis of variance followed by Bonferroni's multiple comparison test (SPSS Statistics version 24, IBM). Differences were considered statistically significant at p < 0.05.

[0275] result The results of simulating gastrointestinal digestion of four honey samples show that 3,6,7-trimethyllumazine from manuka honey is highly stable in the harsh environment of the digestive tract. By the end of the study, i.e., 2 hours of gastric digestion and 4 hours of intestinal digestion, nearly 100% of the initial 3,6,7-trimethyllumazine amount from the four honey samples could be fully recovered in the digestive tract contents. No obvious degradation is observed. The detailed kinetics are shown in Figures 10 and 11, where the first 2 hours are during which 3,6,7-trimethyllumazine is incubated in the simulated gastric fluid, while the subsequent 4 hours represent the intestinal digestion process. The raw data are summarized in Table 3.

[0276] Detailed experimental results

[0277] [Table 3]

[0278] In a subsequent study, we performed gastrointestinal digestion of diluted manuka honey samples to understand the stability of 3,6,7-trimethyllumazine in different concentrations of manuka honey. The stability of 3,6,7-trimethyllumazine (pure compound) was also tested using the same in vitro digestion protocol. Our results show that the stability of 3,6,7-trimethyllumazine is unchanged compared to the stability profile of undigested raw honey, either in 50% (w / w) manuka honey solution (Figures 12 and 13) or when directly exposed to the digestion medium (Figures 14 and 15). No significant degradation is observed. The raw data showing the detailed kinetics are summarized in Table 4.

[0279] [Table 4]

[0280] In vitro studies on the fate of 3,6,7-trimethyllumazine in the gastrointestinal tract tested in diluted (50% dilution) Manuka honey samples A, B, C, and D, as well as in its purest form, clearly demonstrated high stability of the bioactive compound, 3,6,7-trimethyllumazine.

[0281] Example 6 - Effect of 3,6,7-trimethyllumazine on matrix metalloproteinase-9 (MMP-9) in human macrophage cell lines We investigated the efficiency of 3,6,7-trimethyllumazine present in manuka honey to inhibit lipopolysaccharide (LPS)-induced MMP-9 secretion in a human macrophage cell line (THP-1) using enzyme-linked immunosorbent assay (ELISA) technique.

[0282] Macrophages are a potential source of gastric MMPs because they are known to increase MMP-9 secretion in response to both bacterial factors and proinflammatory cytokines, and therefore MMP-9 secretion from THP-1 could be used as a marker of gastric inflammation.

[0283] 3,6,7-trimethyllumazine at concentrations from 2.5 to 40 μg / mL was tested for MMP-9 inhibitory activity. Azithromycin was selected as a positive control. 3,6,7-trimethyllumazine at 40 μg / mL (38% reduction) and 30 μg / mL (23% reduction) significantly (P<0.05) reduced MMP-9 secretion from LPS (1 μg / mL)-treated differentiated THP-1 cells compared to 20 and 5 μg / mL. 3,6,7-trimethyllumazine at 40 μg / mL reduced MMP-9, and above 30 μg / mL, this reduction was slightly higher than azithromycin. However, based on the reported cell viability, 30 μg / mL (13% cell death) is slightly safer than 40 μg / mL (20% cell death).

[0284] method dose: Using differentiated THP-1 cells, 3,6,7-trimethyllumazine was tested at doses ranging from 2.5 to 40 μg / ml for inhibition of MMP-9 inflammatory responses.

[0285] Cell culture: THP-1 cells (ATCC, ATCTIB202) were cultured in RPMI-1640 (Gibco, 11875093) with 0.05 mM 2-mercaptoethanol, 10% fetal calf serum (FCS), and 1% penicillin-streptomycin solution. In the experiments, cells were cultured in RPMI-1640 medium with only 10% fetal bovine serum (FBS).

[0286] THP-1 monocytes were plated in a 96-well plate at 2.5x10 5 The THP-1 cells were seeded at a density of 10 cells / ml and differentiated into macrophages using 10 ng / ml phorbol 12-myristate 13-acetate (PMA) (Bergin et al.) (Sigma, P1585-1MG, Lot No. SLBX889, 100% purity) for 72 hours. The PMA medium was then removed from the differentiated THP-1 cells and the cells were washed once with RPMI-1640 medium and then left to rest for approximately 5 hours.

[0287] LPS stimulation and treatment with 3,6,7-trimethyllumazine: Differentiated THP-1 cells were stimulated with LPS from E. coli 055:B5 (Sigma, L6529; Lot No. 037K4068). LPS was tested at a concentration of 1 μg / ml (Kong et al.). Cells were incubated with LPS alone or in combination with 3,6,7-trimethyllumazine (received from University of Auckland, diluted to a stock concentration of 1 mg / ml in RPMI-1640, stock solution kept in refrigerator for 2 days before use) at concentrations ranging from 2.5 to 40 μg / ml. Azithromycin (Sigma, Cat. No. 75199-25MG, Lot No. 069M4826V) at 6 μM was used as a positive control (Vandooren et al.). Cells were then incubated with the different treatments for 24 h (Kong et al.). After 24 hours, cell culture medium was collected and MMP-9 concentration was measured using MMP-9 ELISA (R&D systems, RDSDY91105 with lot number P239459 and DY008 with lot number P239900).The cells were then incubated with WST-1 and cell viability was measured.

[0288] Two additional 96-well plates were treated as above, the medium was removed and the plates containing the cells were frozen at -80°C for future RT-PCR experiments.

[0289] ELISA specificity: This human MMP-9 assay measures the 92 kDa Pro-MMP-9 and the 82 kDa active MMP-9. It does not measure the 65 kDa form. The assay also recognizes human MMP-9 when complexed with lipocalin-2 / NGAL isolated from human source material.

[0290] The following components prepared at 50 ng / mL were analyzed and showed no cross-reactivity or interference: MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-10, MMP-12, MMP-13, MMP-14, TIMP-2, TIMP-3, TIMP-4, TIMP-4, recombinant mouse MMP-9.

[0291] Recombinant human TIMP-1 does not cross-react in this assay, but does interfere at concentrations above 1.56 ng / ml.

[0292] Cell viability and preliminary MMP-9 secretion assay: 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium, monosodium salt (WST-1) (Roche, 11644807001, lot number 45255800) was used to measure the cytotoxicity of 3,6,7-trimethyllumazine at different doses. WST-1 is a cell proliferation reagent for measuring cell proliferation, viability, and cytotoxicity using a colorimetric assay (Gosert (2011); Peskin (2000)).

[0293] After incubation, a portion of the culture medium was collected and saved for MMP-9 secretion testing using ELISA. The remaining medium in the plate was then removed, and 100 μl of WST-1 (1:10 dilution) in RPMI-1640 medium was added to each cell well and incubated for another 4 hours at 37°C. The plate was then read at a wavelength of 450 nm using a plate reader. Cytotoxicity was calculated as follows: [WST-1 score of each sample / WST-1 score of control] x 100 A WST-1 score below 80% was considered cytotoxic.

[0294] Statistical analysis: To better capture variability, each treatment was performed at least in triplicate on each plate (2 plates). Media from triplicate wells were pooled and analyzed in duplicate for MMP-9 ELISA. The differences between media supplemented with LPS and the different treatments were analyzed using Student's Programming in Excel. t The test was conducted.

[0295] result 3,6,7-trimethyllumazine at a concentration of 40 μg / mL shows a slightly higher % cytotoxicity than the other concentrations (2.5-30 μg / mL) selected in the study (Figure 16). However, it is borderline (79.8) to be considered toxic. 3,6,7-trimethyllumazine at 40 μg / mL (38% reduction) and 30 μg / mL (23% reduction) significantly (P<0.05) reduced MMP-9 secretion from LPS (1 μg / mL) treated differentiated THP-1 cells compared to 20 and 5 μg / mL (Figure 17). 3,6,7-trimethyllumazine reduced MMP-9 to a greater extent than azithromycin at 40 μg / mL than at 30 μg / mL. Adjusted values ​​(with respect to LPS) are shown in Figure 18.

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Claims

1. A composition comprising 3,6,7-trimethyllumazine for use in preventing, ameliorating, or treating inflammation or a condition that can be prevented, ameliorated, or treated by inhibition of MMP-9.

2. The composition of claim 1, wherein the condition that can be prevented, ameliorated or treated by inhibition of MMP-9 is an MMP-9-associated inflammatory condition.

3. The composition of claim 2, wherein the MMP-9 associated inflammatory condition is associated with inflammation of the gastrointestinal tract.

4. 4. The composition of claim 1, wherein the condition that can be prevented, ameliorated or treated by inhibition of MMP-9 is selected from gastrointestinal inflammatory disease, gastric ulcer, peptic ulcer, gastritis, MMP-associated inflammatory conditions, inflammatory bowel disease, Crohn's disease, ulcerative colitis, irritable bowel syndrome, digestive disorders, gastroesophageal reflux disease, heartburn, acid reflux, Helicobacter pylori infection, oral ulcer, stomatitis, pharyngitis, gingivitis, esophageal ulcer, neuropsychiatric disorders, schizophrenia, bipolar disorder, neurodegenerative disorders, traumatic brain injury, multiple sclerosis, Alzheimer's disease, cardiovascular disease, cancer and arthritis.

5. The composition of claim 1 , wherein the inflammation is associated with the gastrointestinal tract of the subject.

6. 2. The composition of claim 1, wherein the inflammation is associated with a condition selected from gastrointestinal inflammatory disease, gastric ulcer, peptic ulcer, gastritis, MMP-associated inflammatory conditions, inflammatory bowel disease, Crohn's disease, ulcerative colitis, irritable bowel syndrome, digestive disorders, gastroesophageal reflux disease, heartburn, acid reflux, Helicobacter pylori infection, oral ulcer, stomatitis, pharyngitis, gingivitis, esophageal ulcer, neuropsychiatric disorders, schizophrenia, bipolar disorder, neurodegenerative disorders, traumatic brain injury, multiple sclerosis, Alzheimer's disease, cardiovascular disease, cancer, and arthritis.

7. The composition according to any one of claims 1 to 6, wherein the source of the 3,6,7-trimethyllumazine is honey.

8. 8. The composition of claim 7, wherein the honey is of floral origin derived from the genus Leptospermum.

9. 9. The composition according to claim 7 or 8, wherein the honey is of floral origin derived from Leptospermum scoparium, Leptospermum polygalifolium, Leptospermum subtenue, and / or combinations thereof.

10. The composition according to any one of claims 1 to 6, wherein the 3,6,7-trimethyllumazine is synthetic.

11. The composition according to any one of claims 1 to 9, further comprising honey or a honey extract.

12. The composition comprises 2.5 μg / mL to 1000 μg / mL of 3,6,7-trimethyllumazine; or 2.5 μg / mL to 80 μg / mL of 3,6,7-trimethyllumazine; or 2.5 μg / mL, 5 μg / mL, 10 μg / mL, 20 μg / mL, 40 μg / mL, 50 μg / mL, 60 μg / mL, 70 μg / mL or 80 μg / mL of 3,6,7-trimethyllumazine The composition according to any one of claims 1 to 11, comprising:

13. The composition comprises 5 mg / kg to 3000 mg / kg of 3,6,7-trimethyllumazine; or 5 mg / kg to 80 mg / kg of 3,6,7-trimethyllumazine; or 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, 50 mg / kg, 55 mg / kg, 60 mg / kg, 70 mg / kg or 80 mg / kg of 3,6,7-trimethyllumazine The composition according to any one of claims 1 to 11, comprising:

14. 14. The composition of any one of claims 1 to 13, wherein the composition comprising 3,6,7-trimethyllumazine is formulated as a liquid formulation, a consumer product, a capsule, a tablet, a chewable tablet, a gel, a lotion, a powder, a suppository, a cosmetic formulation, an intravenous formulation, an intramuscular formulation, a subcutaneous formulation, a solution, a food, a beverage, a dietary supplement or a spray.

15. The composition of any one of claims 1 to 12, wherein the composition has a standardized 3,6,7-trimethyllumazine concentration of 5 to 3000 mg / kg.

16. the composition has a standardized 3,6,7-trimethyllumazine concentration of from 5 mg / kg to 3000 mg / kg; a first composition having a known concentration of 3,6,7-trimethyllumazine; at least one further composition having a known concentration of 3,6,7-trimethyllumazine; Synthetic 3,6,7-trimethyllumazine; isolated 3,6,7-trimethyllumazine; honey extract containing 3,6,7-trimethyllumazine, and / or 3,6,7-trimethyllumazine derived directly from the plant Leptospermum and one or more of:

17. a. testing a first composition comprising honey for 3,6,7-trimethyllumazine concentration; b. testing at least one additional composition comprising honey for 3,6,7-trimethyllumazine concentration; c. selecting a composition comprising honey having a 3,6,7-trimethyllumazine concentration greater than 5 mg / kg; d. selecting at least one additional composition comprising honey having a 3,6,7-trimethyllumazine concentration greater than 5 mg / kg of 3,6,7-trimethyllumazine; e. combining the selected compositions with honey to form a honey composition having a 3,6,7-trimethyllumazine concentration of at least 5-80 mg / kg; A method for making a composition having anti-inflammatory and / or MMP-9 inhibitory activity comprising:

18. 18. The method of claim 17, selected when the composition comprising honey has a concentration of 3,6,7-trimethyllumazine greater than 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 35 mg / kg, 40 mg / kg, 45 mg / kg, 50 mg / kg, 55 mg / kg, 60 mg / kg, 70 mg / kg or 80 mg / kg.

19. a. testing the composition for 3,6,7-trimethyllumazine concentration; i. identifying the composition as having anti-inflammatory and / or MMP-9 inhibitory activity if it contains a concentration of 3,6,7-trimethyllumazine greater than 5 mg / kg; or ii. identifying the composition as having no anti-inflammatory and / or MMP-9 inhibitory activity if it contains a concentration of 3,6,7-trimethyllumazine less than 5 mg / kg; The method of identifying a composition as having anti-inflammatory and / or MMP-9 inhibitory activity comprises:

20. 20. The method of claim 19, wherein the composition comprises honey.

21. Use of a composition comprising 3,6,7-trimethyllumazine for the manufacture of a medicament for preventing, ameliorating, or treating inflammation, or a condition that can be prevented, ameliorated, or treated by inhibition of MMP-9.

22. A composition comprising 3,6,7-trimethyllumazine for use in supporting or maintaining normal digestion in a subject, for supporting or maintaining healthy digestion in a subject, or for supporting or maintaining general intestinal health in a subject.

23. The composition comprising 3,6,7-trimethyllumazine, a. containing a 3,6,7-trimethyllumazine concentration greater than 5 mg / kg; b. comprises a honey extract containing a concentration of 3,6,7-trimethyllumazine that is higher than the concentration of 3,6,7-trimethyllumazine found naturally occurring in the honey from which the extract is derived; and / or c. Synthetic 3,6,7-trimethyllumazine and / or 3,6,7-trimethyllumazine isolated from honey Including, 23. The composition of claim 1 or 22.

24. Use of a composition comprising 3,6,7-trimethyllumazine for the manufacture of a medicament for supporting or maintaining normal digestion in a subject, for supporting or maintaining healthy digestion in a subject, or for supporting or maintaining general intestinal health in a subject.

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