Bioactive Phytochemicals from Plants of the Genus Myrciaria and Guarana

Abstract

Compositions containing selected isolated 4-hydroxymethyl-N-methyl-prolines or pharmaceutically acceptable salts or derivatives thereof are described. Novel 4-hydroxymethyl-N-methyl-prolines are also described, along with processes for their preparation, compositions containing them, and their use as medicines and pharmaceuticals. Also described is a process for producing herbal medicines, including monitoring the quality of the herbal medicines by detecting the presence or absence of, or measuring the amount of, one or more 4-hydroxymethyl-N-methyl-prolines in a sample of the herbal medicine.

Description

Technical Field

[0001] The present invention relates to a composition comprising 4-hydroxymethyl-N-methyl-proline, and their use in medicine including the treatment of energy utilization diseases, blood glucose control, inflammation, bacterial infections, skin disorders, in vivo inhibition of sialidase activity, metabolic syndrome (including any disease or disorder associated with central obesity, elevated triglyceride levels and diabetes (including type 1 diabetes, type 2 diabetes and insulin resistance)), a process for isolating and purifying said composition from various plant sources, and together therewith various products, compounds, compositions, medical uses and methods based thereon.

[0002] The present invention also relates to a method for monitoring the quality of Chinese herbal medicines, supplements, and extracts of the genus Ziziphus and guarana, a process for manufacturing Chinese herbal medicines based on extracts of the genus Ziziphus and guarana, and herbal food additives, foods, and beverages obtained by such a process.

Background Art

[0003] Species of the genus Ziziphus The genus Ziziphus is a genus with many species of thorny shrubs and small trees in the family Rhamnaceae, the buckthorn family. They are distributed in tropical and subtropical regions around the world. The fruits are edible drupes, yellowish-brown, red, or black, often very sweet and sugary, reminiscent of dates in texture and flavor. Well-known species include Z. jujuba, Z. spina-christi, Z. lotus, Z. mauritiana, and Z. joazeiro.

[0004] Zizyphus jujuba, also known as Chinese date, is a fruit, jujube, used in traditional Chinese medicine. It is thought to nourish the heart and calm the mind and is used to treat nervousness, insomnia, and palpitations. Other traditional uses of the jujube species include the treatment of diseases such as digestive disorders, liver diseases, urinary tract diseases, diabetes, skin infections, and fevers. It is used as a treatment for eczema (Khiljee at al., 2011, Journal of Pakistan Association of Dermatologists 21, 112).

[0005] Zizyphus spina-christi is a wild tree that grows in desert areas and is abundant throughout the Middle East. Its leaves are a rich source of protein for desert animals, and its small brown fruits are eaten by local people. All parts of the plant are traditionally used by local people to maintain their health. Antibacterial, antifungal, antioxidant, and anti-inflammatory properties are attributed to the plant, and leaf extracts are used to treat wounds and skin diseases. An infusion of dried leaves is used as a skin cleanser and hair treatment. Its root bark is also used in folk medicine as a treatment for pain.

[0006] Research on the chemical constituents of this plant is well documented (Abdel-Zaher, A. et al., 2005, J. Ethnopharmacol.101,129). Many peptides and cyclopeptide alkaloids, flavonoids, triterpenoid saponin glycosides, and betulinic acid have been identified in various amounts in different species of the genus (Han, B., et al., 1990, Phytochemistry 29, 3315, Solati, J. & Soleimani, N., 2010, Acta Diabetol., 47, 219).

[0007] Recently, the composition, antioxidant activity, and hepatoprotective effect of the aqueous extract of Zizyphus jujuba were evaluated (Liu, N. et al., 2017, RSC Adv.7, 6511). A more detailed analysis of Z. jujuba fruits was carried out by Villanueva, J.R. & Villanueva, L.R. (2017, Phytotherapy Research, 31, 347).

[0008] The literature includes many scientific studies on the use of Z. spina-christi for the treatment of diabetes (e.g., Nesseem, D., et al., 2009, Pharmazie 64, 104). The plant extracts have been reported to have antihyperglycemic activity (e.g., Abdel-Zaher, A. et al., 2005, J. Ethnopharmacol.101,129). The methanol extracts of the roots of Z. spinachristi and Z. jujuba used in the treatment of alloxan diabetic rats have been claimed to have beneficial effects on diabetic rats with decreased hyperglycemia, hyperlipidemia, and lipid peroxidation (Hussein, H., et al., 2006, International Journal of Pharmacology 2, 563). Hussein et al. believe that the activity is due to the strong antioxidant activity, which is not a characteristic of the compounds of the present invention.

[0009] Tanira, M., et al., (1988, International Journal of Pharmacology 2, 563) tested the ethanol extract of the leaves of Z. spina-christi in mice and showed anti-inflammatory and moderate antipyretic effects. Motamedi, H. et al., (2009, Asian Journal of Plant Sciences 8, 187) reported the ethanol and methanol leaf extracts of Z. spina-christi as potential sources of new antibacterial compounds, but it is now clear that a common fatty acid (hexadecanoic acid) in many plants contains strong antibacterial activity. Motamedi et al did not identify 4-hydroxymethyl-N-methyl-proline as a potential active ingredient or suggest the use of fruits.

[0010] Borgi, W. et al.,(2007, Fitoterapia 78, 16) reported that the aqueous and methanol extracts of the root bark of Z. lotus showed significant anti-inflammatory effects in the acute phase of the inflammatory process, but were related to antioxidant effects. U.S. Patent No. 5,849,302 and EP0815842A2, titled "Extracts of Z. spina-christi for cosmetics and psoriasis", describe leaf extracts using a 10% aqueous extract of the leaves of Z. spina-christi, which showed anti-inflammatory activity against the skin of healthy volunteers irradiated with UV, with a 17.5% reduction in erythema in all subjects.

[0011] However, none of the 4-hydroxymethyl-N-methyl-proline (or similar compounds) described herein have been reported from plants of the genus Ziziphus, and these compounds have not been suggested to be involved in any of the biological activity studies. Rather, the interest in this area has focused on structurally unrelated triterpenes, antioxidants, and peptides.

[0012] Guarana The 4-hydroxymethyl-N-methyl-L-proline described herein as a bioactive phytochemical present in the species of the genus Bauhinia has not been reported by other groups, and here, surprisingly, it was also found in guarana, the fruit of Paullinia cupana (family Sapindaceae, not closely related to the genus Bauhinia).

[0013] Paullinia cupana is native to the Amazon basin and is common especially in Brazil. Guarana is best known for its seeds from the fruit, which are about the same size as coffee beans, are used as dietary supplements, and are most famous for their high caffeine content (the seeds contain about twice the concentration of caffeine as that contained in coffee seeds: caffeine is present at levels of about 2% - 4.5% in guarana seeds compared to 1% - 2% in coffee seeds - Bempong, D.K., et al., 1993, Int. J. Pharmacog. 31, 175).

[0014] The first report on the use of guarana as a beverage was in 1669, during a Jesuit expedition to the Amazon, when missionary Joao Felipe Bettendorf observed that the Satere-Mawe indigenous people consumed a stimulating beverage with diuretic effects and therapeutic effects against headache, fever, and convulsions. Guarana is currently used in sweetened or carbonated beverages or energy shots, as an ingredient in herbal teas, or in capsules. The genus Bauhinia does not contain caffeine.

[0015] The therapeutic properties of guarana as a stimulant, tonic, and aphrodisiac have been known worldwide since the first reports of its use. The seeds are the commercially useful part of the plant due to their caffeine (1,3,7-trimethylxanthine) content, which is responsible for guarana's stimulating properties (Kofink, M., et al., 2007, European Food Research and Technology 225, 569, Campos, M.P.D. et al., 2011, Journal of Alternative and Complementary Medicine 17, 505).

[0016] In addition to its psychotropic effects, guarana is thought to have functional properties similar to those of green tea, which is rich in catechins. Therefore, the use of guarana in metabolic disorders has been widely studied. Studies have shown that guarana can have a positive effect on lipid metabolism, increase basal energy and weight loss, and may be useful in the treatment of obesity (e.g., Opala, T., et al., 2006, European Journal of Medical Research 11, 343).

[0017] However, other studies have not shown an effect of guarana-containing preparations on body weight (Sale, C., et al., 2006, Int. J. Obes.(Lond.), 30, 764), which highlights the need to produce quality-controlled extracts that measure compounds shown to have specific activity in vitro or in vivo (as, for example, the inventors have first shown for 4-hydroxymethyl-N-methyl-L-proline).

[0018] None of the 4-hydroxymethyl-N-methyl-proline (and similar compounds) described in this specification have been reported from plants of the genus Guana, nor has it been suggested that these compounds are bioactive phytochemicals. Rather, interest in this area has concentrated on structurally unrelated chemicals, including methylxanthines, catechins, methylbenzenes, cyclic monoterpenes, sesquiterpenes, oleic acid, paulic acid, and methoxyphenylpropene (Avato, P., et al., 2003, Lipids 38, 773-780).

[0019] Herbal Food Additives and Therapies Currently, there is great interest in the use of herbal therapies and supplements, and there is increasing recognition among food manufacturers, healthcare companies, and medical professionals that herbal products have value and can complement established formulations and treatments. Currently, herbal food additives and supplements are widely used.

[0020] However, quality control of herbal food additives is difficult due to the complex nature and inherent heterogeneity of the plant materials. The materials used in herbs and plant-based food additives are usually the whole plant or a part or extract thereof. Since plant materials contain many different chemical components, the materials are complex mixtures. This makes it very difficult to standardize and control the quality of the materials. Furthermore, many herbal food additives are mixtures of two or more plant-based components and are therefore mixtures of mixtures, introducing an additional level of complexity. Additionally, the recipes and manufacturing methods used are often not uniform and may remain undisclosed. Due to these factors, it is very difficult to confirm that two samples of a particular product obtained from different sources and ostensibly identical actually contain the same mixture of components. This problem, which makes it difficult to manage the quality of such materials, has restricted the use of certain herbal extracts even among herbal experts.

[0021] Another problem stems from the fact that the plants used in the practice of herbal food additives are often not locally available and must therefore be obtained from sources remote from the end user. However, the supply of such plants from remote locations can be erratic and inaccurate, especially since there are no detailed monographs, including identity and quality standards for many such plants. The complex mixture of components found in medicinal plants varies widely in type and concentration depending on many factors such as the plant source, where the plant grows, other plants and microorganisms growing nearby, the time when the plant is harvested, the conditions under which the material is stored and processed, and the extraction procedures used.

[0022] Therefore, there is a need for a sensitive process that can profile herbal products and establish standard specifications for herbal medicinal materials that may be related to activity. This enables quality control in the manufacture of herbal food additives and ideally quantifies components that are known or potentially active.

[0023] The inventors have discovered that the plant distribution of a particular 4-hydroxymethyl-N-methyl-proline correlates with plants of the genus Ziziphus and guarana used in the treatment of various diseases. This discovery is of great commercial and medical importance, enabling for the first time the synthesis and / or isolation of a new family of therapeutically active 4-hydroxymethyl-N-methyl-proline, the manufacture of improved crude drugs meeting standard specifications, and a series of therapeutic treatments based thereon.

Summary of the Invention

[0024] The present disclosure includes the following from [1] to

[43] . [1]

Chemical

Chemical

[10] The composition for use as described in the above [8], wherein the energy utilization disorder is selected from (a) disturbance of homeostasis; (b) metabolic diseases; (c) dysfunction of glucose metabolism; (d) eating disorders; (e) insulin resistance; (f) diabetes (e.g., type 1 diabetes or type 2 diabetes); (g) prediabetes; (h) metabolic syndrome; (i) obesity; (j) cachexia (e.g., cancer-related cachexia); (k) muscle disorders; (l) gastrointestinal diseases; (m) growth retardation; (n) hypercholesterolemia; (o) atherosclerosis; (p) age-related metabolic dysfunction; (q) hyperglycemia; (r) impaired glucose tolerance; (s) hyperinsulinemia; (t) glycosuria; (u) metabolic acidosis; (v) cataract; (w) diabetic neuropathy; (x) diabetic nephropathy; (y) diabetic retinopathy; (z) macular degeneration; (aa) glomerulosclerosis; (bb) diabetic cardiomyopathy; (cc) glucose metabolism disorder; (dd) arthritis; (ee) hypertension; (ff) hyperlipidemia; (gg) osteoporosis; (hh) osteopenia; (ii) decreased bone mass; (jj) brittle bone syndrome; (kk) acute coronary syndrome; (ll) infertility; (mm) short bowel syndrome; (nn) chronic fatigue; (oo) eating disorders; (pp) intestinal motility disorder; (qq) glucose metabolism disorder; (rr) fatty liver; (ss) polycystic ovary syndrome; (tt) hemochromatosis; and (uu) seborrheic keratosis.

[11] The composition according to any one of the above [1] to [8] for use in a method of treating an inflammatory disorder.

[12] The inflammatory disorder for which the composition is used as described in

[11] above is selected from: (a) non-localized inflammatory disorders (e.g., systemic lupus erythematosus (SLE), scleroderma, and allergies); (b) chronic prostatitis; (c) glomerulonephritis; (d) inflammatory bowel disease; (e) pelvic inflammatory disease; (f) reperfusion injury; (g) rheumatoid arthritis; (h) graft rejection; (i) vasculitis; (j) asthma; (k) acne; (l) osteoarthritis; (m) inflammation of the oral mucosa and gastrointestinal tract; (n) eye inflammation; (o) nasal inflammation; (p) ear inflammation; (q) steroid-responsive inflammatory diseases; (r) skin inflammatory diseases (e.g., actinic keratosis, acne vulgaris, acne pustulosa, rosacea, and nodulocystic acne, allergic contact dermatitis, angioedema, bullous pemphigoid, cutaneous drug reactions, erythema multiforme, morphea, photosensitivity dermatitis, psoriatic arthritis, scleroderma, and urticaria, psoriasis, dermatitis, atopic dermatitis, scleroderma, steroid-responsive cutaneous inflammatory disorders, urogenital pruritus, skin conditions related to exposure to radiation, chemotherapy, and environmental irritants); (s) inflammatory autoimmune diseases (e.g., ankylosing spondylitis, Crohn's disease, ulcerative colitis, Alzheimer's disease, multiple sclerosis, motor neuron disease, Parkinson's disease, chronic fatigue syndrome, insulin-dependent diabetes mellitus, Addison's disease, Goodpasture syndrome, IgA nephropathy, interstitial nephritis, Sjogren's syndrome, and autoimmune pancreatitis); (t) osteoarthritis; (u) periodontal disease; (v) diabetic nephropathy; (w) chronic obstructive pulmonary disease; (x) joint sclerosis; (y) graft-versus-host disease; (z) chronic pelvic inflammatory disease; (a') endometriosis; (b') chronic hepatitis; (c') tuberculosis, and (d') skin inflammation, e.g., caused by exposure to sunlight, allergens, irritants, or burns).

[13] The inflammatory disorder for which the composition is used as described in

[11] above is an autoimmune disease, asthma, or allergy.

[14] The composition for use as described in

[13] above, wherein the autoimmune disease is selected from Graves' disease; rheumatoid arthritis; Hashimoto's thyroiditis; vitiligo; diabetes (e.g., type I diabetes or type II diabetes); pernicious anemia; multiple sclerosis; glomerulonephritis; systemic lupus erythematosus (SLE, lupus); Sjögren's syndrome; scleroderma; psoriasis; ankylosing spondylitis; myasthenia gravis; pemphigus; polymyositis; dermatomyositis; uveitis; Guillain - Barré syndrome; Crohn's disease; ulcerative colitis and inflammatory bowel disease (IBD).

[15] The composition for use as described in

[13] above, wherein the inflammatory disease is selected from graft - versus - host disease; sarcoidosis; disseminated intravascular coagulation syndrome, atherosclerotic diseases including Kawasaki disease; vasculitis; Sjögren's syndrome; psoriatic arthritis; enteropathic arthritis; reactive arthritis associated with inflammatory bowel disease and arthritis.

[16] The composition for use as described in

[13] above, wherein the allergy is selected from atopic allergy, allergic rhinitis, allergic conjunctivitis, atopic dermatitis, hypereosinophilic syndrome, irritable bowel syndrome, allergen - induced migraine, bacterial allergy, bronchial allergy (asthma), contact allergy (dermatitis), delayed allergy, pollen allergy (hay fever), drug allergy, sting allergy, bite allergy, gastrointestinal allergy; food allergy; and physical allergy such as cold urticaria, angioedema, cholinergic urticaria and photosensitivity.

[17] The composition according to any one of [1] to [8] above for use in a method of treating a neoplasm.

[18] The composition for use as described in

[17] above, wherein the neoplasm is selected from benign, pre - cancerous and malignant neoplasms, hyperplasia, metaplasia and dysplasia.

[19] The composition for use as described in

[17] above, wherein the neoplasm is a malignant neoplasm (cancer).

[20] The composition for use as described in

[19] above, wherein the malignant neoplasm is selected from (a) carcinoma; (b) blastoma; (c) leukemia; (d) lymphoma; (e) myeloma; (f) sarcoma; and (g) mixed - type cancer.

[21] The composition for use as described in

[20] above, wherein the malignant neoplasm is a cancer selected from cancers of the bladder, breast (e.g., primary breast tumors, lymph node-negative breast cancer, invasive ductal adenocarcinoma of the breast, and non-endometrioid breast cancer), colon (e.g., colorectal cancers such as colon adenocarcinoma and colon adenoma), kidney, epidermis (e.g., malignant melanoma), liver, lung (e.g., adenocarcinoma, adrenal cortex, nasopharynx, small cell lung cancer, and non-small cell lung cancer), esophagus, gallbladder, ovary, pancreas (e.g., exocrine pancreatic cancer), stomach, cervix, thyroid, prostate, gastrointestinal system (e.g., gastrointestinal stromal tumor), or skin (e.g., squamous cell carcinoma).

[22] The composition as described in

[20] above, wherein the malignant neoplasm is a leukemia selected from lymphocytic leukemia, such as progenitor cell leukemia, mature B cell leukemia, mature T cell leukemia, and NK cell leukemia, acute myeloid leukemia, chronic myeloproliferative disorders, and myelodysplastic syndromes.

[23] The composition for use as described in

[20] above, wherein the malignant neoplasm is a lymphoma selected from (a) Hodgkin lymphoma; (b) non-Hodgkin lymphoma (e.g., progenitor cell lymphoma, mature B cell lymphoma, mature T cell lymphoma, and NK cell lymphoma); (c) Burkitt lymphoma; and (d) lymphoreticular neoplasms, such as mantle cell lymphoma.

[24] The composition for use as described in

[20] above, wherein the malignant neoplasm is a sarcoma selected from osteosarcoma; chondrosarcoma; leiomyosarcoma; rhabdomyosarcoma; mesothelioma; fibrosarcoma; angiosarcoma or hemangioendothelioma; liposarcoma; glioma; astrocytoma; myxosarcoma, and mesenchymal and mixed mesodermal tumors.

[25] The composition according to any one of [1] to [8] above for use in a method of treating a bacterial or viral infection.

[26] The composition for use as described in

[25] above, wherein the method comprises inhibiting the growth of commensal bacteria and / or pathogenic bacteria in vivo.

[27] The composition for use as described in

[25] above, wherein the method comprises disrupting host-bacterial cell interactions and / or inhibiting or eliminating bacterial biofilm formation in a mammalian (e.g., human) host.

[28] The composition for use as described in

[26] or

[27] above, wherein the method comprises treating or preventing diseases and disorders mediated or characterized by the presence of bacterial biofilms (e.g., subgingival plaque biofilms and fascia biofilms).

[29] The composition according to any one of [1] to [8] above for use in a method of treating periodontal disease, bacterial vaginitis, and / or a disease caused by infection with Tannerella forsythia, Tannerella denticola, Porphyromonas gingivalis, or Gardnerella vaginalis.

[30] The composition according to any one of [1] to [8] above for use in a method of treating atherosclerosis, for example, atherosclerotic arteriosclerosis.

[31] The composition according to any one of [1] to [8] above for use in a method of regulating commensal bacterial growth in a mammalian host.

[32] The composition for use according to

[31] above, wherein the method comprises regulating the composition of commensal bacteria in a mammalian, for example human, host.

[33] The composition according to any one of [1] to [8] above for use in a method of controlling blood glucose in a mammalian, for example human, subject.

[34] The method according to any one of [9] to

[33] above, comprising administering to a subject an effective amount of the composition according to any one of [1] to [8] above.

[35] Use of the composition according to any one of [1] to [8] above for the manufacture of a medicament for the treatment of a disease or for use in the method according to any one of [9] to

[33] above.

[36] A cosmetic, dietary supplement, crude drug, or pharmaceutical composition comprising the composition according to any one of [1] to [8] above and optionally further comprising a cosmetic, dietary supplement, or pharmaceutically acceptable excipient or carrier.

[37] A cosmetic method for reducing skin swelling or erythema, comprising administering to a subject the composition according to any one of [1] to [8] or

[36] above, for example, by topical application to the skin.

[38] The composition, composition for use, method, or use according to any one of the preceding [1] to

[37] , wherein the isolated 4-hydroxymethyl-proline is present in the composition at a level of at least 0.5%, 1% w / w, 5% w / w, 10% w / w, 15% w / w, 20% w / w, 25% w / w, 30% w / w, 35% w / w, 40% w / w, 45% w / w, 50% w / w, 60% w / w, 70% w / w, 80% w / w, 90% w / w, 99% w / w (dry weight basis).

[39] A process for the manufacture of a composition according to any one of [1] to

[38] above, comprising: (a) providing a plant material from a plant source according to any one of [2] to [5] above; (b) extracting 4-hydroxymethyl-proline according to [1] above from the plant material; and then (c) formulating the extracted 4-hydroxymethyl-proline together with a pharmaceutically acceptable excipient to produce a pharmaceutical composition. The process as described above.

[40] A process for the manufacture of a pharmaceutical composition, crude drug, or dietary supplement, comprising monitoring the quality of the pharmaceutical composition, crude drug, or dietary supplement by detecting the presence or absence or measuring the amount of 4-hydroxymethylproline according to [1] above in a sample of the pharmaceutical composition, crude drug, or dietary supplement.

[41] A method for monitoring the quality of a pharmaceutical composition, crude drug, or dietary supplement, comprising: (a) providing a sample of the pharmaceutical composition, crude drug, or dietary supplement; and (b) detecting the presence or absence or measuring the amount of 4-hydroxymethylproline according to [1] above in the sample. The method as described above.

[42] A process for the manufacture of a fortified food or beverage, comprising: (a) providing a composition according to any one of [1] to

[41] above; and (b) adding the composition of step (a) to a food or beverage to produce a fortified food or beverage.

[43] A method for selecting a breeding line and / or variety of a plant according to any one of [3] or [4] above, comprising: (a) providing a material derived from a plant according to [3] or [4] above, for example, a plant part according to [5] above; and (b) determining the presence or absence or measuring the amount of 4-hydroxymethylproline according to [1] or [2] above in the material of step (a). The method as described above. The present invention is based, at least in part, on the surprising discovery that the plant distribution of certain 4-hydroxymethyl-N-methyl-proline correlates with medicinal plants used in the treatment of various diseases. Accordingly, the family of 4-hydroxymethyl-N-methyl-proline has been identified for the first time as an important bioactive component of established crude drugs.

[0025] Accordingly, according to the present invention, there is provided an isolated 4-hydroxymethyl-proline selected from the following:

Chemical formula

[0026] In a preferred embodiment, the 4-hydroxymethyl-proline is

Chemical formula

[0027] Other embodiments of the present invention are defined in the claims appended hereto.

[0028] The specific 4-hydroxymethyl-N-methyl-proline described herein is novel. According to the present invention, also provided are novel 4-hydroxymethyl-N-methyl-proline themselves, their preparation processes, compositions containing them, and their use as medicaments and pharmaceuticals. Some of such 4-hydroxymethyl-N-methyl-proline described herein, as far as is known, have not been recognized as having biological activity and thus have not been claimed as pharmaceuticals themselves.

[0029] Aspects of the herb quality monitoring of the present invention According to another aspect of the present invention, provided is a process for manufacturing a crude drug, comprising the step of monitoring the quality of the crude drug by detecting the presence or absence of one or more 4-hydroxymethyl-N-methylproline in the sample of the crude drug or measuring its amount.

[0030] In another aspect, the present invention provides a method for monitoring the quality of a crude drug, comprising the following steps: (a) providing a sample of the crude drug; and (b) detecting the presence or absence of one or more 4-hydroxymethyl-N-methylproline in the sample of the crude drug or measuring its amount.

[0031] In this context, the term quality is used to define the overall suitability of the crude drug for its intended use and may include, for example, the presence or absence (at an appropriate concentration) of one or more 4-hydroxymethyl-N-methyl-proline in the sample, which indicates an acceptable or unacceptable degree of contamination by specific sources of use, condition, purity, and undesirable supplementary components and / or contaminants.

[0032] In a further aspect, the present invention provides a process for manufacturing a fortified food or beverage, comprising the following steps. (a) Providing plant parts of the genus Saponaria and / or Paullinia cupana; (b) Monitoring the quality of the plant parts by the method of the present invention; and (c) Adding the plant part (or one or more fractions thereof) to a food or beverage to produce the supplemented food or beverage.

[0033] This aspect of the present invention finds wide utility in the production of any supplemented food or beverage and any food or beverage can be used, including refrigerated foods and beverages, warm foods and beverages, sweet foods and beverages, carbonated beverages, alcoholic beverages and non-alcoholic beverages.

[0034] The process and method of the present invention preferably further comprises the step of detecting the presence or absence or measuring the amount of one or more 4-hydroxymethyl-N-methylprolines in a sample of the herbal food additive.

[0035] In embodiments where the presence of one or more 4-hydroxymethyl-N-methyl-prolines is detected by the present invention, one or more 4-hydroxymethyl-prolines selected from the following are

Chemical formula

[0036] In other embodiments where the presence of one or more 4-hydroxymethyl-N-methyl-prolines is detected by the present invention, one or more 4-hydroxymethyl-prolines selected from the following are

Chemical formula

[0037] The present invention also contemplates crude drugs obtained by the methods and processes of the present invention.

Brief Description of the Drawings

[0038]

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Modes for Carrying Out the Invention

[0039] All publications, patents, patent applications, and other references mentioned in this specification are hereby incorporated by reference in their entirety for all purposes as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference and the contents thereof were fully set forth.

[0040] Definitions As used herein, unless otherwise indicated, the following terms are intended to have the following meanings in addition to any broader (or narrower) meanings the terms may enjoy in the art.

[0041] Unless the context requires otherwise, the use of the singular form herein is to be read to include the plural, and vice versa. The terms "a" or "an" used in connection with an entity are to be construed as referring to one or more of that entity. In such cases, the terms "a" (or "an"), "one or more", and "at least one" are used interchangeably herein.

[0042] As used herein, the term "comprising", or variations thereof such as "comprises" or "comprising", are to be read as including any recited integer (e.g., feature, element, property, characteristic, method / process step, or limitation) or group of integers (e.g., feature, element, property, characteristic, method / process step, or limitation), but not excluding any other integer or group of integers. Thus, as used herein, the term "comprising" is open-ended and inclusive of additional, unrecited integers or method / process steps.

[0043] The phrase "consisting essentially of" is used herein to require a specified integer(s) or step(s), as well as those that do not materially affect the characteristics or function of the claimed invention.

[0044] As used herein, the term "consisting of" is used to indicate the presence of only the recited integer (e.g., feature, element, property, characteristic, method / process step or limitation) or group of integers (e.g., feature, element, property, characteristic, method / process step or limitation).

[0045] As used herein, the term "disease" is used to define any abnormal condition that impairs physiological function and is associated with specific symptoms. This term is used broadly to include disorders, illnesses, abnormalities, medical conditions, diseases, states, or syndromes in which physiological function is impaired, regardless of the nature of the causative agent (or whether the causative basis of the disease has actually been established). Thus, conditions resulting from infection, trauma, injury, surgery, radiation burns, poisoning, or nutritional deficiencies are included.

[0046] As used herein, the term "treatment" or "treating" refers to an intervention (e.g., administration of an agent to a subject) that cures, ameliorates, or alleviates the symptoms of a disease, or eliminates (or reduces the impact of) its cause(s) (e.g., a pathological polymorphic condition). In this case, the term is used synonymously with the term "therapy".

[0047] Furthermore, the term "treatment" or "treating" refers to an intervention (e.g., administration of an agent to a subject) that prevents or delays the onset or progression of a disease, or reduces (or eradicates) its incidence within a treated population. In this case, the term treatment is used synonymously with the term "prevention".

[0048] The term "subject" (which should be read to include, where the context permits, "individual", "animal", "patient" or "mammal") defines any subject in need of treatment, particularly a mammalian subject. Mammalian subjects include, but are not limited to, humans, laboratory animals, livestock, zoo animals, sports animals, pet animals. In a preferred embodiment, the subject is a human.

[0049] As used herein, the term "energy utilization disorder" encompasses any disease or disorder resulting from abnormal energy utilization. Thus, this term includes disorders and diseases of homeostasis, metabolic diseases, glucose metabolism dysfunction, and eating disorders. Accordingly, this term includes insulin resistance, various forms of diabetes, metabolic syndrome, obesity, cachexia (e.g., cancer-related cachexia), muscle disorders, gastrointestinal diseases, growth retardation, hypercholesterolemia, atherosclerosis, and age-related metabolic dysfunction. This term also includes conditions associated with metabolic syndrome, obesity, and / or diabetes, such as hyperglycemia, impaired glucose tolerance, hyperinsulinemia, glycosuria, metabolic acidosis, cataracts, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, macular degeneration, glomerulosclerosis, diabetic cardiomyopathy, insulin resistance, glucose metabolism disorder, arthritis, hypertension, hyperlipidemia, osteoporosis, osteopenia, bone loss, brittle bone syndrome, acute coronary syndrome, infertility, short bowel syndrome, chronic fatigue, eating disorders, and intestinal motility disorders.

[0050] As used herein, the term "metabolic syndrome" is used to define a condition characterized by the presence of three or more of the following symptoms: central obesity (waist measurement of 101.6 cm (40 inches) or more in men and 88.9 cm (35 inches) or more in women); high levels of triglycerides (150 mg / dL or more); low levels of HDL (less than 40 mg / dL in men and less than 50 mg / dL in women), and hypertension (130 / 85 mmHg or more).

[0051] Accordingly, this term includes the following conditions defined according to the World Health Organization's definition of metabolic syndrome: (a) fasting plasma glucose exceeding 6.1 mmol / L; (b) blood pressure exceeding 140 / 90 mmHg; and (c) one or more of the following: (i) plasma triglycerides exceeding 1.7 mmol / L; (ii) HDL less than 0.9 and 1.0 mmol / L for men and women, respectively; (iii) body mass index exceeding 30 kg / m 2 exceeds.

[0052] References herein to the treatment of metabolic syndrome are to be construed to include the treatment of any or all of the disorders associated with metabolic syndrome, including, in particular, obesity (e.g., central obesity), as well as elevated serum triglycerides and diabetes (including type 1 and type 2 diabetes and insulin resistance).

[0053] References herein to the treatment of type 1 or type 2 diabetes are to be construed to include the treatment of type 1 and type 2 diabetes themselves, as well as prediabetes (impaired glucose tolerance) and insulin resistance.

[0054] The terms “prediabetes” or “impaired glucose tolerance” define a state in which elevated levels of glucose or glycosylated hemoglobin are present in the absence of diabetes.

[0055] As used herein, an effective amount of a compound or composition is an amount sufficient to provide a desired effect, e.g., treatment or prevention manifested by a permanent or transient improvement in the condition of a subject, without undue toxicity, irritation, allergic response, or other problems or complications commensurate with a reasonable benefit / risk ratio. This amount may vary among subjects depending on, e.g., the individual's age and general condition, mode of administration, and other factors. Accordingly, it is not possible to specify an exact effective amount, but one of ordinary skill in the art will be able to determine an appropriate “effective” amount in each individual case using routine experimentation and the general knowledge of the background. Treatment outcomes in this context include eradication or alleviation of symptoms, reduction of pain or discomfort, extension of survival, improvement of mobility, and other markers of clinical improvement. A treatment outcome need not be a complete cure.

[0056] As used herein, the term "pharmaceutical kit" defines various pharmaceutical compositions in one or more unit doses, optionally accompanied by an administration means (e.g., a measuring device) and / or a delivery means (e.g., an inhaler or a syringe), all contained within a common outer packaging. In a pharmaceutical kit containing a combination of two or more compounds / drugs, the individual compounds / drugs can be in single or multiple formulations. The unit dose(s) may be contained in a blister pack. The pharmaceutical kit may optionally further include instructions for use.

[0057] As used herein, the term "pharmaceutical pack" defines various pharmaceutical compositions in one or more unit doses, optionally contained within a common outer packaging. In a pharmaceutical pack containing a combination of two or more compounds / drugs, the individual compounds / drugs can be in single or multiple formulations. The unit dose(s) may be contained in a blister pack. The pharmaceutical pack may optionally further include instructions for use.

[0058] As used herein, the term "patient pack" defines a package prescribed to a patient that contains pharmaceutical compositions for the entire course of treatment. A patient pack typically contains one or more blister packs. The patient pack is superior to a conventional prescription where a pharmacist divides the supply of a patient's medications from a bulk supply, and the patient always has access to the accompanying documents contained in the patient pack, which are not usually in a patient's prescription. It has been shown that including the accompanying documents improves patient compliance with a physician's instructions.

[0059] The term pharmaceutically acceptable derivative as applied to the compounds of the present invention defines a compound obtained (or obtainable) by chemical derivatization of the parent compound of the present invention. Thus, a pharmaceutically acceptable derivative is suitable for administration to or use in contact with human tissue without undue toxicity, irritation, or allergic response (i.e., commensurate with a reasonable benefit / risk ratio). Preferred derivatives are those obtained (or obtainable) by alkylation, esterification, or acylation of the parent compound. Accordingly, pharmaceutically acceptable derivatives of the compounds of the present invention include N-oxides and their esters.

[0060] The pharmaceutically acceptable derivatives of the present invention may retain some or all of the biological activities described herein. In some cases, the biological activity is increased by derivatization. The derivative may act as a prodrug, and one or more of the biological activities described herein may occur only after in vivo treatment. Particularly preferred prodrugs are ester derivatives that are esterified with one or more free hydroxyls and are activated by hydrolysis in vivo. Derivatization may also enhance other biological activities of the compound, such as bioavailability and / or glycosidase inhibition profile. For example, derivatization may increase CNS penetration (e.g., penetration across the blood-brain barrier).

[0061] The term "pharmaceutically acceptable salt" defines non-toxic organic or inorganic acid addition salts of the free base that are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and that exhibit a reasonable benefit / risk ratio. Suitable pharmaceutically acceptable salts are well known in the art. Examples include salts of inorganic acids (e.g., hydrochloric acid, hydrobromic acid, sulfuric acid, and phosphoric acid), organic carboxylic acids (e.g., acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, malonic acid, succinic acid, fumaric acid, malic acid, tartaric acid, citric acid, ascorbic acid, maleic acid, hydroxymaleic acid, dihydroxymaleic acid, benzoic acid, phenylacetic acid, 4-aminobenzoic acid, 4-hydroxybenzoic acid, anthranilic acid, cinnamic acid, salicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid, and mandelic acid), and organic sulfonic acids (e.g., methanesulfonic acid and p-toluenesulfonic acid).

[0062] These salts and the free base compounds may exist either in hydrated forms or in substantially anhydrous forms. Crystalline forms including all polymorphic forms of the compounds of the present invention are also contemplated, and generally, the acid addition salts of the compounds are soluble in water and various hydrophilic organic solvents and exhibit higher melting points and increased solubilities compared to their free base forms.

[0063] As used herein, the term "alkyl" defines a straight or branched chain saturated hydrocarbon chain. "C 1 -C 6 alkyl" refers to a straight or branched chain saturated hydrocarbon chain having from 1 to 6 carbon atoms. Examples include methyl, ethyl, n-propyl, isopropyl, t-butyl, n-hexyl. The alkyl groups of the present invention can optionally be substituted with one or more halogen atoms.

[0064] As used herein, the term "alkenyl" defines a straight or branched chain hydrocarbon chain containing at least one carbon-carbon double bond. "C 1 -C 6The term "alkenyl" refers to a straight-chain or branched unsaturated hydrocarbon chain having from 1 to 6 carbon atoms. Examples include ethenyl, 2-propenyl, and 3-hexenyl. The alkenyl group of the present invention can optionally be substituted with one or more halogen atoms.

[0065] As used herein, the term "alkynyl" defines a straight-chain or branched hydrocarbon chain containing at least one carbon-carbon triple bond. "C 1 -C 6 The term "alkynyl" refers to a straight-chain or branched unsaturated hydrocarbon chain having from 1 to 6 carbon atoms. Examples include ethynyl, 2-propynyl, and 3-hexynyl. The alkynyl group of the present invention can optionally be substituted with one or more halogen atoms.

[0066] The term phytochemical is used herein in a broad sense to encompass any chemical component of a plant, including macromolecules and small molecules. Important examples include alkaloids (e.g., iminosugars and iminosugar acids, e.g., selected from the structural classes pyrrolidine, piperidine, pyrrolizidine, indolizidine, tropane, and nortropane), carbohydrate analogs, phenolic compounds, terpenoids, enzyme inhibitors, glycosides, nucleotides, amino acids, lipids, and sugars.

[0067] When applied to the compounds of the present invention, the term isolated is used herein to indicate that the compound is present in a physical environment different from its natural environment (or in the case of a synthetic compound, is somewhat purified). For example, an isolated compound can be substantially isolated (e.g., purified) with respect to the complex cellular environment in which it naturally occurs (or with respect to some or all of the starting products, intermediates, buffers, solvents, reactants, and / or by-products when it is synthesized). Thus, an isolated compound can take the form of a concentrated fraction or extract from any of the plant sources described herein.

[0068] When an isolated material (e.g., a synthetic, non-naturally occurring compound) is purified, the absolute level of purity is not critical, and one of ordinary skill in the art can readily determine an appropriate level of purity depending on the use to which the material is to be put. In the case of synthetic materials, the purity level ranges from 85 to 99% w / w and may exceed 99% w / w. However, especially when the material is isolated from a natural source, a purity level of at least 0.1% w / w, 0.2% w / w, 0.3% w / w, 0.4% w / w, 0.5% w / w, 0.6% w / w, 0.7% w / w, 0.8% w / w, 0.9% w / w, 1.0% w / w, 1.1% w / w, 1.2% w / w, 1.3% w / w, 1.4% w / w, 1.5% w / w, 1.6% w / w, 1.7% w / w, 1.8% w / w, 1.9% w / w, or 2.0% w / w is preferred.

[0069] Particularly preferred are purity levels of at least 0.5 to 2.0% w / w, such as at least 0.8 to 1.5% w / w, such as at least about 1.0% w / w. Optionally, by using appropriate concentration techniques such as ion exchange chromatography, when the material is isolated from a natural source, levels of 5 to 10% w / w can be readily obtained.

[0070] In some situations, the isolated compound forms part of a composition (e.g., a cruder or less crude extract containing many other substances) or a buffer system, which may, for example, contain other components. In other situations, the isolated compound can be purified to an essential homogeneity when determined, for example, spectrophotometrically, by NMR or chromatography (e.g., GC-MS of trimethylsilyl derivatives).

[0071] The term "crude drug" is used herein to define a pharmaceutical composition in which at least one active ingredient (e.g., a compound) is not chemically synthesized, but rather is a phytochemical of a plant. In most cases, this non-synthetic active ingredient is not isolated (as defined herein), but rather is present together with other phytochemicals associated with the source plant. However, in some cases, the bioactive ingredient(s) derived from the plant may be included in a concentrated fraction or may be isolated (often involving extensive purification). However, in many cases, a crude drug comprises a more or less crude extract, infusion or fraction of a plant, or the whole (or a part) of the untreated plant, in which case the plant (or part thereof) is usually at least dried and / or powdered. A crude drug can be in the form of a dietary supplement, a beverage, or provided in multiple single doses as a crude drug kit or a crude drug pack.

[0072] The term "herbal food" is used herein to define a composition in which at least one ingredient is not chemically synthesized, but rather is a phytochemical of a plant. In most cases, this non-synthetic ingredient is not purified, but rather is present together with other phytochemicals associated with the source plant. However, in some cases, the ingredient(s) derived from the plant may be included in a concentrated fraction or may be isolated (in some cases to a high purity). However, in many cases, a herbal food additive comprises a more or less crude extract, decoction or fraction of a plant, or the whole (or a part) of the untreated plant, in which case the plant (or part thereof) is usually at least dried and / or powdered. Thus, this term includes herbal foods in the form of additives and supplementary ingredients for use in foods and beverages.

[0073] The term "bioactive ingredient" is used herein to define a phytochemical that is necessary or sufficient for the medicinal efficacy of the crude drug in which it is contained. In the case of the present invention, the bioactive ingredient(s) includes one or more of 4-hydroxymethyl-N-methyl-proline of the present invention.

[0074] The term "nutraceutical" is used herein to define a food (or an isolate thereof) that provides a physiological benefit or protects against a disease. Preferred nutraceuticals of the invention have blood glucose regulating activity and find use in the treatment of energy utilization disorders. Other nutraceuticals of the invention are anti-inflammatory.

[0075] The term "standard specification" is used herein to define the properties or phytochemical profile that correlates with the acceptable quality of a crude drug, cosmetic or nutraceutical. In this context, the term "quality" is used to define the overall suitability of the product for its intended use and includes the presence of one or more of 4-hydroxymethyl-N-methyl-proline of the invention at an appropriate concentration.

[0076] The term "phytochemical profile" is used herein to define a set of properties associated with different phytochemical components.

[0077] In its broadest aspect, the invention contemplates all optical isomers, racemic forms, and diastereomers of 4-hydroxymethyl-N-methyl-proline of the invention. Accordingly, reference to 4-hydroxymethyl-N-methyl-proline of the invention includes 4-hydroxymethyl-N-methyl-proline as a mixture of diastereomers, as individual diastereomers, as a mixture of enantiomers, and in the form of individual enantiomers.

[0078] Chemical synthesis The 4-hydroxymethyl-N-methyl-proline described in this specification can be prepared by conventional methods. Methods for preparing heteroaromatic ring systems are well known in the art. In particular, synthetic methods are described in Comprehensive Heterocyclic Chemistry, Vol. 1 (Eds.: AR Katritzky, CW Rees), Pergamon Press, Oxford, 1984, and Comprehensive Heterocyclic Chemistry II: A Review of the Literature 1982-1995 The Structure, Reactions, Synthesis, and Uses of Heterocyclic Compounds, Alan R. Katritzky (Editor), Charles W. Rees (Editor), E.F.V. Scriven (Editor), Pergamon Pr, June 1996. Other general resources for assisting in the synthesis of the target compound include March’s Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, Wiley-Interscience; 5th edition (January 15, 2001).

[0079] Extraction from plant sources / detection therein The 4-hydroxymethyl-N-methyl-proline of the present invention described in this specification can be isolated from natural sources.

[0080] For example, the compounds of the present invention can be extracted and / or purified from plant sources selected from the following: (a) plants of the genus Ziziphus; and (b) plants of the genus Paullinia. In a preferred embodiment, the plant source is selected from plant species selected from the following: Z. jujuba, Z. spina-christi, Z. lotus, Z. mauritiana, Z. joazeiro, and P. cupana.

[0081] The plant material from the above-mentioned plant source can be used as a starting material for the isolation and purification of 4-hydroxymethyl-N-methyl-proline for use according to the present invention. The 4-hydroxymethyl-N-methyl-proline of the present invention is water-soluble and can be concentrated by anion exchange chromatography or cation exchange chromatography. It can also be concentrated using size exclusion methods. Thus, those skilled in the art will understand that the 4-hydroxymethyl-N-methyl-proline of the present invention can be easily purified and isolated using standard techniques.

[0082] Any suitable part of the plant source can be used. In a preferred embodiment, the source can comprise or consist essentially of plant parts selected from fruits, seeds, and leaves. Other parts such as roots, stems, and bark can also be used.

[0083] Processes suitable for extraction and / or purification include processes comprising the following steps: (a) providing a material, e.g., a plant part selected from: fruits, fruit parts, fruit extracts, fruit juices, seeds, bark, roots and / or leaves from a plant source; (b) extracting a first sample of the material with a polar solvent to produce a polar extract and a non-polar residue; (c) subjecting the polar extract of step (b) to ion exchange chromatography to produce a concentrated extract of ionic and non-ionic residues; (d) fractionating the concentrated extract of step (c) by chromatography to produce one or more polar fractions in which the ionic compounds are concentrated and which contain one or more compounds of the present invention.

[0084] Aspects of Herb Quality Control Sample The herb sample used in the method of the present invention can be a dry plant material or an aliquot of a herb food additive in the form in which it is added to food and beverages. Alternatively, the sample can be pretreated in any of a variety of ways prior to characterization. Pretreatment can include physical or chemical pretreatment, such as pulverization, grinding, freezing, evaporation, filtration, pressing, spray drying, extrusion, supercritical solvent extraction, and tincture production.

[0085] Preferably, the food additive sample is fractionated prior to characterization. Any suitable fractionation method can be used, including solvent extraction(s). In a preferred embodiment, the sample is fractionated by ion exchange chromatography to produce an extract enriched in polar compounds and nonpolar residues. In such an embodiment, the characterization preferably includes gas-liquid chromatography (GC), such as GC-MS. When using GC, the enriched extract is derivatized prior to chromatography.

[0086] If the herb food additive is regulated or sold in the form of the whole plant (or a part thereof), the plant material can be dried prior to use. Any convenient form of drying can be used, including freeze drying, spray drying, or air drying.

[0087] Detection of 4-Hydroxymethyl-N-methyl-proline Any suitable form of characterization of the food additive sample can be used, including functional and / or physical and / or chemical characterizations that are sufficient to detect the presence or absence of 4-hydroxymethyl-N-methyl-proline in the sample or to measure its amount, but are not limited thereto.

[0088] When the sample is physically characterized, the characterization can be selected from the following: (a) quantification of phytochemical(s); and / or (b) measurement of the purity of the component; and / or (c) determination of the molecular weight (or, in the case of a fraction containing multiple different phytochemicals, its molecular weight distribution or various statistical functions thereof); and / or (d) determination of the molecular formula(s) (e.g., by nuclear magnetic resonance); and / or (e) spectral analysis.

[0089] Spectral analysis is particularly preferred and can generate any or all of the following spectra: (a) mass spectrum (e.g., mass-to-charge ratio (m / z) values vs. abundance), and / or (b) chromatographic data (spectra, column retention times, elution profiles, etc.), and / or (c) photodiode array (PDA) spectrum (e.g., both in the UV and visible ranges), and / or (d) electrochemical detection or evaporative light scattering detection, (e) nuclear magnetic resonance (NMR) spectrum (e.g., 1 H and / or 13 a spectral data set obtained via C NMR).

[0090] When used in accordance with the present invention, spectral analysis can be combined with, for example, fractionation and / or derivatization of the sample by use of GC-MS and / or HPLC-PDA-MS.

[0091] Particularly preferred is the use of GC-MS to detect the presence or absence or measure the amount of 4-hydroxymethyl-N-methyl-proline in the sample.

[0092] When the sample is chemically characterized, the characterization can be selected from measurements of the chemical reactivity of the phytochemical(s), the solubility of the phytochemical(s), the stability and melting point of the phytochemical(s), or any combination thereof.

[0093] When the sample is functionally characterized, the characterization may include a biological assay selected from, for example, in vivo or in vitro assays, enzyme inhibition assays (such as sialidase inhibition), receptor binding assays, cell assays (such as cell replication, cell pathogens, cell-cell interactions, and cell secretion assays), immunoassays, antibacterial activity (such as cell binding and / or replication of bacteria and viruses) assays, toxicity assays (such as LD 50 assay) or any combination thereof.

[0094] Solvent extraction Polar solvents suitable for use in the process of the present invention include, but are not limited to, organic solvents such as organic alcohols. Preferred are ethanol and methanol, as well as mixtures of ethanol / water or methanol / water. Preferably, the polar solvent is selected from 51 - 80% ethanol / water, 31 - 50% ethanol / water, and up to 30% ethanol / water. Particularly preferred is a polar solvent of about 50% ethanol / water. Non-polar solvents suitable for use in removing unwanted components in the process of the present invention include, but are not limited to, organic solvents such as hexane and dichloromethane (DCM) or chloroform. Particularly preferred is dichloromethane. The conditions (time, temperature, degree of stirring, etc.) under which the extraction(s) is / are carried out can be readily determined empirically and can vary depending on the nature of the sample, the nature of any pretreatment, and the solvent system selected.

[0095] Chromatographic fractionation Chromatographic fractionation may include gas-liquid chromatography. Gas-liquid chromatography is a process of separating a complex mixture of volatile substances into its components by partitioning the sample between a pressurized inert gas and a thin layer of a non-volatile liquid coated on an inert support in a heated column. To well separate a specific compound in the mixture, it is important to use a column with the correct properties. The nature of the solid support, the type and amount of the liquid phase, the packing method, the overall length, and the column temperature are important factors.

[0096] One of ordinary skill in the art can readily determine appropriate column characteristics through routine trial and error and by using general common knowledge, especially according to the situation including the nature of the extract under study and the solvents used in the extraction and the types of chemical substances expected in those solvents. Particularly preferred and useful in many situations is a capillary column coated with a nonpolar liquid phase (25 m × 0.22 mm id × 0.25 μm BPX5 stationary phase, manufactured by SGE Ltd. or equivalent).

[0097] Many compounds are not suitable for direct injection into a gas chromatograph because they are highly polar, have low volatility, and are thermally unstable. Highly hydroxylated compounds are difficult to vaporize due to intermolecular hydrogen bonding. However, by replacing the hydroxyl hydrogen with other chemical groups, sufficient volatility for GC analysis can be achieved. The two most common means of derivatizing hydroxyl groups are acetylation and silylation, and acetate [CH 3 CO-O-R] or silyl ether, such as trimethylsilyl (TMS) ether [(CH 3 ) 3 Si-O-R] is formed. Thus, in embodiments where the concentrated extract is fractionated by chromatography on an analytical scale, the phytochemical components of the concentrated extract are preferably derivatized, for example, by acylation or silylation. Particularly preferred is trimethylsilyl (TMS) derivatization.

[0098] Chromatographic fractionation can also include ion exchange chromatography. Ion exchange chromatography partially purifies and concentrates ionic species and removes contaminants. One of ordinary skill in the art can readily identify appropriate column packing materials and mobile phase(s) through routine trial and error and by using general common knowledge, especially according to the amount to be fractionated, the extract under study, and the nature of the solvents used in the extraction. + form or ammonium (NH 4 +It is a strongly acidic cation exchange resin that can be used in any of the salt forms. These forms adsorb cations from the solution and release the same number of counterions into the solution (either H + or NH 4 + ions, depending on the form used). Strongly basic anion exchange resins are also preferred when used in the hydroxide form (OH-).

[0099] Characterization of fractions The form of characterization varies depending on the nature of the crude drug under study and the characterization method employed. In general, any or all of the following approaches can be used:

[0100] (a) Functional characterization Functional characterization may include biological assays. Biological assays can be performed in vivo or in vitro and may include enzyme inhibition assays (e.g., sialidase inhibition). Other biological assays include receptor binding assays, cell assays (including cell replication, cell pathogens, and cell-cell interactions, and cell secretion assays), immunoassays, antibacterial activity (e.g., cell binding and / or replication of bacteria and viruses) assays, and toxicity assays (e.g., LD 50 assays).

[0101] Functional characterization can also be performed indirectly by forms of characterization that enable the identification of one or more indicators of biological activity.

[0102] (b) Physical characterization This can take the form of quantification of the phytochemical component(s) present in any given fraction or other stage of the process, measurement of the purity of the component, or determination of the molecular weight distribution or various statistical functions thereof in the case of a fraction containing multiple different phytochemical components, determination of the molecular formula(s) (e.g., by nuclear magnetic resonance), and various forms of spectral analysis.

[0103] Particularly useful spectral characterizations include the following: ● Mass spectrum (e.g., mass-to-charge ratio (m / z) value vs. abundance), and / or ● Chromatography data (spectrum, column retention time, elution profile, etc.), and / or ● Photodiode array (PDA) spectrum (e.g., both UV and visible ranges), and / or ● Electrochemical detection or evaporative light scattering detection, and / or ● Nuclear magnetic resonance (NMR) spectrum ( 1 H and / or 13 including spectral datasets obtained via 1H and / or 13C NMR).

[0104] The evaluation of spectral characteristics can be combined with a fractionation step. For example, using GC-MS and HPLC-PDA-MS-ED-ELSD (as described herein), fractions can be combined with the acquisition of mass spectra, UV-visible spectra, electrochemical responses, or fractional mass data and chromatographic spectral data.

[0105] Using any or all of the above characteristics, a "chemical fingerprint" of any sample (or its fraction or phytochemical component) can be defined.

[0106] (c) Chemical characterization This can take the form of, inter alia, the measurement of the chemical reactivity, solubility, stability, and melting point of phytochemical components (s).

[0107] Medical uses of the compounds of the present invention Neoplasm The compounds of the present invention are sialidase inhibitors and thus find use in the treatment or prevention of diseases and disorders mediated by sialidase activity and / or sialic acid.

[0108] Sialidases are involved in various pathological processes, including bacterial and viral infections and neoplasms, and thus these enzymes have become attractive therapeutic targets. The expression of sialidases Neu1 and Neu3 appears to be altered in diabetes (e.g., Neu1 activity discussed by Natori, Y., et al, 2013, Biol.Pharm., Bull., 36, 1027). Sialidases are also thought to be involved in atherogenesis (Sukhorukov, V.N., et al., 2017, Curr. Pharm. Des., 23, 4696) and osteoarthritis (Katoh, S., et al., 1999, J Immunol., 162, 5058).

[0109] Accordingly, the compounds of the present invention find use in the treatment or prevention of neoplasms / proliferative disorders, as described in more detail below.

[0110] As used herein, the term "neoplasm" is used strictly to define a disease involving abnormal proliferation of neoplastic cells. This term includes benign, pre-cancerous, and malignant neoplasms (as defined above) and is used synonymously with the term "proliferative disorder".

[0111] Neoplasms result from inappropriately high levels of cell division and / or low levels of apoptosis or senescence in neoplastic cells that have acquired genetic or epigenetic changes that liberate them from normal physiological control (i.e., the cells are "transformed"). Neoplasms typically give rise to the following structures known as neoplasms: an abnormal mass of tissue, the growth of which exceeds that of normal tissue and is unregulated, and the growth of which persists in the same excessive manner even after the stimulus that caused the change has ceased. Most neoplasms form large tissue masses (solid tumors), although some neoplasms do not form such discrete tissue masses. These include cervical intraepithelial neoplasia, anal intraepithelial neoplasia, and leukemia.

[0112] Neoplasms can be benign, potentially malignant, or malignant. Benign tumors include uterine fibroids and pigmented nevi (skin moles), which are not invasive and do not change into or progress to malignant neoplasms. Potentially malignant (precancerous) neoplasms include non-invasive intraepithelial carcinomas, which are not invasive but can transform into malignant neoplasms over time.

[0113] Malignant neoplasms are neoplasms (tumors) that can invade and destroy surrounding tissues, form metastases, and ultimately cause the death of the host. The terms "malignant neoplasm" and "cancer" are used synonymously herein.

[0114] The terms "proliferative disorder" and "neoplasm" can be used synonymously herein to define a class of diseases involving the pathological proliferation of cells in vivo.

[0115] Thus, proliferative disorders include cancer, cancer metastases, smooth muscle cell proliferation, systemic sclerosis, cirrhosis, adult respiratory distress syndrome, idiopathic cardiomyopathy, systemic lupus erythematosus, retinopathy (e.g., diabetic retinopathy), cardiac hyperplasia, benign prostatic hyperplasia, ovarian cysts, pulmonary fibrosis, endometriosis, fibromatosis, hamartoma, lymphangiomatosis, sarcoidosis, and desmoid tumors. Neoplasms involving smooth muscle cell proliferation include excessive proliferation of cells in the vasculature (e.g., intimal smooth muscle cell hyperplasia, restenosis, and vascular occlusion including specific stenosis after biological or mechanical mediated vascular injury such as angioplasty). Furthermore, intimal smooth muscle cell hyperplasia can include hyperplasia in smooth muscle outside the vasculature (e.g., obstruction within the kidneys of patients with biliary duct, bronchial airway, and renal interstitial fibrosis). Non-cancerous proliferative diseases also include excessive proliferation of cells in the skin such as psoriasis and its various clinical forms, Reiter's syndrome, actinic keratosis, and hyperproliferative variants of keratinization disorders (including actinic keratosis, seborrheic keratosis, and scleroderma).

[0116] The term "neoplasm" is also used herein in a broad sense to define diseases associated with abnormal cell growth and / or differentiation in vivo, and thus includes hyperplasia, metaplasia, and dysplasia.

[0117] Hyperplasia defines a state in which normal (untransformed) cells within an organ or tissue grow to an abnormal degree. Thus, it can lead to an overall enlargement of the organ, the formation of a benign tumor, or it may only be visible microscopically. Hyperplasia is a physiological response to a specific stimulus, and hyperplastic cells continue to be influenced by normal regulatory control mechanisms (different from neoplastic growth where cells grow in an abnormal way not responsive to normal physiological control). Examples include congenital adrenal hyperplasia, endometrial hyperplasia, benign prostatic hyperplasia (prostatic hypertrophy), breast hyperplasia (including ductal hyperplasia), focal epithelial hyperplasia (Heck disease), sebaceous gland hyperplasia, liver hyperplasia, and the like.

[0118] Metaplasia defines a state in which one type of mature differentiated cell is replaced by another type of mature differentiated cell. Examples include squamous metaplasia of the columnar epithelial cells of the salivary gland ducts (when calculi are present), squamous metaplasia of the transitional epithelium of the bladder (similarly, when calculi are present or in relation to infection), glandular metaplasia of the esophagus in patients with gastroesophageal reflux disease (Barrett esophagus), and osseous metaplasia in connective tissue.

[0119] Dysplasia defines a state characterized by abnormal maturation of cells within a tissue. This generally consists of an expansion of immature cells and a corresponding decrease in the number and arrangement of mature cells. For example, epithelial dysplasia of the cervix is characterized by an increase in the population of immature cells limited to the mucosal surface. Myelodysplastic syndromes, or dysplasia of hematopoietic cells, show an increase in the number of immature cells in the bone marrow and a decrease in the number of mature functional cells in the blood. Other examples include neurofibromatosis.

[0120] Hyperplasia, metaplasia, and dysplasia are generally reversible states and are the result of a stimulus (e.g., an episode or injury). In contrast, neoplasms are generally irreversible and are associated with cell transformation.

[0121] The compounds of the present invention find general use in the treatment of any neoplasm, including proliferative disorders, benign, pre-cancerous and malignant neoplasms, hyperplasia, metaplasia, and dysplasia.

[0122] Accordingly, the present invention finds use in the treatment of proliferative disorders including, but not limited to, cancer, cancer metastasis, smooth muscle cell proliferation, systemic sclerosis, cirrhosis, adult respiratory distress syndrome, idiopathic cardiomyopathy, systemic lupus erythematosus, retinopathy (e.g., diabetic retinopathy), cardiac hyperplasia, benign prostatic hyperplasia, ovarian cysts, pulmonary fibrosis, endometriosis, fibromatosis, hamartoma, lymphangiomatosis, sarcoidosis, and desmoid tumors. Neoplasms associated with smooth muscle cell proliferation include excessive proliferation of cells in the vasculature (e.g., intimal smooth muscle cell hyperplasia, restenosis, and vascular occlusion including specific stenosis after biological or mechanical mediated vascular injury such as angioplasty). Further, intimal smooth muscle cell hyperplasia may include hyperplasia in smooth muscle outside of the vasculature (e.g., occlusion within the kidney in patients with biliary tract, bronchial airway, and renal interstitial fibrosis). Non-cancerous proliferative diseases also include excessive proliferation of cells in the skin such as psoriasis and its various clinical forms, Reiter's syndrome, actinic keratosis, and hyperproliferative variants of keratinization disorders (including actinic keratosis, senile keratosis, and scleroderma).

[0123] Particularly preferred is the treatment of malignant neoplasms (cancer). The present invention finds use in the treatment of all cancers, including those selected from the following major classifications: (a) carcinomas; (b) blastomas; (c) leukemias; (d) lymphomas; (e) myelomas; (f) sarcomas; and (g) mixed cancers.

[0124] Carcinoma refers to a malignant neoplasm of epithelial origin, or cancer of the inner or outer layers of the body. Carcinomas, which are malignant tumors of epithelial tissue, account for 80 - 90 percent of all cancer cases. Epithelial tissue is found throughout the body. It is present not only in the skin, but also in the covering and inner layers of organs and internal passageways such as the gastrointestinal tract. In a preferred embodiment, carcinomas treated according to the present invention are selected from the following carcinomas: colon; rectum; appendix; lung; thymus; breast; cervix; bladder; and eye.

[0125] The present invention finds use in the treatment of all blastomas, including hepatoblastoma (e.g., nephroblastoma, non-epithelial renal tumor, rhabdoid renal tumor, renal sarcoma, and pPNET of the kidney), medulloblastoma, pancreatoblastoma, pulmonary blastoma, pleuropulmonary blastoma, neuroblastoma (including common peripheral nerve cell tumors, as well as ganglioneuroblastoma and retinoblastoma).

[0126] The present invention finds use in the treatment of all leukemias, myeloproliferative diseases, and myelodysplastic diseases, including: lymphocytic leukemia (e.g., precursor cell leukemia, mature B cell leukemia, mature T cell leukemia, and NK cell leukemia); acute myeloid leukemia; chronic myeloproliferative diseases; myelodysplastic syndromes, and other myeloproliferative diseases. Thus, the present invention finds use in the treatment of various leukemias, including lymphocytic leukemia, lymphoblastic leukemia (malignant tumors of the lymphocytic and lymphoblastic blood cell lineages), as well as polycythemia vera or erythremia (malignant tumors of various blood cell products, but with erythrocytes being dominant).

[0127] Lymphomas occur in the network of lymphatic glands or lymph nodes, blood vessels, lymph nodes, and organs (especially the spleen, tonsils, thymus), and produce white blood cells or lymphocytes that purify body fluids and fight infection. Unlike leukemias, which are often called "liquid cancers," lymphomas are "solid cancers." Lymphomas can also occur in specific organs such as the stomach, breast, or brain. These lymphomas are called extranodal lymphomas. Lymphomas are classified into two categories: Hodgkin lymphoma and non-Hodgkin lymphoma. The presence of Reed-Sternberg cells in Hodgkin lymphoma diagnostically distinguishes Hodgkin lymphoma from non-Hodgkin lymphoma. The present invention finds use in the treatment of all such lymphomas and reticuloendothelial neoplasms, including: (a) Hodgkin lymphoma; (b) non-Hodgkin lymphoma (e.g., precursor cell lymphoma, mature B cell lymphoma, mature T cell lymphoma, and NK cell lymphoma); (c) Burkitt lymphoma, and (d) other lymphoreticular neoplasms including mantle cell lymphoma.

[0128] Accordingly, the present invention finds use in the treatment of a wide range of lymphomas, including, for example, tumors of lymphoid glands or nodules (including the spleen, tonsils, and thymus), and extranodal lymphomas of the stomach, breast, and brain.

[0129] Multiple myeloma is a cancer that occurs in the plasma cells of the bone marrow. Accordingly, the present invention finds use in the treatment of hematopoietic tumors and blood malignancies of lymphoid origin (e.g., leukemia, acute lymphoblastic leukemia, chronic lymphocytic leukemia, B-cell lymphoma (such as diffuse large B-cell lymphoma), T-cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma (the presence of Reed-Sternberg cells in Hodgkin lymphoma distinguishes Hodgkin lymphoma from non-Hodgkin lymphoma), hairy cell lymphoma, and Burkitt lymphoma), as well as hematopoietic tumors of myeloid origin (e.g., acute myeloid leukemia, chronic myeloid leukemia, myeloid leukemia, and imatinib-sensitive and refractory chronic myeloid leukemia, myelodysplastic syndrome, bortezomib-sensitive and refractory multiple myeloma, myeloproliferative disorders or promyelocytic leukemia, and thyroid follicular carcinoma).

[0130] The present invention finds use in the treatment of all sarcomas. A sarcoma refers to a cancer that occurs in supportive or connective tissues such as bone, tendon, cartilage, muscle, and fat. The most common sarcomas that generally occur in young adults often present as bone tumors accompanied by pain. Sarcoma tumors usually resemble the tissues in which they grow. Exemplary sarcomas for treatment according to the present invention include osteosarcoma (or osteogenic sarcoma); chondrosarcoma; leiomyosarcoma (smooth muscle); rhabdomyosarcoma (skeletal muscle); mesothelioma or mesothelioma (the membranous inner layer of body cavities); fibrosarcoma (fibrous tissue); angiosarcoma or hemangioendothelioma (blood vessels); liposarcoma; glioma; astrocytoma; myxosarcoma (primary embryonic connective tissue) as well as mesenchymal or mixed mesodermal tumors (mixed connective tissue type). Fibrosarcomas include peripheral nerve sheath tumors and other fibrous neoplasms, such as fibroblastic and myofibroblastic tumors, schwannomas and other fibromatous neoplasms. Kaposi sarcoma is also included. Soft tissue sarcomas, such as soft tissue Ewing's tumors and Askin tumors, soft tissue pPNET, extra-renal rhabdoid tumors, fibrous histiocytic tumors; synovial sarcoma; osseous and chondrosarcomatous neoplasms of soft tissue and alveolar soft part sarcoma are also included. Osteosarcoma (malignant bone tumor) includes malignant fibrous neoplasms of bone; malignant chordoma and odontogenic malignant tumors. Gliomas include oligodendroglioma, mixed and indeterminate gliomas, neuroepithelial gliomas.

[0131] The present invention finds use in the treatment of mixed cancers including, for example, adenocarcinoma, mixed mesodermal tumors, carcinosarcoma and teratoma. Thus, the present invention finds use in the treatment of various CNS, PNS and other intracranial and intraspinal neoplasms including astrocytoma, neuroblastoma, glioma, schwannoma, epithelioma and choroid plexus tumors (e.g., epithelioma and choroid plexus tumors); intracranial and intraspinal germ cell tumors (e.g., medulloblastoma, primitive neuroectodermal tumors (PNET), medulloepithelioma, atypical teratoid / rhabdoid tumors and other intracranial and intraspinal tumors (e.g., generally, pituitary adenomas and carcinomas, tumors in the sellar region (craniopharyngioma), nerve and mixed nerve gliomas, medulloblastoma, and intracranial and intraspinal neoplasms).

[0132] Accordingly, the present invention finds particular use in the treatment of intracranial and intraspinal germ cell tumors; intracranial and intraspinal germinomas; intracranial and intraspinal teratomas; intracranial and intraspinal embryonal carcinomas; intracranial and intraspinal yolk sac tumors; intracranial and intraspinal choriocarcinomas, as well as mixed intracranial and intraspinal tumors.

[0133] The present invention also finds use in the treatment of various germ cell tumors, trophoblastic tumors, and gonadal neoplasms. Accordingly, the present invention finds use, for example, generally in the treatment of malignant germ cell tumors at extracranial and extragonadal sites, malignant teratomas at extracranial and extragonadal sites, embryonal carcinomas at extracranial and extragonadal sites, yolk sac tumors at extracranial and extragonadal sites; choriocarcinomas at extracranial and extragonadal sites, and malignant mixed germ cell tumors at extracranial and extragonadal sites. The present invention also finds use, for example, in the treatment of malignant gonadal germ cell tumors including malignant gonadoblastomas, seminomas, malignant gonadal teratomas, gonadal embryonal carcinomas, gonadal yolk sac tumors, gonadal cholangiocarcinomas, malignant gonadal tumors of mixed morphology, and malignant gonadal gonadoblastomas.

[0134] Infectious diseases The compounds of the present invention are sialidase inhibitors and accordingly find use in the treatment or prevention of diseases and disorders mediated by sialidase activity and / or sialic acid. Such diseases and disorders include infectious diseases (including bacterial and viral infections).

[0135] The compounds of the present invention can have anti-infective (e.g., pathogen-blocking or pathogen-killing) activity against any infective agent. Accordingly, the compounds of the present invention can target (i.e., be active against) a wide range of different infectious agents. Accordingly, the present invention finds broad use in the treatment or prevention of any infectious disease or infectious disorder, including infectious diseases in which viral, bacterial, fungal, protozoal, prion, or metazoal agents are involved.

[0136] Accordingly, the present invention finds wide use in the treatment or prevention of viral infections; the treatment or prevention of bacterial infections; the treatment or prevention of protozoal infections; the treatment or prevention of fungal infections; the treatment or prevention of prion infections; and / or the treatment or prevention of infection or infestation by metazoans (e.g., helminths). The compounds of the present invention can also find use in the treatment or prevention of infection or infestation by chronic, dormant or latent viruses, bacteria, protozoa, fungi, prions or metazoans (e.g., helminths).

[0137] ● Viral targets include, but are not limited to, the following viruses (or virus classes): Retroviridae (e.g., human immunodeficiency virus including HIV-1); Picornaviridae (e.g., poliovirus, hepatitis A virus, enterovirus, human coxsackievirus, rhinovirus, echovirus); Caliciviridae (e.g., strains causing gastroenteritis); Togaviridae (e.g., equine encephalitis virus, rubella virus); Flaviviridae (e.g., dengue virus, encephalitis virus, yellow fever virus); Coronaviridae (e.g., coronavirus); Rhabdoviridae (e.g., vesicular stomatitis virus, rabies virus); Filoviridae (e.g., Ebola virus); Paramyxoviridae (e.g., parainfluenza virus, mumps virus, measles virus, respiratory syncytial virus); Orthomyxoviridae (e.g., influenza virus); Bunyaviridae (e.g., hantavirus, bunyavirus, phlebovirus, and nairovirus); Arenaviridae (hemorrhagic fever virus); Reoviridae (e.g., reovirus, orbivirus, and rotavirus); Birnaviridae; Hepadnaviridae (hepatitis B virus); Parvoviridae (parvovirus); Papillomaviridae (papillomavirus, polyomavirus); Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus (HSV) 1 and 2, varicella-zoster virus, cytomegalovirus (CMV), herpesvirus); Poxviridae (smallpox virus, vaccinia virus, poxvirus); and Iridoviridae (e.g., African swine fever virus); as well as unclassified viruses (e.g., causative agents of spongiform encephalopathies, pathogen of delta hepatitis (thought to be a defective satellite of hepatitis B virus), HCV virus (causing non-A, non-B hepatitis), Norwalk and related viruses, and astrovirus).Particularly preferred among these are HIV, hepatitis A virus, hepatitis B virus, hepatitis C virus, rabies virus, poliovirus, influenza virus, meningitis virus, measles virus, mumps virus, rubella, pertussis, encephalitis virus, papillomavirus, yellow fever virus, respiratory syncytial virus, parvovirus, chikungunya virus, hemorrhagic fever virus and herpes virus, in particular varicella, cytomegalovirus and Epstein-Barr virus.

[0138] ● The bacterial targets include, but are not limited to, both Gram-negative and Gram-positive bacteria. Examples of bacteria that the compounds of the present invention can target include, but are not limited to: Helicobacter pylori, Borelia burgdorferi, Legionella pneumophilia, Mycobacterium species (e.g., M. tuberculosis, M. leprae, M. avium, M. intracellulare, M. kansaii, and M. gordonae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group A streptococcus), Streptococcus agalactiae (Group B streptococcus), Streptococcus viridans, Streptococcus faecalis, Streptococcus bovis, anaerobic species of the genus Streptococcus, Streptococcus pneumoniae, Campylobacter species, Enterococcus species, Haemophilus influenzae, Bacillus anthracis, Corynebacterium species (including C. diphtheriae), Erysipelothrix rhusiopathiae, Clostridium perfringens, Clostridium tetani, Enterobacter aerogenes, Klebsiella species (including K. pneumoniae), Pasturella multocida, Bacteroides species, Fusobacterium nucleatum, Streptobacillus monilijormis, Treponema pallidium, Treponema pertenue, Leptospira species, Rickettsia species, and Actinomyces species (including A. israelii).Bacteria that form biofilms in vivo are specific targets of the compounds of the present invention, and these include Tannerella forsythia, Tannerella denticola, Porphyromonas gingivalis, and Gardnerella vaginalis.

[0139] ● Targets of fungi include, but are not limited to, Cryptococcus neoformans, Histoplasma capsulatum, Coccidioides immitis, Blastomyces dermatitidis, Chlamydia trachomatis, and Candida albicans.

[0140] ● Targets of protozoa include, but are not limited to, Plasmodium species (including Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, and Plasmodium vivax), Toxoplasma species (including T. gondii and T. cruzii), Leishmania species, Cryptosporidium species (including C. parvum), Cyclospora species (including C. cayetanensis), Entamoeba species (including E. histolytica), and Giardia species (including G. lamblia).

[0141] ● Targets of metazoans include parasites or pathogens such as helminths (e.g., Schistosoma species).

[0142] Inhibition of Bacterial Growth in Vivo The sialidase inhibitory properties of the compounds of the present invention also find use in disrupting host-bacterial cell interactions, including inhibition of commensal and / or pathogenic bacterial growth in vivo, particularly inhibition or elimination of bacterial biofilms in a mammalian (e.g., human) host.

[0143] Accordingly, these compounds find use in the treatment or prevention of diseases and disorders mediated or characterized by the presence of bacterial biofilms (such as subgingival plaque biofilms and fascial biofilms).

[0144] Such diseases include periodontal diseases, bacterial vaginitis, and diseases caused by infections with Tannerella forsythia, Tannerella denticola, Porphyromonas gingivalis, and Gardnerella vaginalis (the latter species is associated with bacterial vaginitis and preterm birth).

[0145] Atherogenesis The compounds of the invention are sialidase inhibitors and, since sialidase is involved in this process, find use in the treatment or prevention of atherogenesis (Sukhorukov, V.N., et al., 2017, Curr. Pharm. Des., 23, 4696) and osteoarthritis (Katoh, S., et al., 1999, J Immunol., 162, 5058). Accordingly, the compounds of the invention find use in the treatment and prevention of atherosclerotic diseases.

[0146] Regulation of commensal bacterial growth The sialidase inhibitory properties of the compounds of the invention also find use in the regulation of the composition of the microbiota (and in particular commensal bacteria) in a host, for example, in the regulation of the composition of commensal bacteria in a mammalian (such as human) host. Particularly preferred is the regulation of the gut microbiota.

[0147] Inflammation The compounds of the present invention inhibit sialidase, which is thought to be involved in the TNF-α-induced inflammatory process in osteoarthritis (Gee, K. et al., 2003, J Biol Chem., 37275). Furthermore, the compounds of the present invention can suppress or inhibit TNF-α activity. Thus, they find use in any disorder in which inflammation plays a role in the dysfunction and / or symptoms and / or pain of physiological functions. For example, the compounds of the present invention can be used as anti-inflammatory agents, for example, to reduce or eliminate acute, chronic, local, or systemic inflammation.

[0148] Inflammation occurs when tissues are damaged by microorganisms, trauma, chemicals, heat, cold, sunburn, or other harmful events. Endogenous chemicals (e.g., bradykinin, histamine, and serotonin) are released upon injury or damage, and such chemicals activate and attract tissue macrophages and other white blood cells. In this process, chemical mediators such as TNF-α are released, causing inflammation.

[0149] Inflammatory disorders are those in which the inflammation is persistent or chronic. In such situations, long-term inflammation causes tissue destruction, leading to extensive damage and eventual failure of the affected tissues and / or organs.

[0150] Accordingly, the compounds of the present invention find use in the treatment of non-localized inflammatory disorders, for example, those that affect multiple organs. Such disorders include those resulting from immune dysfunction (and thus may have an autoimmune component). Such conditions include systemic lupus erythematosus (SLE), scleroderma, allergies, and the like.

[0151] The compounds of the present invention also find use in the treatment of localized inflammatory disorders, including skin inflammation and chronic prostatitis, glomerulonephritis, inflammatory bowel disease, pelvic inflammatory disease, reperfusion injury, rheumatoid arthritis, graft rejection, vasculitis, asthma, acne, osteoarthritis, oral mucosa, gastrointestinal inflammation, eye, nose, and ear inflammation, and other steroid-responsive inflammatory diseases.

[0152] In particular, the compounds of the present invention find use in the treatment of inflammatory skin diseases. These include, for example, actinic keratosis, acne (including acne vulgaris, pustular acne, rosacea, and nodulocystic acne), allergic contact dermatitis, angioedema, bullous pemphigoid, cutaneous drug reactions, erythema multiforme, dermatomyositis, photodermatitis, psoriatic arthritis, scleroderma and urticaria, psoriasis, dermatitis (e.g., atopic dermatitis), scleroderma, steroid-responsive cutaneous inflammatory disorders (e.g., uremic pruritus), and skin conditions associated with exposure to sunlight, radiation, chemotherapy, and environmental irritants.

[0153] The compounds of the present invention also find use in the treatment of inflammatory autoimmune diseases. Such diseases can include specific tissues or organs (such as musculoskeletal tissues like rheumatoid arthritis and ankylosing spondylitis), the gastrointestinal tract (e.g., such as Crohn's disease and ulcerative colitis), the CNS (e.g., such as Alzheimer's disease, multiple sclerosis, motor neuron diseases, Parkinson's disease, and chronic fatigue syndrome), pancreatic beta cells (e.g., insulin-dependent type 1 diabetes), the adrenal glands (e.g., Addison's disease), the kidneys (e.g., Goodpasture's syndrome, IgA nephropathy, and interstitial nephritis), exocrine glands (e.g., Sjögren's syndrome and autoimmune pancreatitis), and the skin (e.g., psoriasis and atopic dermatitis).

[0154] Other inflammatory disorders treatable according to the present invention include conditions such as osteoarthritis, periodontal disease, diabetic nephropathy, chronic obstructive pulmonary disease, arthrosclerosis, graft-versus-host disease, chronic pelvic inflammatory disease, endometriosis, chronic hepatitis, and tuberculosis.

[0155] Energy utilization diseases The compounds of the present invention have glucose-regulating activity. Accordingly, the present invention finds use in the treatment of various energy utilization diseases. These diseases include a wide range of diseases and disorders, for example, disorders of homeostasis, metabolic diseases, dysfunctions of glucose metabolism, and disorders of appetite.

[0156] Thus, examples of energy utilization disorders include insulin resistance, various forms of diabetes (including type 1 and type 2 diabetes), metabolic syndrome, obesity, cachexia syndromes (e.g., cancer-related cachexia), myopathy, gastrointestinal diseases, growth retardation, hypercholesterolemia, atherosclerosis, and age-related metabolic dysfunction.

[0157] Energy utilization disorders also include conditions related to metabolic syndrome, obesity, and / or diabetes, such as hyperglycemia, glucose intolerance, hyperinsulinemia, glycosuria, metabolic acidosis, cataracts, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, macular degeneration, glomerulosclerosis, diabetic cardiomyopathy, insulin resistance, glucose metabolism disorders, arthritis, hypertension, hyperlipidemia, osteoporosis, osteopenia, bone loss, brittle bone syndrome, acute coronary syndrome, infertility, short bowel syndrome, chronic fatigue, eating disorders, and intestinal motility disorders.

[0158] Insulin Resistance, Metabolic Syndrome, Diabetes In healthy individuals, blood glucose levels are maintained within a narrow range by two pancreatic hormones, insulin (produced by pancreatic β-cells) and glucagon (produced by pancreatic α-cells). Pancreatic β-cells sense an increase in blood glucose levels and respond by secreting insulin. Insulin promotes the uptake of glucose by body tissues, thereby returning blood glucose concentrations to the physiological range. Glucagon acts reciprocally and raises fasting blood glucose levels mainly by stimulating glucose production in the liver.

[0159] Insulin resistance is characterized by a decrease in the action of insulin in skeletal muscle, adipocytes, and hepatocytes. As a result, a normal amount of insulin becomes insufficient to elicit a normal insulin response from the cells of these tissues. In adipocytes, insulin resistance causes stored triglycerides to be hydrolyzed, leading to an increase in free fatty acids in the plasma. In muscle, insulin resistance reduces glucose uptake, and in hepatocytes, it reduces glucose storage. In both of the latter cases, an increase in blood glucose concentration occurs.

[0160] High plasma levels of insulin and glucose due to insulin resistance often progress to metabolic syndrome and type 2 diabetes.

[0161] Metabolic syndrome is a collection of abnormalities and disorders that increase the risk of cardiovascular disease and diabetes. The incidence is very high in many developed countries: some studies have shown a prevalence in the United States of up to 25% of the population. This disorder is also known as (metabolic) syndrome X, insulin resistance syndrome, Reaven's syndrome, and CHAOS. Metabolic syndrome is diagnosed by the presence of three or more of the following symptoms: central obesity (waist measurement of 101.6 cm (40 inches) or more in men and 88.9 cm (35 inches) or more in women); high levels of triglycerides (150 mg / dL or more); low levels of HDL (less than 40 mg / dL in men and less than 50 mg / dL in women), and high blood pressure (130 / 85 mmHg or more). Associated diseases and symptoms are as follows: fatty liver (often progressing to non-alcoholic fatty liver disease), polycystic ovary syndrome, hemochromatosis (iron overload), and acanthosis nigricans (black skin patches).

[0162] The primary treatment for metabolic syndrome is lifestyle changes (calorie restriction and physical activity). However, drug treatment is often necessary. Generally, the individual diseases that make up metabolic syndrome are treated separately (e.g., diuretics and ACE inhibitors for high blood pressure). Cholesterol medications can be used to lower LDL cholesterol and triglyceride levels when they are elevated and to raise HDL levels when they are low. The use of medications to reduce insulin resistance (e.g., metformin and thiazolidinediones) has been controversial. Aerobic exercise is therapeutic in less than 31% of cases and generally does not cause a reduction in fasting plasma glucose or insulin resistance.

[0163] Accordingly, there is a need for new and / or alternative treatments for metabolic syndrome, particularly treatments effective against obesity and / or elevated triglyceride levels.

[0164] Type 2 diabetes is a chronic disease characterized by a persistent elevation in blood glucose levels (hyperglycemia). Insulin resistance, along with impaired insulin secretion from pancreatic β-cells, is characteristic of this disease. Progression of insulin resistance to type 2 diabetes is characterized by the onset of postprandial hyperglycemia when pancreatic β-cells can no longer produce sufficient insulin to maintain normal blood glucose levels (euglycemia).

[0165] The most important drugs currently used in the treatment of type 2 diabetes are metformin (Glucophage, Diabex, Diaformin, Fortamet, Riomet, Glumetza, Cidophage, etc.). Metformin is an oral antihyperglycemic agent of the biguanide class. Other biguanides include phenformin and buformin (now discontinued). Metformin functions mainly by reducing the hepatic release of glucose from glycogen stores, but also has some effect of increasing glucose uptake. Other widely used drug classes include the sulfonylurea group of drugs (including glyburide and gliclazide). These drugs increase glucose-stimulated insulin secretion by the pancreas. New drug classes include thiazolidinediones (e.g., rosiglitazone, pioglitazone, and troglitazone) that act by binding to peroxisome proliferator-activated receptors (PPARs), a group of receptor molecules in the cell nucleus. Other classes include α-glucosidase inhibitors (acarbose), meglitinides (stimulate insulin release and include nateglinide, repaglinide, and their analogs), peptide analogs (e.g., incretin mimetics that act as insulin secretagogues, glucagon-like peptide analogs (e.g., exenatide)), dipeptidyl peptidase-4 (DPP-4) inhibitors (increase incretin levels (e.g., sitagliptin)), and amylin agonist analogs (slow gastric emptying and suppress glucagon (e.g., pramlintide)).

[0166] However, existing therapies for the various forms of type 2 diabetes do not appear to improve the function of the major endogenous factors of β cells. All existing therapies cannot halt the progression of the disease, normalize blood glucose levels over time, and / or prevent subsequent complications. Existing therapies are also associated with undesirable side effects. For example, insulin secretagogues and insulin injections can cause hypoglycemia and weight gain. Patients may also become unresponsive to insulin secretagogues over time. Metformin and α-glucosidase inhibitors often cause gastrointestinal problems, and PPAR agonists tend to cause weight gain and increased edema. Exenatide has also been reported to cause nausea and vomiting.

[0167] Glycosylation plays an important role in regulating protein properties and is associated with many diseases. Itoh, N. et al., 2007 (Am J Physiol Endocrinol Metab 293: E1069-E1077) reported the serum N-glycan profile of human subjects with type 2 diabetes and found that the amount of branched N-glycans with α1,6-fucose along with bisecting N-acetylglucosamine was increased. Copeland, R.J. et al., 2008 (Am J Physiol Endocrinol Metab 295: E17-E28) reviewed the importance of O-linked N-acetylglucosamine in diabetes and concluded that there is a strong positive correlation between GlcNAcylation and the development of insulin resistance. O-linked-β-N-acetylglucosamine (O-GlcNAc) is a dynamic post-translational modification that cycles on and off serine and / or threonine hydroxyl groups, similar to phosphorylation. The cycling of O-GlcNAc is regulated by the coordinated action of O-GlcNAc transferase and O-GlcNAcase. GlcNAcylation is involved in the etiology of glucose toxicity and chronic hyperglycemia-induced insulin resistance, which are the major features of type 2 diabetes. Hexosaminidase activity has been shown to be elevated in the sera of diabetic patients (e.g., Agardh, C.D. et al., 1982, Acta Med Scand.212:39-41).

[0168] Type 1 diabetes (or insulin-dependent diabetes) is characterized by the loss of insulin-producing beta cells in the pancreatic islets of Langerhans, causing insulin deficiency. The main cause of this beta cell loss is a T cell-mediated autoimmune attack. There are no known preventive measures that can be taken against type 1 diabetes, which accounts for up to 10% of diabetes cases in North America and Europe. Most affected people are otherwise healthy and of normal weight at the time of onset. Sensitivity and responsiveness to insulin are usually normal, especially in the early stages.

[0169] The primary treatment for type 1 diabetes is insulin replacement, combined with careful monitoring of blood glucose levels using a blood test monitor, even from the early stages. Without insulin, ketoacidosis and diabetic ketoacidosis can develop, leading to coma or death. In addition to the common subcutaneous injection, it is also possible to deliver insulin with a pump, which allows for continuous infusion of insulin at pre-set levels 24 hours a day and programming of insulin doses (boluses) as needed at mealtimes. Exubera, an inhaled form of insulin, was recently approved by the FDA.

[0170] The treatment of type 1 diabetes needs to be continued indefinitely. The treatment does not impair normal activities, but sufficient awareness of tests and medications, appropriate care, and compliance with discipline are required.

[0171] Therefore, there is a need for new and / or alternative antidiabetic therapies, particularly those that can restore β-cell function. In particular, there is a real and substantially unmet clinical need for effective drugs that can treat both type 2 and type 1 diabetes and related conditions with fewer side effects than existing drug therapies.

[0172] The present invention finds wide application in the treatment of all energy utilization diseases.

[0173] Therefore, the diseases that can be treated by the present invention include, for example, disorders of homeostasis, metabolic diseases, dysfunction of glucose metabolism, and eating disorders.

[0174] In a preferred embodiment, the present invention finds application in the treatment of insulin resistance, various forms of diabetes, metabolic syndrome, obesity, cachexia syndromes (such as cancer-related cachexia), muscle disorders, gastrointestinal diseases, growth retardation, hypercholesterolemia, atherosclerosis, and age-related metabolic dysfunction.

[0175] The present invention can also be used for the treatment of metabolic syndrome, obesity, and / or conditions associated with diabetes, such as hyperglycemia, impaired glucose tolerance, hyperinsulinemia, glycosuria, metabolic acidosis, cataract, diabetic neuropathy, diabetic nephropathy, diabetic retinopathy, macular degeneration, glomerulosclerosis, diabetic cardiomyopathy, insulin resistance, glucose metabolism disorder, arthritis, hypertension, hyperlipidemia, osteoporosis, osteopenia, bone loss, brittle bone syndrome, acute coronary syndrome, infertility, short bowel syndrome, chronic fatigue, eating disorder, intestinal motility disorder, and sugar metabolism disorder.

[0176] The present invention can also be used to suppress appetite. Without wishing to be bound by theory, this may even affect the maintenance of blood glucose levels and eliminate the large slumps that cause fatigue / tiredness and the desire to eat sweet things (i.e., by promoting an "improved energy balance").

[0177] Particularly preferred is the treatment of insulin resistance, metabolic syndrome, obesity, and diabetes (especially type 2 diabetes).

[0178] The present invention finds use in the treatment of insulin resistance. Insulin resistance is characterized by a decrease in the action of insulin in skeletal muscle, adipocytes, and hepatocytes, such that a normal amount of insulin becomes insufficient to elicit a normal insulin response from the cells of these tissues. In adipocytes, insulin resistance causes stored triglycerides to be hydrolyzed, resulting in an increase in free fatty acids in the plasma. In muscle, insulin resistance reduces glucose uptake, and in hepatocytes, it reduces glucose storage. In both of the latter cases, an increase in blood glucose concentration occurs. The high plasma levels of insulin and glucose due to insulin resistance often progress to metabolic syndrome and type 2 diabetes.

[0179] The present invention finds use in the treatment of metabolic syndrome (as defined herein). This disorder is also known as (metabolic) syndrome X, insulin resistance syndrome, Reaven's syndrome, and CHAOS.

[0180] The present invention finds use in the treatment of diseases associated with metabolic syndrome, including, for example, fatty liver (which often progresses to non-alcoholic fatty liver disease), polycystic ovary syndrome, hemochromatosis (iron overload), and acanthosis nigricans.

[0181] The present invention finds use in the treatment of diabetes, including type 1 and type 2 diabetes. Type 2 diabetes is a chronic disease characterized by a persistent elevation of blood glucose levels (hyperglycemia). Insulin resistance, together with impaired insulin secretion from pancreatic beta cells, is characteristic of this disease. The progression of insulin resistance to type 2 diabetes is characterized by the onset of postprandial hyperglycemia when pancreatic beta cells can no longer produce sufficient insulin to maintain normal blood glucose levels (euglycemia).

[0182] Type 1 diabetes The present invention finds use in the treatment of type 1 diabetes (or insulin-dependent diabetes). Type 1 diabetes is characterized by the loss of insulin-producing beta cells in the pancreatic islets of Langerhans, which causes insulin deficiency. The main cause of this beta cell loss is autoimmune attack mediated by T cells. There are no known preventive measures that can be taken against type 1 diabetes, which accounts for up to 10% of diabetes cases in North America and Europe. Most affected individuals are otherwise healthy and of normal weight at the time of onset. Sensitivity and responsiveness to insulin are usually normal, especially in the early stages.

[0183] As described below, the compounds of the present invention may have anti-diabetes-inducing virus activity. Since diabetes-inducing viruses are etiological factors for type 1 diabetes, compounds having anti-diabetes-inducing virus activity are particularly preferred for use in the treatment or prevention of type 1 diabetes, or in the following: (a) the treatment of virus-induced type 1 diabetes; (b) the delay or prevention of the development of viral diabetes; (c) the treatment of type 1 diabetes; (d) the treatment or prevention of virus-induced β-cell lysis (e.g., by non-immune cell lysis or immune-mediated cell lysis); (e) the prevention, reduction or elimination of virus-mediated endogenous interferon production; (f) the prevention, reduction or elimination of virus-mediated bystander activation of autoreactive T cells targeting β-cells; (g) the prevention, reduction or elimination of virus activation and / or expansion of autoreactive T cells targeting β-cells; and / or (h) the prevention or reduction of virus-mediated loss of regulatory T cells exposing β-cells to immune-mediated cell lysis.

[0184] Such patients are at high risk of progressing to type 1 diabetes and may be infected with diabetes-inducing viruses as described above, so such uses are particularly useful in the treatment of subjects having pancreatic islet autoantibodies.

[0185] The present invention also finds use in the treatment and management of mitochondrial diseases, particularly acquired mitochondrial dysfunction associated with the aforementioned energy utilization disorders.

[0186] Pharmacokinetics The compounds of the present invention can be administered by topical, oral or parenteral routes, including intravenous, intramuscular, intraperitoneal, subcutaneous, transdermal, airway (aerosol), rectal, vaginal, and topical (including buccal and sublingual) administration.

[0187] The amount administered can vary widely depending on the particular dosage unit used, the duration of treatment, the age and sex of the patient being treated, the nature and extent of the disorder being treated, and the particular compound selected.

[0188] Generally, the effective amount of the administered compound is generally in the range of about 0.01 mg / kg to 500 mg / kg per day. The unit dosage can contain 0.05 to 500 mg of the compound and can be taken more than once a day. The compound can be administered with a pharmaceutical carrier using any conventional dosage unit form, either orally, parenterally, or topically, as described below.

[0189] The preferred route of administration is oral administration. Generally, a suitable dosage is in the range of 0.01 to 500 mg per kilogram of body weight per day of the recipient, preferably in the range of 0.1 to 50 mg per kilogram of body weight per day, most preferably in the range of 1 to 5 mg per kilogram of body weight per day.

[0190] The desired dosage is preferably provided as a single dose for daily administration. However, it is also possible to use 2, 3, 4, 5, or 6, or more sub-doses administered at appropriate intervals throughout the day. These sub-doses can be administered, for example, in unit dosage forms containing 0.001 to 100 mg, preferably 0.01 to 10 mg, most preferably 0.5 to 1.0 mg of the active ingredient per unit dosage form.

[0191] Formulations The compounds for use according to the present invention can be in any form. It can be from synthetic or natural sources (e.g., plants of the Rhamnaceae and Rutaceae families (e.g., plants of the Ziziphus or Paullinia genera, e.g., plants of the species Zizyphus jujuba or Paullinia cupana)). Particularly preferred as plant sources are the fruits, parts of the fruits, fruit extracts, and fruit juices of the aforementioned plants.

[0192] When isolated from natural sources, the compound can be purified. However, the compositions of the present invention can be in the form of crude drugs, as described above. Such crude drugs are preferably analyzed to determine whether they meet the standard specifications before use.

[0193] The crude drug for use according to the present invention can be a dried plant material. Alternatively, the crude drug can be a processed plant material including physical or chemical pretreatment such as pulverization, grinding, freezing, evaporation, filtration, pressing, spray drying, extrusion, supercritical solvent extraction, and tincture production. When the crude drug is administered or sold in the form of the whole plant (or a part thereof), the plant material can be dried before use. Any convenient form of drying including freeze drying, spray drying, or air drying can be used.

[0194] The compounds of the present invention can be separated from high molecular weight components such as proteins and polysaccharides by extraction with a polar solvent (ethanol / water mixture, for example, an ethanol / water mixture of 50% v / v or more (for example, up to about 70% v / v)). Other suitable techniques include various membrane techniques. These include microfiltration, ultrafiltration, and nanofiltration. Alternatively, or additionally, electrodialysis can be used to concentrate charged compounds. These methods use membranes with pore sizes that allow only molecules smaller than a certain size to pass through, or that allow molecules to pass through or not pass through the membrane depending on the charge of the molecule. Anion and cation exchange resins can also be used to concentrate the compounds.

[0195] When isolated from natural sources, the compounds for use according to the present invention can be purified. In embodiments where the compounds are formulated with pharmaceutically acceptable excipients, any suitable excipients can be used, including, for example, inert diluents, disintegrants, binders, lubricants, sweeteners, flavoring agents, coloring agents, and preservatives. Suitable inert diluents include sodium carbonate and calcium carbonate, sodium and calcium phosphates, and lactose, while corn starch and alginic acid are suitable disintegrants. Binders can include starch and gelatin, while lubricants, when present, are generally magnesium stearate, stearic acid, or talc.

[0196] The pharmaceutical composition can take any suitable form and may include, for example, tablets, elixirs, capsules, solutions, suspensions, powders, granules, and aerosols.

[0197] The pharmaceutical composition can take the form of a kit of parts, which may include the composition of the present invention together with instructions for use and / or a plurality of different components in unit dosage forms.

[0198] Tablets for oral use may contain a compound for use according to the present invention, mixed with pharmaceutically acceptable excipients such as inert diluents, disintegrants, binders, lubricants, sweetening agents, flavoring agents, coloring agents, and preservatives. Suitable inert diluents include sodium carbonate and calcium carbonate, sodium and calcium phosphates, and lactose, while corn starch and alginic acid are suitable disintegrants. Binders may include starch and gelatin, while lubricants, when present, are generally magnesium stearate, stearic acid, or talc. Optionally, the tablets can be coated with materials such as glyceryl monostearate or glyceryl distearate to delay absorption in the gastrointestinal tract. Oral capsules include hard gelatin capsules in which the compound used in the present invention is mixed with a solid diluent, and soft gelatin capsules in which the active ingredient is mixed with an oil such as water or peanut oil, liquid paraffin, or olive oil.

[0199] Formulations for rectal administration can be provided as suppositories having a suitable base, for example, containing cocoa butter or salicylates. Formulations suitable for vaginal administration may be provided as pessaries, tampons, creams, gels, pastes, foams, or spray formulations containing a carrier as is known to be suitable in the art in addition to the active ingredient.

[0200] For use in muscle, intraperitoneal, subcutaneous, and intravenous applications, the compounds of the invention are generally provided as a sterile aqueous solution or suspension buffered to an appropriate pH and isotonicity. Suitable aqueous vehicles include Ringer's solution and isotonic sodium chloride. Aqueous suspensions according to the invention may contain suspending agents such as cellulose derivatives, sodium alginate, polyvinylpyrrolidone, and tragacanth gum, and wetting agents such as lecithin. Preservatives suitable for aqueous suspensions include ethyl and n-propyl p-hydroxybenzoate.

[0201] The compounds of the invention may also be provided as liposome formulations.

[0202] For oral administration, the compound or compounds may be formulated into solid or liquid preparations such as capsules, pills, tablets, troches, lozenges, melts, powders, granules, solutions, suspensions, dispersions, or emulsions (these solutions, suspensions, dispersions, or emulsions may be aqueous or non-aqueous). Solid unit dosage forms can be, for example, capsules of the normal hard shell or soft shell gelatin type containing surfactants, lubricants, and inert fillers such as lactose, sucrose, calcium phosphate, and corn starch.

[0203] In another embodiment, the compounds of the invention are combined with binders such as acacia, corn starch, or gelatin, disintegrants such as potato starch, alginic acid, corn starch, guar gum, etc. for the purpose of assisting in the disintegration and dissolution of the tablets after administration, lubricants such as talc, stearic acid, or magnesium stearate, calcium stearate, or zinc stearate for the purpose of improving the flow of the tablet granules and preventing the adhesion of the tablet material to the surfaces of the die and punch of the tablet, and dyes, colorants, and flavoring agents for the purpose of enhancing the aesthetic quality of the tablets and making them more acceptable to the patient, and are tableted on a conventional tablet base such as lactose, sucrose, and corn starch.

[0204] Excipients suitable for use in oral liquid dosage forms include diluents such as water and alcohols, for example ethanol, benzyl alcohol, and polyethylene alcohol, with or without the addition of pharmaceutically acceptable surfactants, suspending agents, or emulsifying agents.

[0205] The compounds of the present invention can also be administered parenterally, i.e., subcutaneously, intravenously, intramuscularly, or intraperitoneally.

[0206] In such embodiments, the compounds are provided as injectable dosages in a physiologically acceptable diluent, together with a pharmaceutical carrier, which can be a sterile liquid or a mixture of liquids. Suitable liquids include water, saline, aqueous dextrose and related sugar solutions, alcohols (such as ethanol, isopropanol, or hexadecyl alcohol), glycols (such as propylene glycol or polyethylene glycol), glycerol ketals (such as 2,2-dimethyl-1,3-dioxolan-4-methanol), ethers (such as poly(ethylene glycol) 400), oils, fatty acids, fatty acid esters or glycerides, or acetylated fatty glycerides, with or without the addition of pharmaceutically acceptable surfactants (such as soap or detergent), suspending agents (such as pectin, carrageenan, methylcellulose, hydroxypropylmethylcellulose, or carboxymethylcellulose), or emulsifying agents and other pharmaceutical adjuvants. Suitable oils for use in the parenteral formulations of the present invention are of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, sesame oil, cottonseed oil, corn oil, olive oil, petroleum, and mineral oil. Suitable fatty acids include oleic acid, stearic acid, and isostearic acid. Suitable fatty acid esters are, for example, ethyl oleate and isopropyl myristate.

[0207] Suitable soaps include fatty alkali metal, ammonium, and triethanolamine salts, and suitable detergents include cationic detergents such as dimethyldialkylammonium halides, alkylpyridinium halides, and alkylamine acetates; anionic detergents such as alkyl, aryl, and olefin sulfonates, alkyl, olefin, ether, and monoglyceride sulfates, and sulfosuccinates; nonionic detergents such as fatty amine oxides, fatty acid alkanolamides, and polyoxyethylene polypropylene copolymers; and amphoteric detergents such as alkyl-beta-aminopropionates and 2-alkylimidazoline quaternary ammonium salts, and mixtures thereof.

[0208] The parenteral composition of the present invention typically contains from about 0.5 to about 25 weight percent of the compound for use in accordance with the present invention in solution. Preservatives and buffers can also be used. To minimize or eliminate irritation at the injection site, such compositions can include a nonionic surfactant having a hydrophilic-lipophilic balance (HLB) of about 12 to about 17. The amount of surfactant in such formulations ranges from about 5 to about 15 weight percent. The surfactant can be a single component having the above-described HLB or a mixture of two or more components having the desired HLB. Examples of surfactants used in the parent formulation are the class of polyethylene sorbitan fatty acid esters such as sorbitan monooleate and high molecular weight adducts of ethylene oxide with hydrophobic bases formed by the condensation of propylene oxide with propylene glycol.

[0209] The compound or compounds for use in accordance with the present invention can also be administered topically, and when so done, the carrier can suitably include a solution, an ointment, or a gel base. The base can include, for example, petrolatum, lanolin, polyethylene glycol, beeswax, mineral oil, diluents such as water and alcohol, and one or more of emulsifiers and stabilizers. The topical formulation can contain a concentration of the compound of from about 0.1 to about 10% w / v (weight per unit volume).

[0210] When used adjunctively, the compound or compounds for use in accordance with the present invention can be formulated for use in combination with one or more other drug(s). Thus, adjunctive use can be reflected in a particular unit dosage designed to be compatible (or synergistic) with other drug(s), or in a formulation in which the compound or compounds are admixed with one or more enzymes. Adjunctive use can also be reflected in the composition of a pharmaceutical kit of the invention in which the compound of the invention is co-packaged with an enzyme (e.g., as part of a series of unit dosages). Adjunctive use can also be reflected in information and / or instructions regarding co-administration of the compound or compounds and / or enzyme.

[0211] Cosmetic formulation The cosmetic composition of the present invention can be selected, for example, from a moisturizing composition, a cleansing composition, or any composition that can provide a benefit to the skin. The cosmetic composition of the present invention can include, for example, a cosmetically acceptable excipient or carrier selected from those described below.

[0212] In one embodiment, the cosmetic composition is a cleansing composition. Suitable cleansing compositions are solid or semi-solid at room temperature. Examples of useful cleansing compositions include, but are not limited to, fatty acid soaps including glycerin soap, synthetic detergents, and mixtures thereof. Solid cleansing compositions are widely taught in Soap Technology for the 1990’s, the content of which is incorporated herein by reference. The cleansing composition is desirably fluid.

[0213] In one embodiment of the present invention, the cleansing composition includes glycerin soap. Examples of glycerin soap useful in the present invention include, but are not limited to, those disclosed in U.S. Patent Nos. 4,405,492 and 4,879,063, the content of which is incorporated herein by reference.

[0214] Examples of suitable fatty acid soaps include soaps derived from hydrocarbon chain lengths of about 10 to 22 (including the carboxyl carbon), which can be saturated or unsaturated. The soap can be, for example, a sodium salt, potassium salt, ammonium salt, triethanolammonium salt, and mixtures thereof.

[0215] Suitable synthetic detergents include those known in the art for the desired purpose. Examples of detergents useful for personal cleansing include isethionates, sarcosinates, and glyceryl ether sulfonates, which may be pure chain length variants or derived from commercially available oils such as coconut oil. Other suitable surfactants include anionic acyl sarcosinates, methyl acyl taurates, N-acyl glutamic acids, alkyl sulfosuccinates, alkyl phosphate esters, ethoxylated alkyl phosphate esters, trideceth sulfates, protein condensates, mixtures of ethoxylated alkyl sulfates and alkyl amine oxides, betaines, sultaines, and mixtures thereof. Included are alkyl ether sulfates having 1 to 12 ethoxy groups, particularly ammonium lauryl ether sulfate and sodium lauryl ether sulfate.

[0216] The cosmetic composition may be a moisturizing composition.

[0217] Other optional components of the cosmetic composition of the present invention include, but are not limited to, perfumes, fragrances, preservatives, colorants, dyes, anti-caking agents, and personal care components including, but not limited to, skin care components and hair care components.

[0218] Examples of suitable personal care ingredients useful in the present invention include, but are not limited to, a safe and effective amount of a humectant, a sunscreen active, a skin soother, an anti-irritant, an anti-inflammatory agent, a skin softener, a conditioning agent, a moisturizer, a deodorant, an antiperspirant, a self-tanning agent, an antibacterial agent, an anti-acne agent, an anti-wrinkle agent, an anti-skin atrophy agent, a skin hardening agent, an anti-itch agent, an antifungal agent, a local anesthetic, a skin tone evening agent, an active natural ingredient, an agent for minimizing the appearance of unwanted hair or delaying regrowth, a skin texture modifier, and an additional cleansing agent.

[0219] In one embodiment, the compound can be used from an aqueous or hydroalcoholic extract by using, for example, a water-in-oil (w / o) emulsion such as that used for the treatment of dry skin and for use as a skin softener.

[0220] Skin softeners function by staying on the skin surface or stratum corneum and acting as lubricants, reducing peeling, and improving the appearance of the skin. Typical skin softeners include fatty esters, fatty alcohols, mineral oils, polyether siloxane copolymers, etc. Examples of suitable skin softeners include, but are not limited to, polypropylene glycol (“PPG”)-15 stearyl ether, PPG-10 cetyl ether, steareth-10, oleth-8, PPG-4 lauryl ether, vitamin E acetate, PEG-7 glyceryl cocoate, lanolin, and combinations thereof. Vitamin E acetate, PEG-7 glyceryl cocoate, and combinations thereof are preferred.

[0221] Examples of suitable humectants include polyhydric alcohols. Suitable polyhydric alcohols include glycerol (also known as glycerin), polyalkylene glycols, alkylene polyols and their derivatives (including propylene glycol, dipropylene glycol, polypropylene glycol, polyethylene glycol and their derivatives), sorbitol, hydroxypropyl sorbitol, hexylene glycol, 1,3-dibutylene glycol, 1,2,6-hexanetriol, ethoxylated glycerol, propoxylated glycerol and mixtures thereof, but are not limited thereto.

[0222] Suitable skin soothers include, but are not limited to, panthenol, bisabolol, allantoin, aloe, and combinations thereof.

[0223] Suitable conditioning agents include, but are not limited to, dimethylpropyl PG-betaine, dimethicone copolyol, polyquaternium-10, guar, guar derivatives, and combinations thereof. Suitable anti-acne active ingredients include, but are not limited to, salicylic acid, sulfur, lactic acid, glycolic acid, pyruvic acid, urea, resorcinol, N-acetylcysteine, retinoic acid, benzoyl peroxide, octopirox, triclosan, azelaic acid, phenoxyethanol, phenoxypropanol, flavonoids, their derivatives, and combinations thereof. Salicylic acid and benzoyl peroxide are preferred.

Examples

[0224] Here, the present invention will be described with reference to specific examples. These are merely illustrative and for the purpose of explanation only: they are in no way intended to limit the claimed exclusive scope or the described invention. These examples constitute the best mode currently contemplated for practicing the present invention.

[0225] Example 1: Isolation and Identification of Compounds 1-3 All three 4-hydroxymethyl-N-methyl-L-proline compounds (1 - 3) were observed, isolated, and their structures elucidated from the fruits of Z. jujuba, the seeds of guarana, and the leaves of Z. spina-christi.

[0226] The seeds of guarana mainly contained compound 1 (about 0.1% w / w), with small amounts of 2 and 3. Ziziphus species typically had a ratio of compounds 1 and 3 of about 2:1 (about 0.1% w / w) respectively. Compounds 1 and 2 were isolated from the fruits of Z. jujuba, and 3 was isolated from Z. spina-christi.

[0227] None of these compounds have been isolated from any other source.

[0228] Preparation of extracts from Ziziphus plants A sample (200 g) of dried leaves of Z. spina-christi (BEZ01) was extracted overnight with 4 liters of 50% aqueous ethanol. The filtered extract was passed through a cation exchange column (H + type Amberlite IR120), and the retained fraction was eluted with ammonia. This was then subjected to anion exchange column chromatography (OH - type Amberlite CG400), the bound fraction was eluted with acetic acid, and applied to a CG400 acetate form column.

[0229] Analysis of the fractions enabled the identification of compound 1 (HS0810 / 146 / 36 - 50 mg) and 3 (HS0810 / 146 / 41 - 44 mg). Using the same method, compounds 1 (JH0806 / 42 / 4) and 2 (LJ746 / 18 / 7) were also isolated from the fruits of Z. jujuba, and 1 was also isolated from guarana (JH0420 / 8 / 20).

[0230] The extract of guarana (Blue Sky Botanics batch 150701A) differed from the extract of the genus Ziziphus in that it had a major compound that matched caffeine by GCMS of the trimethylsilyl derivative (major ion 194 amu) with a retention time of 9.9 minutes under the GC conditions described below, and that it had a novel N-methylated pipecolic acid (N-methyl-4R,5S-dihydroxy-2S-pipecolic acid) (JH0806 / 90 / 5; retention time 8.4 minutes).

[0231] GC analysis of extracts and pure compounds All samples were lyophilized before derivatization. The trimethylsilyl (TMS) derivative was prepared using a mixture of hexamethyldisilazane (HMDS) and trimethylchlorosilane (TMCS) in pyridine (Pierce "Tri-Sil" silylation reagent, HMDS:TMCS:pyridine in a ratio of 2:1:10). The samples were heated at 60 °C for 15 minutes and then left at room temperature for at least 60 minutes. The insoluble reaction products were precipitated by centrifugation and the supernatant was transferred to a new vial using a syringe.

[0232] The analysis was performed by GC-MS using a Perkin Elmer AutosystemXL gas chromatograph equipped with a highly polar fused silica column (Varian ‘Factor Four’ VF-5ms column, 25 m × 0.25 mm i.d., 0.25 mm film thickness). The flow rate of the carrier gas (helium) was 1 ml / min. The trimethylsilyl (TMS) derivatives were separated using a temperature program starting at 160 °C for 5 minutes and then linearly increasing to 300 °C at a rate of 10 °C / min. The temperature was held at 300 °C for an additional 10 minutes. The total analysis time was 29 minutes. Electron impact mass spectrometry of the column eluate was performed using a Perkin Elmer TurboMassGold mass spectrometer equipped with a quadrupole ion filter system that was always run at 250 °C during analysis. The mass range of the detector was set to 100 - 650 amu. The temperature of the transfer line (from GC to MS) was maintained at 250 °C. Samples were injected into the column via a split vent (split ratio 50:1) through a fused silica narrow bore injection liner filled with deactivated quartz wool. The inlet temperature was maintained at 200 °C. The injection volume was 1 μl. System control, data collection, and mass spectrum analysis were performed using Perkin Elmer TurboMass software (TurboMass v. 4.4).

[0233] Compounds 1 - 3, as the trimethylsilyl derivatives prepared as described above, exhibit characteristic mass spectra with different retention times by GCMS under the conditions described below. Compound 1, characteristic ions 186 (100%), 260 (10%), and 288 (10%) amu (retention time 4.8 minutes). Compound 2, characteristic ions 170 (10%) and 186 (100%) amu (retention time 5.6 minutes). Compound 3, characteristic ions 170 (10%), 274 (100%), 288 (15%), and 376 (10%) amu (retention time 7 minutes).

[0234] Structure determination The compound purified by ion exchange was analyzed on a Bruker Avance 500 MHz instrument using heavy water as the solvent. The elucidation by NMR is as follows.

[0235] Elucidation of the structure of Compound 1 [Chemical formula] [Table 1] 1 1H NMR (500 MHz, D 2 2O) δ / ppm 3.89 (1H, t, J = 9.14 Hz, 2), 3.48 - 3.58 (2H, m, 5), 3.46 - 3.50 (1H, m, 6), 3.29 (1H, dd, J = 12.0, 8.8 Hz, 5), 2.87 (3H, s, 7), 2.65 - 2.77 (1H, m, 4), 2.60 (1H, dt, J = 13.6, 8.4 Hz, 3), 1.71 - 1.82 (1H, m, 3) 13 13C NMR (126 MHz, D 2 2O) δ / ppm 173.6 (8), 71.2 (2), 62.8 (6), 58.2 (5), 41.4 (7), 38.6 (4), 32.2 (3)

[0236] The proton attachments to the individual carbon atoms were assigned from the pendant and HMQC spectra. The COZY correlations show correlations from 2 to 3 to 4, and correlations from 4 to both 5 and 6. The hydrogen chemical shifts of CH(2) at δ 3.89 ppm, methyl (7) at δ 2.87 ppm, and methylene (5) at δ 3.29 ppm and δ 3.52 ppm all indicate that they are bonded to nitrogen and form an N - methylpyrrolidine ring structure.

[0237] The hydrogen chemical shift of methylene at δ3.50 ppm (6) indicates that the methylene is bonded to a hydroxyl group, thus forming a hydroxymethyl bonded to 4. The HMBC correlations from 2 and 3 to the quaternary at δ173.6 ppm indicate that the acid is bonded to 2.

[0238] The NOESY correlation between the protons at δ3.89 ppm (2) and δ2.70 ppm (4) indicates that they are on the same side of the ring and form an S,S configuration. Based on these interpretations, the structure is assumed to be N-methyl-4S-(hydroxymethyl)-2S-pyrrolidinecarboxylic acid (Compound 1).

[0239] The chemical shifts of the protons are in good agreement with those predicted from the standard table of substituent effects, and the carbon shifts are in exact agreement with the predicted values (ACD CNMR predictor). The elucidated structure has not been reported so far.

[0240] Elucidation of the Structure of Compound 2

Chemical Structure

Table 2

[0241] The attachment of protons to individual carbon atoms was assigned from the pendant and HMQC spectra. The COZY correlations show correlations from 2 to 3 to 4, and from 4 to both 5 and 6. The hydrogen chemical shifts of CH(2) at δ3.01 ppm, methyl (7) at δ2.22 ppm, and methylene (5) at δ2.91 ppm and δ2.51 ppm all indicate that they are bonded to nitrogen and form an N-methylpyrrolidine ring structure. The hydrogen chemical shift of methylene (6) at δ3.47 ppm indicates that the methylene is bonded to a hydroxyl, thus forming a hydroxymethyl bonded to 4. The N-methylpyrrolidine ring structure is confirmed by the HMBC correlations from 7 to 2 and 5. The HMBC correlations from 2 and 3 to the quaternary at δ173.6 ppm indicate that the acid is bonded to 2. There is no NOESY correlation between the protons at δ3.01 ppm (2) and δ2.34 ppm (4), indicating that they are on opposite sides of the ring and have an S,R configuration. Based on these interpretations, the structure is assumed to be N-methyl-4R-(hydroxymethyl)-2S-pyrrolidinecarboxylic acid (Compound 2).

[0242] The chemical shifts of the protons are in good agreement with those predicted from the standard table of substituent effects, and the carbon shifts are in exact agreement with the predicted values (ACD CNMR predictor).

[0243] The elucidated structure has not been reported so far.

[0244] Elucidation of the Structure of Compound 3

Chemical Structure

Table 3

[0245] The proton attachments to the individual carbon atoms were assigned from the pendant and HMQC spectra. The COZY correlations show the linkage from 2 to 3. The hydrogen chemical shifts of CH(2) at δ 4.21 ppm, methyl (7) at δ 2.99 ppm, and methylene (5) at δ 3.14 ppm and δ 3.81 ppm all bond to nitrogen, indicating the formation of an N-methyl ring structure. The HMBC correlations from the methyl at δ 2.99 pm (7) to 2 and 5 confirm this arrangement. The singlet at δ 3.58 ppm is integrated as two protons for hydroxymethyl from the carbon and hydrogen chemical shifts. This results in HMBC correlations to 3, 4, and 5. The fact that it is a singlet means it is attached to a quaternary, thus. The carbon chemical shift of 4 means that a hydroxyl unit is attached.

[0246] Based on these interpretations, the structure is assumed to be N-methyl-4-hydroxy-4-(hydroxymethyl)-2-pyrrolidinecarboxylic acid. There is a NOESY correlation between 2 and 6, meaning that the hydroxymethyl is on the same side as the protons of the ring, thus the structure is assumed to be N-methyl-4-hydroxy-4S-(hydroxymethyl)-2S-pyrrolidinecarboxylic acid (Compound 3).

[0247] The chemical shift of the proton is in good agreement with that predicted from the standard table of substituent effects, and the carbon shift is in exact agreement with the predicted value (ACD CNMR predictor). The elucidated structure has not been reported so far.

[0248] Comparison of the proton spectra of Compounds 1 and 3 with the proton NMR spectrum of the extract of Z. spina-christi (NMR file K101613) confirmed that the mixture of the two compounds contains a major N-methylproline component together with amino acids.

[0249] Example 2: Disaccharide Loading Test in Mice Using Compounds 1 and 3 and the Extract of Z. spina-christi Introduction The purified Compounds 1 and 3 were studied for their blood glucose-regulating activity in mice together with the extract of Z. spina-christi containing both compounds.

[0250] Method The animal experiment protocol for this study was approved by the Animal Experiment Committee of Toyama University (S-2010UH-2). Male ddy mice (29 - 33 g) after an overnight fast were used for the acute disaccharide loading test. Maltose (2.5 g / kg body weight) or sucrose (2.5 g / kg body weight), as well as the test samples, were dissolved in 0.9% NaCl solution and administered to the mice via a gastric tube. The control group was loaded with physiological saline only.

[0251] Blood samples for glucose measurement were collected from the tail vein at 0, 15, 30, 60, and 120 minutes after disaccharide loading. Blood glucose levels were measured using the portable kit Antsence II (trademark) (Sankyo Co. Ltd., Tokyo, Japan). The extract HS0810 / 142 / 41 of the jujube plant and the purified compounds were tested by this method.

[0252] [Table 4]

[0253]

Table 5

[0254]

Table 6

[0255]

Table 7

[0256] Conclusion The mixture of the plants of the genus Ziziphus had an effect of reducing the blood glucose level after the disaccharide challenge with maltose, similar to that of compound 1. Compound 3 seemed to have a more complex effect of decreasing more rapidly at 30 minutes, but the blood glucose level clearly increased at an early stage of the sucrose challenge, and by 2 hours it dropped below the higher starting point than the other groups and probably showed better regulation than the control with lower DMDP and blood glucose level.

[0257] Example 3: Absence of Glucosidase Inhibition by Compounds 1 and 3 Introduction The glucosidase assay was performed with compounds 1 and 3 and the extract of the plants of the genus Ziziphus used in the study of the disaccharide-loaded mice to determine whether glucosidase inhibition could explain the effect on blood glucose. A panel of glucosidases was used to confirm that the activity found was not only non-specific binding to the enzyme but also selective inhibition.

[0258] Enzyme Assay The glycosidase assay was performed using a p-nitrophenyl substrate (except for sialidase using methylumbelliferyl-N-acetylneuraminic acid as the substrate) as described by Watson et al. (Phytochemistry 1997, 46, 255). The assay was performed in microtiter plates. The enzyme was assayed at the pH optimal for the enzyme in 0.1 M citric acid / 0.2 M disodium hydrogen phosphate (McIlvaine) buffer. All assays were performed at 20 °C. All enzymes and substrates were purchased from Sigma Aldrich Chemicals Limited. The incubation mixture consisted of 10 μl of enzyme solution, 10 μl of iminosugar solution, and 50 μl of an appropriate 5 mM p-nitrophenyl substrate composed of McIlvaine buffer at the pH optimal for the enzyme. During the exponential phase of the reaction, the reaction was stopped with 0.4 M glycine (pH 10.4). Absorbance was read at 405 nm. Water was used instead of the blank alkaloid. The reaction was performed three times.

[0259] Enzyme inhibitor solution - The compound was dissolved in distilled water at a concentration of 1 mg mL -1 and then, after dilution with the assay volume of the substrate, screened at a concentration of 0.14 mg mL -1 . -1

[0260] In the sialidase assay, 1 mM inhibitor (or water without inhibitor) and 2.5 nM sialidase (including NanH of T. forsythia) were incubated in the presence of 0.1 mM methylumbelliferyl-N-acetylneuraminic acid in 20 mM sodium phosphate buffer, pH 7.2. The reaction was stopped at 30 seconds and 60 seconds by the addition of 60 mM sodium carbonate buffer, pH 10.5. The release of fluorescently generated MU was quantified by measuring fluorescence emission at 450 nm, excitation 350 nm. The percentage of sialidase activity was expressed as the change in fluorescence between 30 seconds and 60 seconds compared to that of the reaction without inhibitor. The reaction was performed three times.

[0261] Results The results are expressed as % inhibition. No glucosidase inhibition was observed with the extracts of the jujube plants or with compounds 1 and 3 from the jujube plants and guarana. The glucosidase inhibitors DMDP and DNJ (1-deoxynojirimycin) showed the expected strong inhibition of various glucosidases. Among the other glucosidases tested, the only inhibition seen with the isolated compounds was by compound 1 on sialidase from the bacterium T. forsythia (compounds 3 and the extracts of the jujube plants were not available for that assay).

Table 8

[0262] The bold numbers indicate strong inhibition at the concentration tested at 0.4 mM.

[0263] Negative values probably indicate an increase in enzyme activity due to stabilization or improvement of folding.

[0264] DMDP and DNJ are the most commonly occurring plant iminosugars and both are potent inhibitors of α-glucosidase (Watson, A. A., Fleet, G. W. J., Asano, N., Molyneux, R. J.; Nash, R. J. Phytochemistry 2001, 56, 265 - 295). DNJ (1-deoxynojirimycin) was first isolated from mulberry and has been used in traditional Chinese medicine for diabetes.

[0265] Example 4: Reduction of TNF-α by Compound 1 Introduction Measurement of TNF-α production in human blood predicts the action of any drug administered systemically with respect to TNF-α levels. That is, the effects in blood ex vivo or in vitro accurately mimic the effects in vivo. This is particularly useful when it is difficult to conduct direct research in humans in vivo due to either ethical concerns or more important safety considerations. Whole blood incubation has been shown to be useful for demonstrating the stimulation of pro-inflammatory mediators in vitro in humans (Finch-Arietta, M.B. & Cochran, F.R., 1991, Agents and Actions 34, 49). This approach is also useful for monitoring the bioavailability of any drug administered systemically (Song, H.J. et al., 2005, European Journal of Clinical Nutrition 59, 508).

[0266] There are several approaches to suppressing inflammation, and the two most commonly used therapeutically at present are a) steroids (glucocorticoid analogs) and b) agents that neutralize the action of TNF-α such as etanercept (soluble TNF-α receptor fusion protein) and infliximab (monoclonal antibody against TNFα). Both of these approaches target the suppression of pro-inflammatory cytokines, the production by steroids, and the prevention of the action of TNF-α by etanercept and infliximab. This also shows the importance of targeting TNF-α to suppress the inflammatory response (Feldmann, M. & Maini, R.N., 2001, Annual Review of Immunology 19, 163). Similarly, glucocorticoid analogs can also suppress the production of cytokines and dexamethasone, and specifically, can reduce the production of TNF-α by LPS stimulation from human monocytes (Waage, A. & Bakke, O., 1988, Immunology 63: 299).

[0267] The aim of this study was to evaluate the anti-inflammatory activity of Compound 1 with respect to its ability to regulate TNF-α levels in human whole blood.

[0268] Materials Blood and buffy coat fractions were provided by the Scottish National Blood Transfusion Service (SNBTS), Glasgow, UK. Ficoll Histopaque (1.077 g / l), lipopolysaccharide (from Salmonella abortus equi) were purchased from Sigma-Aldrich Co. Ltd. (UK). PGE was obtained from Cayman Chemical Co. (Ann Arbor, MI). The human TNFα-antibody pair for TNFα-ELISA assay was obtained from Invitrogen / Life Sciences Europe. All drugs were dissolved in RPMI 1640 medium from Gibco BRL, UK.

[0269] Methods In experiments using whole blood incubation, blood was used without further treatment after donation. Blood was provided by healthy donors when defined by the Scottish National Blood Transfusion Service as being negative for all parasites such as HIV, hepatitis B and C, CMV, malaria (tests by the National Blood Transfusion Service). Also, the absence of acute inflammatory diseases at the time of blood collection was confirmed in our laboratory by measuring basal levels of TNF-α that were always less than 50 pg / ml.

[0270] Cell stimulation and TNF-α measurement An aliquot (800 μl) of whole blood was incubated for an appropriate pre-incubation period with the compound dissolved in RPMI 1640 as shown in the results, then LPS was added and incubation was continued at 37 °C for 20 h in an atmosphere of 5% CO2 in humidified (100%) air. At the end of the incubation period, the supernatant, consisting of either plasma or medium, was collected by centrifugation at 10,000 g for 30 s at room temperature, and the TNF-α level was measured using a human TNF-α ELISA system (supplied by BioSource Europe S.A., Belgium, Invitrogen) according to the manufacturer's instructions as previously described (Brown et al., 2013).

[0271] Results and conclusions Compound 1 showed a tendency for the mean value to decrease LPS-stimulated TNFα-production in a concentration-dependent manner in human blood.

[0272] Figure 1 shows the effect of compound 1 (μM) on LPS-stimulated TNF-α production in human blood. Whole blood was incubated for 24 h in the absence (open symbols) or presence (black circles) of LPS (10 μg / ml) with varying concentrations of the compound. After incubation at 37 °C (5% CO2, 100% humidity), plasma was collected from the blood by centrifugation and the level of TNF-α in the plasma samples was measured by ELISA. Values represent the mean ± standard deviation for n = 3.

[0273] Although not important at the low μM concentrations used here, the concentrations of jujube extracts can be high depending on how they are produced, and this and the other 4-hydroxymethyl-N-methyl-L-prolines (compounds 1, 2, and 3) may help to explain the anti-inflammatory effects claimed for the plants.

[0274] Example 5: Effect of 4-hydroxymethyl-N-methyl-L-proline on the growth of sialidase and periodontal pathogens Periodontal disease is a major health burden with an estimated 700 million patients worldwide and approximately 10-20% of the UK population affected. Recent research has shown that in addition to being a cause of tooth loss, an increased incidence of periodontal disease is associated with various important systemic diseases such as cardiovascular disease, diabetes, and rheumatoid arthritis. The incidence increases with age.

[0275] As part of the disease progression, the ability of periodontal bacteria to form subgingival plaque biofilm and the ability of periodontal bacteria to stimulate innate immunity through interaction with oral epithelial cells are considered key to the etiology. Pathogenic sialidases from periodontal pathogens Tannerella forsythia, T. denticola, and Porphyromonas gingivalis have been shown to be involved in this process. Therefore, sialidase inhibition represents a promising treatment or prevention for periodontitis. The related enzyme exists as a major pathogenic factor of influenza virus (i.e., the N of H1N1 represents neuraminidase, the target of Tamiflu).

[0276] The aim of this study was to investigate the ability of 4-hydroxymethyl-N-methyl-L-proline to inhibit the sialidase activity of periodontal bacteria and their purified sialidases, and the effect of extracts containing the compound (e.g., the fruit of the genus Ziziphus). Additionally, preliminary studies on in vitro models of biofilm formation and host-bacteria binding were performed to evaluate the effect of the compound and extracts on the virulence of periodontal pathogens. This is because these are considered important for the survival and growth of oral pathogens.

[0277] The effects in these assays may also indicate the impact of the compound and extracts containing them on the human microbiome, which more generally has potential relevance to the prevention or cure of other conditions involving sialidase activity.

[0278] Sialidase activity assay In these assays, methylumbelliferyl-N-acetylneuraminic acid (MUNANA), a fluorogenic substrate, was used. This is cleaved by the action of sialic acid, releasing N-acetylneuraminic acid (Neu5Ac) and methylumbelliferone (MU), the latter of which exhibits maximal fluorescence at 450 nm upon excitation at 350 nm. The release of MU can be used to quantify sialidase activity.

[0279] Whole bacteria or purified sialidase were exposed to MUNANA in the presence or absence of compound 1 (Example 3) or a 50% aqueous ethanol extract of the fruits of the genus Terminalia containing 1% N-methyl-proline.

[0280] 1 mM inhibitor (or water without inhibitor) and 2.5 nM sialidase (including NanH from T. forsythia) were incubated in the presence of 0.1 mM MUNANA, pH 7.2, 20 mM sodium phosphate buffer. The reaction was stopped at 30 seconds and 60 seconds by the addition of 60 mM sodium carbonate buffer at pH 10.5. The release of fluorogenic MU was quantified by measuring fluorescence emission at 450 nm with excitation at 350 nm. The percentage of sialidase activity was expressed as the change in fluorescence between 30 seconds and 60 seconds compared to that of the reaction without inhibitor. The reaction was performed three times.

[0281] An initial screening for N1Neu inhibition was performed using various compounds and the commercially available sialidase (neuraminidase) inhibitor zanamivir (sold as the pharmaceutical formulation "Relenza (trademark)" as a treatment for influenza).

[0282] Initial screening of compounds and plant extracts for Neu1 activity A sialidase assay was performed where the reaction contained 0.1 mM MUNANA, pH 7.4, 50 mM Tris, 5 mM CaCl2, 200 mM NaCl, plant extract or the indicated concentrations of the control sialidase inhibitor zanamivir, or no putative inhibitor. N1Neu was present at 0.025 μg / ml during the reaction. These 50 μl reactions were quenched at 5 and 10 minutes using 100 μl of 100 mM sodium carbonate buffer, pH 10.5. Sialidase activity was evaluated using the 10-minute time point. The data shown represent the mean of one experiment where each condition was repeated 3 times. MU present in the quenched reactions was quantified using a TECAN M200 Infinite microplate reader with excitation 340 nm and emission 430 nm and represented as a percentage of sialidase activity (MU fluorescence) relative to the condition without inhibitor.

[0283] Host cell binding Bacteria can form biofilms and in addition to being present on mucosal surfaces, can bind (adhere and invade) host cells. This confers several advantages to the bacteria, including persistence when faced with antibacterial therapy, the immune system, or mechanical removal of biofilms, and access to nutrients within host cells. Bacterial binding to host cells also affects immune regulation. That is, increased cell invasion can lead to upregulation of inflammation and contribute to periodontal disease. Sialidase (and other products) are important during the binding of P. gingivalis and T. forsythia to host cells.

[0284] To test the inhibition of host cell attachment and invasion by P. gingivalis and T. forsythia, an antibiotic protection assay was used. This involved growing two sets of host cell monolayers, which were then infected with bacteria at a host cell:bacteria ratio of 1:100 (multiplicity of infection). After a 1.5-hour incubation, one set was lysed and the bacteria were plated onto agar to obtain the number of viable bacteria. This yielded the total number of bacteria that had attached. The second set of infected monolayers was incubated with the antibiotic metronidazole for 1 hour. Metronidazole kills external (attached) bacteria, leaving only the invaded (internalized) bacteria. The cells were then lysed and plated onto agar as described above. The number of invaded bacteria was subtracted from the total bound to obtain the number of attached (external) bacteria.

[0285] Biofilm assay Periodontal pathogens are present in oral biofilms (as part of plaque), and both normal oral hygiene practices and periodontitis treatment aim to disrupt the biofilm. Thus, potential treatments or prevention should also be able to disrupt the biofilm of periodontal pathogens, which can be modeled in vitro as single- or mixed-species biofilms. Various different approaches were taken as the effects of plant extracts and compounds may not be directly antibacterial but act to prevent attachment to host glycoproteins or nutrient acquisition from glycoproteins. These included culturing P. gingivalis and T. forsythia in nutrient-rich medium (tryptic soy broth - TSB) on surfaces coated with serum (fetal bovine serum - FBS), and culturing P. gingivalis in nutrient-depleted medium containing only serum (10% v / v FBS) as a carbon and nitrogen source. Considering the apparent activity as a sialidase inhibitor, extracts from the genus Diospyros were tested for their ability to prevent biofilm formation.

[0286] To quantify biofilm formation, two approaches were used: crystal violet staining, which is high-throughput but less sensitive, and direct bacterial counting, which is more sensitive than crystal violet staining but has low throughput. Crystal violet staining can also be used to image biofilms for qualitative observation.

[0287] P. gingivalis and T. forsythia were cultured for 2 days and 5 days respectively in supplemented TSB in microtiter plates pre-coated with FBS in the presence or absence of 1500 μg / ml of the extract of the jujube plant. In this series of experiments, quantification of biofilm formation was enabled by bacterial counting, and visualization of biofilms was enabled by crystal violet staining. Under these conditions, biofilm formation by P. gingivalis was not significantly inhibited. Biofilm formation by T. forsythia decreased by at least 2-fold under all conditions, from 2.2x10 7 bacteria / ml to 1.0x10 7 CFU / ml.

[0288] Biofilm Inhibition Assay - P. gingivalis + F. nucleatum (25586) Mixed-Species Biofilms in Rich Medium Effect of the Extract of the Jujube Plant on T. forsythia and P. gingivalis Cultured in TSB on Serum-Coated Surfaces P. gingivalis or T. forsythia was seeded at an OD600 of 0.05 in TSB containing 0.1% w / v yeast extract, 5 μg / ml hemin, 1 μg / ml menadione, and 50 μg / ml gentamicin in wells of a 96-well polylysine-coated tissue culture plate pre-coated with FBS (100% FBS in 50 μl was added and incubated overnight at 4°C, then washed once with PBS to remove unbound serum). Both species were cultured anaerobically at 37°C for 5 days for T. forsythia and 48 hours for P. gingivalis. The supernatant was removed and the wells were washed twice with PBS. The biofilm was vigorously resuspended in PBS (if appropriate) before dilution and counted using a hemocytometer and phase contrast microscopy at a magnification of 400.

[0289] Wells for imaging were stained with crystal violet. The supernatant was removed and the wells were incubated with 0.1% (w / v) crystal violet at room temperature for 15 minutes. The dye was removed and the wells were washed three times using PBS. The biofilm was imaged at a magnification of 400 using an inverted microscope and imaging software. The image capture parameters (light intensity and exposure time) were constant for each image. The data shown represent the average of three experiments and each condition was repeated three times per experiment.

[0290] A possible explanation for the different effects of the extract of the plant of the genus Ligustrum on biofilm formation is the difference in the nutritional requirements of these two organisms, and T. forsythia has more stringent growth requirements than P. gingivalis. For example, T. forsythia usually requires an exogenous source of N-acetylneuraminic acid (NAM), which can be replaced by sialic acid during biofilm formation (in this experiment, this source was provided by FBS), which may be destroyed by sialidase, while P. gingivalis may be able to obtain nutritional requirements from the supplemented TSB medium even in the presence of sialidase inhibition. Therefore, a defined medium containing only serum (FBS) as a source of carbon and nitrogen was also used to evaluate the effect of the extract of the plant of the genus Ligustrum on biofilm formation.

[0291] Biofilm Inhibition Assay - P. gingivalis in Defined Medium Since T. forsythia is more stringent than P. gingivalis, attempts to culture it as a single-species biofilm using a defined medium failed. Therefore, the extract of the plant of the genus Ligustrum was tested against P. gingivalis itself cultured in a defined medium. P. gingivalis was seeded at an OD600 of 0.05 in a defined medium further supplemented with 10% v / v FBS, 5 μg / ml hemin, and 50 μg / ml gentamicin in wells of a 96-well polylysine-coated tissue culture plate pre-coated with FBS (50 μl of 100% FBS was added, incubated overnight at 4°C, and then washed once with PBS to remove unbound serum). P. gingivalis was cultured anaerobically at 37°C for 5 days.

[0292] The supernatant was removed and the wells were incubated with 0.1% (w / v) crystal violet at room temperature for 15 minutes. The dye was removed and the wells were washed with a stream of water. A) Quantification of crystal violet staining - Crystal violet was extracted from the biofilm into a new 96-well microtiter plate using ethanol:acetone (80:20) and quantified by measuring the absorbance at 590 nm.

[0293] Results The data shown represent the average of two experiments and each condition was repeated 10 times per experiment.

[0294] N1 neuraminidase inhibition [Table 9] [Table 10] N.B. 1 mM of compound 1 resulted in 50% inhibition of NanH from T. forsythia (Example 3)

[0295] [Table 11] [Table 12] [Table 13] 1500 μg / ml of Actinidia plants showed a decrease in biofilm formation compared to without inhibitor. [Table 14] N.B. In rich medium, there was no obvious effect on P. gingivalis biofilm using crystal violet staining.

[0296] Discussion Compound 1 (Example 3) and extracts of plants of the genus Ziziphus containing 1% of 4-hydroxymethyl-N-methyl-L-proline 1-3 inhibited the sialidase of Tannerella forsythia. Although the extracts of plants of the genus Ziziphus did not appear to have a direct antibacterial effect, they were able to inhibit biofilm formation by preventing nutrient acquisition or limiting attachment to serum-coated surfaces, despite their high sugar content. Biofilm formation of P. gingivalis in TSB was not significantly inhibited by the plant extracts, but was inhibited when cultured in a defined medium (where serum-FBS is the sole source of carbon and nitrogen).

[0297] This suggests that the extracts containing compounds 1-3 were interfering with nutrient acquisition by P. gingivalis through inhibition of its sialidase and possibly other enzymes, which may limit the protein catabolism of P. gingivalis. This is particularly detrimental to nutrient acquisition from serum, but less so from TSB where the protein source (soybean meal) has already undergone enzymatic digestion. Presumably, the defined medium better represents the in vivo situation where P. gingivalis acquires nutrients by catabolism of host glycoproteins in gingival crevicular fluid (GCF, similar in composition to serum) and other glycoproteins in blood and saliva.

[0298] Since T. forsythia is more fastidious than P. gingivalis, attempts to culture it as a single-species biofilm using a defined medium have so far failed. However, it can form biofilms on serum-coated surfaces of TSB. In this series of experiments, the extracts of plants of the genus Ziziphus appeared to inhibit the biofilm formation of T. forsythia.

[0299] These findings also indicate that the compounds of the present invention can find use in various therapeutic treatments based on the inhibition of other bacterial sialidases, including Gardnerella vaginalis, a species associated with bacterial vaginitis and preterm birth.

[0300] Example 6: Mouse test of 50% aqueous ethanol extract of Zizyphus jujuba fruit The fruit extract of Zizyphus jujuba was prepared with 50% aqueous ethanol and dried. Analysis by gas chromatography-mass spectrometry (GCMS) of the extract showed a high monosaccharide sugar content (>5%) and the presence of 1% of compounds 1-3. Previous studies on the extraction of Z. spina-christi extract were conducted in male ddy mice in maltose and sucrose loading tests (Example 2).

[0301] In this study, male C57BL / 6J mice were used in glucose, maltose, and sucrose loading tests.

[0302] Method The animal experiment protocol was approved by the Animal Experiment Committee of Toyama University. The animals were fasted overnight. Glucose (2.5 g / kg body weight) or sucrose (2.5 g / kg body weight), and the extract were dissolved in 0.9% NaCl solution and administered to the mice via a gastric tube. The control group was loaded with only physiological saline. Blood samples for glucose measurement were collected from the tail vein at 0, 15, 30, 60, and 120 minutes after sugar loading. Blood glucose levels were measured using the portable kit Antsence II (trademark) (Sankyo Co. Ltd. Tokyo, Japan). The tests were as follows.

[0303] ● Glucose loading test 1000 mg / kg body weight (Figure 2, Table 1) ● Glucose loading test 500 mg / kg body weight (Figure 3, Table 2) ● Sucrose loading test 1000 mg / kg body weight (Figure 4, Table 3) ● Maltose loading test 500 mg / kg body weight (Figure 5, Table 4)

[0304] Results Figure 2 shows the effect of the extract of the jujube plant on blood glucose levels. Blood glucose concentration in male C57BL / 6J mice after oral administration of 2.5 g / kg body weight of glucose containing the extract of the jujube plant (each 1000 mg / kg body weight) (white circles). The control group was administered physiological saline (black circles). Each value represents mean ± SEM (n = 5).

[0305] Figure 3 shows the effect of the extract of the jujube plant on blood glucose levels. Blood glucose concentration in male C57BL / 6J mice after oral administration of 2.5 g / kg body weight of glucose containing the extract of the jujube plant (each 500 mg / kg body weight) (white circles). The control group was administered physiological saline (black circles). Each value represents mean ± SEM (n = 5).

[0306] Figure 4 shows the effect of the extract of the jujube plant on blood glucose levels. Blood glucose concentration in male C57BL / 6J mice after oral administration of 2.5 g / kg body weight of sucrose containing the extract of the jujube plant (each 1000 mg / kg body weight) (white circles). The control group was administered physiological saline (black circles). Each value represents mean ± SEM (n = 5).

[0307] Figure 5 shows the effect of the extract of the jujube plant on blood glucose levels. Blood glucose concentration in male C57BL / 6J mice after oral administration of 2.5 g / kg body weight of maltose containing the extract of the jujube plant (each 500 mg / kg body weight) (white circles). The control group was administered physiological saline (black circles). Each value represents mean ± SEM (n = 5).

[0308]

Table 15

Table 16

Table 17

Table 18

[0309] Conclusion The fruit extract of Zizyphus jujub containing additional monosaccharides suppressed blood glucose in both glucose and sucrose tolerance tests. The effects on maltose or sucrose loading conditions may have been caused by maltase or sucrase inhibition, except that the extract did not show significant glucosidase inhibition. The results of the glucose tolerance test suggest a different mechanism independent of glucosidase inhibition and probably an effect on insulin release or other mechanisms.

[0310] equivalent The foregoing description has detailed presently preferred embodiments of the invention. Considering these descriptions, those skilled in the art would expect numerous modifications and variations to occur in practice. These modifications and variations are intended to be included within the scope of the claims appended hereto.

Claims

1. 【Fig. 1】 An isolated composition comprising 4-hydroxymethyl-proline selected from those pharmaceutically acceptable salts thereof, said composition being a composition for use in a method of treating a bacterial or viral infection, said composition.

2. Said isolated 4-hydroxymethyl-proline is synthetic or purified from a plant source selected from (a) plants of the genus Ziziphus, and (b) plants of the genus Paullinia, Said plant source being (i) selected from the plant species of Z. jujuba, Z. spina-christi, Z. lotus, Z. mauritiana, Z. joazeiro, and Paullinia cupana, and / or (ii) may contain fruits, fruit parts, fruit extracts, fruit juices, seeds, barks, roots, and / or leaves, the composition according to claim 1.

3. A composition in the form of a pharmaceutical composition further comprising a pharmaceutically acceptable excipient, The composition according to claim 1 or 2, which may be in the form of (a) a pharmaceutical pack, kit, or patient pack, or (b) a unit dosage form.

4. Use of the composition according to any one of claims 1 to 3 for the manufacture of a medicament for treating a bacterial or viral infection.

5. A composition according to any one of claims 1 to 3, which may further comprise a cosmetic, dietary supplement, or pharmaceutically acceptable excipient or carrier, the composition being in the form of a cosmetic, dietary supplement, crude drug, or pharmaceutical composition.

6. Said isolated 4-hydroxymethyl-proline is present in the composition at a level of at least 0.5%, 1% w / w, 5% w / w, 10% w / w, 15% w / w, 20% w / w, 25% w / w, 30% w / w, 35% w / w, 40% w / w, 45% w / w, 50% w / w, 60% w / w, 70% w / w, 80% w / w, 90% w / w, 99% w / w (dry weight basis), the composition according to any one of claims 1 to 3 or 5.

7. The use according to claim 4, wherein the isolated 4-hydroxymethyl-proline is present in the composition at a level of at least 0.5%, 1% w / w, 5% w / w, 10% w / w, 15% w / w, 20% w / w, 25% w / w, 30% w / w, 35% w / w, 40% w / w, 45% w / w, 50% w / w, 60% w / w, 70% w / w, 80% w / w, 90% w / w, 99% w / w (dry weight basis).

8. A method for the manufacture of a composition, comprising: (i) the composition is as defined in any one of claims 1 to 3 or 5, and the method comprises: (a) providing plant material from the plant source as defined in claim 2; (b) extracting 4-hydroxymethyl-proline as defined in claim 1 from the plant material; and then (c) formulating the extracted 4-hydroxymethyl-proline with a pharmaceutically acceptable excipient to produce a pharmaceutical composition. The method as described above.

9. A method for the manufacture of a fortified food or beverage for use in a method of treating a bacterial or viral infection, comprising: (a) providing a composition as defined in any one of claims 1 to 3 or 5; and (b) adding the composition of step (a) to a food or beverage to produce a fortified food or beverage.

Images