WO2020079641A1

WO2020079641A1 - Compositions comprising essential oils and/or hydrolates from plants of italian origin and the use thereof for the treatment of gastrointestinal and genitourinary disorders

Info

Publication number: WO2020079641A1
Application number: PCT/IB2019/058869
Authority: WO
Inventors: Heide DE TOGNI; Maura DI VITO; Paola MATTARELLI; Francesca BUGLI
Original assignee: Pegaso S.R.L.; Alma Mater Studiorum - Universita' Di Bologna; Universita' Cattolica Del Sacro Cuore
Priority date: 2018-10-17
Filing date: 2019-10-17
Publication date: 2020-04-23
Also published as: IT201800009540A1

Abstract

The present invention regards a composition comprising a mixture (M) comprising or, alternatively, consisting of at least one essential oil and at least one hydrolate obtained from a plant or at least one part of a plant of Italian origin. Furthermore, the present invention regards the use of said composition in the treatment of symptoms or disorders caused by or related with alteration of the microbiota balance and/or of the immune system, in particular caused by or related with a disorder of the intestinal or genitourinary tract, both in diseased subjects and in healthy subjects.

Description

“Compositions comprising essential oils and/or hydrolates from plants of Italian origin and the use thereof for the treatment of gastrointestinal and genitourinary disorders”

The present invention refers to a composition comprising a mixture (M) comprising or, alternatively, consisting of at least one essential oil or at least one hydrolate obtained from a plant or at least one part of a plant of Italian origin, or of a mixture of two or more of said essential oils and/or hydrolates. Furthermore, the present invention regards said composition for use in a method for the treatment of symptoms or disorders caused by or related with an alteration of the microbiota balance and/or of the immune system, in particular caused by or related with a disorder of the gastrointestinal or genitourinary tract, both in diseased subjects and in healthy subjects.

In the context of the present invention, the expression "essential oils” (or etheric oils) is used to indicate the products obtained by extraction starting from aromatic plant material, i.e. rich in "essences". Essential oils are oily mixtures of different organic substances, which can be obtained by distillation or by squeezing from a single type of plant, whose characteristics such as taste and odour they preserve. Thus, essential oil is a selective phytochemical extract, in the sense that a particular phytochemical group is selectively removed from the plant (the extract isolates a minority component of the plant, from 0.01% to 2%).

In the context of the present invention, the expression "hydrolates” (or floral waters) is used to indicate secondary products obtained from steam distillation of parts of plants such as, for example, flowers, leaves and aerial parts of the plant. Hydrolates contain water-soluble plant ingredients. During the distillation process, the plant material is mixed with water and heated or directly exposed to steam. Essential oils in plants and other volatile substances rise with the steam. The steam is captured in the distillation device and cooled. The cooled condensate contains extracted water and essential oil, with the latter floating upwards, where it can be separated. The aqueous phase separated from oil is known as hydrolate or floral water.

In the context of the present invention, the expression "microbiota” o or human microbiota is used to indicate the set of symbiotic micro-organisms that live with the human organism without damaging it. The expression microbiota in general (or human microbiota) is used to indicate the set of gut microbiota (i.e. symbiotic micro-organisms of the intestinal tract), of gastric microbiota and other microbiota such as the microbiota of the mouth, throat, etc. The expression "gastrointestinal microbiota” is used in the present application to indicate the microbiota of the gastric tract and of the intestinal tract. The immune system is a complex integrated network of chemical and cellular mediators, of biological structures and processes structures having the function of defending the organism against any form of chemical attack, traumatic or infectious attack to the wholeness thereof. In order to operate correctly, an immune system must be capable of detecting the pathogenic agents and eliminating them from the organism. Furthermore, the immune system is capable of recognising and destroying abnormal cells deriving from the tissues of the guest. The skin, the cornea, the mucosa of the respiratory system, the gastrointestinal and genitourinary tract form a physical barrier which constitutes the first defensive line of the organism. Some of these barriers actively take part in the functions of the immune system.

The scientific community hypothesised that the alteration of the microbiota and/or of the immune system, in particular the alteration thereof extended over time, cause several diseases or symptoms or disorders in subjects suffering from such alterations, such as dysfunctions of the functionality of the intestinal or gastro intestinal tract, for example the irritable bowel syndrome (IBS), the chronic inflammatory bowel diseases (IBD) or infections or inflammations from pathogenic agents, for example candidosis or cystitis.

The irritable bowel syndrome (abbreviated as IBS) is a disorder of the bowel function characterised by abdominal pain related with changes of the small bowel transit (either constipation-wise or diarrhoeic-wise) and with signs of altered defecation and tympanites. The irritable bowel syndrome belongs to the group functional gastrointestinal disorders (FGD), a diagnostics category definable solely based on the presentation of symptoms and characterised by the absence of an evident pathogenetic substrate. Although various theories have been formulated, the pathogenesis of the IBS still remains unclear. Just like in the case of the chronic inflammatory bowel diseases (abbreviated as IBD), it is assumed that the irritable bowel syndrome (IBS) is related with: alteration of the intestine-central system axis, altered microbiota and persistent alteration of the immune system. Recent studies show that patients suffering from IBD have a microbiota different from that of healthy subjects with greater presence of some microbial species rather than others. In particular, Sokol et al (2016) proved that in patients suffering from IBD, some fungal species ( Ascomiceti , in particular Saccharomyces cerevisiae) reduce by percentage with respect to healthy subjects, while others increase ( Basidiomiceti , in particular Mallasizia sympodialis). Furthermore, in the sick subjects, the species of the Candida genus increase by percentage even though they do not cause infectious diseases. Trials conducted by the Applicant have proven that the faecal samples of patients suffering from IBS are characterised by fungal strains belonging to the Candida genus.

The therapies currently known for the irritable bowel syndrome (IBS) are: a food diet with restriction of the short-chain carbohydrates (Fodmap) scarcely absorbed in the small bowel, such as fructose, sorbitol and mannitol; administration of kaolin-based preparations such as diosmectite or preparations with low concentrations of polyethylene glycols/mineral salts, in particular in subjects with mainly constipated small bowel transit and with presence of tympanites; the use of anxiolytics such as benzodiazepines or antidepressants such as the SSRIs to reduce the state of anxiety of the subject; therapies aimed at controlling pain, for example the administration of spasmolytics; and lastly, the tympani syndrome can be reduced using scarcely absorbable antibiotics and probiotics that regulate the gut flora (or gut microbiota).

However, the aforementioned therapies are often ineffective and in most cases they cause adverse effects, in particular the administration of anxiolytics, SSRI, spasmolytics and antibiotics.

Chronic inflammatory bowel diseases (IBD) are characterised by the inflammation of the intestine which often causes recurrent abdominal cramps and diarrhoea. The two main IBDs are: Crohn's disease and ulcerative colitis.

The therapies currently known for chronic inflammatory bowel diseases (IBD) are: drugs, diets and stress management, surgery (for serious cases). Despite there being no cure for IBD, many drugs (drugs that reduce the intestinal inflammation caused by Crohn's disease and drugs that reduce the intestinal inflammation caused by ulcerative colitis) such as, for example, aminosalicylates, corticosteroids, immunomodulatory drugs, biological and antibiotic agents can help to reduce the inflammation and alleviate the symptoms of the IBD.

However, the aforementioned therapies are often ineffective and in most cases they cause adverse effects.

Candidosis, also referred to as candidiasis or moniliasis, is a fungal infection of the Candida genus, whose most common species is Candida albicans. Candidosis comprises the infections of the surface level, such as for example the thrush (oral or vaginal), vaginitises and intestinal candidosis. The surface infections of the skin and of the mucosa caused by Candida sp. cause local inflammations, unconformable sensations and systemic disorders on the gastrointestinal tract such as bloating, constipation, diarrhoea, halitosis, abdominal pain in many groups of population. The disease is clearly caused by the presence of opportunistic pathogenic agents of the Candida genus, but candidosis describes a number of different pathological syndromes that often differ in terms of the causes and diagnosis thereof.

In the clinical field, candidosis is commonly treated using antimycotics such as, for example clotrimazole, nistatin, topical fluconazole and ketoconazole. The administration of high doses of fluconazole (=/> 400 mg/day orally) was however related with teratogenesis. Furthermore, Candida sp. strains, in particular Candida albicans, the main cause of candidosis, can develop a resistance against antimycotic drugs, making the use thereof in the treatment ineffective. Lastly, antimycotics solely allow the treatment of vaginitises caused by yeasts and they are ineffective in vaginitises related with combined causes.

The main types of infections of the urinary tract are: cystitis (an infection of the lower urinary tract, bladder and urethra), urethral syndrome and pyelonephritis (infection of the upper urinary tract, with kidney infection). The agent that is most often involved in the various types of infections of the urinary tract is Escherichia coli, even though, less frequently, other bacteria, virus or fungi, such as for example Candida can be involved

The therapies currently known for treating infections of the urinary tract, in particular cystitis, are based on antibiotics. Alternatively, to prevent and limit the recurrent infections of the urinary tract consists in restoring the vaginal microbiota. Probiotics were used for restoring the natural environment of the vaginal saprophytic bacteria and thus making a bacterial, virus or pathogenic fungi infection more unlikely.

However, the aforementioned therapies are often ineffective at permanently solving the problem and relapse events occur. Furthermore, the use of antibiotics extended over time or at close intervals causes unwanted adverse effects.

The technical problem of providing a valid solution for the effective therapeutic and non-therapeutic treatment of symptoms or disorders related with an alteration of the microbiota balance and/or of the immune system, in particular of the gastro-intestinal or genitourinary tract, capable of overcoming the drawbacks of the prior art, in particular as regards the adverse effects, is yet to be resolved.

In particular, the technical problem of providing a valid solution for the therapeutic and non-therapeutic treatment, that is effective and without adverse effects of functional disorders, infection or gastro-intestinal inflammations, in particular of the irritable bowel syndrome (IBS), and/or functional disorders, infection or genitourinary inflammations, in particular of candidosis and of cystitis, is yet to be resolved.

In order to overcome the aforementioned technical problems, the present invention provides a composition comprising at least one essential oil or at least one hydrolate obtained from a plant of Italian origin, preferably originating from Sicily, or mixture of two or more of said essential oils and/or hydrolates, which is capable of effectively and quickly treating, both in diseased subjects and in healthy subjects, the symptoms or disorders caused by or related with an alteration of the microbiota balance and/or of the immune system, in particular caused by or related with a disorder of the gastrointestinal or genitourinary tract. Furthermore, the present invention provides a composition without the adverse effects present in the treatments of the prior art, but that is easy to prepare and economically advantageous.

These and other objects, which will be clear from the detailed description that follows, are attained by the compositions and the mixtures of the present invention due to the technical characteristics claimed in the attached claims.

Description of the figures:

Figure 1 and figure 2: MFC (Minimum Fungicidal Concentration) measurement, on solid agar and stained with Alamar blue, of the content of each well, containing essential oils and both Candida and Saccharomyces genera incubated in the seeded growth medium.

Figures 3-6: measurement of the quantity respectively of four cytokines (IL-10, IL-8, IL-6, TNF-alpha) after incubation of gradual dilutions of hydrolates (from 1/4 to 1/32) in PBMC ( Peripheral Blood Mononuclear Cells).

Figure 7: histogram chart representing the inhibition of S. cerevisiae and/or C. albicans by combinations at different % between the hydrolate of V. vinifera Italian variety (HY) and the essential oil of C. aurantium, biter variety (EO).

Figure 8: histogram chart representing the differentiated count of the CFUs of C. albicans and of S. cerevisiae following the inhibition thereof by combinations at different % between the hydrolate of V. vinifera Italian variety (HY) and the essential oil of C. aurantium, bitter variety (EO).

Figure 9: optical microscope images of an aliquot of the content of the wells containing C. albicans treated with essential oil of C. aurantium, bitter variety, 0.125% combined with the hydrolate of V. vinifera Italian variety (0.125% and 0.06%).

Figure 10: chart of inhibition of cytokines IL-10 and TNF-a by PBMC grown in presence of the pro- inflammatory stimulus (LPS) by combinations at different % between the hydrolate of V. vinifera Italian variety (HY) and the essential oil of C. aurantium, bitter variety (EO).

Figure 11 : GC-MS analysis of evaluation of the composition of the essential oil of C. aurantium bitter variety (EO) and hydrolate of V. vinifera Italian variety (HY).

Figure 12: chart of the inhibitory capacity of C. aurantium, biter variety EO towards C. albicans and S. cerevisiae (expressed in CFUs / mL).

Figure 13: histogram of the checkerboard BMD test of C. aurantium, bitter variety EO (from 0.50% v / v to 0.06% v / v) and V. vinifera cv Italia HY (from 25% v / v to 6.25% v / v) against C. albicans and S. cerevisiae. Following an intense and extensive research activity, the Applicant found out, as described in detail hereinafter, that the anti-microbial effect of the essential oils of the composition (C) of the invention (described hereinafter) combined or not combined with the immunomodulatory effect, in particular of the hydrolates of the composition (C) of the invention, can lead to mixtures, compositions or formulations of great interest in the therapeutic or non-therapeutic treatment of symptoms or diseases or disorders caused by or related with an alteration of the microbiota and/or of the immune system of a subject, in particular caused by or related with a functional gastrointestinal disorder or caused by or related with an infection or inflammation of the gastrointestinal tract or alternatively caused by or related with a genitourinary disorder or caused by or related with an infection or inflammation of the genitourinary tract, more in particular in the treatment of the irritable bowel syndrome (IBS), of the chronic inflammatory bowel diseases (IBD) and in the treatment of candidosis or cystitis. Furthermore, it was proven that the essential oils and/or the hydrolates comprised in the composition (C) of the invention are active in the inhibition of pathogenic agents, such as for example strains of Candida sp., at lower concentrations with respect to the concentrations at which said essential oils and/or hydrolates are lethal for the probiotic strains used in commercial formulations.

Forming an object of the present invention is a composition (C) (abbreviated as composition of the invention) comprising (i) a mixture (M) comprising or, alternatively, consisting of at least one essential oil or at least one hydrolate obtained from a plant or from at least one part of a plant selected from among the group comprising or, alternatively, consisting of:

(a) Citrus limon,

(b) Citrus limon femminiello variety,

(c) Citrus reticulata,

(d) Citrus aurantium, bitter variety,

(e) Citrus aurantium, sweet variety,

(f) Monarda didyma,

(g) Cinnamomum zeylanicum,

(h) Vitis vinifera, Italian variety,

(i) Vitis vinifera, Red globe variety

or a mixture of two or more of said (a)-(i) essential oils and/or hydrolates, and, optionally, (ii) at least one pharmaceutical or food grade additive and/or excipient (abbreviated as composition (C) or composition (C) of the invention or composition of the invention).

Said plants (a)-(i) are plants of Italian origin, preferably of Sicilian origin. Citrus limon, (or lemon) (a), present in the composition of the invention mixed with (b), (c), (d), (e), (f), (g), (h) and/or (i), is a fruit-tree belonging to the family of Rutaceae. The common name lemon may equally refer to the plant as well as to its fruit.

Citrus limon, femminiello variety (b), present in the composition of the invention mixed with (a), (c), (d), (e), (f). (9). (h) and/or (i), is cultivar femminello referable to the Citrus limon botanic species.

Cultivar (abbreviated as cv. according to the International code of nomenclature for cultivated plants)is the term used in agronomy to indicate a variety with cultivated plant, obtained with genetic improvement, which summarises a set of specific morphological, physiological, agronomic and commodity characters of particular interest and that can be transmitted with propagation, both for the seed and for parts of the plant. From a practical point of view, cultivar would be similar to the race of an animal species obtained through taming and selection. Thus, cultivar is identified in a particular genotype, artificially isolated through the massal selection or the individual selection, whose characters are fixed and repeatable with the gamic propagation for at least 3-4 generations.

Citrus reticulata (or tangerine) (c), present in the composition of the invention mixed with (a), (b), (d), (e), (f). (9). (h) and/or (i), is a fruit-tree belonging in the family of Rutaceae.

Citrus aurantium (or orange) (d) or (e), present in the composition of the invention mixed with (a), (b), (c),

(d), (e), (f), (g), (h) and/or (i), also called bitter orange, is a fruit-tree belonging to the Citrus genus, gathering the citrus fruits. It is an ancient hybrid, probably between the pomelo and the tangerine, but for centuries it has been growing as an autonomous species and it propagates by grafting and scion.

Citrus aurantium, bitter variety (d).

Citrus aurantium, sweet variety (e).

The Monarda didyma (f) species, present in the composition of the invention mixed with (a), (b), (c), (d),

(e), (g), (h) and/or (i), is part of the Lamiaceae family and it is of the herbaceous type.

Cinnamomum verum (J.Pres) synonym Cinnamomum zeylanicum (von Blume) (or cinnamon) (g), present in the composition of the invention mixed with (a), (b), (c), (d), (e), (f), (h) e/o (i), is an evergreen tree of the Lauraceae family, of Sri Lankan origin, from which the spice bearing the same name widely present in Europe and Asia alike is obtained. Various plants are equally called cinnamon. The two most frequently used as spices are Cinnamomum verum and Cinnamomum cassia. Vitis vinifera (or common grape) (h) o (i), present in the composition of the invention mixed with (a), (b),

(c), (d), (e), (f), (g), (h) and/or (i), is a climbing shrub of the Vitaceae family. It is the most widely cultivated vine in broad sense currently present in all continents except for the Antarctica.

Vitis vinifera, Italian variety (h)

Vitis vinifera, Red globe variety (i)

Advantageously, said at least one essential oil or at least one hydrolate can be obtained by means of common techniques for obtaining essential oils and/or hydrolates from a plant or from at least one part of said plant or from two or more parts of said plant, such as for example leaves, fruits or any other aerial part. Preferably, the essential oils and/or hydrolates of the present invention are obtained by means of the squeezing and/or distillation method according to methods known to a man skilled in the art, preferably, from fruits and/or tops with fruits and leaves.

Preferably, the essential oil of (d) is obtained by squeezing and the hydrolate of (h) is obtained by distillation.

In a preferred embodiment, said mixture (M) comprises or, alternatively, consists of at least one essential oil or at least one hydrolate obtained from a plant or from at least one part of a plant selected from among the group comprising or, alternatively, consisting of:

(d) Citrus aurantium, bitter variety,

(e) Citrus aurantium, sweet variety,

(f) Monarda didyma,

(g) Cinnamomum zeylanicum,

(h) Vitis vinifera, Italian variety,

or a mixture of two or more of said (d)-(h) essential oils and/or hydrolates.

In a preferred embodiment, said mixture (M) comprises or, alternatively, consists of at least one essential oil obtained from (d) Citrus aurantium, bitter variety, alone or combined with one or more from among an essential oil or a hydrolate obtained from (a), (b), (c), (e), (f), (g), (h) or (i).

In a preferred embodiment, said mixture (M) comprises or, alternatively, consists of at least one essential oil obtained from (d) Citrus aurantium, bitter variety, and at least one hydrolate obtained from (h) Vitis vinifera Italian variety, alone or combined with one or more from among an essential oil or a hydrolate obtained from (a), (b), (c), (e), (f), (g) or (i). In an embodiment, the composition (C) of the invention comprising said at least one essential oil or at least one hydrolate or said mixture of two or more of said essential oils and/or hydrolates (a)-(i) at a volume amount comprised from 0.01% to 10%, preferably comprised from 0.01% to 1%, more preferably from 0.05% to 0.5%, wherein said amounts are with respect to the total volume of the composition (C).

In the aforementioned preferred embodiment in which the mixture (M) comprises or, alternatively, consists of at least one essential oil obtained from (d) Citrus aurantium, bitter variety and at least one hydrolate obtained from (h) Vitis vinifera, Italian variety, alone or combined with one or more from among an essential oil or a hydrolate obtained from (a), (b), (c), (e), (f), (g) or (i), the essential oil obtained from (d) is present at a concentration at a volume amount comprised from 0.01% to 1%, preferably comprised from 0.06% to 0.25%, more preferably comprised from 0.06% to 0.125%, and the hydrolate obtained from (h) is present at a concentration at a volume amount comprised from 1% to 25%, preferably comprised from 6.25% to 12.5%, wherein said amounts are with respect to the total volume of the composition (C). For example, in a composition of the invention the amount of essential oil of (d) Citrus aurantium, bitter variety, is equal to 80 mg (0.5%), the amount of hydrolate of (h) Vitis vinifera, Italian variety, is equal to 8000 mg (50%) and the amount of carrier is equal to 7920 mg, for a total of 16000 mg of product to be administered in a single daily dose or in two doses.

In the aforementioned preferred embodiment in which the mixture (M) comprises or, alternatively, consists of at least one essential oil obtained from (d) Citrus aurantium, bitter variety, and at least one hydrolate obtained from (h) Vitis vinifera, Italian variety, alone or combined with one or more from among an essential oil or a hydrolate obtained from (a), (b), (c), (e), (f), (g) or (i), said essential oil obtained from (d) Citrus aurantium, bitter variety, and said hydrolate obtained from (h) Vitis vinifera, Italian variety, are at a (d):(h) by weight ratio comprised in the range from 1 :200 to 1 :10, preferably from 1 :150 to 1 :50, more preferably about 1 :100.

Furthermore, forming an object of the present invention is the composition (C) of the invention according to the various embodiments of the invention, preferably the composition (C) comprising the mixture (M) comprising or, alternatively, consisting of said essential oil obtained from (d) Citrus aurantium, bitter variety, and said hydrolate obtained from (h), for use as a medicament.

Furthermore, forming an object of the present invention is the composition (C) of the invention according to the various embodiments of the invention, preferably the composition (C) comprising the mixture (M) comprising or, alternatively, consisting of said essential oil obtained from (d) Citrus aurantium, bitter variety, and said hydrolate obtained from (h), for use in a method for the preventive and/or curative treatment (therapeutic treatment) of symptoms or diseases caused by or related with the definition outlined below or, alternatively, the non-therapeutic use of the composition (C) of the invention for modulating or reducing disorders caused by or related with the definition outlined below, in a needy subject.

Generally, the treatments of the present invention are effective and applicable both to diseased subjects (patients) who have been diagnosed with a disease or a symptom/disorder related with a disease, and to healthy subjects. Furthermore, the composition (C) of the present invention shall be for human or veterinarian use indistinguishably, i.e. as a preparation to be applied to animals with the uses and methods known to the man skilled in the art.

In particular, forming an object of the present invention is the composition (C) of the invention according to the various embodiments of the invention, preferably the composition (C) comprising the mixture (M) comprising or, alternatively, consisting of said essential oil obtained from (d) Citrus aurantium, bitter variety, and said hydrolate obtained from (h), for use in a method for the therapeutic (preventive and/or curative) or non-therapeutic treatment of symptoms or diseases or disorders caused by or related with the alteration of the microbiota balance (or human microbiota) of a subject.

Preferably, the composition (C) of the invention according to the various embodiments of the invention, preferably the composition (C) comprising the mixture (M) comprising or, alternatively, consisting of said essential oil obtained from (d) Citrus aurantium, bitter variety, and said hydrolate obtained from (h), is for use in a method for the treatment of symptoms or disorders caused by or related with alteration of balance of the gastrointestinal microbiota, of the genitourinary microbiota or of the microbiota of any of the mucosa of a subject, such as, for example, of the stomach mucosa, of the oesophagus mucosa, of the upper respiratory tract mucosa, in particular of the oral cavity, nasal cavity, larynx and pharynx, or of the ear cavity.

Furthermore, forming an object of the present invention is the composition (C) of the invention according to the various embodiments of the invention, preferably the composition (C) comprising the mixture (M) comprising or, alternatively, consisting of said essential oil obtained from (d) Citrus aurantium, bitter variety, and said hydrolate obtained from (h), for use in a method for the therapeutic (preventive and/or curative) or non-therapeutic treatment of symptoms or diseases or disorders caused by or related with the alteration of the immune system. Preferably, the composition (C) of the invention according to the various embodiments of the invention, preferably the composition (C) comprising the mixture (M) comprising or, alternatively, consisting of said essential oil obtained from (d) Citrus aurantium, bitter variety, and said hydrolate obtained from (h) is for use in the treatment of symptoms or disorders caused by or related with alteration of balance of the gastrointestinal immune system, of the genitourinary immune system or of the specific immune system of any other system or tract of the body of a subject, such as, for example, of the stomach, of the oesophagus, of the upper respiratory tract, in particular of the oral cavity, nasal cavity, larynx and pharynx, or of the ear cavity.

In an embodiment of the present invention, the composition (C) of the invention according to the various embodiments of the invention, preferably the composition (C) comprising the mixture (M) comprising or, alternatively, consisting of said essential oil obtained from (d) Citrus aurantium, bitter variety, and said hydrolate obtained from (h), is for use as a coadjuvant in a method for the therapeutic or non-therapeutic treatment of the aforementioned symptoms or diseases or disorders.

In a preferred embodiment, the composition (C) of the invention according to the various embodiments of the invention, preferably the composition (C) comprising the mixture (M) comprising or, alternatively, consisting of said essential oil obtained from (d) Citrus aurantium, bitter variety, and said hydrolate obtained from (h), is for use in a method for the treatment of the symptoms or diseases or disorders caused by or related with a functional gastrointestinal disorder or caused by or related with an infection or inflammation of the gastrointestinal tract, wherein said functional intestinal or gastrointestinal disorders or said infections or inflammations of the gastrointestinal tract are related with an alteration of the gastrointestinal microbiota balance and/or with an alteration of the immune system in general or of the gastrointestinal immune system in particular.

Preferably, the composition (C) of the invention according to the various embodiments of the invention, preferably the composition (C) comprising the mixture (M) comprising or, alternatively, consisting of said essential oil obtained from (d) Citrus aurantium, bitter variety, and said hydrolate obtained from (h), is for use in a method for the treatment of symptoms or disorders caused or related with the irritable Bowel Syndrome (IBS).

Furthermore, preferably, the composition (C) of the invention according to the various embodiments of the invention, preferably the composition (C) comprising the mixture (M) comprising or, alternatively, consisting of said essential oil obtained from (d) Citrus aurantium, bitter variety, and said hydrolate obtained from (h), is for use in the treatment of symptoms or diseases or disorders, caused by or related with temporary inflammations or chronic inflammatory bowel diseases (IBD), such as for example, adverse reactions to foodstuffs or other food or pharmaceutical grade substances.

In a preferred embodiment, the composition (C) of the invention according to the various embodiments of the invention, preferably the composition (C) comprising the mixture (M) comprising or, alternatively, consisting of said essential oil obtained from (d) Citrus aurantium, bitter variety, and said hydrolate obtained from (h), is for use in a method for the treatment of the symptoms or diseases or disorders caused by or related with a functional genitourinary disorder or caused by or related with an infection or inflammation of the genitourinary tract, wherein said functional genitourinary disorders or said infection or inflammation of the genitourinary tract are related with an alteration of the genitourinary microbiota balance and/or with an alteration of the immune system in general or of the genitourinary immune system in particular.

Preferably, the composition (C) of the invention according to the various embodiments of the invention, preferably the composition (C) comprising the mixture (M) comprising or, alternatively, consisting of said essential oil obtained from (d) Citrus aurantium, bitter variety, and said hydrolate obtained from (h), is for use in a method for the treatment of symptoms or disorders caused by or related with candidosis.

Preferably, the composition (C) of the invention according to the various embodiments of the invention, preferably the composition (C) comprising the mixture (M) comprising or, alternatively, consisting of said essential oil obtained from (d) Citrus aurantium, bitter variety, and said hydrolate obtained from (h), is for use in a method for the treatment of symptoms or disorders caused or related with acute or recurrent cystitis.

In a preferred embodiment, the composition (C) of the invention according to the various embodiments of the invention, preferably the composition (C) comprising the mixture (M) comprising or, alternatively, consisting of said essential oil obtained from (d) Citrus aurantium, bitter variety, and said hydrolate obtained from (h), is for use in a method for the treatment of symptoms or diseases or disorders caused by or related with at least one pathogenic agent of the genus selected from among the group comprising or, alternatively, consisting of: Candida, Basidiomycetes, or mixtures of two or more of said pathogenic agents.

Preferably, said pathogenic agent belongs to the Candida genus, more preferably said pathogenic agent belongs to the Candida albicans species. Candida albicans is a fungus belonging to the family of Saccharomycetes. They are normally found in the oral cavity, in the gastrointestinal tract and in the vagina. Candida albicans is present in the human organism at saprophyte state, but it can turn pathogenic under specific conditions (opportunism) causing candidosis. Candida albicans is present at high amounts both on the mucosa and on the skin. Candida albicans usually multiplies abnormally in organisms with some form of imbalance, and markedly greater in debilitated and immunodeppressed subjects such as children, the elderly and the sick. These forms of superinfections by Candida also affect subjects under long antibiotics therapies (tetracycline and neomycin, which damage the intestinal flora), extended and intense stress or hormonal excursion. Thus, candidosis is manifested above all as a vaginal disease (vaginitis), of the oral cavity (thrush) or a skin disease but also pharynx disease, oesophagus disease or gastrointestinal disease or urine bladder disease. Through the intestine, it can reach the blood where it releases its toxins causing the candidemia. This event leads to several symptoms such as abdominal bloating, slowing of the digestion, bowel disorders (constipation or diarrhoea), food intolerances, tiredness, irritability, insomnia, loss of memory, headache and depression. Candidosis also causes poor absorption of the nutritional substances and, in the long-term, a state of malnutrition.

The aforementioned treatment methods provide for the administration of an effective amount of a composition according to the invention to a needy subject. Said effective amount will be established by the man skilled in the art based on the conditions and the needs of the treated subject.

The composition (C) as defined above according to the various embodiments of the invention can, optionally, comprise at least one pharmaceutical or food grade additive and/or excipient, i.e. a substance without therapeutic activity, suitable for pharmaceutical or food purposes. In the context of the present invention the acceptable ingredients for pharmaceutical or food use comprise all auxiliary substances known to the man skilled in the art such as, by way of non-limiting example, diluents, solvents (including water, glycerine, ethyl alcohol), solubilisers, thickeners, sweeteners, flavourings, dyes, lubricants, surfactants, antimicrobials, antioxidants, preservatives, pH stabilisation buffers and the mixtures thereof. Non-limiting examples of such substances are maltodextrins, phosphate buffers, bases such as sodium hydroxide, xanthan gum, guar gum, fructose, natural or artificial flavours.

The composition (C) according to the present invention, besides the mixture (M) and, optionally, said pharmaceutical or food grade additives and/or excipients, may further comprise other active ingredients such as, by way of non-limiting example, anti-inflammatories, antimycotics, antiviral, mucosa disinfectants, antacids, products for the treatment of disorders of the gastrointestinal tract or of the genitourinary tract and the mixtures thereof. In the context of the present invention, the expression composition/s is deemed to include the pharmaceutical compositions, the medical device compositions, the dietary supplement compositions and the food composition (e.g. nutraceutical food products).

In particular, forming an object of the present invention is a pharmaceutical composition, a medical device composition, a dietary supplement or food product, preferably a nutraceutical food product, comprising or, alternatively, consisting of the composition (C) of the present invention.

The expression "medical device” in the context of the present invention is used according to the meaning laid down by the Italian Legislative Decree n° 46, dated 24 February 1997 or according to the new Medical Devices Regulation (UE) 2017/745 (MDR), i.e. it indicates a substance or another product, used alone or in combination, designated by the manufacturer to be used in humans for the diagnosis, prevention, control, therapy or attenuation of a disease, the product not exercising the main action, in or on the human body, for which it is designated, neither using pharmacological or immunology means nor by means of a metabolic process but the function thereof can be coadjuvated by such means.

Nutraceutics are nutrient principals contained in food products with beneficial effects on health. They are found in nature, but industrial transformation tends to nullify them. Nutraceutics can be extracted, synthesised and used for dietary supplements, or added to food products. It is rarer to find them in food products in natural form and at amounts sufficient to obtain beneficial effects. The minimum amounts of nutrients required to obtain beneficial effects are regulated by European regulations (e.g. Regulation 432/2012).

"Nutraceutical food products”, also referred to as functional food products or pharma foods are defined - in the context of the present invention- as food products containing said nutrient principles (nutraceutics) in the minimum amounts required by the regulations.

The composition (C) of the present invention according to the various embodiments of the invention, preferably the composition (C) comprising the mixture (M) comprising or, alternatively, consisting of said essential oil obtained from (d) Citrus aurantium, bitter variety, and said hydrolate obtained from (h), can be in liquid form, such as, by way of non-limiting example, solution, two-phase liquid system, suspension or syrup, in a semi-solid form, such as, for example, gel, cream or foam, or in a solid form, such as, for example, powder, granules, chips, aggregates, capsules, pills, bars and equivalent forms. The composition (C) of the present invention according to the various embodiments of the invention, preferably the composition (C) comprising the mixture (M) comprising or, alternatively, consisting of said essential oil obtained from (d) Citrus aurantium, bitter variety, and said hydrolate obtained from (h), can be administered through topical route, through enteral route, preferably through oral route (or gastroenteric route) or through sublingual route (or buccal route) or through parenteral route, preferably through inhalation route.

For the sake of clarity, in order to achieve the object of the present invention, the essential oils or hydrolates obtained from the plants or parts of plants (a), (b), (c), (d), (e), (f), (g), (h) and/or (i) can also be administered separately and following any order but, preferably, the essential oils or hydrolates obtained from the plants or parts of plants (a), (b), (c), (d), (e), (f), (g), (h) and/or (i) are administered to a subject simultaneously, even more preferably in a single composition to obtain a quicker effect and for ease of administration.

When the essential oils or hydrolates obtained from the plants (a), (b), (c), (d), (e), (f), (g), (h) and/or (i) are administered in a single composition, said single composition corresponds to the composition (C) of the present invention.

Unless specified otherwise, the indication that a composition "comprises” one or more components or substances means that other components or substances can be present besides the one, or the ones, indicated specifically.

The essential oils and the hydrolates of the present invention are obtained from plant species for human use included in the list of botanic species identified by the Italian Ministry under the Ministerial Decree dated 9 July 2012 (and subsequent amendment through the Decree dated 27 March 2014) and in the BELFRIT list (annex 1 bis of the Decree dated 27 March 2014) as plant substances and preparations that can be used in formulations of dietary supplements for human use; furthermore, Vitis vinifera is monographed by EMA (European Medicines Agency -London, 13 January 2010 Doc. Ref.: EMA/HMPC/16635/2009 Rev. 1).

As illustrated in the experimental part, the compositions of the invention, according to any one of the preferred embodiments described above, exercise an immunomodulatory action on polymorphonucleated cells of the peripheral blood and simultaneously prevent the virulence of the fungal strains of the Candida genus without interfering with the growth of probiotic, bacterial and fungal strains belonging to the gastrointestinal or genitourinary physiological microbiota. This helps our organism to recover the physiological gastrointestinal and/or genitourinary balance stimulating the immune response and fighting the growth of pathogenic microbial strains in favour of the beneficial microbiota.

In particular, the essential oils and/or the hydrolates obtained from (a), (b), (c), (d), (e), (f), (g), (h) and/or (i), comprised in the compositions of the invention according to any one of the preferred embodiments described above, synergically act on the balance of the gastrointestinal and/or genitourinary microbiota balance of the immune system.

Unless specified otherwise, the expression composition or mixture comprising a component at an amount "comprised in a range from x to y” is used to indicate that said component can be present in the composition or mixture at all the amounts present in said range, even though not specified, extremes of the range comprised.

Embodiments (FRnr) of the present invention are indicated below:

FR1. A composition (C) comprising

(i) a mixture (M) comprising or, alternatively, consisting of at least one essential oil or at least one hydrolate obtained from a plant or from at least one part of a plant selected from among the group comprising or, alternatively, consisting of:

(a) Citrus limon,

(b) Citrus limon femminiello variety,

(c) Citrus reticulata,

(d) Citrus aurantium, bitter variety,

(e) Citrus aurantium, sweet variety,

(f) Monarda didyma,

(g) Cinnamomum zeylanicum,

(h) Vitis vinifera, Italian variety,

(i) Vitis vinifera, Red globe variety,

or a mixture of said essential oils and/or hydrolates,

and, optionally,

(ii) at least one pharmaceutical or food grade additive and/or excipient.

FR2. The composition (C) according to FR1, wherein said mixture (M) comprising or, alternatively, consisting of at least one essential oil or at least one hydrolate obtained from a plant or from at least one part of a plant selected from among the group comprising or, alternatively, consisting of:

(d) Citrus aurantium, bitter variety,

(e) Citrus aurantium, sweet variety, (f) Monarda didyma ,

(g) Cinnamomum zeylanicum,

(h) Vitis vinifera, Italian variety,

or a mixture of said (d)-(h) essential oils and/or hydrolates,

preferably wherein said mixture (M) comprising or, alternatively, consisting of at least one essential oil and a hydrolate obtained from (d) Citrus aurantium, bitter variety.

FR3. The composition (C) according to FR1 or FR2, wherein said composition (C) comprise said at least one essential oil or at least one hydrolate or said mixture of said essential oils and/or hydrolates at a volume amount comprised from 0.01% to 10%, preferably comprised from 0.01% to 1%, more preferably comprised from 0.05% to 0.5%, wherein said amounts are with respect to the total volume of the composition (C).

FR4. The composition (C) according to any one of FR1 to FR3, wherein said composition (C) is

- for use in a method for the preventive and/or curative treatment of the symptoms or diseases caused by or related with alteration of the microbiota balance, preferably of the gastrointestinal microbiota or of the genitourinary microbiota; or

- for use in a method for the preventive and/or curative treatment of the symptoms or diseases caused by or related with alteration of the immune system, preferably of the gastrointestinal immune system or of the genitourinary immune system; preferably for use in a method for the preventive and/or curative treatment of said symptoms or diseases as a coadjuvant.

FR5. The composition (C) for use according to FR4, wherein said composition (C) is

- for use in a method for the preventive and/or curative treatment of the symptoms or diseases caused by or related with a functional gastrointestinal disorder or caused by or related with an infection or inflammation of the gastrointestinal tract, preferably caused by or related with an irritable bowel syndrome or caused by or related with inflammatory, chronic or temporary diseases of the bowel; or

- for use in a method for the preventive and/or curative treatment of the symptoms or diseases caused by or related with a functional genitourinary disorder or with an infection or inflammation of the genitourinary tract, preferably caused by or related with candidosis or cystitis.

FR6. The composition (C) for use according to FR4 or FR5, wherein said composition (C) is for use in a method for the preventive and/or curative treatment of the symptoms or diseases caused by or related with at least one pathogenic agent belonging to the Candida genus; more preferably said pathogenic agent belongs to the Candida albicans species. FR7. Non-therapeutic use of the composition (C) according to any one of FR1 to FR3 for modulating or reducing disorders caused by or related with

- alteration of the microbiota balance, preferably of the gastrointestinal microbiota or of the genitourinary microbiota and/or

- alteration of the immune system, preferably of the gastrointestinal immune system or of the genitourinary immune system;

preferably non-therapeutic use of the composition (C) as a coadjuvant for modulating or reducing said disorders.

FR8. Non-therapeutic use of the composition (C) according to FR7 for modulating or reducing disorders caused by or related with

- a functional gastrointestinal disorder or caused by or related with an infection or inflammation of the gastrointestinal tract, preferably caused by or related with the irritable bowel syndrome or caused by or related with inflammatory, chronic or temporary diseases of the bowel; and/or

- a functional genitourinary disorder or caused by or related with an infection or inflammation of the genitourinary tract, preferably caused by or related with candidosis or caused by or related with cystitis.

FR9. Non-therapeutic use of the composition (C) according to FR7 or FR8, for modulating or reducing disorders caused by or related with at least one pathogenic agent belonging to the Candida genus; more preferably said pathogenic agent belongs to the Candida albicans species.

FR10. Pharmaceutical composition, medical device, dietary supplement or food product comprising the composition (C) according to any one of FR1 to FR3.

EXPERIMENTAL TESTS SECTION 1

Material

Table 1 shows the essential oils (abbreviated as EO) and the hydrolates (abbreviated as HY) used in the tests indicated below. In particular, table 1 shows the plants from which the essential oils and the hydrolates were obtained.

Table 1

In vitro test n° 1

Purpose a): Identifying the MFCs regarding the essential oils indicated in table 1 exploring the activity thereof on a wider group of fungal strains isolated from faecal samples of patients suffering from IBS. Purpose b): Identifying the MFCs regarding the 4 selected essential oils (C. Aurantium, bitter variety, C. aurantium, sweet variety, M. didyma and C. zeylanicum) exploring the activity thereof on a wider group of fungal strains isolated from patients suffering from IBS.

Method:“Micro broth dilution" (MBD) tests were conducted in compliance with the EUCAST ( European . Committee on Antimicrobial Susceptibility Testing) guidelines using the essential oils and the hydrolates n° 1-9 indicated in table 1 with respect to 54 fungal strains (38 Candida spp., 8 Galactomyces spp., 4 S. cerevisiae) to identify the most active essential oils. Each test was repeated at least three times.

Results a): table 2.1 shows the MIC values of the 38 species of Candida isolated from faecal samples of patients suffering from IBS against the EO of C. lemon, C. lemon femminiello variety, C. reticulata, C. aurantium bitter variety, C. zeylanicum and M. didyma tested at concentrations ranging from 0.50% v / v to 0.06% v / v. The MIC50 values were > 0.50% v / v for C. lemon, C. lemon var. emminello and C. reticulata, 0.50% v / v for C. aurantium, bitter variety, <0.06% v / v for C. zeylanicum and 0.25% v / v for M. didyma.

Table 2.2 shows the MIC values of the 7 probiotic strains and of the 8 isolated species of Galactomyces and 4 of S. cerevisiae against the EOs of C. aurantium, bitter variety, C. zeylanicum and M. didyma (the only three EOs showing a better activity in table 2.1) tested at concentrations ranging from 4.0% v / v to 0.06% v / v. It was opted to test lower concentrations of EOs for isolates from pathogenic species (i.e. Candida species) and higher concentrations of EOs for isolates from beneficial species (i.e. probiotic species or Galactomyces I S. Cerevisiae). This choice would guarantee the activity towards pathogenic species and non-toxicity towards beneficial species possibly present in the gut microbiota of patients suffering from IBS.

The MIC50 values were > 4.0% v / v for C. aurantium, bitter variety, 0.50% v / v for M. didyma and <0.06% v / v for C. zeylanicum, as indicated briefly in table 2.2. The data indicates that both the tested probiotics and the tested beneficial species are generally more resistant to the action of the EOs with respect to pathogenic fungal isolates. In particular, the EO of C.aurantium, bitter variety has an MIC50 (MIC50 > 4% v / v) value at least 3 dilutions higher with respect to the beneficial clinical isolates with respect to the clinical isolates of the Candida (MIC50 = 0.50% v / v) species. Thanks to the different MIC values between probiotic and pathogenic strains, probiotics and EOs can be administered together to cure pathogenic infections of the human bowel.

Results b): table 2.3 shows the results obtained for oils with greater antimycotic activity (nos. 4, 5, 6 and 7 of table 1) towards strains of: Candida spp., Galactomyces sp. and of Saccharomyces cerevisiae. The MFC90 values ( Minimal Fungicidal Concentration values for 90% of the present cells) show that, in the case of the essential oils of C. aurantium bitter variety and of M. didyma, a higher concentration of essential oil is required so as to obtain cytocidal effects on beneficial strains and strains belonging to the gut microbiota ( Galactomyces sp. and S. cerevisiae) rather than on pathogenic strains which - on the contrary - are more sensitive.

Table 2.1 : MIC = equal to the value of MFC for all tested strains. CL= Citrus limon, CL-f = Citrus limon femminiello variety, CR= Citrus reticulata, CA-a = Citrus aurantium bitter variety, MD= Monarda didyma, CZ= Cinnamomum zeylanicum,

Table 2.2: MIC = equal to the value of MFC for all tested strains. CA-a = Citrus aurantium bitter variety , MD= Monarda didyma, CZ= Cinnamomum zeylanicum,

Table 2.3. The concentration of the essential oils is expressed as % volume on volume. In vitro test n° 2.1

Purpose: to study the modulating action of the 4 essential oils (C. aurantium bitter variety, C. aurantium sweet variety, M. didyma and C. zeylanicum) on the growth of 5 strains of Candida spp. (pathogenic strains) and 4 strains of Saccharomyces cerevisiae (strains belonging to the physiological gut microbiota). Methods: a“Micro broth dilution" (MBD) test in compliance with the EUCAST ( European . Committee on Antimicrobial Susceptibility Testing) guidelines, combined with the CFU / ml (CFU, colony-forming units) count, was used for studying the modulating effect of the four essential oils nos. 4, 5, 6 and 7 of table 1 , selected based on the in vitro test n°. 1 indicated above, with respect to 5 strains of Candida spp. and 4 strains of S. cerevisiae (table 3). Each test was repeated at least three times.

Results: as shown in table 3 sub-MIC concentrations were identified as pertains to the EOs of C. aurantium bitter variety (0.25%), M. didyma (0.25%) and C. zeylanicum (0.01 %) in which the essential oils exercise almost identical modulating actions with respect to the two strains. A similar sub-MIC concentration could not be identified for the essential oil of C. aurantium sweet variety.

Table 3: the first concentration with ower inhibitory action towards both fungal classes was indicated for each essential oil.

In vitro test n° 2.2

The inhibitory capacity of C. aurantium bitter variety EO was further studied by counting the CFU / mL of both C. albicans and S. cerevisiae recovered after treatment thereof with sub-MIC (<2% v / v) concentrations of EO. Figure 12 shows that the concentration of 2% of C. aurantium bitter variety EO was capable of fully suppressing both organisms. It is worth noting that S. cerevisiae revealed an exponential trend of greater tolerance to the EO (R² = 0.71) that was statistically significant and different (p <0.05) from the linear trend of C. albicans (R² = 0.65).

In vitro test n° 3

In order to evaluate the effect of the essential oils on a fungal mix characterised by 1 strain of C. albicans and 1 strain of S. cerevisiae present in co-culture, OD (Optical Density) was acquired in each well at 450 nm so as to identify the MIC (Minimum Inhibition Concentration) and the MFC (Minimum Fungicidal Concentration) was evaluated by appropriately seeding the content of the wells of the essential oils nos. 4, 6 and 7 of table 1 (table 4). Simultaneously, in order to confirm the MFC values, the content of each well was initially seeded in solid agar and stained using Alamar blue (figure 1 and figure 2).

Table 4

Results: table 4 shows that the most active essential oil in terms of cytocidal action is that of C. zeylanicum, while those of M. didyma and C. aurantium, exercising a more moderate action, seem to be more appropriate for our purpose. As shown below in table 5, the most effective modulator is seemingly the essential oil of C. aurantium bitter variety for concentrations lower than or equal to the concentration of 0.25% with maximum action at concentration of 0.125%. The other essential oils exercise a less differentiated modulating action given that a greater action on the strain of C. albicans or on the strain of S. cerevisiae could not be observed.

The data reveals that, if subjected to co-culture, the two fungi (C. albicans and S. cerevisiae) have the same MFC but, by analysing the fungal growth in presence of the sub-MIC of C. aurantium bitter variety EO, there can be identified a different modulating action of this EO towards the two clinical strains (C. albicans and S. cerevisiae). In particular, for concentrations equal to or less than 2% v / v of C. aurantium bitter variety EO, the strain of C. albicans is more inhibited than that of S. cerevisiae (p<0.05) (Fig. 12). Thus, this activity could be useful towards the attempt to balance the relative abundance of each clinical species in patients suffering from IBS.

In vitro test n° 4

Portions of the content of each well obtained in the in vitro test n° 3, after incubation, were diluted 1 : 10,000. 10pl, of what was obtained were seeded on chromagar with the aim of distinguishing the fungicidal activity of the essential oils towards the two genera ( Candida and Saccharomyces) (table 5).

Table 5. Sc: S.cerevisiae, Ca: C. albicans; the values are expressed as mean x 10⁴ UFC/ml; the standard deviation is between brackets.

In vitro test n° 5

In order to evaluate the immunomodulatory effect of the hydrolates (HY) nos. 4, 6, 8 and 9 of table 1 , the PBMCs ( Peripheral Blood Mononuclear Cells) obtained from two donors were incubated overnight in an RPMI ( Rosewell Park Memorial Institute) growth medium containing gradual dilutions of the hydrolates (from 1/4 to 1/32). Thus, the growth medium was collected and the amount of four cytokines (IL-10, IL-8, IL-6, TNF-alpha) was collected using the ELISA kit (figures 3-6).

Results: After 24 hours, the cells treated using hydrolates (HY) of Vitis vinifera Red globe variety or V. vinfera Italian variety did not show any viability variation with respect to the control (for all the tested conditions) while for all the others (HY) the treated cells with the highest percentage of (HY) (0.25% v/v) seemed less viable as compared to the control. Furthermore, the result obtained using the hydrolate of V. vinfera Italian variety, seemed worth noting. The samples treated using gradual dilutions of this hydrolate, showed a slight increase of the expression of L-10 (anti-inflammatory) and a simultaneous decrease of the expression of IL-8 (pro-inflammatory). These variations are worth noting given that they are opposite from those observed in vivo in subjects suffering from IBS.

In vitro test n° 6

Purpose: a trial was conducted on the action of the essential oils of interest of the Applicant on the probiotic strains commonly used in dietary supplements.

Methods: 7 probiotic strains were considered: Saccharomyces boulardii CBS5926, Lactobacillus acidophilus LA3SACCO, Lactobacillus acidophilus LA-14, Lactobacillus plantarum ATCC 14917, Lactobacillus rhamnosus, Lactobacillus easel R0215, Lactobacillus helveticus R0052. The action of the essential oils of C. aurantium, M. didyma and C. zeylanicum was tested on probiotic strains according to the micro-broth dilution method as described under point A, with the following changes: the test was conducted using the BH (Bushnell and Haas) liquid medium given that it is more appropriate for the growth of the probiotics; the probiotic strains were incubated at 37°C for 24 hours under microaerophilic conditions.

Results: as observable from table 6, all probiotic strains, except Lactobacillus helveticus R0052, were sensitive at concentrations higher than the sub-MIC concentrations of our interest. In particular, all tested strains are sensitive at concentrations of EO of C. aurantium bitter variety higher than the concentration of 0.125% previously identified as most active on the strain of C. albicans.

Table 6. DAS: Department of Agricultural Sciences Department, University of Bologna; firm Pegaso srl (Arbizzano, VR).

Results of the in vitro test n° 1-6

The data of the in vitro tests n° 1-5 indicated above showed that Candida spp. and Saccharomyces cerevisiae are equally modulated by essential oils obtained from Monarda didyma, Citrus aurantium bitter variety and Cinnamomum zeylanicum. From among these essential oils, the Citrus aurantium bitter variety essential oil, when used at sub-MIC concentrations and tested on a mixture characterised by both genera (' Candida spp. and Saccharomyces cerevisiae), is capable of further inhibiting the Candida strains with respect to Saccharomyces cerevisiae. Based on the indications of Sokol et al (2016), i.e. that in patients suffering from IBD, the Saccharomyces cerevisiae fungal species reduces in percentage with respect to healthy subjects, while the Candida genus species increase in percentage even though they do not cause infectious diseases, the aforementioned characteristic of the essential oil obtained from Citrus aurantium bitter variety shows the capacity of a composition comprising said essential oil, according to the concentration (C) of the present invention, to restore the right microbiota balance, in particular the gut microbiota.

Furthermore, it was observed that the active concentrations of the essential oils are lower than the ones lethal for the probiotic strains subject of analysis and which correspond to the probiotic strains used for commercial formulations (in vitro test n° 6).

Lastly, the data of the in vitro test n° 6 indicated above show the immunomodulatory action on PBMC of the hydrolates obtained from Citrus aurantium bitter variety, Monarda didyma, Vitis vinifera Italian variety and Vitis vinifera Red globe variety.

In vitro test n° 7: synergic action of the essential oil of C. aurantium bitter variety and hydrolate of V. vinifera Italian variety

Purpose: To study the synergic action between the hydrolate of V. vinifera Italian variety (HY) and the essential oil of C. aurantium. bitter variety (EO)

In vitro test n° 7.1. a

Method: The strains of C. albicans and of S. cerevisiae were seeded at gradual concentrations of hydrolate (HY) (from 0.25% to 0.06%) and of essential oils (EO) (from 0.5% to 0.06%), as indicated in table 7, with the aim of testing the combinations thereof. All tests were conducted three times.

Table 7: NOTES: S: S.cerevisiae, C: C. Albicans. Example of plate used for he test.

Results: The combinations study showed that S. cerevisiae is inhibited as pertains to all concentrations of hydrolate when combined with the concentrations of essential oil of 0.5% and 0.25%, while at the concentrations of essential oil of 0.125% and 0.06% both S. cerevisiae and C. albicans are not inhibited in none of the 3 combinations examined with the hydrolate. Similarly, the absence of significant inhibition is observed in the co-cultures (C+S). Thus, combinations of essential oil comprised between 0.125% and 0.06% combined with concentrations of hydrolate comprised between 12.5% and 6.25% do not reveal significant inhibitions on the growth of each species (figure 7).

In vitro test n° 7.1.b

Figure 13: shows the results of the checkerboard BMD test of C. aurantium, bitter variety EO (from 0.50% v / v to 0.06% v / v) and V. vinifera cv Italia HY (from 25% v / v to 6.25% v / v) against C. albicans (clinical isolate 3.1) and S. cerevisiae (clinical isolate 14.3). As proven by the ANOVA test, concentrations equal to or lower than 0.12% v / v for EO, irrespectively of the concentration of ID, did not reveal any statistically significant inhibition for the two isolates, while for concentrations of EO higher than 0.12% v / v, the isolate of S. cerevisiae shows a statistically significant growth inhibition.

In vitro test n° 7.2

Method: 10pl of the content of the wells treated with essential oil of C. aurantium bitter variety 0.125% combined with the hydrolate of V. vinifera Italian variety (0.125% and 0.06%), as described in the in vitro test n° 7.1, were examined on chromagar so as to conduct the differentiated count of the CFUs of C. albicans and of S. cerevisiae.

Results: the data obtained from the CFU count confirms the observations subject of the combinations study, as shown in figure 8. No significant variations were observed in the growth of the treated strains as compared to those of the controls.

In vitro test n° 7.3

Method: an aliquot of the content of the wells treated with essential oil of C. aurantium bitter variety 0.125% combined with the hydrolate of V. vinifera Italian variety (0.125% and 0.06%), as described in the in vitro test n° 7.1, was observed under the optical microscope to study the fungal conformation.

Results: of particular interest is the data obtained from the microscopic analysis given that the morphology of the Candida fungi at the concentrations of our interest (essential oil 0.125% combined with hydrolate 0.125% and 0.06%) is strongly inhibited in the capacity of forming hyphae (figure 9). This action exercised on the strains of C. albicans would be such to inhibit the virulence with ensuing inhibition of the capacity of forming biofilms.

Tests conducted to identify the best combination of C. aurantium bitter variety EO and V. vinifera Italian variety HY proved that the serial dilution of a mixture obtained with a 1 : 100 ratio of the two natural products (starting from a concentration of 0.50% v / v of C. aurantium bitter variety EO and 50% v / v of V. vinifera Italian variety HY) caused an interesting inhibition of the capacity of the species of C. albicans to form hyphae (table 8). As stated in a recent review, the morphological plasticity of Candida albicans cells makes this fungus capable of adapting to the various niches present in the bowel of the guest organism. Generally, the production of hyphae and pseudohyphae of C. albicans is typical of virulent cellular models. The above matches what was observed on C. albicans isolated from patients suffering from IBS in whom there is found the gut microbiota characterised by greater abundance of cells of C. albicans, more virulent than in healthy subjects. In this context, the use of one of the effective serial dilutions based on C. aurantium bitter variety EO and V. vinifera Italian variety ID, capable of inhibiting the production of hyphae and pseudohyphae of strains of C. albicans makes the effectiveness of the composition according to the invention at treating IBS plausible.

In addition, no morphological alterations were observed for the tested S. cerevisiae. Furthermore, none of the concentrations of mixture EO / HY was capable of affecting the 7 isolated probiotics (Test in vitro n° 6 paragraph) and the isolated A muciniphila and F. prausnitzii (that can be potentially used as probiotics).

Table 8

In vitro test n° 7.4

The same serial dilution of C. aurantium bitter variety EO and V. vinifera Italian variety HY used in paragraph n° 7.3 has the capacity to linearly inhibit - depending on the concentration - the production of cytokines 11-10 and TNF-a from the PBMCs grown in the presence of pro-inflammatory stimulus (LPS) (figure 10). These activities on cytokines show the effectiveness of the composition of the invention in that they induce the reduction of the pro-inflammatory picture generally present in patients and capable, said pro-inflammatory picture, of facilitating the growth of the Candida species and promoting the creation of a micro-environment appropriate for the development of beneficial probiotic strains including A. muciniphila and F. prausnitzii recently proposed as markers of the IBD syndrome.

In particular, as illustrated in figure 10, it was evaluated whether the concentrations of the EO / HY mixture had immunomodulatory effect on human PBMCs if used combined with LPS and the levels of IL-10 and TNF-a were 0.64 ± 0.14 pg / mL and 9.00 ± 31 ,25 pg / mL, respectively, in the PBMCs treated with 50% v / v HY and 0.50% v / v EO (Fig. 10, letter i in the CTR circle); 119.00 ± 0.46 pg / mL and 977.91 ± 16.60 pg / mL, respectively, in the PBMCs treated with 25% v / v HY and 0.25% v / v EO; and 269.53 ± 4.07 pg / mL and 1901.90 ± 0 pg / mL, respectively, in the PBMCs treated with 12.50% v / v HY and 0.12% EO; 403.23 ± 37.65 pg / mL and 20143.01 ± 25.09 pg / m 316 L, respectively, in the PBMCs treated with 6.25 % v / v HY and 0.625% v / v EO (Fig. 10, letters I, m and n in Mix + LPS circle).

In vitro test n° 8

Purpose: to evaluate the toxicity of the single products and of the compound on human gingival fibroblast lines (obtained as described in Radicioni G. et al, Characterization of the cell penetrating properties of a human salivary proline-rich peptide. Biochim Biophys Acta. 2015; 1848: 2868-77).

Methods: after appropriate informed consent, human gingival fibroblasts were obtained from healthy patients subjected to gingivectomy of the molar region. Immediately after removal, the tissues were placed in HBSS solution with penicillin (250 U/mL), streptomycin 0.25 mg/mL) and amphotericin B (0.0025 mg/mL). The epithelial layer was mechanically detached and the sub-epithelial samples were plated in flasks with DMEM, supplemented with 50% FCS, L-glutamine (2mM), sodium pyruvate (1 mM), penicillin (50 UI/mL) and streptomycin (50 pg/mL) at 37°C in an atmosphere humidified to 5% in CO2. After the first passage, the cells were cultured in DMEM supplemented with 10% of FCS and they were not used beyond the fifth passage. The cytotoxicity of gradual dilutions of the essential oil mixture of C. aurantium bitter variety (concentrations comprised between 0.5% and 0.03%) and hydrolate of V. vinifera Italian variety (50% and 3.25), as well as dilutions of the essential oil or of the hydrolate alone for 24 and 48 hrs. with MTS assay (Promega, Madison, Wl, USA). The solutions were diluted in base medium. 1 x 10⁴ cells were resuspended in 200 ml of base medium and then plated in 96-well plates and cultured to confluence for 24 hrs. Cell viability was evaluated after adding the gradual dilutions described above at 37°C for 24 and 48 hrs. The MTS analysis was conducted according to the manufacturer's protocols. Each test was triplicated.

Results: As shown in figure 11 neither the action of the single components nor that of the mix is of the cytotoxic type, this to support the safety of the components as well as of the compositions of the invention when intended for human use. In vitro test n° 9

Purpose: to evaluate the composition of the mixture of essential oil of C. aurantium bitter variety (EO) and hydrolate of V. vinifera Italian variety (HY).

Method: GC-MS analysis conducted on EO and on the organic fraction of ID.

Results: the GC-MS analysis showed that the main component of EO is limonene (93.93%) and the main components of the HY are limonene (17.68%), cis-geraniol (25.40 %) and b-linalool (38.81%) (table 9). Thus, the mixture of essential oil of C. aurantium bitter variety and hydrolate of V. vinifera Italian variety according to the present invention seems appropriate for oral use in patients suffering from IBS in that, besides not showing in vitro cytotoxic activity (figure 11), it seems to mainly consist of monoterpene limonene given that all the other chemical constituents are present as traces (table 9). As known, limonene is designated as a compound with low oral toxicity based on the lethal dose thereof (LD50) and repeated dose toxicity trials.

Table 9: Chemical composition (%) of C. aurantium EO and V. vinifera HY

¹ Elution in polar column, ² LRI (linear retention index) measured on polar column; LRI^Mt: LRI from literature; 4 Normal alkane Rl; CA-EO= essential oil of_C. aurantium bitter variety, W-HY = hydrolate of V. vinifera Italian variety. The % W HY refer to fractions of organic components extracted from 50ml of hydrolate. Statistical analysis

The data indicated above was obtained triplicated by each test as mean ± standard deviation (SD). The response of S. cerevisiae and C. albicans at the concentration of EO of C. aurantium bitter variety was obtained through mathematical models, using the Sigma Plot 10 software (Systat Software Inc., Chicago, IL, USA). The most appropriate model was selected from among the linear decay and exponential function. The two respective equations are:

y = y₀ + ax (1)

y = a x exp < ^bx) (2)

where: yo, intercepts; a, slope (Eq. (1)), initial level (Eq. (2)); b, constant of speed.

The results obtained from the test of sensitiveness to broth micro-dilution (BMD) are indicated as relative values, assuming the mean of S. cerevisiae and C. albicans as the positive control (Ctrl +) = 100%. The data was processed using the GraphPad V 4.0 software. A two-way ANOVA test for clinical isolate factors (2 levels) and the combined treatments of EO-ID (13 levels included Ctrl +). The least significant difference (LSD) test at P < 0.05 was used to separate the levels of significant ANOVA sources. The CoStat 6.3 (CoHort Software, Berkeley, CA, USA) package was used for the ANOVA test and the subsequent LSD test. The results, obtained from the cytotoxicity trial, were expressed as mean viability percentage and the data processed using the GraphPad V 4.0 software. The means of the treated groups were compared by means of variance analysis (ANOVA) followed by a multiple comparison of the means using the Dunnett test. Values with p <0.05 were deemed significant.

EXPERIMENTAL TESTS SECTION 2

Effectiveness clinical trial of a composition according to the invention comprising (d) essential oil of Citrus aurantium and (h) hydrolate of Vitis vinifera (in short, combination in trial) in patients suffering from irritable bowel syndrome (IBS):

- Sponsor: Fondazione Policlinico Universitario A. Gemelli, Largo Agostino Gemelli 8, 00168 Roma RM.

- Type of trial: non-pharmacological, monocentric, randomised, double blind intervention pilot trial.

- Action mechanism of the composition subject of trial: facilitating the gut microbial balance of the patients.

- Primary goal of the trial: to evaluate the effect of the combination subject of trial when administered alone or combined with probiotics in patients suffering from IBS. -End-points: to evaluate whether the proposed treatment induces a beneficial variation of the following clinical parameters:

- Primary end-points:

1. Microbiota variation of the recruited patients (reduction of the microbial population of strains belonging to the Candida genus);

2. Change of clinical parameters (abdominal pain, constipation and/or diarrhoea)

- Secondary end-point:

1. Variation of the state of relative wellness of the patient.

- Inclusion criteria:

1. Patients capable of giving informed consent;

2. Patients suffering from IBS characterised by abdominal pain, who could be treated with probiotic therapy.

- Exclusion criteria:

1. minors below 18 years of age and people above 60 years of age;

2. pregnant or breastfeeding women;

3. serious concurrent diseases (cancer, type I or II diabetes mellitus, blood, renal or heart diseases and hyper - or hypothyroidism);

4. gastrointestinal surgery or abdominal surgery in the previous 6 months;

5. organic disorder of the small bowel (e.g. Ulcerative colitis, Crohn's disease);

6. treatment with antibiotics in the month prior to screening;

7. diagnosis of any medical condition related with constipation (different from IBS);

8. mental illness

At the time of registration, the following parameters must be absent:

1. rectal bleeding,

2. anaemia,

3. unexplainable weight loss,

4. Nocturnal diarrhoea and family anamnesis of organic gastrointestinal diseases (e.g. Colon cancer).

5. severe hypertension

- Effectiveness parameters:

- Primary end-point:

1. Reduction of colonisation of Candida spp in the treated group with respect to the placebo.

- Secondary end-points:

1. Improvement of the intestinal motility; 2. Improvement of the quality of life.

- Treatment:

GROUP I (14 pts): 10 days of treatment with the composition subject of trial +15 days of capsules (ops) of placebo probiotics

GROUP II (14 pts): 10 days of treatment with the composition subject of trial +15 days of probiotics (commercial product Candinorm manufactured by Pegaso srl based on Saccharomyces boulardii CNCM 1-3799 and Lactobacillus acidophilus LA-14 -ATCC SD5212).

GROUP III (14 pts): 10 days of placebo mixture (packaging containing water with citrus fruit-flavoured cap) + 15 days of placebo probiotics ops.

The patient suitable for the inclusion/exclusion criteria must take: 1 vial of composition subject of trial in the evening before going to bed to be taken with a little amount of water, and 2 probiotics ops (1 one in the morning and one in the evening away from meals) for a total of 15 days, and follow a correct diet as instructed.

- Study design:

This is a non-pharmacological, monocentric, randomised, double blind intervention pilot trial, for evaluating the effectiveness of the composition subject of study in 42 patients suffering from irritable bowel syndrome (IBS). Eligible patients based on the inclusion / exclusion criteria were randomly allocated to one of the three groups, at a 1 : 1 : 1 ratio.

The trial envisages 4 check-ups:

(a) Screening check-up (VI, day 0)

(b) V2 check-ups (days 10)

(c) V3 check-ups (days 15, end of treatments)

(d) V4 check-up (end of trial).

(a) Screening check-up (VI):

Full patient admissibility evaluation, based on compliance with the inclusion / exclusion criteria of the trial, is conducted prior to the administration of the informed consent. Once the patient has accepted to take part in the trial and once the informed consent form has been signed, the patient is recruited and the tester starts to conduct the following specific trial procedures:

- Collection of demographic data, including date of birth and sex

- Physical examination and vital signs recording including the height, weight, blood pressure, heart rate.

- Collection of anamnesis data.

- Collection of data on concurrent treatments. - Collection of faecal samples.

- Pregnancy test.

- Dispense of natural and probiotic products,

- IBS-SSS.

- IBS-QoL.

- AE report.

- randomisation.

A unique subject identification number is allocated to all subjects who have signed the informed consent.

(b) Check-up 2 (days 10)

The following procedures and evaluations are conducted during these check-ups:

- Telephone interview.

- End of treatment with natural products.

- AE report (if present).

(c) Check-up 3 (days 15)

The following procedures and evaluations are conducted during these check-ups:

- Collection of faecal samples.

- IBS-SSS.

- IBS-QoL.

- End of treatments with probiotics

- AE report.

(d) Check-up 4 (days 30, end of study)

The following procedures and evaluations are conducted during these check-ups:

- Collection of faecal samples.

- IBS-SSS.

- IBS-QoL.

- AE report.

- Returning / responsibility of natural and probiotic products.

- Duration of the trial:

The trial lasts for 9 months. The registration period lasts 6 months and the treatment period lasts 15 days. - Data analysis and data statistics:

- Determination of sample size

Given that the prevalence of IBS, in Italy, varies between 5 and 10% (Lovell RM. 2017, P. Enrk, 2016), the sample group should be made up of 42 patients to be divided into 3 groups (14 patients per group). The choice of the sample size is acceptable for a pilot clinical trial with the aim of having a force equivalent to 95% and significance equivalent to 0.09.

- Statistical analysis method

Descriptive statistics were conducted with the aim of describing the population subject of study. The continuing variables are described as mean and standard deviation or as median interval and interquartile, where deemed appropriate. The category variables are described as number and percentages. The missing data is described in all summaries. Change of clinical parameters prior to and after treatment is compared using a t-test for paired groups or a Wilcoxon signed-rank test, where deemed appropriate.

The variation of the subjective state of wellness of the patient prior to and after the treatment is compared through a McNemar test.

The microbiota variation of the recruited patients is evaluated using the Linear Discriminant Analysis (LDA) Effect Size (LEfSe) method, a tool for identifying and interpreting biomarkers on large data sets, focusing on metagenomics. LEfSe corresponds to a test for the identification of taxa, whose abundance is significantly different among the classes taken into account for elements capable of verifying the biological consistency within single classes. The dimension of the effect of each otherwise abundant taxa is subsequently measured using linear discriminant analysis (LDA). All the tests are on two sides and a p value smaller than 0.05 is deemed statistically significant.

- Randomisation:

Randomisation is conducted by means of a GCP-compliant Web-based and centralised randomisation.

The process is of the double blind type, with identical appearance active treatments. Eligible patients were randomised during the screening check-up, the patients are randomly allocated at a 1 : 1 : 1 ratio to receive treatments. The list of treatment codes is generated using a computer programme at the coordination unit. Packaging with the active ingredients is conducted blindly according to the requirements pursuant to Annex 13 "Test medicinal products" EU guidelines of good manufacturing practices for medicinal products for human and veterinary use).

Claims

1. A composition (C) comprising

(i) a mixture (M) comprising or, alternatively, consisting of an essential oil obtained from a plant or from at least one part of a plant identified as (d) Citrus aurantium, bitter variety, and a hydrolate obtained from a plant or from at least one part of a plant identified as (h) Vitis vinifera Italian variety,

and, optionally,

(ii) at least one pharmaceutical or food grade additive and/or excipient.

2. The composition (C) according to claim 1, wherein said essential oil obtained from (d) Citrus aurantium, bitter variety, and said hydrolate obtained from (h) Vitis vinifera Italian variety are at a (d):(h) by weight ratio comprised in the range from 1 :200 to 1 :10, preferably from 1 :150 to 1 :50, more preferably about 1 100

3. The composition (C) according to claim 1 or 2, wherein said essential oil obtained from (d) is present at a concentration at a volume amount comprised from 0.01% to 1%, preferably comprised from 0.06% to 0.25%, more preferably comprised from 0.06% to 0.125%, and said hydrolate obtained from (h) is present at a concentration at a volume amount comprised from 1% to 25%, preferably comprised from 6.25% to 12.5%, wherein said amounts are with respect to the total volume of the composition (C).

4. The composition (C) according to any one of claims 1-3, wherein said composition (C) is for use as a medicament.

5. The composition (C) for use according to claim 4, wherein said composition (C) is for use in a method for the preventive and/or curative treatment of the symptoms or diseases caused by or related with a functional gastrointestinal disorder or caused by or related with an infection or an inflammation of the gastrointestinal tract.

6. The composition (C) for use according to claim 5, wherein said composition (C) is for use in a method for the preventive and/or curative treatment of the symptoms or diseases caused by or related with irritable bowel syndrome (IBS) or caused by or related with chronic inflammatory bowel diseases (IBD) or temporary inflammatory bowel diseases.

7. The composition (C) for use according to claim 6, wherein said chronic inflammatory bowel diseases (IBD) are selected from among adverse reactions to foodstuffs or other food or pharmaceutical grade substances.

8. The composition (C) for use according to claim 4, wherein said composition (C) is for use in a method for the preventive and/or curative treatment of the symptoms or diseases caused by or related with a functional genitourinary disorder or caused by or related with an infection or inflammation of the genitourinary tract.

9. The composition (C) for use according to claim 8, wherein said composition (C) is for use in a method for the preventive and/or curative treatment of the symptoms or diseases caused by or related with candidosis or caused by or related with cystitis.

10. The composition (C) for use according to claim 8 or 9, wherein said composition (C) is for use in a method for the preventive and/or curative treatment of the symptoms or diseases caused by or related with at least one pathogenic agent belonging to the Candida genus; more preferably, said pathogenic agent belongs to the Candida albicans species.

11. The composition (C) for use according to any one of claims 5-10, wherein said composition (C) is for use in a method for the preventive and/or curative treatment of said symptoms or diseases as a coadjuvant.

12. A pharmaceutical composition, medical device composition, dietary supplement or food product comprising the composition (C) according to any one of claims 1 to 3.