WO2018116238A1

WO2018116238A1 - Composition for use in the treatment of disorders of the urogenital apparatus

Info

Publication number: WO2018116238A1
Application number: PCT/IB2017/058284
Authority: WO
Inventors: Umberto DI MAIO; Antonino BAGNULO
Original assignee: Neilos SRL
Current assignee: Neilos SRL
Priority date: 2016-12-22
Filing date: 2017-12-21
Publication date: 2018-06-28
Anticipated expiration: 2019-06-22
Also published as: IT201600130012A1; RU2019122801A3; CN110300616A; EP3558459A1; RU2019122801A

Abstract

The present invention relates to a composition comprising a mixture of at least three plant extracts and at least one monosaccharide for use in the therapeutic or preventive treatment of the symptoms of pathologies and/or disorders caused by or associated with infections of the urogenital apparatus, preferably of the bladder and/or urethra, and a pharmaceutical composition or food supplement or medical device comprising said mixture.

Description

DESCRIPTION of an invention having the title:

"Composition for use in the treatment of disorders of the urogenital apparatus"

The present invention relates to a composition that comprises a mixture, wherein said mixture comprises, or, alternatively, consists of at least the following components:

a) an extract of at least one plant of the genus Vaccinium;

b) mannose;

c) an extract of at least one plant of the genus Arctostaphylos;

d) an extract of a plant belonging to the genus O/ea, preferably Olea europaea L;

wherein said composition is for use in the therapeutic or preventive treatment of the symptoms of pathologies and/or disorders caused by or associated with infections of the urogenital apparatus, preferably of the bladder and/or urethra, and a pharmaceutical composition or food supplement or medical device comprising the mixture of said components a), b), c) and d).

Urinary tract infections (UTI) are very widespread and affect a large portion of the world population.

Every year, 13 million women in the United States develop at least one urinary tract infection, and 150 million people worldwide are affected by this type of pathology. The costs associated with urinary tract infections amount to around 3.5 billion dollars a year in the United States alone.

Urinary tract infections occur when the concentration of bacteria in urine exceeds a threshold value of 10⁵ bacteria/mL. The main types of urinary tract infections are: cystitis (a lower urinary tract infection, characterised by the presence of bacteria in urine), urethral syndrome and pyelonephritis (an upper urinary tract infection, with infection of the kidneys).

The urinary tract is made up of the ureters, bladder and urethra. The ureters are two ducts that place the kidney, the organ that forms urine by filtering blood, in communication with the bladder, the organ that collects and functions as a urine "reservoir". Urine is expelled to the outside via another small duct, the urethra, through the act of urination.

Cystitis is a bladder inflammation caused in most cases by a bacterial infection of the urinary tract (bladder and urethra).

The main etiological agent of cystitis is the bacterium Escherichia coli. In order to be able to colonise the bladder, this pathogen produces various virulence factors, including adhesins, siderophores and toxins. Fimbriae are the main organelles of the bacterium involved in adhesion to the epithelium. The main fimbriae used by the bacterium are known as "type 1 pili" and are expressed in 90% of £ coli strains isolated in the bladder. The distal part of these organelles is responsible for adhesion and consists of FimH adhesin, which has the ability to recognise and bind mannose. This adhesin imparts to the bacterium the ability to adhere to various glycoproteins and non-glycosylated proteins: very probably, the proteins most used by the bacterium for the adhesion process are integrins, via the a3 and β1 subunits.

Cystitis is indicated as "acute" when the symptomatic inflammatory episode manifests itself, and as "recurrent" when the inflammatory episode recurs repeatedly in a given period of time (3 episodes in the last 12 months or 2 episodes in the last 6 months) (Guida uso farmaci, AIFA, Agenzia Italians del Farmaco, 2008).

Acute cystitis is a pathology caused by an infection of the lower urinary tract, which may display signs and symptoms such as dysuria, i.e. painful urination, an increased frequency of urination, urinary urgency, haematuria and suprapubic pain. Pyuria (presence of pus in the urine) and bacteriuria are quite frequently found in this pathology.

Like all urinary tract infections, acute cystitis can be classified as uncomplicated, if found in patients with a urinary tract that is normal from both a structural and functional viewpoint, or as complicated if there are anatomic or functional abnormalities in the urinary tract or if a patient has pathologies such as diabetes mellitus or AIDS, which may predispose him or her to urinary tract infections.

Uncomplicated acute cystitis is observed mainly in women.

Recurrent cystitis is common in young women, even if they generally have a urinary tract that is normal from an anatomical and functional viewpoint.

Recurrent cystitis is one of the main infectious pathologies in premenopausal women, has a high morbidity and every year it is associated with enormous health costs. The incidence in sexually active premenopausal women is 0.5-0.7 infections/person/year.

The percentage of women who will be subject to cystitis at least once in their lifetimes ranges from 20 to 40%.

The likelihood of occurrence of a urinary tract infection increases during winter months.

With regard to pathogenesis, the main etiological agents are the following:

Escherichia coli (responsible for 70-95% of the cases of cystitis);

Staphylococcus saprophyticus (responsible for 5-20% of the cases);

occasionally other pathogens such as Proteus mirabilis, Klebsiella spp. or Enterococcus spp. It is believed that most cases of recurrent cystitis are associated with the "ascending" path of the infection. The first step consists in the colonisation of periurethral tissue by uropathogenic bacteria. This is followed by colonisation of the urethra and bladder. Considering the bacterial origin of this pathology, the main line of therapy consists in the use of antibiotics in the case of non-recurrent acute infections.

The therapeutic approaches used acute recurrent cystitis are the following:

Changes in lifestyle;

Chronic therapy with low doses of antibacterials, such as co-trimoxazole (Trimethoprim + sulfamethoxazole) and fluoroquinolones.

Other antibacterials approved for use in uncomplicated cystitis of bacterial origin are nitrofurantoin, β- lactams and fosfomycin. Antibiotic treatment is effective for prophylaxis, as has been demonstrated by various studies, but it has the drawback of completely losing its effectiveness once the treatment is interrupted. Furthermore, there are numerous side effects associated with long-term therapies with antibiotics, including vaginal itchiness, skin rash and/or nausea, as well as a possibility of individual hypersensitivity towards the specific active principles;

Intermittent antibiotic therapy, with high doses of antibacterials. In this case as well, numerous side effects are encountered;

Products based on cranberry (Vaccinium macrocarpon). Cranberry extract is one of the most widely used treatments of an herbal type. Cranberry contains proanthocyanidins, in particular A-type proanthocyanidins, which are capable of inhibiting the adhesion of Escherichia coli to the wall of urothelial cells. A meta-analysis has demonstrated the effectiveness of using cranberry in the treatment of cystitis (A. B. Howell, H. Botto, C. Combescure, A.-B. Blanc-Potard, L. Gausa, T. Matsumoto, P. Tenke, A. Sotto, and J. -P. Lavigne, "Dosage effect on uropathogenic Escherichia coli anti-adhesion activity in urine following consumption of cranberry powder standardized for proanthocyanidin content: a multicentric randomized double blind study' BMC Infect. Dis., vol. 10, p. 94, 2010). Ten studies were analysed, with a total of 1494 participating patients. The relative risk of cystitis developing in patients who made use of cranberry compared to those that did not was 0.62 (95% CI, 0.49- 0.80).

The use of mannose for acute urinary tract infections has recently been proposed (Domenici, L. et al. Eur. Rev. Med. Pharmacol. Sci. 2016, 20, 2920), however, this study did not reveal any positive effects of the treatment with mannose as far as symptoms like back pain and pathological conditions like haematuria are concerned.

Similar results were reported by Vicariotto, F. {J. Clin. Gasteroenterol, 2014, Nov/Dec, 48 (1), S96-S101) in relation to the association of mannose, cranberry extract and two probiotics.

Chronic cystitis is characterised by the same symptoms as the acute form, the difference being that the latter are milder and extended over time. Since there is a subtle difference between recurrent acute cystitis and chronic cystitis, the therapeutic approaches used in this case are practically the same as those used for the acute, recurrent and relapsing forms of cystitis.

Treatment with active ingredients capable of restoring the glycosaminoglycan layer of the urothelium has been proposed as therapy for chronic cystitis, given that damage to the GAGs of the urothelium which does not heal may lead to chronic damage of the bladder epithelium, neurogenic inflammation, peripheral neuropathy and central sensitisation (Cicione, A. et al. Int. J. Urol' 2014, 21 , 763-768); however, the experimental results have not been judged to be wholly convincing.

There is thus a felt need to provide a treatment for disorders of the urogenital apparatus, in particular cystitis, which is effective both in treating and preventing them, easy to use and basically free of the side effects of the antibiotic-based therapies presently used.

In order to satisfy this need, the present invention provides the composition for use as disclosed in the independent claim set forth below.

The present invention relates to a composition which comprises a mixture, wherein said mixture comprises, or, alternatively, consists of at least the following components:

a) an extract of at least one plant of the genus Vaccinium;

b) Mannose;

c) an extract of at least one plant of the genus Arctostaphylos; and

d) an extract of a plant belonging to the genus O/ea, preferably Olea europaea L.

wherein said composition is for use in the therapeutic or preventive treatment of the symptoms of pathologies and/or disorders caused by or associated with infections of the urogenital apparatus, preferably of the bladder and/or urethra.

The present invention further relates to a pharmaceutical composition, a food supplement or a medical device that comprises at least one inert ingredient suitable for dietary or pharmaceutical use and a mixture, wherein said mixture comprises or, alternatively, consists of at least the following components: a) an extract of at least one plant of the genus Vaccinium;

b) Mannose

c) an extract of at least one plant of the genus Arctostaphylos; and

d) an extract of a plant belonging to the genus O/ea, preferably Olea europaea L. Preferred embodiments will be illustrated below, with no intention of limiting their scope and content in any way.

Unless otherwise specified, the content of an ingredient in a composition relates to the percentage by weight of that ingredient relative to the total weight of the composition.

Unless otherwise specified, the indication that a composition "comprises" one or more components means that other components can be present in addition to the one or ones specifically named and the indication that a composition "consists" of given components means that the presence of other components is ruled out.

For example, the composition for use as indicated above may consist of a), b), c) and d) or may comprise other ingredients in addition to a), b), c) and d), which may be other active ingredients, bacteria belonging to genera other than Lactobacillus and/or to species other than Lactobacillus acidophilus or excipients such as the ones commonly used in the pharmaceutical and food industries and for food supplements.

The inventors have found that the association between the specific plant extracts and mannose as disclosed in the appended independent claim have a synergistic action that makes it possible to obtain fast, lasting results in the treatment and prevention of diseases and disorders of the urogenital apparatus, such as cystitis (acute, also recurrent, or chronic), urethral syndrome and pyelonephritis.

The present invention comprises both the administration of the ingredients specified above in the same composition, i.e. simultaneously, and their administration in sequence, i.e. within a given period of time: by way of non-limiting example, within a few minutes to within 3-6 hours, in separate compositions.

The composition for use according to the present invention can be administered orally or topically, for example intravaginally, preferably orally.

The present invention allows to obtain, simultaneously:

- anti-adhesive action against uropathogenic strains of £ coli;

- antibacterial action;

- probiotic and prebiotic action;

- anti-inflammatory action.

Extract of Vaccinium macrocarpon Alton (cranberry), thanks to its content of A-type proanthocyanidins, is capable of inhibiting the adhesion of uropathogenic strains of Escherichia coli by modifying the conformation of PapG adhesins, expressed on the P-fimbriae, and impeding interaction between the fimbriae and the galactosyl-galactose residues present on the surface of the bladder epithelium. By impeding adhesion, it reduces the proliferation of the bacterium, since the latter needs to adhere to a surface in order to be able to proliferate. D-mannose has the ability to interact with the FimH adhesins present on the terminal part of type 1 fimbriae, expressed by some uropathogenic strains of Escherichia coli. As in the case of A-type proanthocyanidins, inhibiting adhesion of the bacterium to the urothelium contributes to reducing its proliferation. A-type proanthocyanidins and D-mannose can show a synergistic action, since they bind different adhesins (PapG and FimH, respectively), present on different fimbriae, and can thus inhibit the adhesion of different strains of Escherichia coli.

It has been found that the extract of Arctostaphylos uva-ursi (L.) Spreng. (bearberry), thanks to its content of arbutin and methylarbutin, is capable of inhibiting bacterial growth. The local antibacterial action of these two secondary bearberry metabolites derives from the fact that these two compounds are mainly excreted in urine and thus have the possibility of preventing bacterial growth in the bladder. Extract of Olea europaea L, thanks to the presence of oleuropein and its metabolite hydroxytyrosol, has the ability to contribute further to the antibacterial activity of the formulation, mainly in the intestine.

D-mannose is a monosaccharide that can be obtained from various natural and synthetic sources. Within the scope of the present invention, unless other specified, the term "mannose" indicates, indistinctly, D- mannose, L-mannose or the racemic or scalemic mixture of the two isomers. The composition of the present invention preferably contains D-mannose.

Since the main adhesin of E. coli recognises mannose, the use of this monosaccharide seems to impede the adhesion of the bacterium to the bladder endothelium. A fundamental aspect is given by evidence that the A-type cranberry proanthocyanidins, as identified below, inhibit the adhesion of Escherichia coli by blocking class II P-fimbriae, whose terminal part consists of PapG adhesin, capable of recognising galactosyl-(a1-4')-galactose residues: their mechanism is thus different from that of D-mannose and, without wishing to be limited by this theory, the two active ingredients could act in synergy at least for this reason.

Another important aspect of the action of mannose is its inhibition of the adhesion of several Escherichia coli strains to natural killer (NK) cells. The pathogens use the FimH adhesin also to bind to NK cells and cause their death by haemolysin-A. By blocking the adhesion of E. coli to NK cells, it can be ensured that the latter will be free to contribute to the pathogen's removal.

Since D-mannose is metabolised in scant quantities and is prevalently excreted through urine, it does not interfere with glycaemia. This makes it useful in the treatment of urinary tract infections also in diabetic subjects. The American cranberry (Vaccinium macrocarpon) is a small evergreen shrub belonging to the family of the Ericaceae, which grows in boreal climate zones, in particular in North America and in some northern regions of Europe and Asia, and produces red fruits similar to berries, with a dense pulp and typically tart flavour.

In the past, among the Indians of North America, the cranberry was considered a sacred fruit and was used both as a food and as a treatment for kidney stones and various urinary tract problems.

Today the American cranberry is used worldwide for its countless beneficial properties, due fundamentally to the high content of proanthocyanidins (PACs), in particular A-type proanthocyanidins.

American cranberry extracts are traditionally used above all in the prevention and treatment of urinary tract disorders, but, in more recent times, they have been applied as prevention against the adhesion of

Helicobacter pylori, a bacterium that is often a cause of stomach and duodenal ulcers, to the walls of the stomach, or the adhesion of bacteria which populate the oral cavity and are responsible for the formation of plaque or the adhesion of potentially pathogenic bacteria such as E. Coli at an intestinal level. Other pharmacological activities of cranberry extract are antioxidant and anti-inflammatory activities. All of these pharmacological activities are associated with the presence of proanthocyanidins.

Proanthocyanidins are condensed tannins: oligomers and polymers of flavan-3-ol monomers, such as catechin and epicatechin. Two types of proanthocyanidins exist: A-type proanthocyanidins, which derive from the formation of two bonds between monomers, and B-type proanthocyanidins, which derive from the formation of a single bond between monomers.

The differences lie not only in their molecular structure, but also in their biological activity. In fact, A-type proanthocyanidins are capable of inhibiting the adhesion of Escherichia coli, whereas B-type ones do not possess this activity. The last difference consists in the different concentration of proanthocyanidins in different plant species. In fact, whilst the majority of plants, such as Vitis vinifera, Camellia sinensis and Theobroma cacao, contain mainly B-type proanthocyanidins, the cranberry (Vaccinium macrocarpon) contains almost exclusively A-type proanthocyanidins, which are capable of countering urinary tract infections.

The mechanism of action of A-type proanthocyanidins consists in impeding the adhesion of Escherichia coli ^' to the bladder epithelium. In this specific case, A-type proanthocyanidins mainly inhibit the adhesion of uropathogenic strains of E. coli endowed with P fimbriae (which express PapG adhesins, capable of binding galactosyl-galactose residues).

Furthermore, it has been hypothesised that some chemical compounds present in cranberry extract are capable of inhibiting the gene expression of the proteins that form P-fimbriae. In a preferred embodiment, in the composition for use according to the present invention, extract (a) is from the fruits of at least one plant of the genus Vaccinium and subgenus Oxycoccus, such as Vaccinium macrocarpon or Vaccinium oxycoccus, or subgenus Vaccinium, such as Vaccinium arboreum, Vaccinium crassifolium, Vaccinium boreale or Vaccinium myrtillus or mixtures thereof, preferably wherein extract (a) is from the fruits of Vaccinium macrocarpon.

The bearberry is a plant native to North America, belonging to the family Ericaceae.

Bearberry (Arctostaphylos uva-ursi (L.) Spreng.) leaf extract has traditionally been used for the treatment of urinary tract infections (Schindler, G. et al. J. Clin. Pharmacol. 2002, 42(8), 920-927).

The main chemical compounds believed to be responsible for the pharmacological action of the extract of this plant, in particular of the leaves, are the phenolic glycosides arbutin and methyl arbutin. After oral administration, the arbutin is hydrolysed in the intestine into glucose and hydroquinone. Hydroquinone is absorbed and subsequently conjugated with glucuronic acid in the liver. The metabolite conjugated with glucuronic acid is excreted in urine. In infections of the urogenital apparatus, there is an increase in the urine pH, which favours the proliferation of £ coli, otherwise hindered at acidic pHs; however, with the increase in urine pH, the metabolite conjugated with glucuronic acid is hydrolysed and releases hydroquinone, which is able to perform antibacterial activity.

In a preferred embodiment, in the composition for use according to the present invention, extract (c) is from a part, preferably leaves, of at least one plant of the species Arctostaphylos (L.) Spreng.

In a preferred embodiment, the composition according to the invention is for use in the treatment of a pathology or disorder of the urogenital apparatus caused by, or correlated with, at least one pathogenic microorganism belonging to one of the genera Listeria, Shigella, Salmonella, Escherichia, Bacillus, Staphylococcus and Pseudomonas; more preferably, said at least one pathogenic microorganism belongs to one of the species Staphylococcus aureus, Salmonella enteritidis, Escherichia coli, Shigella sonnei, Pseudomonas fluorescens, Listeria monocytogenes and Bacillus subtilis. Even more preferably, said at least one pathogenic microorganism belongs to one of the species Salmonella enteritidis and Listeria monocytogenes.

In a preferred embodiment, the composition is for use in the treatment of a pathology or disorder of the urogenital apparatus which is acute, recurrent or chronic cystitis.

Olea europaea (olive) is known in traditional medicine for numerous pharmacological activities. The phenolic compounds to which antimicrobial activity is mainly attributed have shown high bioavailability. Oleuropein, belonging to the family of the secoiridoids, is the main polyphenol present in olive leaves, where it is present in a free form and in a form bonded to glycoside. Oleuropein has shown strong antibacterial activity against Gram-positive and Gram-negative bacteria. Different mechanisms whereby oleuropein might express its antimicrobial action have been proposed, although the actual mechanism is not yet known. It has been hypothesised that it might cause damage to the bacterial membrane and/or cell peptidoglycans. Or else oleuropein might hinder the formation of some amino acids necessary for the growth of specific microorganisms. One study has demonstrated, moreover, that oleuropein also acts by stimulating phagocytosis, and thus the endogenous immune response against the pathogens (Omar, S.H. Scientia Pharmaceutica 2010, 78(2), pp. 133-154). Oleuropein and other phenolic compounds present in Olea europaea (such as vanillic acid and p-coumaric acid) partially inhibit the growth of some bacteria such as E. coli and K. pneumoniae at a concentration of 0.3 mg/mL. Furthermore, they completely inhibit the growth of E. coli, K. pneumoniae and B. cereus at a concentration of 0.4 mg/mL.

Oleuropein is the ester of hydroxytirosol, which represents its main metabolite. The absorption of hydroxytirosol, tirosol and oleuropein aglycone is about 55-60% in humans. Low concentrations of free hydroxytirosol and oleuropein are found in urine.

A qualitative and quantitative determination of oleuropein and hydroxytirosol was carried out by liquid chromatography (electrospray ionization tandem mass spectrometry), using plasma and urine samples taken from rats following administration of a single oral dose (100 mg/kg) of oleuropein.

About 100% of the compounds were found in plasma and 60% in urine. A maximum peak of oleuropein was identified 2h after administration (200 ng/mL), and a small portion of hydroxytirosol. In urine the compounds were mainly found as glucuronides (Del Boccio, P. et al. J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 2003, 785 (1) 47-56).

In another study (Pereira, A.P. Molecules 2007, 12(5), 1153-1162), the content of 7 phenolic compounds present in the aqueous leaf extract of Olea europaea was calculated by HPLC-DAD. The content of oleuropein in this extract was 73% of the total compounds identified. The extract was subjected to an assessment of antimicrobial activity against several bacteria, including Escherichia coli and Staphylococcus aureus, known to be a cause of intestinal infections in humans. The extract inhibited the growth of all tested bacteria in a concentration-dependent manner. The antimicrobial capacity was the following (in descending order): S. cereus - C. albicans > E. coli > S. aureus > C. neoformans - K. pneumoniae - P. aeruginosa > B. subtilis.

Furthermore, Olea europea leaves might be useful for combating opportunistic infections promoted by extended use of antibiotics. Based on the data obtained from the study, it was concluded that the use of Olea europea extracts in nutraceutics could reduce the risk of bacterial infections, particularly in the intestinal and respiratory tracts.

Preferably, in the composition of the present invention, the extract (d) is from Olea europaea leaves.

In a preferred embodiment, the composition for use according to the present invention comprises, in addition to a), b), c) and d), at least one among:

e) at least one prebiotic dietary fibre, preferably inulin and/or fructo-oligosaccharides (FOS);

f) at least one probiotic bacterial strain, preferably Lactobacillus acidophilus, or mixtures thereof.

Bacterial urinary tract infections can be caused by various uropathogenic bacterial strains that have the ability to colonise the vagina and rise along the urinary tract.

Probiotics are defined by the World Health Organization and the FAO as "live microorganisms which, when administered in adequate amounts, confer a health benefit on the host", as stated, by way of non- limiting example, in the "Report of a Joint FAO/WHO Expert Consultation on Evaluation of Health and Nutritional Properties of Probiotics in Food Including Powder Milk with Live Lactic Acid Bacteria, Health and Nutritional Properties of Probiotics in Food including Powder Milk with Live Lactic Acid Bacteria (PDF)", in Food and Agriculture Organization of the United Nations, World Health Organization, October 2001.

Probiotics of the genus Lactobacillus have been studied for years as possible prophylaxis of urinary tract infections. In fact, various probiotic bacterial strains have been found among vaginal bacterial flora and it is believed that they play an important role in preventing colonisation of the epithelium by uropathogenic bacterial strains.

There are various mechanisms whereby probiotics are able to prevent urinary tract infections:

1. Replenishment of normal intestinal and vaginal bacterial flora following antibiotic therapy for the treatment of cystitis;

2. Inhibition of the proliferation of uropathogenic bacteria in the intestine;

3. Reduction in the pH of the vaginal epithelium to values below 4.5, by producing lactic acid;

4. Production of substances with bactericidal action, such as hydrogen peroxide.

The tyndallization process consists in subjecting bacteria to heat treatment cycles which bring about their inactivation. In one study, it was demonstrated that tyndallized bacteria are capable of forming a barrier against intestinal pathogenic bacteria, albeit in a less efficient manner than viable probiotics. In a preferred embodiment, the composition according to the invention comprises probiotic bacteria in tyndallized form.

A prebiotic is a non-digestible dietary fibre that selectively stimulates the proliferation of some species of bacteria which can have a positive effect on the health of individuals who include them in their diet.

Fructo-oligosaccharides (FOS), sometimes also called oligofructose or oligofructans, are short-chain fructans (oligosaccharides) present in various plant species, where they perform a role as an energy reserve. Structurally considered oligo- and polysaccharides of fructose, they are joined by β (1-2) glycosidic bonds, at the end of which there is a unit of a-D-Glucose: GF_n (where G=glucose, F=fructose, n=number of fructose molecules).

Molecules having a degree of polymerisation comprised from 3 to 10 are considered fructo- oligosaccharides and those with a degree of polymerisation greater than 10 are considered fructans. Inulin, one of the most used prebiotics, is a polymer of β-D-fructose, in which the monomers are joined by β-1 ,2- glycosidic bonds. After oral administration, inulin reaches the intestine intact: this enables it to be hydrolysed by the bacteria present in the intestine, in particular bifidobacteria and lactobacilli. The prebiotic activity of inulin, fructans, fructo-oligosaccharides and galacto-oligosaccharides has been confirmed in numerous studies, both in vitro and in vivo.

It has been found that the selective proliferation of strains of the genera Lactobacillus and Bifidobacterium contribute to reducing the proliferation of pathogenic bacteria, including various E. coli strains that can lead to the occurrence of urinary tract infections.

Lactobacillus acidophilus has the ability to colonise the intestine and inhibit the growth of uropathogenic bacteria by impeding their adhesion to the intestinal epithelium and producing substances with a bactericidal action, such as hydrogen peroxide.

In a preferred embodiment, the composition for use according to the present invention contains probiotic bacteria belonging to at least one strain of the species Lactobacillus acidophilus.

The tyndallized bacterium, too, still has the ability to compete with uropathogenic bacteria with respect to adhesion to the intestinal membrane even though, not being viable, it cannot produce substances with bactericidal action. Thanks to their ability to selectively favour the proliferation of bacterial strains of the genera Lactobacillus and Bifidobacterium, inulin and fructo-oligosaccharides can further contribute to reducing the proliferation of uropathogenic bacterial strains in the intestine. The inhibition of the proliferation of these bacteria, even only in the intestine, can contribute to reducing the occurrence of urinary tract infections caused by colonisation of the vaginal epithelium and subsequent colonisation of the lower urinary tract by uropathogenic intestinal bacteria.

As previously described, in the present invention the synergistic action occurs between Vaccinium macrocarpon Alton (cranberry), D-mannose, Arctostaphylos uva-ursi (L.) Spreng., Olea europaea L, and, if present, inulin (or FOS) and Lactobacillus acidophilus.

It has been found that an optimal synergy is obtained when Vaccinium macrocarpon Alton (cranberry) extract is present in an amount comprised between 50 mg and 600 mg, D-mannose is present in an amount comprised between 50 mg and 3000 mg, Arctostaphylos uva-ursi (L.) Spreng. extract is present in an amount comprised between 10 mg and 400 mg, Olea europaea L. extract is present in an amount comprised between 50 mg and 300 mg, inulin, if present, and/or fructo-oligosaccharides, if present, are in an amount comprised between 100 mg and 3000 mg and Lactobacillus acidophilus is present in an amount comprised between 1 x 10⁷ and 1 x 10¹² CFU (colony forming units).

In a preferred embodiment, in the composition for use according to the present invention, each component, independently of the others, is present as follows: extract a) is present in an amount comprised from 100 mg to 400 mg, preferably 120 to 250 mg, mannose is present in an amount comprised from 400 to 2000 mg, preferably 450 to 550 mg, extract c) is present in an amount comprised from 80 to 250 mg, extract d) is present in an amount comprised from 80 to 150 mg, and, when present, the prebiotic fibre e) is present in an amount comprised from 100 to 3000 mg, preferably 1500 to 2000 mg, and/or the probiotic bacteria f) are present in an amount comprised from 1 to 10 x 10⁹ CFU, preferably 3.5 to 4.5 x 10^s CFU.

The composition for use according to the present invention can be solid, liquid or semi-solid, for example as a suspension or gel, and it can be in any form known to the person skilled in the art of food, pharmaceutical or nutraceutical formulations, such as a powder, tablet, capsule, gel, syrup, suspension, solution etc.

Non-limiting examples of the formulation are the following:

Formulation I

Active ingredient Dose

Inulin 2,000 mg

D-mannose 500 mg

Vaccinium macrocarpon Alton (cranberry) 200 mg Arctostaphylos uva-Ursi (L.) Spreng. 200 mg

Olea europaea L. 100 mg

Lactobacillus acidophilus 1 x 10¹⁰ (10 BLN) /CFU

Formulation II

The present invention relates to a pharmaceutical composition, a food supplement or a medical device which comprises at least one inert ingredient suitable for food or pharmaceutical use and a mixture, wherein said mixture comprises or, alternatively, consists of at least the following components:

a) an extract of at least one plant of the genus Vaccinium;

b) Mannose;

c) an extract of at least one plant of the genus Arctostaphylos; and

d) an extract of a plant belonging al genus Olea, preferably Olea europaea L.

In the context of the present invention, the term "medical device" is used with the meaning as per Italian Legislative Decree no. 46 of 24 February 1997, i.e. it indicates a substance or another product, whether used alone or in combination, intended by the manufacturer to be used for human beings for the purpose of diagnosis, prevention, monitoring, treatment or alleviation of disease, and which does not achieve its principal intended action in or on the human body for which it is intended by pharmacological, immunological or metabolic means, but which may be assisted in its function by such means.

The pharmaceutical composition, food supplement or medical device of the present invention can be solid, liquid or semi-solid, for example as a suspension or gel, and it can be in any form known to the person skilled in the art of food, pharmaceutical or nutraceutical formulations, by way of non-limiting example, in the form of a capsule, tablet or powder, at least partially orally dissolvable or water-soluble, granules, pellets or microparticles, optionally contained in a sachet or in a capsule or tablet (mini-tablet), liquid or semi-solid preparation, gel, suspension, solution, two-phase liquid system and equivalent forms. "Inert ingredient" means any substance, or combination of substances, auxiliary to the production of a pharmaceutical, food or nutraceutical form found in the finished product that is not the active ingredient, even if it can modify its stability, release or other characteristics.

Non-limiting examples of such ingredients, as known to the person skilled in the art of pharmaceutical, food or nutraceutical formulations, are excipients such as diluents, absorbents, lubricants, colourants, surfactants, antioxidants, sweeteners, binders, disintegrating agents and the like.

In a preferred embodiment, in the pharmaceutical composition, food supplement or medical device of the present invention, extract a) is a Vaccinium macrocarpon extract and/or extract c) is an Arctostaphylos uva-ursi (L.) Spreng extract, and/or extract d) is an Olea europaea L. extract.

In a preferred embodiment, the pharmaceutical composition, food supplement or medical device of the present invention further comprises at least one between:

In a preferred embodiment, in the pharmaceutical composition, food supplement or medical device of the present invention, wherein extract a) is present in an amount comprised from 50 mg to 600 mg, preferably 120 to 250 mg, mannose is present in an amount comprised from 50 to 3000 mg, preferably 400 to 2000 mg, and/or extract c) is present in an amount comprised from 10 to 400 mg, preferably 80 to 250 mg and extract d) is present in an amount comprised from 50 to 300 mg, preferably 80 to 150 mg, and, when present, the prebiotic fibre e) is present in an amount comprised from 100 to 3000 mg, preferably 1500 to 2000 mg, and/or the probiotic bacteria f) are present in an amount comprised from 1 x 10⁷ to 1 x 10¹² CFU, more preferably 1 x 10⁹ to 1 x 10¹⁰ CFU.

EXPERIMENTAL PART

Studies conducted on the composition of the present invention for the treatment of acute and recurrent cystitis.

The effect of a composition of the present invention and of the individual constituents was assessed (in order to observe the synergistic effect between the individual components) (i) on the inhibition of bacterial adhesion, (ii) antibacterial action, and (iii) anti-inflammatory action. INHIBITION OF BACTERIAL ADHESION

The activity of inhibiting bacterial adhesion can be assessed on strains of Escherichia coli endowed with P-fimbriae that express the PapG adhesin and type 1 fimbriae that express the FimH adhesin, as previously described [A. B. Howell et al., "Dosage effect on uropathogenic Escherichia coli anti-adhesion activity in urine following consumption of cranberry powder standardized for proanthocyanidin content: a multicentric randomized double blind study.," BMC Infect. Dis., vol. 10, p. 94, 2010]. In order to enable microscopic observation of bacterial adhesion, the bacterial strain is genetically modified so as to express green fluorescent protein (GFP), with one of the experimental procedures described in the literature [J. Amalric, P. H. Mutin, G. Guerrero, A. Ponche, A. Sotto, and J. -P. Lavigne, "Phosphonate monolayers functionalized by silver thiolate species as antibacterial nanocoatings on titanium and stainless steel," J. Mater. Chem., vol. 19, no. 1 , p. 141 , 2009].

The bacteria are cultured in tryptic soy broth or agar gel containing colonisation factor in order to increase the expression of P-fimbriae [A. B. Howell et al., "Dosage effect on uropathogenic Escherichia coli anti- adhesion activity in urine following consumption of cranberry powder standardized for proanthocyanidin content: a multicentric randomized double blind study.," BMC Infect. Dis., vol. 10, p. 94, 2010].

In order to test the anti-adhesion activity of our formulation, the ability to suppress the agglutination of erythrocytes incubated with Escherichia coli endowed with P-fimbriae was measured [A. B. Howell, J. D. Reed, C. G. Krueger, R. Winterbottom, D. G. Cunningham, and M. Leahy, "A-type cranberry proanthocyanidins and uropathogenic bacterial anti-adhesion activity," Phytochemistry, vol. 66, no. 18 SPEC. ISS., pp. 2281-2291 , Sep. 2005]. The bacteria are suspended in a phosphate buffer solution at pH 7. The different formulations to be tested are solubilised in PBS. Dilutions of the starting solutions are prepared. 30 mL of each diluted solution are added to 10 mL of the bacterial suspension on a polystyrene plate with 24 wells. A 3% V/V solution of erythrocytes in PBS is prepared and 10 mL of this suspension is added to the suspension to be tested. The resulting suspension is incubated for 20 minutes in a rotary shaker at a temperature of 21 °C and examined under an optical microscope in order to assess the ability of the formulations to prevent agglutination.

The minimum concentration of the formulation at which the suppression of erythrocyte agglutination can be observed is recorded.

The wells containing the bacterial suspension in PBS, the suspension of erythrocytes in PBS, the suspension of bacteria with the solutions of the formulations to be tested and the suspension of erythrocytes with the formulations to be tested represent the negative controls. The well containing the suspension of bacteria and erythrocytes represents the positive control. Every experiment is carried out in triplicate.

ANTIBACTERIAL ACTIVITY

Antibacterial activity can be assessed with the agar diffusion method [M. Balouiri, M. Sadiki, and S. K. Ibnsouda, "Methods for in vitro evaluating antimicrobial activity: A review," J. Pharm. Anal., vol. 6, no. 2, pp. 71-79, 2016], [D. P. S. Leitao, A. C. M. Polizello, I. Y. Ito, and A. C. C. Spadaro, "Cranberry Juice," J. Med. Food, vol. 8, no. 1 , pp. 36-40, 2005]. Culture media consisting of brain-heart infusion agar or Mueller Hinton agar are used in the tests for screening the different bacterial strains. These agar media are superimposed on culture media seeded with the bacteria to be tested and the equivalence turbidity is brought to a McFarland index with a value of 0.5. Seventeen wells are obtained from each culture medium using a sterile stopper borer.

The formulations are solubilised in a saline phosphate buffer solution (PBS) to create solutions at different concentrations.

Using a sterile pipette, 50 μΙ_ of each solution are cautiously applied on the wells and allowed to diffuse for 2 hours.

The wells are subsequently incubated at a temperature of 37°C for a time of 24 h for most of the bacteria. For some bacterial strains it is necessary to use different conditions, for example: temperature of 37°C for 48 hours, in an atmosphere containing 5% CO2.

A 0.12% chlorhexidine solution is used as a positive control.

The antibacterial activity is tested in triplicate and, following incubation, the average diameter of the bacterial growth inhibition area is calculated.

ANTI-INFLAMMATORY ACTIVITY

Anti-inflammatory activity can be assessed by means of an immunoenzymatic assay of pro-inflammatory cytokines.

In the literature it is reported that interleukin 6 (IL-6), methylhistamine and histamine show a considerable increase in patients with interstitial cystitis [L. M. Lamale, S. K. Lutgendorf, M. B. Zimmerman, and K. J. Kreder, "lnterleukin-6, histamine, and methylhistamine as diagnostic markers for interstitial cystitis," Urology, vol. 68, no. 4, pp. 702-706, 2006], and could thus be considered markers that could be evaluated as an index of the degree of inflammation.

Aquino et al. report the following method of measuring cytokines. Use is made of 1x10^s cells, which are allowed to adhere to the plate and are then treated with the extract whose activity it is intended to measure for 2 hours, and stimulated with TNF-a for 14 hours. The release of IL-6 is measured by means of the ELISA test and the cytokine concentration is expressed as pg/ml/10⁶ cells and expressed as a percentage of the control in the absence of any stimulation or treatment [R. P. Aquino, A. Santoro, L. Prota, T. Mencherini, E. Esposito, M. V. Ursini, P. Picerno, S. Nori, F. Sansone, and P. Russo, "Composition and anti-inflammatory activity of extracts from three Paeonia species," Pharmacologyonline, vol. 1 , pp. 137— 147, 2014].

Histamine and methylhistamine can be assessed by means of radioimmunological assay kits and by normalising the result relative to urine creatinine levels [L. M. Lamale, S. K. Lutgendorf, M. B. Zimmerman, and K. J. Kreder, "lnterleukin-6, histamine, and methylhistamine as diagnostic markers for interstitial cystitis," Urology, vol. 68, no. 4, pp. 702-706, 2006].

A further marker that may be used is interleukin 1 β. Based on data in the literature, this cytokine is more expressed in patients with bacterial cystitis, whereas it remains unchanged in patients with interstitial cystitis; this enables one pathology to be distinguished from the other. Measurement of this cytokine is performed with a specific ELISA kit [S. M. Martins, D. J. Darlin, P. M. Lad, and P. E. Zimmern, "Interleukin- 1 B: a clinically relevant urinary marker," J. Urol., vol. 151 , no. 5, pp. 1198-1201 , 1994].

Tests and experimental results

The antimicrobial activity of the constituents is assessed by analysing the effect of each substance individually and in a formulation. An assessment is made of the antimicrobial activity against target strains of the following bacterial genera: Listeria, Salmonella, Staphylococcus and Pseudomonas.

ASSESSMENT OF ANTIMICROBIAL ACTIVITY

The antimicrobial activity of the substances was assessed by incubating various target strains (specified in paragraph 1.1) with the individual substances and with the two formulations (described in paragraph 1.2). An assessment was then made as to whether or not the target strain has the capacity to grow and duplicate in the presence of the aforesaid substances, as described in detail in paragraph 2.0.

1 MATERIALS AND METHODS

1.1 : target strains used to assess antimicrobial activity

1 ) Salmonella enteritidis

2) Listeria monocytogenes

1.2: preparation of substances

The individual substances Inulin, D-mannose, Vaccinium Macrocarpon Aiton (cranberry),

Arctostaphylos Uva-Ursi (L.) Spreng (bearberry), Olea Europaea L. and Lactobacillus acidophilus were dissolved in water to assess their solubility.

For some of these, such as cranberry, Uva-Ursi and Olea Europaea, water solubility problems were encountered and it was therefore decided to use dimethyl sulfoside (DMSO) as a solvent. The latter was used in a concentration of 20% to avoid problems of toxicity of the solvent itself against the target strains to be used in the experiment.

Data from the literature and our preliminary data indicate that cranberry is one of the substances that have shown antimicrobial activity against various bacterial species; it was thus chosen as the starting point to assess what was the maximum concentration that could be used based on its solubility.

The maximum concentration of cranberry that showed to be soluble in DMSO 20% is 10mg/ml. This concentration was therefore chosen as a reference for obtaining the concentrations of the other substances on the basis of the two formulations according to the invention (table 1), maintaining the proportions between the different components.

Formulation 1 contains all of the substances indicated, whereas formulation 2 contains only the substances marked with an asterisk (table 1).

The substances contained in the formulations ("dosage of formulations 1 and 2" column), the concentration of the solutions prepared for the experiment ("initial concentration" column) and the concentration of each substance obtained in the experimental samples ("final concentration" column) are all indicated in table 1.

substances in the starting solutions; Concentration of the substances in the final solutions.

* substances solubilised in 20% DMSO

2 EXPERIMENTAL PROTOCOL

The target bacterial strains were cultured for 20 hours in LB (Luria-Bertani) culture medium and the optical density thereof was subsequently measured at 600nm using a spectrophotometer.

The bacterial culture was then diluted in nutrient broth until obtaining a cell concentration of about 5*10⁵ cfu/ml (corresponding to ~ 0.15 OD). 180 μΙ aliquots of the cell suspension were added in each well of a 96-well plate in order to prepare the experimental samples for assessing the antimicrobial activity of the various substances and formulations.

20 μΙ of the individual substances (using the initial solutions indicated in Table 1) and formulations were added and the plate was incubated at 37°C for 24 hours. The next day the presence or absence of bacterial growth was assessed by measuring the absorbance at 600 nm by means of a plate reader. Appropriate controls of the experiment consisted in:

duplicate wells containing only the culture medium - nutrient broth - an indicator of the sterility of the culture medium used;

duplicate wells containing the target microbial cultures in the absence of the substances, which represent the positive control of the experiment;

duplicate wells containing the target microbial cultures in the presence of the 20% DMSO solvent alone, in order to confirm that the presence of the solvent did not interfere with microbial growth;

duplicate wells containing the substances alone (absence of bacteria). This control was necessary since some substances were coloured and thus their presence brought about an increase in optical density which in this case was not an indicator of microbial growth.

3 EXPERIMENTAL RESULTS

The experimental results obtained for each target bacterial strain are shown in the graphs in figures 1 and 2 obtained using the values shown in table 2, corresponding to the mean of the data obtained. OD

Target strain A B C D E F G H I

T = 0

Salmonella 0.1530 0.4358 0.3506 0.4465 0.4351 0.3226 0.4534 0.3055 0.1493 -

Listeria 0.1525 0.4916 0.4365 0.4333 0.2905 0.2735 0.4610 0.2514 - 0.1375

Table 2: mean optical density obtained for the various target strains and under the different experimental conditions at the start (T = 0) and after 24 h

A: OD T 24 (control)

B: OD target strain + INULIN T 24

C: OD target strain + OLEA T 24

D: OD target strain + UVA URSI T 24

E: OD target strain CRANBERRY T 24

F: OD target strain + MANNOSE T 24

G: OD target strain + L acidophilus T 24

H: OD target strain + FORMULATION 1 T 24

I: OD target strain + FORMULATION 2 T 24

Each graph in figures 1-3 shows, in sequence, the optical density of the target strain that is obtained after 24 hours of incubation in the absence (control sample) and in the presence of the individual substances or the formulation, as indicated on the X-axis.

The horizontal lines in figures 1-3 represent the optical density of the strain at the start of the experiment (T = 0) and after 24 hours of incubation (T = 24).

Figure 1 : the graph shows the optical density of the cultures obtained after incubating the cells of the genus Salmonella for 24 hours in the presence of the substances indicated. The point T 0 indicates the starting optical density of the target strain, inoculated into all of the test tubes. The "control" sample is represented by the bacterial cells incubated in the culture medium in the absence of substances (-). The data are shown in the histograms and the horizontal lines represent the initial (T 0) and final (T 24) OD obtained under these experimental conditions. Figure 2: The graph shows the optical density of the cultures obtained after incubating the cells of the genus Listeria in the presence of the substances indicated. The point T 0 indicates the starting optical density of the target strain, inoculated into all of the test tubes. The "control" sample is represented by the bacterial cells incubated in the culture medium in the absence of substances (-). The data are shown in the histograms and the horizontal lines represent the initial (T 0) and final (T 24) OD obtained under these experimental conditions.

The results obtained are also shown in table 3 as a percentage of inhibition of microbial growth calculated using the following formula (Sivasankar et al. Microb Pathog 2017, 110, 66-72):

O ^'Ό strain in the culture medium - O D strain with the substance ,, ...

■ HJU

ΟΌ strain in the culture medium

Table 3: percentage of inhibition of bacterial growth

Antimicrobial activity estimated as % of inhibition using the following formula: [Control CD - Treated O.D/Control CD] * 100. The higher the value, the greater the inhibitory activity. The symbol - indicates the absence of inhibition.

4 CONCLUSIONS It was established that microbial activity would be defined as absent if less than 10%, low if comprised between 10% and 20%, moderate if comprised between 20% and 40%, and high if greater than 40%.

4.1 Analysis of the antimicrobial activity of the individual components

• Inulin: it showed a low inhibitory activity against Salmonella (13%) and Listeria (11%).

• Olea: it showed a low inhibitory activity against Listeria (12%).

• Bearberry: it showed a high inhibitory activity against Listeria (41%).

• Cranberry: it showed a moderate inhibitory activity against Salmonella (26%), and high inhibitory activity against Listeria (44%).

• L. acidophilus: it showed a moderate inhibitory activity against Salmonella (30%) and high inhibitory activity against Listeria (49%).

4.2 Analysis of the antimicrobial activity of the formulations

• Formulation 1 according to the present invention: it showed a high inhibitory activity against Salmonella (66%).

The values found, when compared to those obtained with the individual components, indicate that Formulation 1 showed synergistic activity against the bacteria belonging to the genus Salmonella (66%).

• Formulation 2 according to the present invention: it showed a high inhibitory activity against Listeria (60%). The results, when compared to those obtained with the individual components, indicate that Formulation 2 showed synergistic activity against Listeria (60%).

Claims

1. A composition which comprises a mixture, wherein said mixture comprises, or, alternatively, consists of at least the following components:

a) an extract of at least one plant of the genus Vaccinium;

b) mannose;

c) an extract of at least one plant of the genus Arctostaphylos; and

2. The composition for use according to claim 1 , wherein extract (a) is from the fruits of at least one plant of the genus Vaccinium and subgenus Oxycoccus, such as Vaccinium macrocarpon or Vaccinium oxycoccus, or subgenus Vaccinium, such as Vaccinium arboreum, Vaccinium crassifolium, Vaccinium boreale or Vaccinium myrtillus or mixtures thereof, preferably wherein extract (a) is from the fruits of Vaccinium macrocarpon.

3. The composition for use according to claim 1 or 2, wherein extract (c) is from a part, preferably leaves, of at least one plant of the species Arctostaphylos (L.) Spreng.

4. The composition for use according to any one of the preceding claims, which comprises, in addition to a), b), c) and d), at least one between:

e) at least one prebiotic dietary fibre, preferably inulin and/or fructo-oligosaccharides (FOS); and f) at least one probiotic bacterial strain, preferably Lactobacillus acidophilus, or mixtures thereof.

5. The composition for use according to any one of the preceding claims, wherein the pathology and/or disorder of the urogenital apparatus is acute, recurrent or chronic cystitis.

6. The composition for use according to any one of the preceding claims, wherein extract a) is present in an amount comprised from 50 mg to 600 mg, preferably 120 to 250 mg, mannose is present in an amount comprised from 50 to 3000 mg, preferably 400 to 2000 mg, and/or extract c) is present in an amount comprised from 10 to 400 mg, preferably 80 to 250 mg, and/or extract d) is present in an amount comprised from 50 to 300 mg, preferably 80 to 150 mg and, when present, the prebiotic fibre e) is present in an amount comprised from 100 to 3000 mg, preferably 1500 to 2000 mg, and/or the probiotic bacteria f) are present in an amount comprised from 1 x 10⁷ to 1 x 10¹² CFU, more preferably 1 x 10⁹ to 1 x 10¹⁰ CFU.

7. The composition for use according to any one of the preceding claims in the treatment of a pathology or disorder of the urogenital apparatus caused by, or correlated with, at least one pathogenic microorganism belonging to one of the genera Listeria, Shigella, Salmonell, Escherichia, Bacillus, Staphylococcus and Pseudomonas.

8. The composition for use according to claim 7, wherein said at least one pathogenic microorganism belongs to one of the species Staphylococcus aureus, Salmonella enteritidis, Escherichia coli, Shigella sonnei, Pseudomonas fluorescens, Listeria monocytogenes or Bacillus subtilis.

9. The composition for use according to claim 8, wherein said at least one pathogenic microorganism belongs to one of the species Salmonella enteritidis or Listeria monocytogenes.

10. A pharmaceutical composition, food supplement or medical device which comprises at least one inert ingredient suitable for food or pharmaceutical use and a mixture, wherein said mixture comprises, or, alternatively, consists of at least the following components:

a) an extract of at least one plant of the genus Vaccinium;

b) mannose;

c) an extract of at least one plant of the genus Arctostaphylos;

d) an extract of a plant belonging to the genus Olea, preferably Olea europaea L.

11. The pharmaceutical composition, food supplement or medical device according to claim 10, wherein a) is an extract of Vaccinium macrocarpon and/or c) is an extract of Arctostaphylos uva-ursi (L.) Spreng.

12. The pharmaceutical composition, food supplement or medical device according to either of claims 10 and 11 , further comprising at least one between:

e) at least one prebiotic dietary fibre, preferably inulin and/or fructo-oligosaccharides (FOS); (FOS), f) at least one probiotic bacterial strain, preferably Lactobacillus acidophilus, or mixtures thereof.

13. The pharmaceutical composition, food supplement or medical device according to any one of claims 10-12, wherein extract a) is present in an amount comprised from 50 mg to 600 mg, preferably 120 to 250 mg, mannose is present in an amount comprised from 50 to 3000 mg, preferably 400 to 2000 mg, and/or extract c) is present in an amount comprised from 10 to 400 mg, preferably 80 to 250 mg and/or extract d) is present in an amount comprised from 50 to 300 mg, preferably 80 to 150 mg and, when present, the prebiotic fibre e) is present in an amount comprised from 100 to 3000 mg, preferably 1500 to 2000 mg, and/or the probiotic bacteria f) are present in an amount comprised from 1 x 10⁷ to 1 x 10¹² CFU, more preferably 1 x 10⁹ to 1 x 10¹⁰ CFU.