Salicylic acid compositions and uses thereof

Abstract

The present invention relates to compounds, compositions, uses and methods useful in preventing or treating urinary tract infections, particularly E. coli infections, and especially recurrent urinary tract infections.

Description

[Technical field]

[0001] The present invention relates to the prevention or treatment of urinary tract infections, including E. coli infections, and in particular to compositions useful in the prevention or treatment of urinary tract infections. [Background technology]

[0002] Urinary tract infections (UTIs) are one of the most common infectious diseases affecting millions of people every year. Women are particularly affected, as they have a 50% lifetime risk of developing the disease (compared to 5% for men), and UTIs account for over 1 million hospitalizations and over $1.6 billion in medical costs annually in the United States (Ulett et al., 2013, Cur Opin Microbiol., 16:100-107). Over half of women experience a recurrence of infection within 6 months, and 70-95% of reported UTI cases are primarily caused by uropathogenic Escherichia coli (E. coli) (Foxman, 2002, Am J Med., 113, Suppl 1A:5S-13S). Infection occurs through the following steps: E. coli interacts with the urothelium via bacterial I fimbrial adhesion; epithelial defense molecules are released. However, invasion of host cells protects E. coli from defenses that allow E. coli to replicate. An additional defense is the detachment of E. coli-infected epithelial cells to clear the infection via urination, at the expense of host cells, a defense called "exfoliation." Incomplete clearance leaves E. coli in a filamentous form that is highly infectious and protected from phagocytosis. Because UTIs require antimicrobial treatment, such as antibiotics, which often leads to the development of resistant microflora, new approaches to the prevention and treatment of UTIs are needed (Hooton et al., 2004, Clin Infect Dis. 39(1):75-80).

[0003] Symptoms of uncomplicated UTIs are painful urination (dysuria), frequent urination (frequency), inability to start urination (hesitation), a sudden need to urinate (urgency), and blood in the urine (hematuria).

[0004] Phenolic acids are typically divided into hydroxybenzoic acids and hydroxycinnamic acids (Valanciene et al., 2020, Biomolecules, 10, 874). Salicylic acid (SA) and its derivatives, such as salicylic acid methyl ester and monotropitoside (methyl salicylate O-beta-D-xylopyranosyl-(1->6)-O-beta-D-glucopyranoside), belong to the hydroxybenzoic acids and their derivatives. Salicylic acid (SA) acts as a key hormone in plant innate immunity, including resistance in both local and systemic tissues to biological attacks, hypersensitive responses, and cell death (Ding et al., 2020, Trends Plant Sci. 25(6), 549-565). Salicylic acid has demonstrated a broad spectrum of biological activities including antithrombotic, anti-inflammatory, antitumor and antibacterial properties (Wu, 2007, Anti-Inflam Anti-Allerg Agents Medicin Chem, 6, 278).

[0005] Oenothein B is an ellagitannin dimer with a macrocyclic structure present in medicinal Oenothera, Epilobium, and Eucalyptus species and has been found to have various biological activities (Yoshida et al., Molecules, 2018, 23(3), 552).

[0006] There is a significant need for new strategies to prevent and / or treat urinary tract infections, especially E. coli infections. In particular, targeting a single enzyme with an anti-infective drug can induce resistance mechanisms, so multi-target action is desirable in the antibacterial field. Summary of the Invention

[0007] The present invention relates to the unexpected discovery that salicylic acid and some of its metabolites formed in vivo after ingestion of salicylic acid have beneficial activity against E. coli bacterial adhesion on bladder cells, as well as bactericidal and diuretic effects that are beneficial in the prevention and / or treatment of UTIs, particularly E. coli infections. Even more surprisingly, it has been found that oenothein B and extracts of Epilobium parviflorum containing oenothein B complement the activity of salicylic acid and some of its metabolites, and play a role in inhibiting E. coli growth in vivo, which would be advantageous in the prevention and / or treatment of UTIs, particularly E. coli infections.

[0008] A first aspect of the present invention provides salicylic acid (SA) or a metabolite thereof or a mixture thereof or a pharmaceutical composition thereof for use by oral route in the prevention and / or treatment of urinary tract infections, wherein said metabolites are metabolites formed in vivo following ingestion of salicylic acid.

[0009] According to another further aspect, the present invention provides plants of the species Filipendula ulmaria and / or parts of these plants and / or extracts of these plants or pharmaceutical compositions thereof for use by the oral route according to the present invention in the prevention and / or treatment of urinary tract infections.

[0010] In another embodiment of the present invention, there is provided a use of salicylic acid (SA) or a metabolite thereof, or a mixture thereof or a pharmaceutical composition thereof for the preparation of an oral pharmaceutical composition for the prevention, inhibition and / or treatment of urinary tract infection.

[0011] In another embodiment of the invention, the invention relates to a formulation comprising about 1 to about 550 mg, e.g., 10 to about 200 mg, dry weight of a Meadowsweet extract and at least one pharma- ceutically or cosmetically acceptable carrier, diluent or excipient thereof, in particular for use for the prevention, inhibition and / or treatment of urinary tract infections.

[0012] In another embodiment of the present invention, the present invention relates to a formulation comprising salicylic acid (SA) or a metabolite thereof, or a mixture thereof or a pharmaceutical composition thereof, in combination with at least one co-agent useful in the prevention and / or treatment of urinary tract infections, and at least one pharma- ceutically or cosmetically acceptable carrier, diluent or excipient thereof.

[0013] In another embodiment of the present invention, the present invention relates to a formulation comprising an extract of Meadowsweet in combination with at least one co-agent useful in the prevention and / or treatment of urinary tract infections, and at least one pharma- ceutically or cosmetically acceptable carrier, diluent or excipient thereof.

[0014] In another embodiment of the present invention, there is provided a method for preventing and / or treating a urinary tract infection in a subject, said method comprising administering an effective amount of salicylic acid (SA) or a metabolite thereof, or a mixture thereof or a pharmaceutical composition thereof to a subject in need thereof by oral route.

[0015] In another embodiment of the present invention, there is provided a method for preventing and / or treating a urinary tract infection in a subject, said method comprising administering an effective amount of a Meadowsweet extract or a formulation thereof to a subject in need thereof by oral route. [Brief description of the drawings]

[0016] [Figure 1A]FIG. 1 shows the effect on E. coli adhesion to human bladder cells, quantifying the number of viable bacteria attached (cfu) by plate count agar culture of compounds of the invention and extracts described in Examples 1-3. The inhibitory effect is shown compared to a negative control with no inhibitor (=100%, on the y-axis). Cranberry juice extract is used as a control, as it is known to have beneficial effects in vivo. The figure shows the effect of meadowsweet extract, which was extracted with an aqueous solution (0% ethanol) (1), a 20% ethanol solution (2), and a 70% ethanol solution (3). [Figure 1B] FIG. 1 shows the effect on E. coli adhesion to human bladder cells, quantifying the number of viable bacteria attached (cfu) by plate count agar culture of compounds and extracts of the invention described in Examples 1-3. The inhibitory effect is shown compared to a negative control with no inhibitor (=100%, on the y-axis). Cranberry juice extract is used as a control, as it is known to have beneficial effects in vivo. The figure shows the effect of an extract of Ipomoea batatas, which was extracted with an aqueous solution (0% ethanol) (1), a 20% ethanol solution (2), and a 70% ethanol solution (3). [Figure 1C] FIG. 1 shows the effect on E. coli adhesion to human bladder cells, quantifying the number of viable bacteria attached (cfu) by plate count agar culture of the compounds and extracts of the invention described in Examples 1-3. The inhibitory effect is shown compared to a negative control with no inhibitor (=100%, on the y-axis). Cranberry juice extract is used as a control, as it is known to have beneficial effects in vivo. Salicylic acid (SA) (1) and its two metabolites, namely 2,3-dihydroxybenzoic acid (2) and salicyluric acid (3), are shown. [Figure 1D]FIG. 1 shows the effect on E. coli adhesion to human bladder cells, quantifying the number of viable bacteria attached (cfu) by plate count agar culture of the compounds and extracts of the invention described in Examples 1-3. The inhibitory effect is shown compared to a negative control with no inhibitor (=100%, on the y-axis). Cranberry juice extract is used as a control, as it is known to have beneficial effects in vivo. A mixture of extracts from Meadowsweet (F) and from Euphorbia gracilis (E) is shown. [Figure 1E] FIG. 1 shows the effect on E. coli adhesion to human bladder cells, quantifying the number of viable bacteria attached (cfu) by plate count agar culture of the compounds and extracts of the invention described in Examples 1-3. The inhibitory effect is shown in comparison to the negative control with no inhibitor (=100%, on the y-axis). Cranberry juice extract is used as a control since it is known to have beneficial effects in vivo. The anti-adhesive activity of each of the extracts of the invention alone and combined as a mixture of the invention (30% (E) + 70% (F) of a total of 5 mg / mL) at ((E) 2.5 mg / mL, (F) 2.5 mg / mL) is shown, compared to cranberry extract (C) (2.5 mg / mL) and D-mannose (D) (2.5 mg / ml). [Figure 1F] FIG. 1 shows the effect on E. coli adhesion to human bladder cells, quantifying the number of viable bacteria attached (cfu) by plate count agar culture of compounds and extracts of the invention described in Examples 1-3. Inhibitory effects are shown compared to a negative control with no inhibitor (=100%, on the y-axis). Cranberry juice extract is used as a control since it is known to have beneficial effects in vivo. The prevention of E. coli growth (MIC) of each of the extracts or substances alone ((F) 0.9 mg / mL, (E) 2.7 mg / mL, 1.3 mg / mL oenothein B (O), 2.5 mg / mL DHBA (2), 2.5 mg / mL SUA (3), 5 mg / mL SA (1)) compared to cranberry extract (C) (2.5 mg / mL) is shown. [Diagram 2] Figure 1 represents the phytochemical characterization of the extracts described in Example 4. A: HPLC profile of extract of Meadowsweet and detection of salicylic acid by HPLC-UV analysis (tR 19.8 min). A SunFire C18 column (3.5 μm, 3.0 mm x 150 mm) with a guard column (3.0 mm x 10.0 mm) was used. The mobile phase consisted of HO (A) and MeCN (B) both containing 0.2% formic acid. A gradient of 15-40% B for 30 min, 40-100% B for 1 min, and 100% B for 10.00 min was applied at a flow rate of 0.4 mL / min. B: Chromatographic profile of extract of Susukiyaki and detection of oenothein B. A SunFire C18 column (3.5 μm, 3.0 mm × 150 mm) with a guard column (3.0 mm × 10.0 mm) was used. The mobile phase consisted of HO (A) and MeCN (B) both containing 0.1% formic acid. A gradient of 5 to 50% B was applied for 30 min at a flow rate of 0.4 mL / min. Peak 1 to peak 3: oenothein B, myricetin-hexoside, and hyperoside, respectively. ELSD: evaporative light scattering detection. [Figure 3A] Figure 1 shows the detection and content of salicylic acid and its metabolites in urine samples collected from mice treated with meadowsweet extract as described in Example 3. Comparison of RT and peaks from analytical standards of salicylic acid (SA) (1) and its metabolites 3-dihydroxybenzoic acid (DHBA) (2) and salicyluric acid (SUA) (3) found in urine samples collected from the group treated with meadowsweet extract (Fil). Analysis was performed on a SunFire™ C18 (3.5 μm, 3.0 mm x 150 mm) column (Waters). 5% MeCN (A) and MeCN (B), both containing 0.1% formic acid, were used as mobile phases. A gradient of 0-100% B was applied in 20 min at a flow rate of 0.8 mL / min. [Figure 3B]FIG. 1 shows the detection and content of salicylic acid and its metabolites in urine samples taken from mice treated with meadowsweet extract as described in Example 3. Concentrations, amounts (in samples taken over 3 hours), and cumulative amounts of salicylic acid (SA) (1) and its metabolites 2,3-dihydroxybenzoic acid (DHBA) (2) and salicyluric acid (SUA) (3) found in urine samples collected from the group treated with meadowsweet extract (F). The cumulative amounts are the amount of each compound accumulated in the urine over time up to the indicated time point. The numbers on the x-axis indicate the collection time. Analysis was performed by HPLC-MRM-MS using the chromatographic conditions described in A. [Figure 4] FIG. 1 represents the amount of urine collected in 24 hours using a mouse model and a combination of extracts according to the present invention compared to a control, as described in Example 3. [Diagram 5] Figure 1 shows the in vivo anti-adhesive effect of the substances and extracts of the invention against E. coli in the mouse model described as described in the examples, expressed in colony forming units (cfu) and percentage of variation compared to the control. Each value was calculated from a total of 6 mice. 106 means 1'000'000 cfu. [Figure 6] Figure 1 shows the results of an efficacy study of the extract combination in women with recurrent UTI as described in Example 6. A: Improvement of urinary symptoms in untreated patients (light grey) compared to patients treated with the extract combination (dark grey), B: Mean duration (days) of urinary tract infection symptoms in untreated patients (light grey) compared to patients treated with the extract combination (dark grey), C: Satisfaction rate among treated patients. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] The term "pharmacologically acceptable" refers to a carrier composed of materials that are not biologically or otherwise unsuitable.

[0018] The term "cosmetically acceptable" refers to a carrier composed of materials that are not biologically or otherwise unsuitable for topical application to the skin or mucous membranes.

[0019] The term "carrier" refers to any ingredient, other than an active agent, present in a pharmaceutical formulation and thus includes diluents, binders, lubricants, disintegrants, fillers, coloring agents, wetting or emulsifying agents, pH buffering agents, preservatives, etc.

[0020] As used herein, "treatment" and "treating" and the like generally refer to obtaining a desired pharmacological and physiological effect. The effect may be prophylactic, in that it prevents or partially prevents a disease, its symptoms or pathology, and / or it may be therapeutic, in that it partially or completely cures a disease, pathology, symptom, or adverse effects caused by a disease. The term "treatment", as used herein, encompasses any treatment of a disease in a mammal, particularly a human, and is not necessarily intended to imply a cure or complete elimination of symptoms, but refers to any type of treatment that benefits a patient, and includes (a) preventing the onset of a disease in a subject who may be predisposed to a disease, for example, based on family history, obesity status, or age, but has not yet been diagnosed as such, and (b) inhibiting the disease, i.e., halting its onset, or relieving the disease, i.e., causing regression of the disease and / or its symptoms or pathology, such as ameliorating or curing damage.

[0021] In particular, the prevention and / or treatment of urinary tract infection according to the present invention includes reducing an individual's susceptibility to E. coli infection, for example, bacteriostatic (preventing bacterial growth), anti-adhesion, and diuretic effects. The term "treatment" refers to any type of treatment or prevention that benefits a subject suffering from or at risk of developing a urinary tract infection, including improving the subject's condition (e.g., in one or more physiological conditions) or slowing down the progression of the infection.

[0022] According to one embodiment, the effect of treatment or application according to the present invention may be observed by a reduction in urinary tract infection symptoms such as pain or burning when urinating (dysuria), the need to urinate more frequently than usual (urinary frequency), abdominal pain, and hematuria.

[0023] The term "efficacy" of a treatment, application or method according to the invention can be measured based on the number of E. coli bacteria measured in bladder samples. Bacterial colonies are developed by inoculating the sample into culture medium and then incubating at 37° C. for one or several days. Characteristics such as number, morphology and color of the colonies allow the diagnosis of E. coli infection.

[0024] Use according to the invention According to a further aspect, the present invention provides salicylic acid (SA) or a metabolite thereof or a pharmaceutical composition thereof for use by oral route in the prevention and / or treatment of urinary tract infections, said SA metabolite being selected from 2,3-dihydroxybenzoic acid and salicyluric acid.

[0025] According to a further aspect, the SA or metabolite thereof for use according to the invention is provided in a composition comprising between about 0.75 μg and 2.6 mg of SA.

[0026] According to a further aspect, there is provided an oral formulation for use in preventing, suppressing and / or treating a urinary tract infection comprising salicylic acid (SA) or a metabolite or mixture thereof, said oral formulation comprising from about 0.75 μg to 2.6 mg of SA and at least one pharma- ceutically or cosmetically acceptable carrier, diluent or excipient thereof, and wherein said metabolite is a metabolite formed in vivo following ingestion of salicylic acid.

[0027] According to a further embodiment, the SA is provided in the form of an extract of Meadowsweet.

[0028] According to another further aspect, the present invention provides plants of the Meadowsweet species and / or parts of these plants and / or extracts of these plants or pharmaceutical compositions thereof for use by oral route in the prevention and / or treatment of urinary tract infections.

[0029] According to another further embodiment, the meadowsweet extract is an extract from the flower tops of meadowsweet.

[0030] According to another further embodiment, the meadowsweet extract is an alcoholic or hydroalcoholic extract.

[0031] According to another further embodiment, the meadowsweet extract is an aqueous extract.

[0032] In another embodiment of the present invention, there is provided a use of salicylic acid (SA) or a metabolite thereof or a pharmaceutical composition thereof for the preparation of a pharmaceutical composition for the prevention, inhibition and / or treatment of urinary tract infections by oral route.

[0033] Another aspect of the present invention relates to the use of plants of the Meadowsweet species and / or parts of these plants and / or extracts of these plants or pharmaceutical compositions thereof for the preparation of a herbal medicine for the prevention and / or treatment of urinary tract infections by oral route.

[0034] According to a further aspect, the present invention provides salicylic acid (SA) or a metabolite thereof or a pharmaceutical composition thereof for use in the prevention and / or treatment of urinary tract infections by the oral route, wherein SA or said SA metabolite is in combination with oenothein B.

[0035] According to a further embodiment, oenothein B is provided in a composition comprising about 15 μg to about 65 mg of oenothein B.

[0036] According to a further embodiment, oenothein B can be provided in the form of an extract of the genus Onacara, in particular Onacara officinalis.

[0037] According to another further aspect, the present invention provides a plant of the Meadowsweet species and / or parts of these plants and / or extracts of these plants or pharmaceutical compositions thereof for use in the prevention and / or treatment of urinary tract infections by the oral route, said plant of the Meadowsweet species and / or parts of these plants and / or extracts of these plants or pharmaceutical compositions thereof being administered in combination with a plant of the Eupatorium species and / or parts of these plants and / or extracts of these plants or pharmaceutical compositions thereof.

[0038] According to another further embodiment, the extract of Onosmodium is an extract from the aerial parts of Onosmodium.

[0039] According to another further aspect, the extract of Isahaya is obtained from Isahaya leaves.

[0040] According to another further aspect, the extract of Isahaya japonica is a hot water extract.

[0041] According to another further embodiment, the extract of Isahaya japonica is an alcoholic or hydroalcoholic extract.

[0042] According to another further aspect, the extract of Isahaya japonica is an aqueous extract.

[0043] In another embodiment of the present invention, there is provided a use of the formulations described herein for the preparation of an oral pharmaceutical composition for the prevention, inhibition, and / or treatment of urinary tract infection.

[0044] In another embodiment of the present invention, there is provided a method for preventing and / or treating a urinary tract infection in a subject, comprising administering an effective amount of salicylic acid (SA) or a metabolite thereof or a pharmaceutical composition thereof to a subject in need of prevention and / or treatment of a urinary tract infection by oral route, wherein said SA metabolite is selected from 2,3-dihydroxybenzoic acid and salicyluric acid.

[0045] In another embodiment of the invention, there is provided a method according to the invention, wherein said salicylic acid (SA) or a metabolite thereof or a pharmaceutical composition thereof is administered in combination with oenothein B.

[0046] In another embodiment of the present invention, there is provided a method for preventing and / or treating a urinary tract infection in a subject, comprising administering an effective amount of a Meadowsweet extract or a formulation thereof via oral route to a subject in need of prevention and / or treatment of a urinary tract infection.

[0047] In another embodiment of the invention there is provided a method according to the invention, wherein said extract of Meadowsweet is administered in combination with an extract of Onosmodium or a preparation thereof.

[0048] In another embodiment of the present invention, a method according to the present invention is provided in which a formulation described herein is administered.

[0049] extract Extracts of the plants according to the invention are prepared by standard methods involving maceration at 70° C. for 1 hour with a material to water ratio of 1 / 10, followed by percolation, filtration and concentration.

[0050] In certain embodiments, the meadowsweet extract of the invention comprises from about 0.1 to about 2.5% w / w salicylic acid (SA) or a metabolite thereof, for example from about 0.5 to about 2% w / w salicylic acid (SA) or a metabolite thereof, such as from about 1 to about 1.5% w / w.

[0051] In certain embodiments, a therapeutically or prophylactically effective daily dose of an extract of Meadowsweet may be from about 1 to about 550 mg, from about 1 to about 200 mg dry weight, especially from about 1 to about 100 mg, in humans based on the active dose in mice.

[0052] In further specific embodiments, a therapeutically or prophylactically effective daily dose of an extract of Meadowsweet may be from about 10 to about 200 mg dry weight, especially from about 20 to about 100 mg, in humans based on the active dose in mice.

[0053] In certain embodiments, a therapeutically or prophylactically effective daily dose of an extract of Isogaiella can be from about 10 to about 1000 mg dry weight, particularly about 10 to 500 mg, in humans based on the active dose in mice.

[0054] In further specific embodiments, a therapeutically or prophylactically effective daily dose of an extract of Isogaiella can be from about 15 to about 1000 mg dry weight, especially from about 15 to 500 mg, in humans based on the active dose in mice.

[0055] In another further specific embodiment, a therapeutically or prophylactically effective daily dose of an extract of Isogai japonica may be from about 50 to about 1000 mg dry weight, particularly from about 100 to about 500 mg, in humans based on the active dose in mice.

[0056] In certain embodiments, the present invention comprises from about 0.2 to about 20% w / w oenothein B, for example from about 1 to about 18% w / w oenothein B, such as from about 5 to about 15% w / w.

[0057] In certain embodiments, the present invention comprises from about 0.5 to about 20% w / w oenothein B, for example from about 1 to about 18% w / w oenothein B, such as from about 5 to about 15% w / w.

[0058] Extracts may be characterized by the percentage of dry matter they contain. Dry matter is the solid residue remaining after removal of the carrier or solvent by drying, such as drying the solution or suspension in an oven, freeze-drying or evaporation under reduced pressure. Dry matter may be expressed in %, and may also be referred to as plant solids concentration. Thus, 100 g or 100 mL of a solution or suspension containing 5% dry matter by weight will result in 5 grams of solid or residue after drying. Alternative methods of drying may result in slightly different values ​​of the weight percentage of dry matter, and as a result, all such values ​​stated herein are necessarily approximate. Dry matter values ​​include suspended solids and precipitated solids. The dry matter values ​​of the extracts of the present invention are determined according to the European Pharmacopoeia, 8th Edition, Chapter 2.8.17.

[0059] composition According to certain embodiments, there is provided a formulation comprising about 10 to about 200 mg dry weight of an extract of Meadowsweet, particularly useful for preventing and / or treating urinary tract infections, and at least one pharma- ceutically or cosmetically acceptable carrier, diluent or excipient thereof.

[0060] According to another embodiment, the present invention relates to an oral formulation comprising salicylic acid (SA) or a metabolite thereof or a pharmaceutical composition thereof in combination with at least one co-agent useful in the prevention and / or treatment of urinary tract infections, and at least one pharma- ceutically or cosmetically acceptable carrier, diluent or excipient thereof.

[0061] In another embodiment of the invention, the present invention relates to a formulation comprising an extract of Meadowsweet in combination with at least one co-agent useful in the prevention and / or treatment of urinary tract infections and at least one pharma- ceutically or cosmetically acceptable carrier, diluent or excipient thereof.

[0062] According to certain embodiments, the co-agent is selected from oenothein B, and spiroside, or a mixture thereof.

[0063] According to another particular embodiment, the co-agent is an extract of Isobium orbiculare.

[0064] According to certain embodiments, a formulation is provided that includes a combination of an extract of meadowsweet and an extract of onychophos. Typically, the combination formulation according to the present invention includes about 30 to about 70% w / w of an extract of meadowsweet and about 70 to about 30% w / w of an extract of onychophos. According to further specific embodiments, the combination formulation according to the present invention includes about 30 to about 50% w / w of an extract of meadowsweet and about 70 to about 50% w / w of an extract of onychophos.

[0065] According to another embodiment, the combination preparation according to the invention comprises about 15 to about 85% w / w of an extract of Meadowsweet and about 85 to about 15% w / w of an extract of Onagraceae. According to a further particular embodiment, the combination preparation according to the invention comprises about 15 to about 50% w / w of an extract of Meadowsweet and about 85 to about 50% w / w of an extract of Onagraceae.

[0066] According to one embodiment, there is provided a formulation comprising a combination of about 10 to about 200 mg dry weight of an extract of Meadowsweet together with a combination of about 10 to about 200 mg dry weight of an extract of Onosmodium falcatum, and at least one pharma- ceutically or cosmetically acceptable carrier, diluent or excipient thereof.

[0067] According to one embodiment, an oral formulation is provided, the formulation comprising about 1 to about 550 mg dry weight of meadowsweet extract (e.g., 10 to about 200 mg) and at least one pharma- ceutically or cosmetically acceptable carrier, diluent, or excipient thereof.

[0068] According to further specific embodiments, there is provided an oral formulation comprising 1 to about 550 mg dry weight of meadowsweet extract (e.g., about 20-25 mg) and about 10-500 mg of onychophos umbellata extract (e.g., about 100-110 mg), and at least one pharma- ceutically or cosmetically acceptable carrier, diluent, or excipient thereof.

[0069] According to certain embodiments, the formulations of the present invention are for oral use in solid forms, such as tablets (e.g., hard or soft), capsules or powders, semi-liquid forms, such as orally drinkable gels, and liquid forms, such as beverages and syrups.

[0070] The compounds, extracts, uses and methods of the present invention are particularly useful in preventing urinary tract infections, especially E. coli infections. They are advantageous not only in preventing bacterial growth, but also in preventing adhesion to urinary tract cells, which is the major cause of recurrence. In addition, the uses and methods of the present invention are advantageous in avoiding the use of antibiotic treatments, which increase the risk of resistance to pathogenic bacteria.

[0071] In another particular embodiment, the present invention provides compositions according to the present invention for use in the preparation of dietary supplements, cosmetic formulations and medicaments.

[0072] According to a particular embodiment, the composition of the invention is a pharmaceutical composition.

[0073] According to a particular embodiment, the composition of the present invention is a dietary supplement.

[0074] According to a particular embodiment, the composition of the present invention is a cosmetic composition.

[0075] According to a particular embodiment, the composition of the present invention is an oral composition.

[0076] The compositions of the present invention may further comprise one or more additional pharma- ceutically acceptable ingredients, such as alum, stabilizers, antimicrobial agents, buffers, colorants, flavoring agents, adjuvants, and the like.

[0077] The compositions of the present invention, together with conventional adjuvants, carriers, diluents or excipients, may be in the form of pharmaceutical compositions and unit dosages thereof, and in such form may be used as solids, such as tablets or filled capsules, or as liquids, such as solutions, suspensions, emulsions, elixirs, or filled capsules thereof, all for topical use.

[0078] According to the present invention, the compositions of the present invention may be prepared in the form of dietary supplements, medical devices, cosmetic products and pharmaceutical products.

[0079] The compositions of the present invention may be liquid preparations, including, but not limited to, aqueous or oily suspensions, solutions, and emulsions.The compositions may also be prepared as dry products for reconstitution with water or other suitable vehicles before use.Such liquid preparations may contain additives, including, but not limited to, suspending agents, emulsifying agents, non-aqueous vehicles, and preservatives.Suspending agents, dispersing agents, wetting agents, and emulsifying agents include, but are not limited to, polyethylene glycol, glycerol stearate, and sorbitol esters.

[0080] Further materials and formulation processing techniques and the like can be found in "The Science and Practice of Pharmacy" by Remington, 23rd Edition, 2020, Editor: Adeboye Adejare, Academic Press, the contents of which are incorporated herein by reference.

[0081] Administration Method The extracts, compounds or compositions of the present invention may be administered orally.

[0082] The exact dosage of the extracts and compositions can be readily determined by one of skill in the art based on the teachings herein, together with the potency of the extracts and compositions, the age, weight, sex and physiological condition of the subject.

[0083] According to one embodiment, the compound or composition of the invention is applied or administered before or at the beginning of the onset of urinary tract infection symptoms or when at risk for E. coli infection.

[0084] According to another embodiment, the compound or composition of the present invention is applied or administered after symptoms of E. coli infection.

[0085] combination The SA compounds or meadowsweet extracts of the present invention, and pharmaceutical formulations thereof, may be administered alone or in combination with co-medicines useful in the prevention and / or treatment of urinary tract infections, such as co-medicines selected from, for example, D-mannose, cranberry extract, uva ursi extract, zinc, vitamin D3, vitamin C, and antibiotics.

[0086] According to a further aspect, there is provided a formulation comprising about 0.1 to about 50 mg, e.g., about 0.5 to about 20 mg, of salicylic acid (SA) or a metabolite thereof or a mixture thereof, in combination with at least one co-agent useful in the prevention and / or treatment of a urinary tract infection, and at least one pharma- ceutically or cosmetically acceptable carrier, diluent or excipient thereof.

[0087] According to another further aspect, there is provided a formulation comprising about 0.1 to about 50 mg, such as about 0.5 to about 20 mg, of salicylic acid (SA) or a metabolite thereof or a mixture thereof, and at least one pharma- ceutically or cosmetically acceptable carrier, diluent or excipient thereof, in combination with at least one co-agent useful in the prevention and / or treatment of a urinary tract infection, said formulation comprising an extract from Meadowsweet species.

[0088] According to a particular embodiment, the co-agent is selected from oenothein B, and spireoside, or a mixture thereof.

[0089] According to a particular embodiment, the formulation further comprises 0.1 to about 1,000 mg of oenothein B, such as, for example, from about 0.5 to about 80 mg of oenothein B.

[0090] According to another particular embodiment, the co-agent is an extract of Isobium orbiculare.

[0091] According to another further embodiment, the formulation according to the invention further comprises D-mannose, in particular from 500 to about 3'000 mg (eg 700 mg / mL).

[0092] According to another further embodiment, the formulation according to the invention further comprises vitamin D3, in particular 200 to about 2'000 UI (eg 500 UI).

[0093] The present invention encompasses administering the extracts and pharmaceutical formulations thereof of the present invention to an individual prior to, simultaneous with or sequential to other therapeutic / prophylactic regimens or co-medicines in the prevention or treatment of urinary tract infections. The extracts or pharmaceutical formulations thereof administered simultaneously with said co-medicines can be of the same or different composition and administered by the same or different route of administration.

[0094] The dosage administered to an individual as a single or multiple dose will vary depending on a variety of factors, including pharmacokinetic properties, the patient's condition and characteristics (age, weight, health, size), the severity of symptoms, concurrent therapy, frequency of treatment, and the desired effect.

[0095] patient In one embodiment, a patient according to the present invention is a patient suffering from a urinary tract infection, in particular an E. coli infection.

[0096] In another embodiment, a patient according to the present invention is a patient at risk of suffering from a urinary tract infection.

[0097] Patients at risk for developing urinary tract infections include those with recurrent E. coli infections, those with suppressed immune systems, pre- and postmenopausal (estrogen deficient) or pregnant female patients, those with implanted catheters, those who have frequent sexual intercourse, those with a history of infection, those who have used repeated antibiotic treatments, those with congenital malformations, and those with diabetes.

[0098] According to certain embodiments, the formulations of the invention should be administered during the acute (symptomatic) phase of the UTI, and preferably for at least 5 days.

[0099] In the following, illustrative examples of the invention are explained in more detail and with reference to embodiments represented in the drawings. EXAMPLES

[0100] The following abbreviations have the following definitions: ASE (Accelerated Solvent Extraction), cfu / mL (Colony Forming Units per Milliliter), DMEM (Dulbecco's Modified Eagle's Medium), DMSO (Dimethyl Sulfoxide), EDTA ((Ethylenedinitrilo)tetraacetic acid), EGF (Epidermal Growth Factor), FCS (Fetal Calf Serum), HEPES (4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid), PBS (Phosphate Buffered Saline), SDA (Sabouraud Dextrose Agar), SFM (Serum Free Medium).

[0101] Example 1: Effects of salicylic acid and its metabolites The effects of salicylic acid (SA) CAS n° 69-72-7 (1) and two of its metabolites formed in vivo, namely 2,3-dihydroxybenzoic acid CAS n° 303-38-8 (2) and salicyluric acid CAS n° 487-54-7 (3) (Nelson et al. 2019, Godfrank's Toxicologic Emergencies, 11th ed., McGraw-Hill Education; Dachineni et al. 2017, Int. J. Oncol. 51, 1661-1673) have been tested as follows:

[0102] [ka]

[0103] Compounds 1-3 were purchased from Sigma. Cranberry juice extract, which is widely recommended for the prevention of UTI (Gonzalez de Llano et al., 2020, Molecules, 25, 352), was used as a positive control and was obtained as follows: Cranberry fruits were extracted for 1 h using a plant / solvent ratio of 1 / 10, then concentrated to a concentration of 5 g / L through a filtration step and stored at -20 °C. Cranberry juice extract has been claimed to be useful in the treatment of urinary tract infections (Raz et al., 2004, Clinical Infectious Diseases, 38(10), 15, 1413-1419; Wang et al., 2012, Arch Intern Med., 172(13):988-996; Gbinigie et al., 2021, Antibiotics (Basel), 10(1):12).

[0104] In vitro adhesion assay (ADH) - Host-pathogen interactions assessed by in vitro adhesion to human cells Adhesion of E. coli to the bladder epithelium was demonstrated in cell culture. Upon exposure to E. coli, detachment of epithelial cells was observed, similar to exfoliation in vivo. The test was miniaturized by growing epithelial cell cultures in 96-well plates instead of the standard 24-well plates. Miniaturization resulted in an increase in the number of measurements and a decrease in variability from >50% to <30%, thereby meeting the acceptance criteria for reproducibility of cell-based bioassays.

[0105] Before the start of the adhesion process, the test samples were added. Bladder cells grown to confluence in 96-well plates were inoculated with E. coli (1.4*10 in each well). 7 The plates were exposed to 50 μL of E. coli (0.1 cfu / mL) for 1 hour. After 1 hour, non-adherent E. coli were rinsed off, adherent E. coli were detached, and colony forming units (cfu) were enumerated by plate counting. Test variability was further improved by automating the counting using a spiral plate counting device.

[0106] E. coli adhesion studies were performed in the absence (given as 100% adhesion) and presence of various drug concentrations. Criteria for a significant reduction in E. coli adhesion to the host included values ​​<50% of standard adhesion. Human cytotoxicity measurements of mitochondrial activity (MTT) excluded side effects of the drugs.

[0107] The results are shown in Figure 1C, where relative adhesion (ADH) is expressed as a percentage (no inhibition control = 100%, buffer was substituted for treatment). Any adhesion value <50% is defined as significant inhibition.

[0108] The data show that salicylic acid and its metabolites cause a reduction in the adhesion of E. coli CFT073 onto bladder cells. Salicylic acid shows adhesion-reducing efficacy at concentrations of 5 mg / mL, 2.5 mg / mL, and 1.25 mg / mL. Salicyluric acid shows a significant reduction at concentrations of 5 mg / mL and 2.5 mg / mL. The same concentrations also show an effect on incubation with 2,3-dihydroxybenzoic acid.

[0109] Thus, the in vitro antiadhesive activities of salicylic acid and its two metabolites are similar.

[0110] In vitro antibacterial assay Growth of E. coli in solution was monitored in a 96-well plate (diluted E. coli (2.5*10 5 The microbial population was measured in 96-well plates filled with 100 μL of 100 cfu / mL of 100% microbial microbial fungi. MIC (minimum inhibitory concentration) values ​​were determined in the absence and presence of various concentrations of the test drug during 24 hours of exponential growth. Growth was usually measured by spectrophotometric OD 690nm The inhibitory activity is quantified by ELISA. However, due to the colorimetric properties of certain compounds and plant extracts, it interfered with the spectrophotometric method. Therefore, an adaptation was made by counting the number of E. coli by plate counting method (ufc count). The inhibitory activity is shown in Table 1 below.

[0111] [Table 1]

[0112] Thus, the MIC values ​​for salicylic acid and its metabolites (2 and 3) were 5 mg / mL, 5 mg / mL, and 1.25 mg / mL, respectively. Cranberry extract did not show any MIC effect.

[0113] The concentration of salicylic acid required to inhibit E. coli adhesion to human bladder epithelium was 2.5 mg / mL (=0.25 mg / 100 μL) at 1 h, and the minimum inhibitory concentration for bacterial growth was 5 mg / mL (=0.5 mg / 100 μL) at 24 h, so these data suggest that salicylic acid may be useful not only for its direct bactericidal effect but also for inhibiting bacterial adhesion.

[0114] In vivo efficacy For the identification of active plant extracts and compounds, an in vivo test for UTI (urinary tract infection) was constructed and performed. Age-matched female C3H / HeNCrl mice were adapted to clean, non-septic laboratory conditions. The experiment was started with 2 days of glucose-enhanced feeding to promote subsequent UTI infection. Then, the respective compound or plant extract was applied in various concentrations by 8 consecutive intestinal feedings in a gavage apparatus over a period of 24 hours. Each feeding contained a volume of 100 μL of solution. During these 24 hours, the mice were kept in metabolic cages. Urine was collected every 3 hours to analyze the diuretic effect. Subsequent UTI infection * To assess the efficacy of E. coli, isolate CFT073 (commercially available from DSM and derived from human UTI) was applied transurethrally into the bladder and incubated in vivo for 3 h under free movement in normal cages. Mice were euthanized and viable E. coli were counted in bladder homogenates (homogenization of 1 / 2 the bladder for viable count (cfu)) and organ morphology was assessed by histological microscopy. * 5 x 10 implanted in the bladder 7Infection of the UTI with 50 μL of E. coli (CFT073) containing 10 bacteria (in a separate experiment, in vivo infection was performed similarly but with a slightly lower number of bacteria per implant, i.e., 3.3 × 10 6 pcs or 5×10 6 bacteria).

[0115] Conversion of doses used in animal models to human doses (HED, human equivalent dose) was calculated according to the Guidance for Industry for Estimating Maximum Safe Starting Dose for Initial Clinical Trials of Therapeutics in Healthy Adult Volunteers published by FDA (CDER) in July 2005 (https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC4804402 / and https: / / www.fda.gov / media / 72309 / download, Table 1, p. 7).

[0116] Effects on bladder adhesion in vivo 5 × 10 applied transurethrally into the bladder 7 In a representative experiment (n = 6 mice per group) with E. coli at a bacterial concentration of cfu, salicylic acid (0.05 mg / 100 μL) had a -60% inhibitory effect on bacterial infection (adhesion), as did the positive control cranberry (2.5 mg / 100 μL). 6 In a separate experiment with a bacterial concentration of cfu, salicylic acid (0.78 μg / 100 μL) had a -61.3% inhibitory effect on bacterial infection (adhesion), similar to the positive control cranberry (2.5 mg / 100 μL).

[0117] These in vivo data demonstrate the bacteriostatic and anti-adhesive effects of salicylic acid when applied at a concentration of 0.05 mg / 100 μL / gavage by eight gavage sessions over a 24-h period (total of 0.4 mg / 24 h). Furthermore, the cumulative amounts of salicylic acid and its metabolites present in the urine of these mice were 84 μg salicylic acid, 707 μg salicyluric acid, and 68 μg 2,3-dihydroxybenzoic acid (Figure 3B), confirming that in addition to its in vitro inhibitory effects on adhesion (Figure 1C), the direct bactericidal and anti-adhesive effects of salicylic acid likely play a role in E. coli urinary tract infection in vivo.

[0118] diuretic effect Urine was collected from control mice and mice treated with various drugs. The values ​​for urine volume (in mg) produced by the various treatments are shown in Table 2. Table 2 shows the results of administering 100 μL of the test substance orally every 3 hours for 21 hours, followed by intravesical administration of 5×10 6 Figure 1 shows the diuretic effect of various substances and extracts tested in vivo using a mouse model inoculated with E. coli. The experiment lasts for a total of 24 hours, with urine collected and weighed every 3 hours. Results are expressed as percentage of variation compared to a negative control (95% water, 5% glycerol), a cranberry extract as a positive control (0.2 mg in water extract in 5% glycerol for comparison with pure substances or at the same total concentration of the extract or mixture of extracts to be compared), and an extract of meadowsweet and onyx (water extract in 5% glycerol). Each value is calculated from the sum of urine collected from 6 mice.

[0119] [Table 2]

[0120] The concentration of salicylic acid, 0.78 μg / 100 μL, as the pure compound tested was similar to the 1.3% concentration seen in the extract of Meadowsweet (see below) when used at a concentration of 0.5 mg in 100 μL. Salicylic acid given at the very low concentration of 0.78 μg / 100 μL caused an increase in urinary excretion of +10.7% and +24.9% over the negative control (water) and the positive control (cranberry extract), respectively. Salicylic acid given at a concentration of 50 μg / 100 μL caused an increase in urinary excretion of +22.3% and +67.8% over the negative control (water) and the positive control (cranberry extract), respectively.

[0121] Thus, these data support the idea that salicylic acid and its two major metabolites may have beneficial effects on various factors involved in urinary tract infections, namely bladder bacterial cell adhesion, bacterial infection, and may also have beneficial diuretic properties that may aid in the healing of urinary tract infections.

[0122] Example 2: Effect of Oenothein B Oenothein B, CAS n° 104987-36-2 (4), was isolated from the aqueous extract of Ipomoea batatas by a combination of preparative and semi-preparative HPLC on RP18 (1.3% yield). The compound was characterized by mass spectrometry and NMR spectroscopy. The effect of oenothein B has been tested in the same assay as described in Example 1. The in vitro antibacterial activity is presented in Table 3 below and the diuretic activity in Table 2 above.

[0123] [ka]

[0124] [Table 3]

[0125] For oenothein B, the MIC was 1.3 mg / mL.

[0126] In vivo efficacy Effects on bladder adhesion in vivo Density 3.28×5×10 6 Under bacterial inoculation conditions at cfu, the number of adherent E. coli was 75.12% lower in animals treated with oenothein B at 0.065 mg / 100 mL than in controls.

[0127] diuretic effect Oenothein B given at a concentration of 18.2 μg / 100 μL caused a significant increase in urinary excretion of +39.9% and +57.8% over the negative control (water) and the positive control (cranberry extract), respectively. Since the oenothein B concentration in the extract is 13% as explained above, the tested amount of oenothein B corresponds to an extract of Ipomoea batatas which would have been 1.12 mg / 100 μL.

[0128] Oenothein B and its metabolites were not identified by LC-MS in the collected urine samples, suggesting that mechanisms other than direct inhibition of bacterial growth in the bladder may play a role in the activity of oenothein B against E. coli urinary tract infection in vivo, which would imply a potential advantage for combination with SA or its metabolites.

[0129] Example 3: Effects of Meadowsweet and Onosmodium falcatum extracts The effects of various extracts of Meadowsweet and Euphorbia gracilis were tested under the following conditions as explained above.

[0130] The following extracts were prepared: 1. Fu1 and Ep1: Hot water extraction of flower tops at a plant / solvent ratio of 1 / 10 for 1 h, filtration, determination of dry content, addition of maltodextrin (20% of the final weight), freeze-drying, and storage in aluminium pouches under vacuum. 2. Fu2 and Ep2: 20% EtOH extraction of flower tops at a plant / solvent ratio of 1 / 10 for 1 h, filtration, ethanol evaporation, determination of dry content, addition of maltodextrin (20% of the final weight), freeze-drying, and storage in aluminium pouches under vacuum. 3. Fu3 and Ep3: 70% EtOH extraction of flower tops at a plant / solvent ratio of 1 / 10 for 1 h, filtration, ethanol evaporation, determination of dry content, addition of maltodextrin (20% of the final weight), freeze-drying, and storage in aluminium pouches under vacuum. 4. Fu4: hot water extraction from aerial parts (flower tops and top 60 cm of stem) for 1 h at a plant / solvent ratio of 1 / 10 as described, filtration, ethanol evaporation, determination of dry content, addition of maltodextrin (20% of final weight), freeze-drying, and storage under vacuum in aluminum pouches.

[0131] Extracts of meadowsweet flower tops, aerial parts of Eucalyptus japonica and cranberry berries (cranberries) were prepared as follows: Initial extraction in hot water (70°C) for 1 hour with a material to solvent ratio of 1 / 10. The maceration was filtered and the amount of dry material was measured. Maltodextrin DE20 was added to the filtrate at a rate of 20% of the final dry weight, followed by freeze-drying. The dry powder was stored under vacuum in aluminum pouches.

[0132] In vitro antibacterial assay The minimum inhibitory concentrations (MICs) of the different extracts from the different extraction procedures presented in Table 4 (uropathogenic E. coli, centrifugation of plant extracts at 14'000xg, 5 min) indicated that extraction procedure 1 (Fu1, Ep1) was pursued for further evaluation. Thus, the MIC value of the meadowsweet extract alone is 0.9 mg / mL and that of the scutellaria barbata is 2.7 mg / mL.

[0133] [Table 4]

[0134] The growth of E. coli in the solution was measured in 96-well plates. The MIC (minimum inhibitory concentration) values ​​were determined in the absence and presence of various concentrations of the test drug during exponential growth. The number of E. coli was enumerated by plate counting method (ufc) as described above. The data is summarized in Figure 1E and shows that: The anti-adhesion activity is more than 2-fold superior for meadowsweet extract and 8-fold superior for the combination of extracts of the present invention compared to the effect of cranberry extract and D-mannose.

[0135] The minimum inhibitory concentrations of mixtures of hot water extracts of Meadowsweet (Fu1) (F) and Eupatorium officinalis (E) under the same conditions as shown above are presented in Table 5 below.

[0136] [Table 5]

[0137] The MIC value for the 50% + 50% concentration ratio is 10 mg / mL. A 20% decrease in the concentration of the extract of Meadowsweet (% of the total mixture) and a 20% increase in the concentration of the extract of Eupatorium officinalis (70% of the total mixture) reduced the F 50% +E 50% The MIC value is calculated as F 30% +E 70% The ratio of extracts is 10 mg / mL. 70% +E 30% and F 85% +E 15% Similar activity was seen for the 100% glycerol extract and 100% glycerol extract, respectively. The data are summarized in Figure IF and show that the compositions of the present invention are active at concentrations up to 10-fold lower than the cranberry extract.

[0138] In vitro adhesion test (ADH) The anti-adhesion effect is measured in vitro (standard method) using centrifuged extracts (Fu1) of Meadowsweet (F) and Euphorbia gracilis (E) using human epithelial bladder cells (T24 ATCC® HTB-4™ Homo sapiens bladder cell line).

[0139] Figures 1A and 1B confirm that all tested extracts of Meadowsweet (F) and Oncorhynchus gracilis (E), respectively, have an effect on the adhesion of E. coli CFT073 to human bladder cells. Figure 1D confirms that all combined extracts of Meadowsweet (F) and Oncorhynchus gracilis (E) in 50 / 50 and 30 / 70 ratios tested at total concentrations of 5, 10, 15, 20 and 25 mg / mL have an effect on the adhesion of E. coli CFT073 to human bladder cells.

[0140] In vivo efficacy Effects on bladder adhesion in vivo The in-vivo anti-adhesive effect of various substances and extracts of the invention was tested in vivo using the above-mentioned mouse model in which 100 μL of the test substance was given orally every 3 hours for 24 hours, followed by inoculation of the bladder with E. coli 3 hours before measuring E. coli in the bladder. At the end of each assay, the mice were euthanized and their bladders were homogenized for E. coli measurement.

[0141] 5 × 10 Escherichia coli administered transurethrally into the bladder 6 3.3 x 10 cfu 6 In three representative experiments (n = 6 mice per group in each experiment) with similar bacterial concentrations, the combined extract of Meadowsweet and Onyx gracilis showed a dose-ranging effect as bacteriostasis and prevention of adhesion of E. coli of 87.08% (F50% + E50%), 65.65% (F30% + E70%), and 40.87% (F30% + E70%) compared to water control at concentrations of 1 mg, 0.5 mg, and 0.2 mg of total plant extract per 100 μL, respectively (Figure 5).

[0142] This combined extract of Meadowsweet and Onosmodium falciparum at 5mg / 100μL (2.5+2.5mg each) (F50%+E50%) was shown to inhibit 5×10 Escherichia coli cells administered transurethrally into the bladder. 7 Using a higher bacterial concentration of cfu was more effective at reducing the amount of E. coli by 40%, although this effect was less than that observed with cranberry extract in this study (60.24%).

[0143] diuretic effect The extracts were tested as described in Example 1, with the results shown in Table 2. Extract of Meadowsweet at a concentration of 2.5 mg / 100 μL caused a 35.8% increase in urine volume compared to cranberry extract given at the same concentration. No diuretic effect was measured with extract of Ipomoea batatas at a concentration of 2.5 mg / 100 μL alone.

[0144] The combined extract of meadowsweet and onyx (F50%+E50%) showed a dose-ranging diuretic effect with an increase in urine volume of 38.1%, 11.4%, and 8.3% compared to cranberry extract (5mg / 100μl) at total concentrations of combined extract of 10mg, 5mg, and 1mg per 100μl, respectively. In another experiment, the combined extract of meadowsweet and onyx (F50%+E50%) at 5mg / 100μl caused an increase in diuresis compared to the control (5mg / 100μl cranberry extract) that continued throughout the 24 hour treatment as shown in FIG. 4.

[0145] Overall, it can be concluded that the effects are significant when compared to cranberry extract, a well-known alternative treatment for UTIs; the combination of plant extracts prevented both E. coli attachment and growth 8-fold more than cranberry extract, and increased urine volume by 62.4% over a 24-hour period compared to cranberry extract.

[0146] Example 4: Phytochemical characterization of meadowsweet and onyx extracts The extracts prepared as described in Example 3 were characterized as follows.

[0147] Polyphenol profile and spiroside content of meadowsweet

[0148] [ka] Spireoside (quercetin-4'-O-glucoside) was identified as the major flavonoid glycoside in extracts of Meadowsweet by HPLC analysis (SunFireC 18 In addition, quercetin, hyperoside (quercetin-3-O-galactoside), and rutin (quercetin-3-O-rutinoside) were also detected. The identities of these compounds were confirmed by chromatographic comparison with commercially available authentic standards. The spireosides that could be used as marker compounds were analyzed by HPLC-UV analysis (SunFireC column) using a calibration curve prepared with commercially available standards of spireosides (Extrasynthese) (0.125 (Cal1), 0.063 (Cal2), 0.031 (Cal3), 0.016 (Cal4), 0.008 (Cal5), 0.004 (Cal6), and 0.002 (Cal7) mg / mL). 18 The analysis was performed in three independent replicates and provided a spireoside content of 2.8±0.3% in the extracts of Meadowsweet.

[0149] Salicylic acid derivatives in extracts of Meadowsweet. Meadowsweet is known to contain, among many other components, salicylic acid and its derivatives such as salicylic acid methyl ester and monotropitoside (Bijttebier et al., 2016). Salicylic acid was detected by HPLC-UV analysis (SunFireC 18The salicylic acid content was quantified in the extracts by column chromatography (Figure 2A). A calibration curve was constructed with a commercial reference standard (TCI Europe) of salicylic acid (1.0 mg / mL stock solution in DMSO) with concentrations of 0.25 (Cal7), 0.125 (Cal6), 0.063 (Cal5), 0.031 (Cal4), 0.016 (Cal3), 0.008 (Cal2), and 0.004 (Cal1) mg / mL. Besides the content of salicylic acid, the content including salicylic acid glycosides and esters was determined after acid hydrolysis of the extracts with 2 N TFA at 100 °C for 2 h. The experiment was performed with three independent replicates. The identity of salicylic acid was confirmed by HPLC-MS analysis and by comparison with commercial standards.

[0150] A salicylic acid content of 1.27±0.02% was determined in the extract, while the content increased slightly to 1.32±0.01% after acid hydrolysis, indicating that most of the salicylic acid was present in the extract in free form.

[0151] Polyphenol profile and oenothein B content of red onion The tannin oenothein B was by far the major phenolic constituent in the extract of Ipomoea batatas. In addition to this, hyperoside and myricetin-hexoside were found to be the major flavonoids. Oenothein B was found to be the major phenolic constituent in the extract of Ipomoea batatas by HPLC-UV analysis (SunFireC 18 The concentration of 13.1% was determined in the extract of I. canadensis (Figure 2B).

[0152] Analysis of salicylic acid and its metabolites in the urine of treated mice. Urine collected from a control group as described in Example 3 was compared to urine collected from mice treated with various combinations of plant extracts. 18Urine was analyzed using HPLC-UV / MS on a column. 5% MeCN (A) and MeCN (B), both containing 0.1% formic acid, were used as mobile phases. A gradient of 0-100% B was applied for 20 min at a flow rate of 0.8 mL / min. MS scans were recorded in positive and negative ion mode (ESI+ / -). Major additional peaks in the group treated with the meadowsweet-sugar herb mixture were identified as salicylic acid (SA) (1), 2,3-dihydroxybenzoic acid (DHBA) (2), and salicyluric acid (SUA) (3). Figure 3A compares the RT and peaks from analytical standards of salicylic acid (SA) (1) and its metabolites salicyluric acid (SUA) (2) and 2,3-dihydroxybenzoic acid (DHBA) (2) found in urine samples collected from groups treated with Meadowsweet extract. High-resolution mass spectrometry was used to confirm the accurate masses of the three molecules. Furthermore, the three identified metabolites were quantified in the urine samples by MRM with the following transitions (m / z): 136.5 → 65.0, 136.5 → 92.9 (SA), 152.9 → 108.9, 152.9 → 80.9 (DHBA), 194.0 → 150.0, 194.0 → 92.9 (SUA), and pure analytical standards diluted in urine from the negative control for the calibration curve. FIG. 3B shows the cumulative amounts and concentrations of salicylic acid (SA), salicyluric acid (SUA) and dihydroxybenzoic acid (DHBA) in urine samples collected over a 21-hour period from the group treated with 40 mg of meadowsweet extract (8 doses administered at 3-hour intervals).

[0153] As described above (Example 3, in-vivo efficacy study), the amounts of SA, SUA and DHBA were quantified in the urine of mice following absorption of an extract of Meadowsweet containing 1.3% SA at a total dose of 40 mg in eight equally divided doses over a 24 hour period. It was found that 41% of the ingested SA was found in the urine in the form of SA (5.4%), SUA (32.0%) and DHBA (3.9%) (41% total).

[0154] These experiments thus confirm that oral administration of extracts of Meadowsweet makes it possible to measure the presence of SA and its metabolites at the site of action with a recovery of 41% of the intake found with SA, SUA and DHBA.

[0155] In summary, the in vitro data support the beneficial effect of SA and its metabolites formed in vivo after SA ingestion against E. coli UTIs, as they have a direct bactericidal effect as well as an effect on bacterial adhesion to bladder cells. Furthermore, the data suggest that oenothein B has in vivo activity against E. coli UTIs through a mechanism other than direct inhibition of bacterial growth in the bladder, and therefore that the activity of SA and its metabolites may be complementary in efforts to combat E. coli UTIs.

[0156] Furthermore, the data of the present invention also show that the administration of SA and its derivatives 2,3-dihydroxybenzoic acid and salicyluric acid can be successfully achieved by administration of an extract of Meadowsweet, preferably an aqueous extract.

[0157] Based on the active dose in mice, it can be reasonably estimated by extrapolation that the use of a composition containing about 0.25 to 2.6 mg of SA would be beneficial (FDA (CDER) supra, July 2005: https: / / www.fda.gov / media / 72309). This dose can be achieved with about 10 to about 200 mg dry weight, particularly about 20 to about 100 mg dry weight of extract of Meadowsweet.

[0158] SA may be advantageously combined with oenothein B, particularly from about 1.3 to about 65 mg of oenothein B.

[0159] Furthermore, the administration of oenothein B can be achieved by administration of an extract of Isogaiella orbiculatus, preferably an aqueous extract. The above amount of oenothein B can be achieved by administration of an extract of Isogaiella orbiculatus of about 10 to about 1,000 mg dry weight, in particular of about 10 to 500 mg dry weight.

[0160] Finally, Figures 1A, 1B and 1C further support that the combination of extracts of Meadowsweet and Onyx according to the present invention is therapeutic against E. coli infections and that since they contain identified active principles that have been demonstrated to have complementary activities, such a combination is expected to have an advantageous multi-target effect against E. coli urinary tract infections and would be better than each extract alone.

[0161] Example 5: Effect on inflammation The effect of the compositions of the invention can be evaluated on CACO-2 cells in which an inflammatory response is induced by TNF-α. The anti-inflammatory effect can be measured by quantifying the amount of IL-6 released in response to TNF-α (Vitkus et al., 1998, In Vitro Cellular and Developmental Biology, 34, 660-664).

[0162] Example 6: Effect of combined extracts in recurrent UTI The efficacy of the composition of the present invention is evaluated in adult women (15 women aged 18-65 years) with recurrent UTI (more than 3 recurrences per year). Tablets incorporating the mixture of extracts of the present invention are prepared by standard methods using freeze-dried hot water extracts of Meadowsweet and Oncorhynchus gracilis as described above (Example 3) so that each tablet contains 132 mg of the mixture of the two plant extracts mentioned above (23 mg Meadowsweet and 109 mg Oncorhynchus gracilis). The mixture is comprised of F 17% +E 83% is equivalent to.

[0163] Each tablet also contains 700mg D-mannose, 80mf FOS, 1.25mg zinc gluconate, and 500UI vitamin D3.

[0164] As soon as UTI symptoms appeared, the women were asked to take one tablet 20 minutes before meals for five days according to the following schedule: one dose of two tablets in the morning and one dose of two tablets in the evening, even if symptoms disappeared before the end of the treatment. If antibiotics were required for the infection, patients could continue taking the regimen, as no interactions or contraindications have been known to date. Two patients who took antibiotics during the assay were excluded from the statistical analysis, which was performed on 15 patients.

[0165] Outcomes were assessed by questionnaire among women who underwent treatment. 1. On a scale of 0 to 10 (0=none, 10=severe), how severe do you think your usual urinary tract symptoms were during your last two UTIs before treatment, and how severe do you think the urinary tract symptoms you experienced during and after treatment were? A: Pain when urinating B: Frequent urge to urinate C: Bladder heaviness / pain 2. How long your last two UTIs lasted (days / hours) 3. How long the urinary tract infection lasted during the procedure (days / hours) 4. Did you need to take antibiotics in conjunction with the procedure? Yes / No (If no, skip to question 5) A. If yes, which antibiotic / duration of treatment: B. Was there any improvement in the intensity or duration of symptoms when the treatment was combined with antibiotics?

[0166] The results are shown in Figure 6, where pain during urination was reduced by 59%, frequency of urination was reduced by 53%, and pain and bladder discomfort was reduced by 39%. The duration of symptoms was significantly reduced by 45.5% (p<0.05), from a mean value of 6 days (mean value without treatment, individual values ​​2-14 days) to 3.27 days (individual values ​​1-5 days) with treatment. 86.7% of the women in the study were satisfied with the reduction in the duration of symptoms, 73.3% were satisfied with the reduction in symptoms, 86.7% would buy the product again if they had another UTI, and 86.7% of them declared that they would recommend the treatment to a friend suffering from UTI.

[0167] These results are extremely encouraging in the context of increasing antibiotic resistance and the strong need for alternative options for the prevention and treatment of UTIs.

Claims

1. Use of salicylic acid (SA) or a metabolite thereof or a mixture thereof or a pharmaceutical composition thereof in the manufacture of an oral medicament for the prevention and / or treatment of urinary tract infection, wherein the metabolite is a metabolite formed in vivo after ingestion of salicylic acid.

2. 2. The use according to claim 1, wherein the metabolite is selected from 2,3-dihydroxybenzoic acid and salicyluric acid.

3. 3. The use according to claim 1 or 2, wherein the medicament is to be used in combination with oenothein B.

4. 3. The use of claim 1 or 2, wherein the SA or the metabolite thereof is provided in a composition comprising about 0.75 μg to 2.6 mg of SA.

5. 5. The use according to claim 4, wherein the salicylic acid (SA) is in the form of a meadowsweet extract.

6. 3. The use according to claim 1 or 2, wherein the urinary tract infection is an Escherichia coli infection.

7. 3. The use of claim 1 or 2, wherein the medicament is to be used in combination with oenothein B, wherein the oenothein B is provided in a composition comprising from about 15 μg to about 65 mg of oenothein B.

8. 3. The use according to claim 1 or 2, wherein the medicament is to be used in combination with an extract of Ipomoea oryzae.

9. An oral formulation for use in preventing, suppressing, and / or treating a urinary tract infection, comprising salicylic acid (SA) or a metabolite thereof or a mixture thereof, said formulation comprising about 0.75 μg to 2.6 mg of SA and at least one pharmaceutically or cosmetically acceptable carrier, diluent, or excipient thereof, and said metabolite is a metabolite formed in vivo after ingestion of salicylic acid.

10. An oral formulation comprising salicylic acid (SA) or a metabolite thereof or a mixture thereof, said oral formulation further comprising at least one co-drug useful in the prevention and / or treatment of urinary tract infections, and at least one pharmaceutically or cosmetically acceptable carrier, diluent or excipient thereof, wherein said metabolite is a metabolite formed in vivo after ingestion of salicylic acid.

11. 11. The oral formulation of claim 9 or 10, wherein the oral formulation comprises about 1 to about 550 mg dry weight of meadowsweet extract (e.g., 10 to about 200 mg) and at least one pharmaceutically or cosmetically acceptable carrier, diluent, or excipient thereof.

12. 12. The oral formulation of claim 11, wherein the meadowsweet extract is an alcoholic or hydroalcoholic extract.

13. 12. The oral formulation of claim 11, wherein the meadowsweet extract is an extract of the flower tops of meadowsweet.

14. 11. The oral formulation of claim 9 or 10, wherein the oral formulation further comprises oenothein B.

15. 15. The oral formulation of claim 14, wherein the oral formulation comprises about 15 μg to about 65 mg of oenothein B.

16. 15. The oral formulation of claim 14, wherein the oral formulation comprises an extract of the genus Onagraceae, in particular Onagraceae.

17. 17. Oral formulation according to claim 16, comprising 10 to about 1000 mg dry weight, in particular about 10 to 1000 mg, of the extract of Ilex orbicularis, in particular 15 to 500 mg.

18. The oral formulation according to claim 9 or 10, further comprising at least one ingredient selected from D-mannose and vitamin D3.

19. 11. Use of the oral formulation according to claim 9 or 10 for the preparation of an oral pharmaceutical composition for the prevention, inhibition and / or treatment of urinary tract infections.

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