Kit for the treatment and / or prevention of human intestinal dysfunctions

Abstract

A kit of products for the treatment or prevention of intestinal dysfunctions comprising a first product comprising at least 40% catechins, wherein at least 60% of said catechins is formed by (–) epigallocatechin-3-gallate (EGCG), and a second product comprising a mixture of probiotics having a bacterial load of at least 5 billion CFU / g.

Description

[0001] Kit for the treatment and / or prevention of human intestinal dysfunctions

[0002] Description

[0003] Technical field

[0004] The present invention relates to a kit for the treatment and / or prevention of human intestinal dysfunctions. The present invention also relates to a kit for maintaining and / or restoring human intestinal functions. It is further directed to the use of said kit in the prevention and treatment of intestinal inflammations and / or in the maintenance of intestinal functions, in particular in the prevention or treatment of constipation and / or sluggish bowel.

[0005] The present invention finds a preferred, although not exclusive, application in the field of food supplements based on dry extract of green tea leaves. An example of green tea leaves used are those of the species Camellia Sinensis L. Kuntze, to which reference may hereinafter be made without loss of generality.

[0006] Prior art

[0007] In this technical field, food supplements based on catechins are known, which are polyphenols present in tea with antioxidant biochemical properties, that is, capable of protecting cells from damage caused by free radicals.

[0008] In particular, the beneficial properties attributed to epigallocatechin gallate (EGCG) from green tea are numerous: antioxidant properties; antitumor properties; it is beneficial for the cardiovascular system and has neuroprotective effects. Furthermore, several studies on catechins, in particular on epigallocatechin, have investigated the anti-inflammatory properties of the gastrointestinal tract and their beneficial effects on metabolic diseases

[0001] [2], Irritable bowel syndrome with constipation (IBS-C) is a multifactorial functional condition in which alterations of motility, visceral hypersensitivity, dysbiosis, gut-brain axis dysregulation and, in some subgroups, possible changes in the intestinal barrier coexist. The most impactful symptoms include abdominal pain or discomfort, bloating and distension, irregular bowel movements, sensation of incomplete evacuation and worsening of quality of life.

[0009]

[0010] of the fi

[0011] Figure 1. Representation of the layout of the clinical study.

[0012] Figure 2. Comparison between the groups treated with the supplement and with placebo for the 14 variables examined in this study measured at times tO, tl, t2 and t3 as predicted by the LMM models (means and 95% confidence intervals).

[0013] Figure 3. Variation in the frequency of use of rescue systems in the experimental groups (supplement and placebo) in the four scheduled measurements of the study.

[0014] Figure 4. Intestinal colonization of L. rhamnosus in subjects belonging to the treated group (B) and to the placebo group (A) at the different experimental times (TO, T2, T3).

[0015] Detailed description of the invention

[0016] In the present description as well as in the claims appended thereto, certain terms and expressions are intended to assume, unless otherwise explicitly indicated, the meaning expressed in the definitions that follow.

[0017] The term “flavonoids” refers to the set of nutrients that are a source of antioxidant and antiaging substances, capable of counteracting the harmful action of free radicals. In particular, reference is made to the class of flavanols and specifically to catechins.

[0018] The term “kit” means a set composed of at least two products that are packaged and sold together, preferably in a single package. The term “intestinal dysfunctions” means disorders of the gut-brain interaction and includes: Functional Dyspepsia (FD); Irritable Bowel Syndrome (IBS); Functional Abdominal Discomfort; Defecation Disorders; Gastrointestinal Motility Disorders; other non-specific functional gastrointestinal disorders.

[0019] The term probiotics refers to live microorganisms which, when administered in adequate amounts, confer a health benefit on the host (FAO / WHO 2001). They may be used as a synonym for bacteria and may also include fungi in addition to bacteria, for example Saccharomyces boulardii.

[0020] The Applicant has preliminarily observed that the etiological concept of chronic metabolic disease has shifted from tissue- and organ-specific mechanisms to gut-centered pathologies, which include conditions such as mucosal inflammation, intestinal barrier dysfunction, bileacid metabolism and, above all, alterations in intestinal microbial abundance, diversity and metabolic functions. In particular, reciprocal intestinal interactions between the gut microbiome, metabolome and metabolism have emerged as a critical determinant of human health and disease in general [3],

[0021] In particular, the Applicant has verified that such an occurrence becomes highly probable whenever there is an alteration of the intestinal bacterial flora.

[0022] The Applicant has also verified how the intake of flavonoids, in particular catechins derived from green tea, in particular from Camellia Sinensis L. Kuntze, has the anti-inflammatory and metabolic support effect discussed above.

[0023] The Applicant has however observed that the intake of flavonoids alone cannot compensate for dysfunctions due to alterations of the bacterial flora.

[0024] The Applicant has verified the prebiotic role of catechins and their metabolites in increasing beneficial bacteria in the intestine (Lactobacilli and Bifidobacteria), in modulating the immune response, in exerting an anti-inflammatory and antioxidant action at the level of the intestinal epithelium, in exerting a selective bacteriostatic action against pathogenic bacteria and in improving the integrity of the intestinal barrier.

[0025] The Applicant has therefore understood that the combination of catechins and probiotics has a synergistic effect that contributes to the maintenance of intestinal homeostasis, promoting the treatment of gut-brain interaction disorders, including Functional Dyspepsia (FD), Irritable Bowel Syndrome (IBS), Functional Abdominal Discomfort, Defecation Disorders, Gastrointestinal Motility Disorders and other non-specific functional gastrointestinal disorders.

[0026] The Applicant has finally found that products containing catechins could be optimized by combining them with a product containing probiotics, so as to form a kit having a synergistic effect generated by the anti-inflammatory and antioxidant effect of catechins and the regulatory effect provided by probiotics, which also tend to promote the properties of catechins, thus obtaining a greater beneficial effect compared to the effect obtained by the independent intake of catechins or probiotics.Such a solution makes it possible to address a complex clinical picture through the combined intake of products of a single kit, so that catechins and probiotics mutually enhance the beneficial effect on the user.

[0027] Therefore, in a first aspect thereof, the present invention is directed to a kit for the treatment or prevention of intestinal dysfunctions.

[0028] Preferably, said kit comprises a first product.

[0029] Preferably, said first product comprises at least 40% catechins. Preferably, at least 60% of said catechins is formed by (-)-epigallocatechin-3 -gallate (EGCG).

[0030] Advantageously, EGCG comprises one more hydroxyl group compared to other tea catechins, making EGCG a more active antioxidant than other catechins.

[0031] It has been found that epigallocatechin gallate (EGCG) is the most effective among tea catechins as a scavenger of the superoxide anion, the hydroxyl radical and the 1,1-diphenyl-3-picrylhydrazyl radical [4],

[0032] Preferably, said kit comprises a second product.

[0033] Preferably, said second product comprises a mixture of probiotics having a bacterial load of at least 5 billion CFU / g.

[0034] Thanks to these characteristics, catechins and probiotics interact producing a synergistic therapeutic effect better than the intake of catechins alone or probiotics alone.

[0035] In at least one of the aforementioned aspects, the present invention may further present at least one of the preferred characteristics set forth below.

[0036] Preferably, said first product comprises at least 50%. Preferably, said first product comprises between 50% and 60% of said catechins.

[0037] Preferably, said catechins comprise at least 70% of said EGCG.

[0038] Preferably, said kit comprises at least 50% dry extract of green tea leaves.

[0039] Preferably, said kit comprises between 50% and 80%, more preferably 60%, of dry extract of green tea leaves.

[0040] Advantageously, the main components of green tea polyphenols are flavanols (or catechins), among which the most abundant are (-)-epigallocatechin-3 -gallate (EGCG), (-)-epicatechin(EC), (-)-epicatechin-3 -gallate (ECG) and (-)-epigallocatechin (EGC) [5], EGCG represents more than 50-60% of all green tea catechins. It is the catechin containing the highest number of hydroxyl groups and therefore a greater antioxidant capacity [6],

[0041] According to some embodiments, the dry extract of green tea leaves is of the species Camellia Sinensis L. Kuntze.

[0042] Advantageously, the species Camellia Sinensis L. Kuntze is the species with the highest quantity of catechins and in particular EGCG [7],

[0043] Preferably, said second product comprises said mixture of probiotics having a bacterial load of at least 6 billion CFU / g.

[0044] Preferably, said second product comprises said mixture of probiotics having a bacterial load between 6 billion CFU / g and 15 billion CFU / g, more preferably 12 billion CFU / g.

[0045] Preferably, said first product comprises between 100 mg and 600 mg of catechins.

[0046] Preferably, said catechins comprise (-)-epigallocatechin-3 -gallate (EGCG) between 80 mg and 550 mg.

[0047] Preferably, said second product comprises between 100 mg and 600 mg of probiotic mixture to promote and maintain intestinal functions.

[0048] Preferably, said catechins are between 200 mg and 350 mg, more preferably 270 mg.

[0049] Preferably, of said catechins, EGCG is between 190 mg and 300 mg, more preferably 195 mg. Preferably, said dry green tea extract is between 200 mg and 800 mg, more preferably 300 mg. Preferably, said first product has a weight between 100 mg and 1000 mg, preferably 500 mg. Preferably, said second product has a weight between 100 mg and 1000 mg, preferably 500 mg.

[0050] In some embodiments, said catechins are selected from the group consisting of:

[0051] (-)-epigallocatechin-3 -gallate (EGCG),

[0052] (-)-epigallocatechin (EGC),

[0053] (-)-epicatechin-3 -gallate (ECG),

[0054] (+)-gallocatechin-3 -gallate (GCG),

[0055] (-)-epicatechin (EC),(+)-gallocatechin (GC),

[0056] (+)-catechin (C),

[0057] and / or combinations thereof.

[0058] Preferably, said probiotics belong to the genus Lactobacillus and / or Bifidobacterium.

[0059] Preferably, each genus is between 2 billion CFU and 4 billion CFU, more preferably 2 billion CFU.

[0060] Preferably, one or more of said probiotics are selected from the group consisting of Bifidobacterium animalis subspecies lactis BL050, Lactobacillus rhamnosus LRH020, Lactobacillus plantarum PBS067 and / or combinations thereof.

[0061] Preferably, each probiotic is between 2 billion CFU and 4 billion CFU, preferably 2 billion CFU.

[0062] Alternatively, said probiotics are selected from the group consisting of: Bacillus coagulans GBI-30, 6086, Bacillus subtilis var. nattb, Bifidobacterium, Bifidobacterium sp., Bifidobacterium bifidum, Bifidobacterium bifidum Rosell-71, Bifidobacterium breve, Bifidobacterium breve Rosell-70, Bifidobacterium infantis, Bifidobacterium lactis, Bifidobacterium longum, Bifidobacterium longum Rosell-175, Bifidobacterium animalis, Bifidobacterium animalis subsp. lactis BB-12, Bifidobacterium animalis subsp. lactis HN019, Bifidobacterium infantis 35624, Escherichia coli M-17, Escherichia coli Nissle 1917, Lactobacillus acidophilus, Lactobacillus acidophilus LAFTI LIO, Lactobacillus casei, Lactobacillus casei LAFTI L26, Lactobacillus brevis, Lactobacillus bulgaricus, Lactobacillus gasseri, Lactobacillus paracasei, Lactobacillus plantarum, Lactobacillus reuteri ATCC 55730 (Lactobacillus reuteri SD2112), Lactobacillus rhamnosus, Lactobacillus salivarius, Lactobacillus delbrueckii, Lactobacillus fermentum, Lactococcus lactis, Lactococcus lactis subsp., Lactococcus lactis Rosell-1058, Lactobacillus paracasei Stll (or NCC2461), Lactobacillus fortis, Lactobacillus johnsonii Lal (= Lactobacillus LCJ, Lactobacillus johnsonii NCC533), Lactobacillus rhamnosus Rosell-11, Lactobacillus acidophilus Rosell- 52, Streptococcus thermophilus, Diacety lactis, Saccharomyces cerevisiae and / or combinations thereof.Advantageously, probiotics are able to produce metabolites with antipathogenic activity, counteract the adhesion of pathogens to the intestinal mucosa, modulate the immune response, reduce gastrointestinal discomfort, restore the integrity of the intestinal barrier, exert an antiinflammatory action and produce neurohormones.

[0063] With regard to green tea, numerous studies have highlighted the prebiotic role of catechins and their metabolites in increasing beneficial bacteria in the intestine (Lactobacilli and Bifidobacteria), in modulating the immune response, in exerting an anti-inflammatory and antioxidant action at the level of the intestinal epithelium, in exerting a selective bacteriostatic action against pathogenic bacteria and in improving the integrity of the intestinal barrier. Preferably, said first product comprises a capsule shell.

[0064] Preferably, said first product comprises a gelling agent.

[0065] Preferably, said first product comprises a bulking agent.

[0066] Preferably, said first product comprises an anti-caking agent.

[0067] Preferably, said first product comprises a colorant.

[0068] Preferably, said second product comprises a capsule shell.

[0069] Preferably, said second product comprises a gelling agent.

[0070] Preferably, said second product comprises a bulking agent.

[0071] Preferably, said second product comprises an anti-caking agent.

[0072] Preferably, said capsule shell comprises hydroxypropyl methylcellulose.

[0073] Advantageously, hydroxypropyl methylcellulose is gastro-resistant. Gastro-resi stance allows the active ingredient to exert a localized action at the intestinal level.

[0074] Preferably, said gelling agent comprises pectin and / or corn starch and / or gellan gum.

[0075] Advantageously, pectin in the capsule shell performs a gelling action.

[0076] Advantageously, corn starch stabilizes the formulation.

[0077] Preferably, said bulking agent comprises cellulose.

[0078] Advantageously, cellulose stabilizes the formulation.

[0079] Preferably, said anti-caking agent comprises silicon dioxide and / or magnesium salts of fatty acids.Advantageously, silicon dioxide and magnesium salts of fatty acids exert an anti -caking action so as to preserve the extract powder and prevent agglomerates from forming during production.

[0080] Preferably, said colorant comprises copper complexes of chlorophylls and / or iron oxides and / or hydroxides and / or patent blue V.

[0081] Advantageously, copper complexes of chlorophylls impart the green color to the capsule. According to some embodiments, said kit is used for the prevention and treatment of intestinal inflammations and / or for the maintenance of intestinal functions.

[0082] According to some embodiments, said kit is used for the prevention and / or treatment of gutbrain interaction disorders, in particular irritable bowel associated with constipation.

[0083] Preferably, said first product is in the form of a capsule, tablet or powder.

[0084] Preferably, said second product is in the form of a capsule, tablet or powder.

[0085] Preferably, said capsule or tablet has a weight of 250 mg, 500 mg or 1000 mg, more preferably 500 mg.

[0086] Preferably, said first product is green in color.

[0087] Preferably, said second product is white in color.

[0088] According to some embodiments, said first and second products are taken simultaneously or in rapid succession.

[0089] In this manner, said first and second products are mixed when in use.

[0090] Advantageously, substantially simultaneous intake allows combined intake of the products of the kit in a single moment, avoiding incorrect intake due to forgetfulness. Furthermore, in the case of an acute phase of intestinal dysfunctions, in particular constipation, a timely synergistic action between the first and second product is possible.

[0091] Alternatively, said first product is to be taken in the morning and said second product is to be taken in the evening.

[0092] This type of administration is more suitable for chronic situations; in fact, by taking said first product in the morning, the intestine is prepared by reducing inflammation and creating an environment favorable to bacterial proliferation, so that when the second product is taken,probiotics can proliferate and promote the anti-inflammatory and antioxidant effect of the catechin contained in the first product.

[0093] Preferably, said first and second products are taken for at least one week, more preferably for at least four weeks.

[0094] Preferably, said kit is suitable for intake by persons suffering from gut-brain interaction disorders, including Functional Dyspepsia (FD), Irritable Bowel Syndrome (IBS), Functional Abdominal Discomfort, Defecation Disorders, Gastrointestinal Motility Disorders and other non-specific functional gastrointestinal disorders.

[0095] The characteristics and advantages of the invention will be better apparent from the detailed description of a preferred embodiment thereof.

[0096] With reference to the preferred embodiment described herein, made in accordance with the present solution, a kit of products for the treatment or prevention of intestinal dysfunctions comprises a first product having a weight of 500 mg and comprising 300 mg of dry extract of green tea leaves, of which 270 mg are catechins, of which 195 mg are EGCG.

[0097] The kit further comprises a second product having a weight of 500 mg and comprising 6 billion CFU of a probiotic mixture.

[0098] According to the present preferred embodiment, the green tea is of the species Camellia Sinensis L. Kuntze.

[0099] Furthermore, the probiotic mixture comprises 2 billion CFU of Bifidobacterium animalis subspecies lactis BL050, 2 billion CFU of Lactobacillus rhamnosus LRH020 and 2 billion CFU of Lactobacillus plantarum PBS067.

[0100] The first product also comprises hydroxypropyl methylcellulose, gellan gum, com starch, cellulose, silicon dioxide, magnesium salts of fatty acids and iron oxides and / or hydroxides and / or patent blue V.

[0101] The second product also comprises hydroxypropyl methylcellulose, pectin, com starch, silicon dioxide and magnesium salts of fatty acids.

[0102] The first product is in the form of a green capsule and the second product is in the form of a white capsule.According to the preferred embodiment described, the first product is to be taken in the morning and the second product is to be taken in the evening.

[0103] Advantageously, this type of administration is more suitable for chronic situations; in fact, by taking said first product in the morning, the intestine is prepared by reducing inflammation and creating an environment favorable to bacterial proliferation, so that when the second product is taken, probiotics can proliferate and promote the anti-inflammatory and antioxidant effect of the catechin contained in the first product.

[0104] According to other embodiments, in order to treat and / or prevent intestinal dysfunctions, the first and second products are mixed when in use.

[0105] To the above-described kit for treating and / or preventing intestinal dysfunctions, a person skilled in the art, in order to satisfy further and contingent needs, may make numerous further modifications and variants, all of which are in any case included within the scope of protection of the present invention, as defined by the appended claims.

[0106] Examples

[0107] An efficacy study was carried out to demonstrate the usefulness of the food supplement based on a green tea extract (Camellia sinensis (L.) Kuntze - folium) and a pool of probiotics (L. plantarum, L. rhamnosus and A animalis subsp. lactis) for the management of gastrointestinal discomfort and for the maintenance of intestinal function balance in subjects presenting IBS- C.

[0108] In particular, the clinical study aimed to evaluate the efficacy of the food supplement on gastrointestinal discomfort through the use of validated questionnaires measuring subjective global symptomatology, as a target recommended by the guidelines of the competent Authority (EF SA ND A Panel, 2016. Guidance on the scientific requirements for health claims related to the immune system, the gastrointestinal tract and defence against pathogenic microorganisms. EFSA Journal 2016; 14(1):4369, 23 pp. doi:10.2903 / j.efsa.2016.4369). Gastrointestinal discomfort is a target recommended by the competent Authority for efficacy studies on food supplements.A monocentric, randomized, controlled, parallel-arm, double-blind clinical study was conducted, with a treatment period of 56 days and a follow-up period lasting 28 days after the end of treatment.

[0109] Before being included in the study, subjects deemed eligible by the investigator were provided with:

[0110] 1) the information sheet, with clear information regarding the clinical study, the objectives and the implementation methods;

[0111] 2) the informed consent, which was completed and signed by the subjects and countersigned by the investigator in duplicate;

[0112] 3) the information and consent to the processing of personal data;

[0113] 4) the daily bowel function diary.

[0114] In order to ensure maintenance of an adequate sample size until the end of the study, it was planned to enroll approximately 15% more subjects than indicated by the power analysis (total 64 subjects).

[0115] Subjects of both sexes were included in the study:

[0116] - aged between 18 and 70 years;

[0117] - able to understand and sign the informed consent;

[0118] - negative HIV test;

[0119] - negative pregnancy test;

[0120] - with IBS symptoms for at least 3 months (with onset at least 6 months earlier), characterised by recurrent abdominal pain at least 1 day per week, associated with two or more of the following criteria: associated with defecation, associated with a change in stool frequency, associated with a change in stool form (Rome IV diagnostic criteria);

[0121] - presenting fewer than three spontaneous complete bowel movements per week and at least one of the following conditions: straining in more than 25% of defecations, lumpy or hard stools (BSFS type 1 or 2) in more than 25% of defecations, sensation of incomplete evacuation in more than 25% of defecations, sensation of anorectal obstruction / blockage in more than 25% of defecations, or use of manual maneuvers to facilitate evacuation in morethan 25% of defecations;

[0122] - not taking any type of medication throughout the study period (except rescue treatments); - able to understand and comply with the protocol requirements.

[0123] The experimental groups were as follows:

[0124] GROUP 1 (32 subjects): subjects who were required to take the food supplement consisting of a green tea extract (Camellia sinensis (L.) Kuntze - folium) and a pool of probiotics (L. plantarum, L. rhamnosus and B. animalis subsp. lactis).

[0125] GROUP 2 (32 subjects): subjects who were required to take the placebo.

[0126] Each subject in both experimental groups, before, during and after administration of the food supplement or placebo, underwent evaluation of the primary and secondary objectives.

[0127] Primary objective

[0128] Evaluation of the efficacy of the food supplement in improving the overall gastrointestinal symptomatology perceived, as recommended by EF SA guidelines (EFSANDAPanel, 2016 Guidance on the scientific requirements for health claims related to the immune system, the gastrointestinal tract and defence against pathogenic microorganisms. EFSA Journal 2016; 14(1):4369, 23 pp. doi:10.2903 / i.efsa.2016.4369).

[0129] Evaluation method: Validated questionnaire for self-assessment of the severity of global gastrointestinal symptomatology characterizing IBS, the IBS-Severity Scoring System (IBS- SSS) [Irvine et al., 2006],

[0130] The questionnaire consists of five items referring to:

[0131] - abdominal pain,

[0132] - bowel dysfunction,

[0133] - general well-being.

[0134] It is commonly used as an outcome measure in clinical efficacy studies due to its high sensitivity following treatment. The total score obtained allowed determination of the severity of global gastrointestinal symptomatology, whether IBS-related symptoms were mild, moderate or severe, and whether following treatment there was an improvement in global symptomatology in the treated group compared to the placebo group.Secondary objectives

[0135] 1) Impact of constipation on perceived quality of life in the last 4 weeks

[0136] Evaluation methods: Quality of life questionnaire (Short Form Health Survey-12 - SF-12), developed in a multi-year study on patients with chronic diseases. It is a validated questionnaire widely used in clinical practice for self-assessment of quality of life with respect to a generic disorder. The SF-12 questionnaire describes a subject’s health through two summary indices calculated from 12 questions. The index called Physical Component Summary (PCS) concerns physical status, while the index Mental Component Summary (MCS) measures mental status.

[0137] 2) Changes in stool consistency

[0138] Evaluation methods: Bristol Stool Form Scale (BSFS), a validated tool that assesses stool consistency across a spectrum of seven types. Stool types 1 and 2 indicate hard or lumpy stools, while stool types 6 and 7 indicate loose or watery stools. Stools characterizing subjects with intestinal hyperactivity are types 5, 6 and 7. Stool consistency was reported by the subject at visits and recorded during the study period in the bowel function diary

[0139] 3) Changes in bowel movement frequency

[0140] Evaluation methods: Number of bowel movements per week (BM / WK), reported by the subject at visits (average BM / week in the previous four weeks) and recorded during the study period in the bowel function diary.

[0141] 4) Changes in frequency and intensity of symptoms characteristic of IBS

[0142] Evaluation methods: Evaluation of frequency and intensity of symptoms characteristic of constipation-predominant IBS (sensation of bloating, abdominal distension, sensation of fullness, abdominal pain, flatulence) was determined through completion of a 5 -point Likert scale (0 = no discomfort - 5 = maximum discomfort), included in the bowel function diary. Daily recording was useful to avoid recall bias that could occur during questionnaire completion at scheduled visits.

[0143] 5) Changes in frequency of use of rescue treatmentsEvaluation methods: Frequency of use of rescue treatments, such as medications for bowel function, laxatives or antispasmodics, was recorded through completion of the bowel function diary.

[0144] 6) Evaluation of abdominal pain separately from intestinal discomfort

[0145] According to the recommendations reported by Irvine et al. (2016).

[0146] Evaluation methods: Visual Analogue Scale (VAS). This is a pain self-assessment tool used for various purposes and in the evaluation of generic pain in patients presenting to emergency departments. It consists of a 10-cm paper strip with the two endpoints “no pain” and “worst pain imaginable”; the patient marks on the strip the level of pain perceived.

[0147] 7) Evaluation of serotonin, gastrin and cortisol

[0148] Evaluation methods: Investigation carried out on blood samples.

[0149] 8) Evaluation of intestinal inflammatory status through analysis of fecal inflammatory markers calprotectin and zonulin.

[0150] Evaluation methods: Investigation carried out on stool samples. This analysis was performed on a pool of 30 subjects, 15 from the treatment group and 15 from the placebo group.

[0151] 9) Probiotic colonization rate

[0152] Evaluation methods: Relative abundance of microbial species present in the treatment (L. plantarum, L. rhamnosus and B. animalis subsp. lactis) to evaluate the colonization ability of the treatment under study. Investigation carried out on stool samples. This analysis was performed on a pool of 30 subjects, 15 from the treatment group and 15 from the placebo group.

[0153] Composition under study

[0154] The treatment consisted of the supplement Teakine IBS, notified to the Ministry of Health (notification number: 194721).

[0155] Dosage: 2 capsules per day, of which:

[0156] - one capsule based on green tea extract (Camellia sinensis (L.) Kuntze) leaves dry extract, standardized, and- the second capsule based on probiotics (Lactobacillus plantarum PBS067, Lactobacillus rhamnosus LRH020, Bifidobacterium lactis BL050), to be taken in the morning.

[0157] Ingredients of the green tea extract-based capsule:

[0158] Dry green tea extract (Camellia sinensis (L.) Kuntze), leaves, standardized to 90% total catechins, 65% EGCG; capsule (hydroxypropyl methylcellulose, gelling agent: gellan gum (E418), colorants: iron oxides and hydroxides (E172), patent blue V (E131)); corn starch; bulking agent: microcrystalline cellulose, cellulose gel (E460(i)); anti-caking agents: silicon dioxide (E551), magnesium salts of fatty acids (E470b).

[0159] Specifications: _ _

[0160] Active dose per 300 mg dry green tea leaf extract

[0161] capsule per capsule, of which 270 mg total

[0162] catechins and 195 mg EGCG

[0163]

[0164] Dosage form 500 mg capsule

[0165] One probiotic-based capsule to be taken in the evening before bedtime with a glass of water.

[0166] Ingredients of the probiotic-based capsule:

[0167] Corn starch; capsule (hydroxypropyl methylcellulose, gelling agent: pectins (E440));

[0168] Lactobacillus rhamnosus LRH020; Bifidobacterium animalis subsp. lactis BL050;

[0169] Lactobacillus plantarum PBS067; anti-caking agents: silicon dioxide (E551), magnesium salts of fatty acids (E470b).

[0170] Specifications: _ _

[0171] Lactobacillus Actual intake: 33.3 mg equal to 10 billion per capsule rhamnosus (containing 5 x overage)

[0172] LRH020 300 Intake to be reported on label: 2 billion per capsule Mid CI Lfi

[0173] Bifidobacterium Actual intake: 33.3 mg equal to 10 billion per capsule lactis BL050 (containing 5 x overage)

[0174] 300 Intake to be reported on label: 2 billion per capsule

[0175]

[0176] Mid CI LfiLactobacillus Actual intake: 20 mg equal to 10 billion per capsule (containing plantarum 5* overage)

[0177] PBS067 Intake to be reported on label: 2 billion per capsule

[0178] 500 Mid CFU / g

[0179] Forma di 500 mg capsule

[0180]

[0181] dosaggio

[0182] Placebo composition

[0183] Dose: two placebo capsules per day.

[0184] Dosage: two capsules per day as described above for the treatment.

[0185] Ingredients of capsule A:

[0186] Corn starch; capsule (hydroxypropyl methylcellulose, gelling agent: gellan gum (E418), colorants: iron oxides and hydroxides (E172), patent blue V (E131)); bulking agent: microcrystalline cellulose, cellulose gel (E460(i)); anti-caking agents: silicon dioxide (E551), magnesium salts of fatty acids (E470b).

[0187] Ingredients of capsule B:

[0188] Corn starch; capsule (hydroxypropyl methylcellulose, gelling agent: pectins (E440)); anticaking agents: silicon dioxide (E551), magnesium salts of fatty acids (E470b).

[0189] Study duration and experimental design

[0190] The total duration of the study was approximately 5 months, specifically 1 month for patient enrolment, 56 days of treatment and 28 days of follow-up.

[0191] Two parallel arms (Placebo and Supplement) with four measurements, respectively at the beginning of the study (tO), after 28 days of treatment (tl), at the end of treatment after 56 days (t2) and after a 28-day follow-up (t3). For four variables (VAS, cortisol, serotonin and gastrin), only measurements at tO and t2 were performed.

[0192] The analysed sample included 64 patients (34 men and 30 women) with a mean age (± SD) of 45 ± 16 years (men: 45 ± 16; women: 45 ± 17) and a value range between 18 and 70 years (men: 19-69; women: 18-70).The study uses Linear Mixed Models, an appropriate choice when repeated measures on the same subjects are available, it is necessary to model both mean effects (group, time) and individual variability, and it is desired to formally test whether groups behave differently over time. The key point is the Time x Treatment interaction: if significant, it indicates that the trajectory of the supplement group differs from that of the placebo (not only “they are different”, but “they change differently”); if not significant, time may still improve both (time effect), or the treatment may have an overall mean effect, but without a clear divergence in dynamics.

[0193] This type of interpretation is fundamental in IBS, where improvement over time is often also observed in the placebo group (expectation effect, clinical attention, regression to the mean).

[0194] Results:

[0195] Primary objective

[0196] The LMM model for the IBS-SSS score (Tab. 4) identified a significant effect for the main effect of treatment (P < 0.001), measurement (P < 0.001) and also for their interaction (P < 0.001). A significant effect for patient age also emerged (P = 0.029), but not for sex. The result indicates that the IBS-SSS score varies across the four measurements differently between the two experimental groups (Fig. 2).

[0197] In particular, the IBS-SSS score at measurement tO did not differ between experimental groups (P = 2.91 ± 9.31, t238 = 0.313, P = 0.75, Fig. 2). Following the experimental period, the IBS-SSS score in the group treated with the supplement significantly decreased at measurements tl and t2 and remained unchanged at measurement t3 (Fig. 2), as indicated by the comparisons in the table below:

[0198] Comparison B ± SE tl86 P

[0199] to - tl 53.6 ± 8.9 6.047 <0.001

[0200] tl -t2 62.2 ± 8.9 7.021 <0.001

[0201] t2 -t3 10.1 ± 8.9 1.140 0.26

[0202]

[0203] Consequently, there is a significant difference between tO and t2 (P = 115.7 ± 8.9, tl 86 = 13.07, P< 0.001, Fig. 2).

[0204] In the group treated with placebo, by contrast, the IBS-SSS score does not vary significantly between measurements (Fig, 2), as shown by the comparisons in the table below:

[0205] Comparison B ± SE tl86 P

[0206] to - tl 1.8 ± 8.9 0.208 0.84

[0207] tl -t2 8.7 ± 8.9 0.981 0.33

[0208]

[0209] t2 -t3 13.3 ± 8.9 1.506 0.13

[0210] Also the comparison between measurements tO and t2 is not statistically significant (P = 10.5 ± 8.9, tl86 = 1.189, P = 0.23, Fig. 2).

[0211] Consequently, the IBS-SSS score in the group treated with the supplement is significantly lower than that of the group treated with placebo in all measurements subsequent to baseline (tO), and such difference increases as measurements progress (Fig. 2), as also reported in the table below:

[0212] Comparison B ± SE t238 P

[0213] tl 48.8 ± 9.3 5.240 <0.001

[0214] t2 102.3 ± 9.3 10.98 <0.001

[0215]

[0216] t3 125.8 ± 9.3 13.50 <0.001

[0217] Independently of treatment, the IBS-SSS score increases with increasing patient age (P = 5.82 ± 2.60, t60 = 2.240, P = 0.029).

[0218] In conclusion, between the treated group and the placebo group at the beginning of the study there are no statistically significant differences, indicating correct randomization of subjects who, on the basis of the IBS-SSS questionnaire score, can be on average classified as subjects with moderate IBS. Following treatment with the food supplement, the score decreases in a statistically significant manner over time. This trend indicates the clinically significant efficacy of the supplement, whereby subjects, after one month and especially after two months of treatment, present mild IBS.It is also noteworthy that the effect of reduction in IBS symptom severity is maintained even after one month of discontinuation of treatment with the food supplement.

[0219] Secondary objectives

[0220] 1) Impact of constipation on perceived quality of life: The LMM model for the PCS score (physical component of quality of life) identified a significant effect for the main effect of treatment (P < 0.001), measurement (P < 0.001) and also for their interaction (P < 0.001). No significant effects emerged for patient age or sex. The result indicates that the PCS score varies across the four measurements differently between the two experimental groups (Fig. 2). In particular, the PCS score at measurement tO did not differ between experimental groups (P = 1.80 ± 1.73, t224 = 1.043, P = 0.30, Fig. 2). Following the experimental period, the PCS score in the group treated with the supplement significantly increased at measurement tl and remained unchanged at subsequent measurements (Fig. 2), as indicated by the comparisons in the table below:

[0221] Comparison B ± SE tl86 P

[0222] to - tl 9.24 ± 1.58 5.864 <0.001

[0223] tl -t2 0.82 ± 1.58 0.521 0.60

[0224]

[0225] t2 — 13 0.51 ± 1.58 0.329 0.74 Consequently, there is a significant difference between tO and t2 (P = 10.07 ± 1.58, tl86 = 6.385, P < 0.001, Fig. 2).

[0226] In the group treated with placebo, the PCS score significantly increased at measurement tl, then significantly decreased at measurement t2 and remained substantially unchanged at measurement t3 (Fig, 2) as shown by the comparisons in the table below: _

[0227] Comparison B ± SE tl86 P

[0228] to - tl 3.39 ± 1.58 2.152 0.033

[0229] tl -t2 3.97 ± 1.58 2.522 0.012

[0230]

[0231] t2 — 13 2.56 ± 1.58 1.625 0.11 Consequently, the comparison between measurements tO and t2 is not statistically significant (P = 0.58 ± 1.58, tl 86 = 0.369, P = 0.71, Fig. 2).From the comparison between the two groups, it emerges that the PCS score in the group treated with the supplement is significantly higher than that of the group treated with placebo in all measurements subsequent to baseline (tO), and such difference increases as measurements progress (Fig, 1), as also reported in the table below:

[0232] Comparison B ± SE t238 P

[0233] tl 4.05 ± 1.73 2.338 0.020

[0234] t2 8.84 ± 1.73 5.110 <0.001

[0235]

[0236] t3 6.80 ± 1.73 3.930 <0.001

[0237] The LMM model for the MCS score (mental component of quality of life - Tab. 4) identified a significant effect for the main effect of treatment (P < 0.001) and for the interaction treatment x measurement (P = 0.047). No significant effects emerged for the main effect of measurement or for patient age or sex (Tab. 4). The result indicates that the MCS score differs between treatments and that this difference depends on measurement (Fig. 2).

[0238] In particular, the MCS score at baseline (tO) did not differ between experimental groups (P = 2.41 ± 1.71, t211 = 1.410, P = 0.16, Fig. 2). Following the experimental period, the MCS score in the group treated with the supplement does not change significantly between measurements (Fig 2), as indicated by the comparisons in the table below: _

[0239] Comparison B ± SE tl86 P

[0240] to - tl 2.01 ± 1.51 1.333 0.18

[0241] tl -t2 0.66 ± 1.51 0.437 0.66

[0242]

[0243] t2 — 13 1.79 ± 1.51 1.186 0.24

[0244] Consequently, there is also no significant difference between tO and t2 (P = 1.35 ± 1.51, tl 86 = 0.897, P = 0.37, Fig. 2).

[0245] In the group treated with placebo, the MCS score significantly decreases at measurement tl and remains substantially unchanged at subsequent measurements (Fig. 2), as shown by the comparisons in the table below:Comparison B ± SE tl86 P

[0246] to - tl 3.84 ± 1.51 2.549 0.012

[0247] tl -t2 1.71 ± 1.51 1.137 0.26

[0248]

[0249] t2 -t3 0.22 ± 1.51 0.149 0.88

[0250] Consequently, the comparison between measurements tO and t2 is not statistically significant (P = 2.12 ± 1.51, tl86 = 1.412, P = 0.16, Fig. 2).

[0251] From the comparison between the two groups, it emerges that the MCS score in the group treated with the supplement is significantly higher than that of the group treated with placebo in all measurements subsequent to baseline tO (Fig, 2), as also reported in the table below:

[0252] Comparison B ± SE t211 P

[0253] tl 8.27 ± 1.71 4.827 <0.001

[0254] t2 5.89 ± 1.71 3.442 <0.001

[0255]

[0256] t3 4.33 ± 1.71 2.529 0.012

[0257] In conclusion, between the treated group and the placebo group at the beginning of the study there are no statistically significant differences, indicating correct randomization of the subjects who, on the basis of the score relating to the physical and mental components of quality of life, do not differ.

[0258] Following treatment with the food supplement, the scores increase in a statistically significant manner over time.

[0259] This trend indicates the efficacy of the treatment, whereby subjects, after one month and especially after two months of treatment, present an improved quality of life. Unlike the trend observed for the score relating to the severity of IBS symptoms, in this case discontinuation of treatment with the food supplement leads to a decrease in scores, indicating that the effect on quality of life is not maintained.

[0260] 2) Changes in stool consistency: The LMM model for the Bristol score (Tab. 4) identified a significant effect for the main effect of treatment (P < 0.001), measurement (P < 0.001) and also for their interaction (P < 0.001). No significant effects emerged for patient age or sex (Tab. 4). The result indicates that the Bristol score varies across the four measurementsdifferently between the two experimental groups (Fig. 2). In particular, the Bristol score at baseline (tO) did not differ between experimental groups (P = 0.12 ± 0.20, t244 = 0.581, P = 0.56, Fig. 2). Following the experimental period, the Bristol score in the group treated with the supplement significantly increases at measurements tl and t2 and remains unchanged at measurement t3 (Fig, 2), as indicated by the comparisons in the table bel ow :

[0261] Comparison B ± SE tl86 P

[0262] to - tl 0.69 ± 0.20 3.426 <0.001

[0263] tl -t2 1.34 ± 0.20 6.697 <0.001

[0264]

[0265] t2 — 13 0.16 ± 0.20 0.779 0.44

[0266] Consequently, there is also a significant difference between tO and t2 (P = 2.03 ± 0.20, 1186 = 10.12, P < 0.001, Fig. 2).

[0267] In the group treated with placebo, the Bristol score does not vary with respect to baseline at measurement tl, then significantly increases at measurement t2 and remains substantially unchanged at measurement t3 (Fig, 2), as shown by the comparisons in the table b el ow :

[0268] Comparison B ± SE tl86 P

[0269] to - tl 0.09 ± 0.20 0.467 0.64

[0270] tl -t2 0.92 ± 0.20 3.115 0.0021

[0271]

[0272] t2 — 13 0.25 ± 0.20 0.646 0.21

[0273] Consequently, the comparison between measurements tO and t2 is statistically significant (P = 0.72 ± 0.20, tl 86 = 3.582, P < 0.001, Fig. 2). From the comparison between the two groups, it emerges that the Bristol score in the group treated with the supplement is significantly higher than that of the group treated with placebo in all measurements subsequent to baseline (tO), and such difference increases between tl and t2 (Fig, 2), as also reported in the table below:

[0274] Comparison B ± SE t244 P

[0275] tl 0.71 ± 0.20 3.488 <0.001

[0276] t2 1.43 ± 0.20 7.006 <0.001

[0277]

[0278] t3 1.34 ± 0.20 6.547 <0.001In conclusion, between the treated group and the placebo group at the beginning of the study there are no statistically significant differences, indicating correct randomization of subjects who, on the basis of the Bristol scale score, present hard and lumpy stools. Following treatment with the food supplement, the score increases in a statistically significant manner over time. This trend indicates the clinically significant efficacy of the supplement, whereby subjects, after two months of treatment, present on average normal stools.

[0279] It is also noteworthy that the normalizing effect on stool consistency is maintained even after one month of discontinuation of treatment with the food supplement.

[0280] 3) Changes in bowel movement frequency: The LMM model for the variable Bw / wK (bowel movements per week - Tab. 4) identified a significant effect for the main effect of treatment (P < 0.001), measurement (P < 0.001) and also for their interaction (P < 0.001). No significant effects emerged for patient age or sex (Tab. 4). The result indicates that the Bw / wK value varies across the four measurements differently between the two experimental groups (Fig. 2). In particular, the Bw / wK value at baseline (tO) did not differ between experimental groups (P = 0.05 ± 0.18, t235 = 0.293, P = 0.77, Fig. 2). Following the experimental period, the variable in the group treated with the supplement increases in a nearly significant manner at measurement tl and significantly at measurement t2, and remains unchanged at measurement t3 (Fig, 2), as indicated by the comparisons in the table below: _

[0281] Comparison B ± SE tl86 P

[0282] to - tl 0.31 ± 0.17 1.839 0.067

[0283] tl -t2 1.06 ± 0.17 6.252 <0.001

[0284]

[0285] t2 — 13 0.16 ± 0.17 0.492 0.36

[0286] Consequently, there is also a significant difference between tO and t2 (P = 1.38 ± 0.17, 1186 = 8.091, P < 0.001, Fig. 2).

[0287] In the group treated with placebo, the Bw / wK value does not vary significantly between measurements (Fig, 2) _ _ _

[0288] Comparison B ± SE tl86 P

[0289]

[0290] to - tl 0.03 ± 0.17 0.366 0.85tl -t2 0.06 ± 0.17 0.398 0.71

[0291]

[0292] t2 — 13 0.12 ± 0.17 0.736 0.46

[0293] Consequently, the comparison between measurements tO and t2 is also not statistically significant (0 = 0.09 ± 0.17, tl 86 = 0.552, P = 0.58, Fig. 2).

[0294] From the comparison between the two groups, it emerges that the BM / WK value in the group treated with the supplement is higher than that of the group treated with placebo in a nearly significant manner at measurement tl and in a statistically significant manner in subsequent measurements (Fig, 2) as also reported in the table below: _

[0295] Comparison B ± SE t235 P

[0296] tl 0.33 ± 0.18 1.849 0.066

[0297] t2 1.33 ± 0.18 7.384 <0.001

[0298]

[0299] t3 1.61 ± 0.18 8.940 <0.001

[0300] In conclusion, between the treated group and the placebo group at the beginning of the study there are no statistically significant differences, indicating correct randomization of subjects who on average present 1.5 bowel movements per week, in line with the constipation condition required by the inclusion criteria. Following treatment with the food supplement, the value increases in a statistically significant manner over time. This trend indicates the clinically significant efficacy of the supplement, whereby subjects, after two months of treatment, present on average three bowel movements per week. It is also noteworthy that the increase in bowel movement frequency is maintained even after one month of discontinuation of treatment with the food supplement.

[0301] 4) Changes in frequency and intensity of IBS characteristic symptoms.

[0302] - Symptom A: The LMM model for the score of symptom A (Tab. 4) identified a significant effect for the main effect of measurement (P < 0.001) and for the interaction treatment x measurement (P = 0.017). No significant effects emerged for the main effect of treatment or for patient age or sex (Tab. 4). The result indicates that the score of symptom A differs between measurements, with some differences between the two treatments (Fig. 2). In particular, the score of symptom A at baseline (tO) did not differ between experimental groups (0 = 0.23 ±0.28, t242 = 0.827, P = 0.41, Fig. 2). Following the experimental period, the score of symptom A in the group treated with the supplement decreases in a nearly significant manner at measurement tl and significantly at measurement t2, and remains unchanged at measurement t3 (Fig, 2), as indicated by the comparisons in the table below: _

[0303] Comparison B ± SE tl86 P

[0304] to - tl 0.45 ± 0.28 1.730 0.085

[0305] tl -t2 1.09 ± 0.28 4.037 <0.001

[0306]

[0307] t2 — 13 0.25 ± 0.28 0.284 0.36

[0308] Consequently, the value of symptom A measured at t2 is significantly lower than baseline (P = 0.62 ± 0.28, tl 86 = 2.307, P = 0.022, Fig. 2).

[0309] In the group treated with placebo, the score of symptom A does not vary significantly between subsequent measurements (Fig, 2), _ _ _

[0310] Comparison B ± SE tl86 P

[0311] to - tl 0.22 ± 0.28 0.316 0.42

[0312] tl -t2 0.19 ± 0.28 0.347 0.49

[0313]

[0314] t2 — 13 0.03 ± 0.28 0.566 0.91

[0315] Consequently, the comparison between measurements tO and t2 is also not statistically significant (P = 0.41 ± 0.28, tl86 = 1.500, P = 0.13, Fig. 2). From the comparison between the two groups, it emerges that the score of symptom A does not differ significantly between treatments at measurements tl and t2, but is significantly lower in the group treated with the supplement at measurement t3 (Fig, 2), as indicated by the comparisons in the table below:

[0316] Comparison B ± SE T242 P

[0317] tl 0.46 ± 0.28 1.630 0.10

[0318] t2 0.46 ± 0.28 1.611 0.11

[0319]

[0320] t3 0.73 ± 0.28 2.617 0.0094

[0321] Symptom B: The LMM model for the score of symptom B (Tab. 4) identified a significant effect for the main effect of measurement (P < 0.001), while no significant effects emerged fortreatment, treatment x measurement interaction, or patient age or sex (Tab. 4). The result indicates that the score of symptom B varies with measurement without differing between experimental treatments (Fig. 2). In particular, the score of symptom B at baseline (tO) did not differ between experimental groups (P = 0.02 ± 0.27, t244 = 0.060, P = 0.95, Fig. 2). Following the experimental period, the score of symptom B in the group treated with the supplement does not vary significantly with respect to baseline at measurements tl and t2, and significantly decreases at measurement t3 (Fig, 2), as indicated by the comparisons in the table b el ow :

[0322] Comparison B ± SE tl86 P

[0323] to - tl 0.31 ± 0.27 1.160 0.25

[0324] tl -t2 0.22 ± 0.27 0.812 0.42

[0325]

[0326] t2 — 13 1.31 ± 0.27 4.872 <0.001

[0327] However, the value of symptom B measured at t2 is significantly higher than baseline (P = 0.53 ± 0.27, tl 86 = 1.972, P = 0.0501, Fig. 2).

[0328] In the group treated with placebo, the score of symptom B does not vary significantly between measurements, as indicated by the comparisons in the table below: _

[0329] Comparison B ± SE tl86 P

[0330] to - tl 0.06 ± 0.27 0.232 0.82

[0331] tl -t2 0.01 ± 0.27 0.001 0.99

[0332]

[0333] t2 — 13 0.40 ± 0.27 1.508 0.13

[0334] Consequently, the comparison between measurements tO and t2 is not statistically significant (P = 0.06 ± 0.27, tl 86 = 0.232, P = 0.82, Fig. 2). From the comparison between the two groups, it emerges that the score of symptom B does not differ significantly between treatments at any measurement (Fig, 2), as indicated by the comparisons in the table below: _

[0335] Comparison B ± SE t244 P

[0336] tl 0.27 ± 0.27 0.970 0.33

[0337] t2 0.48 ± 0.27 1.767 0.078

[0338]

[0339] t3 0.42 ± 0.27 1.543 0.13The LMM model for the score of symptom C (Tab. 4) identified a significant effect for the main effect of treatment (P < 0.001). Symptom C: The LMM model for the score of symptom C (Tab. 4) identified a significant effect for the main effect of treatment (P < 0.001), measurement (P < 0.001) and also for their interaction (P < 0.001). No significant effects emerged for patient age or sex (Tab. 4). The result indicates that the score of symptom C varies across the four measurements differently between the two experimental groups (Fig. 2). In particular, the score of symptom C at baseline (tO) did not differ between experimental groups (P = 0.24 ± 0.29, t246 = 0.820, P = 0.41, Fig. 2). Following the experimental period, the variable in the group treated with the supplement does not vary at measurement tl and significantly decreases at measurement t2, remaining unchanged at measurement t3 (Fig. 2), as indicated by the comparisons in the table below: _ _

[0340] Comparison B ± SE tl86 P

[0341] to - tl 0.06 ± 0.28 0.220 0.83

[0342] tl -t2 1.19 ± 0.28 4.174 <0.001

[0343]

[0344] t2 — 13 0.19 ± 0.28 0.659 0.51

[0345] Consequently, there is also a significant difference between tO and t2 (P = 1.25 ± 0.28, 1186 = 4.394, P < 0.001, Fig. 2). In the group treated with placebo, the score of symptom C increases in a nearly significant manner at measurement tl, significantly decreases at measurement t2 and remains unchanged at measurement t3 (Fig. 2), as indicated by the comparisons in the table below:

[0346] Comparison B ± SE tl86 P

[0347] to - tl 0.53 ± 0.28 1.867 0.063

[0348] tl -t2 0.56 ± 0.28 0.398 0.049

[0349]

[0350] t2 — 13 0.28 ± 0.28 0.987 0.33

[0351] Consequently, the comparison between measurements tO and t2 is not statistically significant (P = 0.03 ± 0.28, tl 86 = 0.110, P = 0.91, Fig. 2). From the comparison between the two groups, it emerges that the score of symptom C does not differ between treatments at measurement tl but is significantly lower in the group treated with the supplement compared to the grouptreated with placebo at measurements t2 and t3 (Fig. 2), as indicated by the comparisons in the table below:

[0352] Comparison B ± SE t246 P

[0353] tl 0.36 ± 0.29 1.234 0.22

[0354] t2 0.98 ± 0.29 3.397 <0.001

[0355]

[0356] t3 1.07 ± 0.29 3.721 <0.001

[0357] Symptom D: The LMM model for the score of symptom D (Tab. 4) identified a significant effect only for the main effect of treatment (P < 0.001) and a nearly significant effect of measurement (P = 0.065). No relevant effects emerged for the measurement x treatment interaction or for patient age or sex (Tab. 4). The result indicates that the score of symptom D differs between treatments in a manner almost independent of measurement (Fig. 2). In particular, the score of symptom D at baseline (tO) did not differ between experimental groups (P = 0.08 ± 0.29, t239 = 0.279, P = 0.78, Fig. 2). Following the experimental period, the variable in the group treated with the supplement decreases in a non- significant manner between subsequent measurements (Fig, 2) as indicated by the comparisons in the table below:

[0358] Comparison B ± SE tl86 P

[0359] to - tl 0.28 ± 0.27 1.030 0.30

[0360] tl -t2 0.31 ± 0.27 1.145 0.25

[0361]

[0362] t2 — 13 0.12 ± 0.27 0.458 0.65

[0363] Consequently, the decrease observed between tO and t2 is statistically significant (P = 0.59 ± 0.27, tl86 = 2.175, P = 0.031, Fig. 2).

[0364] In the group treated with placebo, the score of symptom D does not vary significantly between measurements (Fig2), as indicated by the comparisons in the table below:

[0365] Comparison B ± SE tl86 P

[0366] to - tl 0.25 ± 0.27 0.916 0.36

[0367] tl -t2 0.34 ± 0.27 1.259 0.21

[0368]

[0369] t2 — 13 0.01 ± 0.27 0.001 0.99Consequently, the comparison between measurements tO and t2 is not statistically significant (P = 0.09 ± 0.27, tl 86 = 0.343, P = 0.73, Fig. 2). From the comparison between the two groups, it emerges that the score of symptom D is significantly lower in the group treated with the supplement compared to the group treated with placebo at all measurements subsequent to tO (Fig. 2) as indicated oy the comparisons in the table below Comparison B ± SE t246 P

[0370] tl 0.61 ± 0.28 2.138 0.034

[0371] t2 0.58 ± 0.28 2.029 0.044

[0372]

[0373] t3 0.70 ± 0.28 2.466 0.014

[0374] Symptom E: The LMM model for the score of symptom E (Tab. 4) identified a significant effect for the main effect of treatment (P < 0.001) and for the interaction treatment x measurement (P = 0.0084). No significant effects emerged for the main effect of measurement or for patient age or sex (Tab. 4). The result indicates that the score of symptom E differs between treatments and that this difference depends on measurement (Fig. 2). In particular, the score of symptom E at baseline (tO) did not differ between experimental groups (P = 0.20 ± 0.28, t232 = 0.734, P = 0.46, Fig. 2). Following the experimental period, the score of symptom E in the group treated with the supplement decreases across measurements but in a non- significant manner (Fig. 2), as indicated by the comparisons in the table below Comparison B ± SE tl86 P

[0375] to - tl 0.40 ± 0.26 1.554 0.12

[0376] tl -t2 0.34 ± 0.26 1.315 0.19

[0377]

[0378] t2 — 13 0.19 ± 0.26 0.717 0.47

[0379] Consequently, the decrease in the score of symptom E between tO and t2 is statistically significant (P = 0.75 ± 0.26, tl86 = 2.868, P = 0.0046, Fig. 2). In the group treated with placebo, the score of symptom E does not vary significantly between measurements (Fig 2) as indicated by the comparisons in the table below:Comparison B ± SE tl86 P

[0380] to - tl 0.06 ± 0.26 0.239 0.81

[0381] tl -t2 0.34 ± 0.26 1.315 0.19

[0382]

[0383] t2 -t3 0.16 ± 0.26 0.717 0.55

[0384] Consequently, the comparison between measurements tO and t2 is not statistically significant (P = 0.28 ± 0.26, tl 86 = 1.076, P = 0.28, Fig. 2). From the comparison between the two groups, it emerges that the score of symptom E in the group treated with the supplement is lower than that of the group treated with placebo in all measurements subsequent to measurement tO, but the difference is statistically significant only for measurements t2 and t3 (Fig. 1), as also reported in the table below. _ _ _

[0385] Comparison B ± SE t232 P

[0386] tl 0.54 ± 0.28 1.960 0.052

[0387] t2 1.23 ± 0.28 4.411 <0.001

[0388]

[0389] t3 1.27 ± 0.28 4.523 <0.001

[0390] In summary, the disorders sensation of bloating, abdominal distension and sensation of heaviness decrease, abdominal pain shows a decrease but not statistically significant. Conversely, flatulence remains unchanged compared to the placebo group.

[0391] 5) Changes in frequency of use of rescue treatments (“salvage treatment”).

[0392] The frequency of use of rescue treatments in the group that took the supplement decreased during the experimental period (Fig. 3), decreasing from 54% at measurement tO, to 34% at measurement tl and to 19% at measurement t2. This value was maintained also after followup (t3). The variation was statistically significant (%2= 11.927, df = 3, P = 0.0076). On the contrary, in the group that took the placebo, the use of rescue treatments did not vary significantly during the experimental period, remaining consistently slightly above 50% (Fig.

[0393] 3). The differences were not statistically significant (%2= 0.597, df = 3, P = 0.90). At measurement tO, the frequency of use of rescue treatments was identical in the two experimental groups (Tab. 3c), whereas at measurement tl the frequency of ST in the group treated with the supplement was lower than that of the group that took the placebo, althoughsuch difference was at the limit of statistical significance (%2= 3.074, df = 1, P = 0.079). At measurements t2 and t3, the group treated with the supplement used ST significantly less frequently compared to the group treated with placebo (t2: %2= 6.787, df = 1, P = 0.0092; t3: X2= 5.610, df= 1, P = 0.018).

[0394] 6) Evaluation of abdominal pain: The LMM model for the VAS (Tab. 4) identified a significant effect for the main effects of treatment (P = 0.0027) and measurement (P < 0.001) but not for their interaction or for patient age and sex (Tab. 4). The result indicates that the VAS value varies with measurement in a similar manner in the two experimental groups (Fig. 2). In particular, the VAS score value at baseline (tO) did not differ between experimental groups (P = 0.20 ± 0.41, t246 = 0.484, P = 0.63, Fig. 2). Following the experimental period, the VAS value in the group treated with the supplement does not vary significantly at measurement tl compared to baseline, significantly decreases at measurement t2 and remains substantially unchanged at measurement t3 (Fig, 2), as indicated by the comparisons in the table below.

[0395] Comparison B ± SE tl86 P

[0396] to - tl 0.46 ± 0.41 1.153 0.25

[0397] tl -t2 0.81 ± 0.41 1.998 0.047

[0398]

[0399] t2 — 13 0.41 ± 0.41 0.999 0.32

[0400] Consequently, the decrease in VAS between tO and t2 is statistically significant (P = 1.28 ± 0.41, tl86 = 3.151, P = 0.0018, Fig. 2).

[0401] In the group treated with placebo, the VAS value does not vary significantly between measurements (Fig, 2) as evidenced by the comparisons in the table below.

[0402] Comparison B ± SE tl86 P

[0403] to - tl 0.12 ± 0.41 0.307 0.76

[0404] tl -t2 0.53 ± 0.41 1.307 0.19

[0405]

[0406] t2 — 13 0.19 ± 0.41 0.461 0.64

[0407] Consequently, the comparison between measurements tO and t2 is not statistically significant (P = 0.66 ± 0.41, tl 86 = 1.614, P = 0.11, Fig. 2). From the comparison between the two groups, it emerges that the VAS value in the group treated with the supplement does not differ fromthat observed in the group treated with placebo at measurement tl, but is significantly lower at subsequent measurements (Fig, 2), as also reported in the table below

[0408] Comparison B ± SE t232 P

[0409] tl 0.54 ± 0.41 1.316 0.19

[0410] t2 0.82 ± 0.41 1.998 0.047

[0411]

[0412] t3 1.04 ± 0.41 2.524 0.012

[0413] In summary, the VAS expressing abdominal pain is lower in the group treated with the food supplement compared to the placebo.

[0414] 7) Evaluation of serotonin, gastrin and cortisol: The LMM model for cortisol did not identify statistically significant effects (Tab. 4). The cortisol value, therefore, does not change across measurements and does not differ between the experimental groups (Fig. 2).

[0415] The LMM model for serotonin did not identify statistically significant effects (Tab. 4). The serotonin value, therefore, does not change across measurements and does not differ between the experimental groups (Fig. 2).

[0416] The LMM model for gastrin did not identify statistically significant effects (Tab. 4). The value, therefore, does not change across measurements and does not differ between the experimental groups (Fig. 2).

[0417] In summary, the blood parameters, serotonin, gastrin and cortisol, do not vary as a function of the treatment undergone by the subject.

[0418] TABLE 3

[0419] a. Descriptive statistics (mean, standard deviation and range of values) for the 14 response variables measured from time tO to time t3 for the subjects who received the TREATMENT.

[0420] tO tl t2 t3

[0421] IBS-SSS 202.0±32.1 148.5±32.7 86.3 ± 30.7 76.2± 31.0

[0422] (123 - 260) (77 - 210) (30 - 156) (20 - 137) PCS 31.8 ± 6.1 41.0 ± 6.1 41.8 ± 7.6 42.3 ± 7.5

[0423] (21.98 - (29.43 - (25.67 - (28.39 48.28) 53.7) 62.62) 60.52)MCS 41.9 ± 5.9 43.9 ± 5.8 43.2 ± 6.2 41.4 ± 7.0 (30.21 (29.57 (33.38 (18.18 50.41) 55.78) 57.73) 54.14) Bristol 1.5 ± 0.5 2.2 ± 0.7 3.5 ± 1.1 3.7 ± 1.1

[0424] (1 - 2) (1 - 3) (2 - 5) (2 - 5) Bw / wk 1.6 ± 0.5 1.9 ± 0.9 2.9 ± 0.9 3.1 ± 0.9

[0425] (1 - 2) (1 - 3) (2 - 4) (2 - 4) SymptomA 3.4 ± 1.2 3.8 ± 1.1 2.8 ± 1.2 2.5 ± 1.1

[0426] (2 - 5) (2 - 5) (1 - 4) (1 - 4) SymptomB 3.4 ± 1.2 3.7 ± 1.3 3.9 ± 0.4 2.6 ± 1.0

[0427] (2 - 5) (2 - 5) (2 - 4) (1 - 4) Symptom C 3.5 ± 1.2 3.4 ± 1.3 2.2 ± 1 2.4 ± 1.2

[0428] (2 - 5) (2 - 5) (1 - 4) (1 - 4) SymptomD 3.2 ± 1.2 3.0 ± 1.1 2.7 ± 1.1 2.5 ± 1.2

[0429] (2 - 5) (2 - 5) (1 - 4) (1 - 4) SymptomE 3.2 ± 1.1 2.8 ± 1.0 2.5 ± 1.1 2.3 ± 1 (2 - 5) (2 - 5) (1 - 4) (1 - 4) VAS 5.5 ± 1.8 5.0 ± 1.4 4.2 ± 1.4 3.8 ± 1.3

[0430] (3 - 8) (3 - 7) (2 - 6) (2 - 6) Cortisol 16.4 ± 6.8 15.3 ± 6.7

[0431] (6 - 25) (5 - 25)

[0432] Serotonin 179.2 ± 53.4 195.9 ± 52.5

[0433] (101 - 276) (107 - 280)

[0434] Gastrin 42.0 ± 31.4 48.4 ± 32.9

[0435] (2 - 97) (0 - 100)

[0436] b. Descriptive statistics (mean, standard deviation and range of values) for the 14 response variables measured from time tO to time t3 for the subjects who received the PLACEBO.to tl t2 t3 IBS-SSS 195.4 ± 193.5 ± 184.8 ± 198.2 ±

[0437] 40.6 39.2 40.3 45.3 (106 - 255) (132 - 288) (111 - 264) (108 - 292) PCS 33.6 ± 6.9 37 ± 6.4 33 ± 6.8 35.6 ± 6.3

[0438] (14.81 (20.58 (20.42 - (22.89 - 50.49) 49.21) 46.1) 47.31) MCS 39.1 ± 7.6 35.2 ± 6.6 37 ± 6.2 36.7 ± 7.8

[0439] (21.84 (22.85 (22.75 - (17.29 - 53.22) 46.92) 55.3) 52.84) Bristol 1.3 ± 0.5 1.4 ± 0.5 2.1 ± 0.9 2.3 ± 0.8

[0440] (1 - 2) (1 - 2) (1 - 3) (1 - 3) Bw / wk 1.6 ± 0.5 1.6 ± 0.5 1.7 ± 0.8 1.5 ± 0.5

[0441] (1 - 2) (1 - 2) (1 - 3) (1 - 2) SymptomA 3.6 ± 1.1 3.4 ± 0.9 3.2 ± 1.0 3.2 ± 1.2

[0442] (2 - 5) (2 - 5) (2 - 5) (2 - 5) SymptomB 3.5 ± 1.2 3.5 ± 1.1 3.5 ± 1.2 3.1 ± 1.0

[0443] (2 - 5) (2 - 5) (2 - 5) (2 - 5) SymptomC 3.2 ± 1.0 3.8 ± 1.3 3.2 ± 1.0 3.5 ± 1.2

[0444] (2 - 5) (2 - 5) (2 - 5) (2 - 5) SymptomD 3.4 ± 1.0 3.7 ± 1.2 3.3 ± 1.2 3.3 ± 1.0

[0445] (2 - 5) (2 - 5) (2 - 5) (2 - 5) SymptomE 3.4 ± 1.3 3.4 ± 1.0 3.7 ± 1.1 3.6 ± 1.2

[0446] (2 - 5) (2 - 5) (2 - 5) (2 - 5) VAS 5.7 ± 1.6 5.6 ± 1.6 5.1 ± 1.9 4.9 ± 1.9

[0447] (3 - 8) (3 - 8) (3 - 8) (3 - 8) Cortisol 15.4 ± 6.6 16.3 ± 5.6(5 - 25) (5 - 25)

[0448] Serotonin 180 ± 50.7 176.2 ±

[0449] 55.1

[0450] (100 - 270) (101

[0451] 280)

[0452] Gastrin 49.7 ± 28.8 44.4 ±

[0453] 34.0

[0454] (4 - 100) (2 - 99)

[0455] c. Frequencies of use of rescue treatments in the two experimental groups at each measurement.

[0456] Treatment tO tl t2 t3 Supplement

[0457] No 15 21 26 26

[0458] (46%) (66%) (81%) (81%)

[0459] Yes 17 11 6 6

[0460] (54%) (34%) (19%) (19%) Placebo

[0461] No 15 13 15 16

[0462] (46%) (41%) (46%) (50%)

[0463] Yes 17 19 17 16

[0464]

[0465] (54%) (59%) (54%) (50%) TABLE 4

[0466] Results of the LMM models for the comparison between treatment and placebo in the 14 variables analysed in this study.

[0467] Variable F gdl P

[0468] IBS-SSS

[0469] Measure 46.481 3,186 <0.001 Treatment 168.16 1,60 <0.001Sex 0.280 1,60 0.60 Age 5.017 1,60 0.029 Measure x Treatment 42.093 3,186 <0.001 PCS

[0470] Measure 14.317 3,186 <0.001 Treatment 17.684 1,60 <0.001 Sex 0.020 1,60 0.89 Age 0.001 1,60 0.98 Measure x Treatment 8.598 3,186 <0.001 MCS

[0471] Measure 0.653 3,186 0.58 Treatment 22.227 1,60 <0.001 Sex 1.035 1,60 0.31 Age 0.253 1,60 0.62 Measure x Treatment 2.700 3,186 0.047 Bristol

[0472] Measure 57.570 3,186 <0.001 Treatment 70.212 1,60 <0.001 Sex 0.078 1,60 0.78 Age 0.159 1,60 0.69 Measure x Treatment 9.265 3,186 <0.001 Bw / wk

[0473] Measure 20.569 3,186 <0.001 Treatment 63.326 1,60 <0.001 Sex 0.675 1,60 0.41 Age 1.704 1,60 0.20 Measure x Treatment 19.859 3,186 <0.001Symptom A

[0474] Measure 7.526 3,186 <0.001 Treatment 2.479 1,60 0.12 Sex 0.057 1,60 0.81 Age 0.024 1,60 0.88 Measure x Treatment 3.498 3, 186 0.017 Symptom B

[0475] Measure 8.051 3,186 <0.001 Treatment 0.358 1,60 0.55 Sex 2.236 1,60 0.14 Age 1.010 1,60 0.32 Measure x Treatment 2.085 3,186 0.10 Symptom C

[0476] Measure 7.665 3,246 <0.001 Treatment 12.975 1,246 <0.001 Sex 0.010 1,246 0.92 Age 0.062 1,246 0.80 Measure x Treatment 4.609 3,246 <0.001 Symptom D

[0477] Measure 2.450 3,186 0.065 Treatment 9.465 1,60 <0.001 Sex 0.753 1,60 0.39 Age 3.560 1,60 0.064 Measure x Treatment 1.061 3,186 0.37 Symptom E

[0478] Measure 1.628 3,186 0.18 Treatment 24.277 1,60 <0.001 Sex 0.032 1,60 0.86Age 0.084 1,60 0.77 Measure x Treatment 4.024 3,186 0.0084

[0479] VAS

[0480] Measure 8.281 3,246 <0.001 Treatment 9.149 1,246 0.0027 Sex 1.902 1,246 0.17 Age 0.352 1,246 0.55 Measure x Treatment 0.805 3,246 0.49 Cortisol

[0481] Measure 0.005 1,122 0.95 Treatment 0.020 1,122 0.89 Sex 0.347 1,122 0.56 Age 0.006 1,122 0.94 Measure x Treatment 0.838 1,122 0.36 Serotonin

[0482] Measure 0.475 1,122 0.49 Treatment 0.612 1,122 0.44 Sex 0.226 1,122 0.64 Age 0.068 1,122 0.80 Measure x Treatment 1.189 1,122 0.28 Gastrin

[0483] Measure 0.012 1,122 0.91 Treatment 0.128 1,122 0.72 Sex 1.422 1,122 0.24 Age 0.378 1,122 0.54 Measure x Treatment 1.072 1,122 0.308) Evaluation of intestinal inflammatory status through the analysis of fecal inflammatory markers Calprotectin and Zonulin.

[0484] Evaluation of fecal zonulin levels by ELISA assay

[0485] Quantification of fecal zonulin was performed using the Human Zonulin ELISA Kit (E-EL-H5560) ELABSCIENCE, based on a sandwich ELISA assay. All reagents contained in the kit were brought to room temperature before use. The microELISA plate provided with the kit was pre-coated with an antibody specific for human zonulin. Samples (or standard zonulin solutions) were added to the wells of the plate and combined with the specific antibody. Subsequently, a biotinylated detection antibody specific for human zonulin and an Avidin-Horseradish Peroxidase (HRP) conjugate were added to each well, followed by an incubation period. Free components were removed by washing. Thereafter, substrate solution was added to each well. Only the wells containing human zonulin, the biotinylated detection antibody and the Avidin-HRP conjugate developed a blue coloration. The enzymatic reaction was stopped by addition of the stop solution, which changed the colour from blue to yellow. The optical density (OD) was then measured spectrophotometrically at a wavelength of 450 nm. The OD value is proportional to the concentration of human zonulin present in the ELISA plate well. The concentration of zonulin in the samples can be calculated by comparing the OD value of the samples with the calibration curve generated using standard zonulin solutions at known concentrations.

[0486] Analysed Samples

[0487] • Type of samples: Stool

[0488] • Number of samples: 30 divided into two treatment groups: A1-A15 (placebo) and B1-B15 (treated with the food supplement)

[0489] • Collection time: TO (baseline), T56d (T2) and T84d (T3)

[0490] • Storage: Samples were stored at -80°C until the time of analysis.

[0491] Extraction Procedure

[0492] For extraction of zonulin from each sample, 1 g of stool, accurately weighed, was solubilized in 9 mL of lysis buffer containing PBS IX and 0.05% EDTA. Samples were kept on ice forapproximately 20 minutes in order to facilitate buffer action. Subsequently, they were centrifuged at 5000*g for 10 minutes. The resulting supernatant was collected and used for performance of the ELISA assay, according to the instructions reported in the kit manual. RESULTS of stool samples collected at TO

[0493] The following table reports the concentrations of fecal zonulin in the analyzed samples measured by ELISA assay at time TO. Values are expressed in ng / mL of fecal zonulin and reported as the mean of two technical replicates. The coefficient of variation (% CV), standard deviation (SD) and standard error of the mean (SEM) for each sample are also reported.

[0494]

[0495] By comparing these values with the reference ranges of fecal zonulin according to which a sample is considered:

[0496] • Negative, if the concentration is < 61 ng / mL

[0497] • Weakly positive, if the concentration is between 61 and 100 ng / mL

[0498] • Positive, if the concentration is > 100 ng / mL

[0499] the fecal zonulin values detected in all analysed samples are lower than 61 ng / mL and are considered negative and therefore physiological.In conclusion, since values higher than those determined may indicate an increase in intestinal permeability to be monitored over time and concentrations higher than 100 ng / mL are generally indicative of a significant impairment of the intestinal barrier, compatible with a condition of leaky gut, associated with chronic inflammatory intestinal diseases, such as Crohn’s disease, but also with unhealthy lifestyles or imbalances of the intestinal microbiota, the results of the analyses of the samples collected at TO indicate that fecal zonulin concentrations are clearly lower than the pathological threshold of 100 ng / mL.

[0500] All values fall within the range considered normal (< 61 ng / mL) according to the parameters provided by the kit manufacturer.

[0501] The mean concentrations detected in treatment group A range from a minimum of 0.07 ng / mL (sample Al) to a maximum of 2.551 ng / mL (sample A14), thus remaining well below the threshold value.

[0502] With regard to treatment group B, all concentration values are lower than the concentrations used for construction of the standard curve and therefore show a fecal zonulin concentration lower than 0.78 ng / mL.

[0503] These results indicate that, before the beginning of the treatment, the subjects involved in the study overall presented an intact intestinal barrier, without evidence of epithelial dysfunction or signs attributable to a condition of leaky gut, in line with the exclusion criteria that provided for not enrolling subjects affected by organic intestinal diseases.

[0504] The following table reports the concentrations of fecal zonulin in the samples analyzed by ELISA assay at time T2. Fecal zonulin concentrations in the samples analyzed by ELISA assay at time T2 (T56d). Values are expressed in ng / mL of fecal zonulin and reported as the mean of two technical replicates. The coefficient of variation (% CV), standard deviation (SD) and standard error of the mean (SEM) for each sample are also reported.

[0505]

[0506] By comparing these values with the reference ranges of fecal zonulin according to which a sample is considered:

[0507] • Negative, if the concentration is < 61 ng / mL

[0508] • Weakly positive, if the concentration is between 61 and 100 ng / mL

[0509] • Positive, if the concentration is > 100 ng / mL the fecal zonulin values detected in all analysed samples are lower than 61 ng / mL and are therefore considered negative and physiological.

[0510] In conclusion, all subjects showed fecal zonulin values lower than 0.78 ng / mL. Overall, the data demonstrate the good tolerability of the food supplement at the end of the 8-week treatment.

[0511] The following table reports the results of the stool samples collected at T3 (end of follow-up, that is, 30 days after the end of treatment).

[0512] Fecal zonulin concentrations in the samples analysed by ELISA assay at time T84. Values are expressed in ng / mL of fecal zonulin and reported as the mean of two technical replicates. Thecoefficient of variation (% CV), standard deviation (SD) and standard error of the mean (SEM) for each sample are also reported.

[0513] ID Uac % SB SEM ID % SD SEM samplenu wlcv 1 sample CV

[0514] ■ 1 Averagt?) t Vvera&tO

[0515] Al 0,432 11,5 7,043 0.0352 | 81 1 557 133 0383 i 0,1 W 0356 163 i 0.0576 i 0,0407 1 82 1302 I 1 ' s\03f t>,014 A3 0343 6,0^ i 0,0 M3 s 03105 ] S3 3,520 1 <3 MJl A4 0377 A , ' COLA coin 1 84 U.442 1 . 20 S A5 016 . 23 ■■ 'V 85 0375 1V.” 040 V At' 0,356 12 ' O OtCb 0.2SS 2, / 5 t'Jjn’ t’.vt A7 W Cl'* \ A i jsK tHtji) AS 0367 293 i 0,227 | 0, 161 1 \ \ SS 0371 ■ ” 4 O i sc" 4 < s-A'

[0516] \A j <? ’

[0517] 20 -1 8

[0518]

[0519] AO om 1 89 i 0,265 \~ 0,0' 1

[0520] By comparing these values with the reference ranges of fecal zonulin according to which a sample is considered:

[0521] • Negative, if the concentration is < 61 ng / mL

[0522] • Weakly positive, if the concentration is between 61 and 100 ng / mL

[0523] • Positive, if the concentration is > 100 ng / mL

[0524] the fecal zonulin values detected in all analysed samples are lower than 61 ng / mL and are therefore considered negative and physiological.

[0525] The data obtained at follow-up (T84) show that intake of the food supplement under examination did not determine a clinically relevant increase in fecal zonulin.

[0526] CALPROTECTIN: is a heterodimeric protein (S100A8 / S100A9) released mainly by neutrophils during inflammatory reactions. It is considered a reliable marker for evaluating intestinal inflammation and is commonly used to monitor chronic inflammatory diseases (e.g., Crohn’s disease and ulcerative colitis). The purpose of the present efficacy study is to demonstrate that the food supplement based on a green tea extract (Camellia sinensis (L.) Kuntze - folium) and a pool of probiotics (L. plantarum, L. rhamnosus and B. animalis subsp.lactis) is useful for the management of gastrointestinal discomfort and for maintaining the balance of intestinal function in subjects presenting IBS-C. At the clinical level, determination of calprotectin is considered useful to exclude that subjects enrolled in the study are affected by an inflammatory bowel disease.

[0527] Evaluation of fecal calprotectin levels by ELISA assay

[0528] For performance of the ELISA assay, the Human CALP (Calprotectin) ELISA Kit (E-EL-H2357) ELABSCIENCE was used, based on a sandwich ELISA method, for quantification of calprotectin in stool. Before use, all reagents provided in the kit were allowed to reach room temperature. Standard calprotectin solutions at known concentration and blank controls were then prepared, necessary for construction of the calibration curve. The samples to be analysed, together with the standards, were incubated in a multi-well plate with the specific primary antibody. Subsequently, a secondary antibody conjugated to an enzyme was added. Addition of the enzyme substrate generated a coloured product, the intensity (absorbance), measured at 450 nm, being proportional to the amount of calprotectin present in the samples.

[0529] Analysed Samples

[0530] • Type of samples: Stool

[0531] • Number of samples: 30 divided into two treatment groups: A1-A15 (placebo) and B1-B15 (food supplement)

[0532] • Collection time: TO (baseline), T56d (T2) and T84d (T3)

[0533] • Storage: Samples were stored at -80°C until the time of analysis.

[0534] Extraction Procedure

[0535] For extraction of calprotectin from each sample, 1 gram of stool, accurately weighed, was solubilized in 9 mL of lysis buffer containing PBS IX and 0.05% EDTA. Samples were kept on ice for approximately 20 minutes in order to facilitate buffer action. Subsequently, they were centrifuged at 5000 xg for 10 minutes. The resulting supernatant was collected and used for performance of the ELISA assay, according to the instructions reported in the kit manual. RESULTS of stool samples collected at TOThe results (Table 8) were reported in ng / mL and converted into pg / g using a conversion factor derived from the extraction ratio (Table 9).

[0536] In detail, to perform the experiment, 1 g of stool was homogenized in 9 mL of extraction buffer; therefore, 1 mL of “protein extract” corresponds to 111.1 mg of stool.

[0537] Considering sample Al, the ELISA kit yielded a concentration of 2.717 ng of calprotectin per mL of solution. This means that in 1 mL of protein extract there are 2.717 ng of calprotectin. It is also known that 1 mL of extract represents 111.1 mg of stool.

[0538] Therefore, 2.717 ng of calprotectin are present in 111.1 mg of stool.

[0539] Conversion into ng / g was then performed.

[0540] To determine how many ng of calprotectin are present in 1 g (1000 mg) of stool, the following proportion was calculated:

[0541] >- 2.717 ng : 111.1 mg = x : 1000 mg

[0542] >- 24.5 ng / g of stool (0.0245 pg / g)

[0543] Using this conversion factor derived from the extraction ratio (1 ng / mL in the protein extract corresponds to 50 pg / g in stool), the fecal calprotectin concentrations obtained range between 0.003 and 0.073 pg / g.

[0544] By comparing these values with the reference ranges of fecal calprotectin according to which a sample is considered:

[0545] • Negative, if the calprotectin concentration is < 50 pg / g

[0546] • Weakly positive, if the calprotectin concentration is between 50 and 120 pg / g • Positive, if the calprotectin concentration is > 120 pg / g the results indicate low levels of fecal calprotectin at time TO in all analysed subjects. This suggests that all samples are negative, indicating the absence of an intestinal inflammatory condition, consistently with what was expected and in compliance with the inclusion and exclusion criteria of the study.

[0547] Table 8. Fecal calprotectin concentrations in the samples analysed by ELISA assay at time TO (baseline). Values are expressed in ng / mL of fecal calprotectin and reported as the mean of two technical replicates. The coefficient of variation (% CV), standard deviation (SD) and standard error of the mean (SEM) for each sample are also reported.Cour. SI):SFM SD SEM

[0548] t V

[0549] sample BJl Hl! sample i HAASI cv

[0550] t km Ago f Uet age)

[0551] 1

[0552] Al 0. m ; .06 o

[0553] 0,113 O.OSO. B2

[0554] A3 24.9 O Jt. OA 8?' 9.L.1 M3

[0555] A4 ‘8J IM 0

[0556] AS 3: V L'l V 2 OS

[0557] AS pK 5,39 0.6889 O.®29

[0558] A7 2 SO 0" i sMMO 1 116 M2 (MJ?

[0559] A8 27,1 i 0,445 1 0,315 B8 2,635 7,69 0,293 99,143

[0560] AO 3 JO 23,3 1 0,787 I aS56 B9 t * < .:;■■> e

[0561] A10 79 0 0 B10 6

[0562] Ail 0,39 to 0 0

[0563] A J: o 0.192 Bi 0J36 0A98S

[0564] A13 3,055 0.39 B13 6.40: 5,99 0383 0,271

[0565] Al 4 6,818 iOJ 0,691 0.489 B14 4,405 1,68 0,0739

[0566] Al 5 8,977 i0,4 0,936 0.662 B15 3,389 11,4 0387 0,273

[0567]

[0568] Table 9. Results of the ELISA assay for fecal calprotectin, expressed both in ng / mL and in pg / g. m CMS? IB .SiM’ SQM

[0569] sample sampleHgfW^

[0570] MI Mi

[0571] AI 0.025 Bi MJ6 001

[0572] A2 0.035 B7 p Curve

[0573] A3 0,4663 0.004 B3 J L759 0,02

[0574] A4 3.407 0.031 B4 0,991 0.01

[0575] A5 1 28'1 0.012 B5 | < Curve

[0576] A6 3A31 0.055 B6 | 1,65 0.015

[0577] A7 2.106 0.0 M B? | 0.5444 0.05

[0578] AS 1.644 0.0 i BS j 2,635 0.024

[0579] A9 3.106 0.028 B9 < Curve

[0580] A 10 5,279 0.048 B10 | 0,2966 0.003

[0581] AH 3,055 0.027 BH J 8,124 0.073

[0582] AM 3.30? 0.03 B12 | 7,933 0.071

[0583] A 13 3,035 0.03 B13 | 6,402: 0.06

[0584] AM 6.818 0.061 BM | 4,405 0.04

[0585]

[0586] Al 5 8,977 0.081 Bi 5 s 0.031RESULTS of stool samples collected at T2 (end of treatment - after 56d)

[0587] The results (Table 10) were reported in ng / mL and converted into pg / g using a conversion factor derived from the extraction ratio (Table 11), as reported above.

[0588] Table 10. Fecal calprotectin concentrations in the samples analysed by ELISA assay at time T2 (after the end of treatment). Values are expressed in ng / mL of fecal calprotectin and reported as the mean of two technical replicates. The coefficient of variation (% CV), standard deviation (SD) and standard error of the mean (SEM) for each sample are also reported.

[0589] ID Cone, ID Cone,

[0590] ng / mL % CV SD SEM ng / mL % CV SD SEM sample sample

[0591] (Average) (Average)

[0592] Al 184,7 0,0947 0,484 0,342 Bl 136,2 1,97 2,69 1,9 A2 179,7 0,141 0,254 0,18 B2 24,6 12,8 3,14 2,22 A3 184,6 0,284 0,524 0,371 B3 4,564 10,3 0,47 0,332 A4 183 2,79 2,32 1,64 B4 89 6,93 6,17 4,36 A5 182,1 1,27 2,32 1,64 B5 33,17 5,57 1,85 1,31 A6 177,5 2,3 4,09 2,89 B6 87,14 3,69 3,22 2,27 A7 183,9 1,84 3,39 2,4 B7 120,2 8,71 10,5 7,4 A8 182,3 2,46 4,48 3,16 B8 73,01 0,736 0,537 0,38 A9 185,1 4,12 7,62 5,39 B9 144,4 2,44 3,52 2,49 A10 184,1 1,23 2,26 1,6 B10 65,76 3,89 2,55 1,81 All 163,2 2,37 3,87 2,74 Bll 168,5 0,467 0,787 0,556 A12 186,8 1,42 2,66 1,88 B12 44,93 2,09 0,937 0,663 A13 172,6 1,31 2,26 1,6 B13 145 2,58 3,74 2,64 A14 176,3 1,27 2,24 1,58 B14 173,8 0,0938 0,163 0,115

[0593]

[0594] A15 136,2 1,97 2,69 1,9 B15 166,9 1,68 2,8 1,98Table 11. Results of the ELISA assay for fecal calprotectin, expressed both in ng / mL and in hg / g

[0595] ID f / W- ro IM

[0596] sample sample

[0597] Al {S4.7 1,64 ni 136,3 1,23

[0598] A2 U9 ,7 1,62 B2 24.6 0,22

[0599] A3 IMfi 06 B3 4.564 0,041

[0600] ....

[0601] A4 L63 K i 09 0,80

[0602] A5 182,1 1,64 BS 33,17 O

[0603] A6 177,5 1,60 86 37,14 0,78

[0604] A? 123,0 1,66 B? 130,2 1,08

[0605] AS IM, 3 l,f?4 BO 73,01 0,86

[0606] AS H5.1 1,87 BO 144,4 1,3

[0607] AIS 1M,1 BIO 6A78 0,50

[0608] Al 1 ia,2 1,47 S A i<A < 1,53

[0609] A12 iso i, a 812 403 0.40

[0610] A.13 172,6 1,55 813 l i ' 1,31

[0611] A 14 176,3 1,57 BIA >v1,56

[0612] w.s

[0613] Al 5 1 .1 I J 5 B15 1,50

[0614]

[0615] By comparing these values with the reference ranges of fecal calprotectin according to which a sample is considered:

[0616] • Negative, if the calprotectin concentration is < 50 pg / g

[0617] • Weakly positive, if the calprotectin concentration is between 50 and 120 pg / g

[0618] • Positive, if the calprotectin concentration is > 120 pg / g

[0619] the results indicate that after 56 days of treatment with a food supplement based on a green tea extract (Camellia sinensis (L.) Kuntze - folium) and a pool of probiotics (L. plantarum, L. rhamnosus and B. animalis subsp. lactis) in subjects presenting irritable bowel syndrome (Irritable Bowel Syndrome - IBS) with constipation predominance (IBS-C), analysis of fecal calprotectin by ELISA assay showed a slight increase compared to the baseline value (TO). However, the levels remained well below the threshold of 50 pg / g, confirming negative values and consistent with the absence of active intestinal inflammation. This finding supports theintestinal safety profile of the treatment and reinforces the non-inflammatory nature of the condition.

[0620] RESULTS of stool samples collected at T3 (end of follow-up, that is, 30 days after the end of treatment)

[0621] The results (Table 12) were reported in ng / mL and converted into pg / g using a conversion factor derived from the extraction ratio (Table 13), as reported above.

[0622] Table 12. Fecal calprotectin concentrations in the samples analyzed by ELISA assay at time T84. Values are expressed in ng / mL of fecal calprotectin and reported as the mean of two technical replicates. The coefficient of var|i 11 iation (% CV), standard deviation (SD) and standard error of the mean (SEM) for each sample are also reported.

[0623] 1 i ..

[0624] CUK- SB SEXI ID SB SEXI

[0625] sample <:V

[0626] es-S f»U sample!iCV

[0627] SA

[0628] « VUW

[0629] At A Bl 3,3 3,62 3,07

[0630] A: 7J 2 B2 168,2 422 2,1 M2. :

[0631] AS 4,34 ?:A> B3 j*>3 8433 0.332

[0632] AS 25 .'.'A 1,5 i Mil 84 170,8 5,53 MS Mi

[0633] AS 134,3 3,34 4:,48 Lit B3 W 1,76 4,7i 3,33

[0634] A6 122,8 7,22 8,83 6,25 86 23.45 L24 2,17 1,33

[0635] A" 84,7:7 ■i * < S: X •1 xx B7 18,43 2,05 0,380 0,282

[0636] AS 103 J 3,00 X ■ > L.S it 12

[0637] 75,86 ^45 M

[0638] AH’ 65 J BH 0,343 0,330

[0639] Ail ISO e < 1 <45 Bll 75,78 j,M 0,804

[0640] A S3 MM Mi 812 M2.S 0,53" 0,38

[0641] AS 1 HO." 0,342 <1,378 S,2f» Bl 3 2MJ 0,463 I Mi

[0642] AM 63.6 s.M >.u 2,77 BM 38,47 4,H 1,33 M2

[0643] Ai< ISSA *.7S 8,30 3,93 Bii 38,78 3,73 7, Bl 1,53

[0644]

[0645] Table 13. Results of the ELISA assay for fecal calprotectin, expressed both in ng / mL and in Fg / g-ID sample Cone. Cone. ID sample Cone. Cone.

[0646] ng / mL Pg / g ng / mL Jig / g (Average) (Average)

[0647] Al 211,9 1,91 Bl 170,1 1,53

[0648] A2 194,3 1,75 B2 168,2 1,51

[0649] A3 63,53 0,57 B3 193 1,74

[0650] A4 58,25 0,52 B4 170,8 1,54

[0651] A5 134,3 1,21 B5 170,3 1,53

[0652] A6 122,6 1,10 B6 23,45 0,21

[0653] A7 84,79 0,76 B7 19,43 0,17

[0654] A8 103,2 0,93 B8 107,6 0,97

[0655] A9 75,66 0,68 B9 103,5 0,93 A10 65,1 0,59 B10 54,88 0,49 All 186,4 1,68 Bll 75,78 0,68 A12 203,8 1,83 B12 162,8 1,47 A13 110,7 1 B13 216,7 1,95 A14 68,6 0,62 B14 38,47 0,35

[0656]

[0657] A15 108,3 0,97 B15 58,78 0,53 By comparing these values with the reference ranges of fecal calprotectin according to which a sample is considered:

[0658] • Negative, if the calprotectin concentration is < 50 pg / g

[0659] • Weakly positive, if the calprotectin concentration is between 50 and 120 pg / g

[0660] • Positive, if the calprotectin concentration is > 120 pg / g the absence of significant alterations in calprotectin levels during follow-up (T84) confirms the non-inflammatory nature of irritable bowel syndrome (IBS), distinguishing it from chronic inflammatory bowel diseases (IBD), in which calprotectin is generally elevated. Furthermore, the results suggest that treatment with the supplement under examination did not induce any inflammatory response at the intestinal level, strengthening its safety profile in terms of mucosal tolerability.In conclusion, treatment with the food supplement did not modify intestinal inflammatory status, as demonstrated by the consistently negative levels of fecal calprotectin. This finding supports the safety of the product in a context of IBS-C, a functional disorder by definition not associated with active mucosal inflammation.

[0661] 9) Probiotic colonization rate

[0662] An in vitro molecular analysis was conducted aimed at evaluating intestinal colonization of the probiotic strains administered to subjects under treatment compared to the placebo group. For this purpose, relative quantification of the three probiotic bacterial species — L. plantarum, L. rhamnosus and B. animalis subsp. lactis — was performed by Real-Time quantitative PCR (qPCR) on fecal DNA samples from the two groups enrolled in the study:

[0663] • Group A (placebo): subjects who received a treatment devoid of the supplement ingredients.

[0664] • Group B (treated): subjects who took the supplement containing the probiotic pool — L. plantarum, L. rhamnosus and B. animalis subsp. lactis — and green tea extract, at three different observation times:

[0665] • TO: before the beginning of treatment;

[0666] • T2: after 56 days of intake;

[0667] • T3 : follow-up time, corresponding to 84 days from the beginning of the study.

[0668] The analysis aimed to evaluate the effective intestinal colonization induced by intake of the supplement and to determine persistence of each strain over time compared to the placebo group through fecal analysis.

[0669] MATERIALS AND METHODS DNA extraction from fecal samples

[0670] All fecal samples were stored at -80 °C until the time of analysis, to ensure stability of the genetic material and prevent degradation of microbial DNA.

[0671] Genomic DNA extraction from fecal samples was performed using the Quick-DNA™ Fecal / Soil Microbe Miniprep Kit (Zymo Research, USA), following the manufacturer’s instructions.For each sample, 150 mg of stool were transferred into ZR BashingBead™ Lysis Tubes and subjected to mechanical homogenization using the FastPrep® system, in order to ensure complete lysis of microbial cells present in the sample.

[0672] Subsequently, lysates were subjected to purification, washing and DNA elution steps according to the standard kit protocol, until obtaining DNA of high purity and integrity suitable for subsequent amplification analyses.

[0673] Extracted DNA from each sample was quantified by spectrophotometry (absorbance at 260 / 280 nm) and stored at -20 °C until use for Real-Time qPCR analyses.

[0674] Quantification of probiotic strains

[0675] Relative quantification of the three probiotic strains L. plantarum, L. rhamnosus and B. animalis subsp. lactis contained in the supplement was performed by Real-Time quantitative PCR (qPCR) on DNA extracted from fecal samples of enrolled subjects at different time points:

[0676] 1. TO (before the beginning of treatment),

[0677] 2. T2 (after 56 days of intake of supplement / placebo),

[0678] 3. T3 (follow-up corresponding to 84 days from the beginning of the study).

[0679] Amplification reactions were set up using the QuantiTect SYBR Green PCR Kit (QIAGEN) in a total volume of 20 pL, containing SYBR Green master mix, specific primers (final concentration 0.5 pM), and 10 ng of DNA for each sample.

[0680] Reactions were performed in a Real-Time thermocycler under the following cycling conditions (40 total cycles): 15 min at 95 °C for enzyme activation, 15 s at 94 °C for denaturation, 30 s at 58 °C for annealing and 30 s at 72 °C for extension. Melting curve analysis was also performed to verify specificity of amplified products.

[0681] Data were analyzed according to the AACt (comparative Ct) method. For each sample, the housekeeping gene, in this case 16S rRNA, was also analyzed and used as reference for data normalization.Relative expression of the gene of interest was calculated by comparing its expression level with that of the housekeeping gene. As internal control, the expression value at time TO was used, considered equal to 1-fold induction (reference value).

[0682] Results were expressed as Fold change, representing the relative increase of bacterial DNA quantity at subsequent time points (T2 and T3) compared to baseline (TO).

[0683] Statistical analysis was performed using the Mann-Whitney U test, comparing the mean fold induction values between the treated group (B) and the placebo group (A) for each experimental time point. Differences were considered significant for p < 0.05.

[0684] RESULTS

[0685] Quantitative analysis by Real-Time qPCR showed a significant increase in the relative amount of Lactobacillus rhamnosus DNA in fecal samples of subjects belonging to the treated group (B) compared to the placebo group (A) at observation times T2 and T3. As illustrated in the graph (Figure 10), in both groups an increase in fold change compared to baseline (TO) is observed, but in the treated group (B) such increase is marked and statistically significant already at T2 (p < 0.01) and remains elevated at follow-up (T3) (p < 0.01). Conversely, in the placebo group (A), L. rhamnosus levels remain essentially unchanged over time, suggesting absence of spontaneous colonization. These results indicate that treatment with the supplement containing the probiotic pool and green tea extract induced effective and persistent intestinal colonization of the strain L. rhamnosus LRH020, confirming the ability of this microorganism to survive and establish itself in the human gastrointestinal tract. Under the same experimental conditions, only L. rhamnosus showed evidence of intestinal colonization. Indeed, for the other two probiotic strains, L. plantarum and B. animalis subsp. laclis. no amplification of the target region was detected, suggesting that their presence in fecal samples was below the detection limit of the method. These results may reflect a different capacity of survival, adhesion or persistence of the strains in the intestinal tract, or may be influenced by technical variables (for example extraction conditions or primer amplification efficiency). Overall, the results obtained indicate that L. rhamnosus has intestinal colonization capability.Figure 4 reports data relating to intestinal colonization of L. rhamnosus in subjects belonging to the treated group (B) and the placebo group (A) at different experimental times (TO, T2, T3).

[0686] Data are expressed as Fold Change normalized with respect to time TO (reference value). Thin lines represent individual subject values, while thicker lines indicate the mean for each group. Statistical analysis was performed using the Mann-Whitney U test, comparing mean values between treated group (B) and placebo group (A) for each experimental time point.

[0687] Differences were significant (p < 0.01, indicated with ** at times T2 and T3) in the treated group compared to placebo.

[0688] In addition to the statistical analysis reported above, which compared results obtained from the two groups (placebo and treated) at the same time point (TO-treated vs TO placebo), a second analysis was performed taking into account simultaneously the time of fecal sample collection (T0-T2 and T3) and the treatment condition (treated with supplement and placebo). This analysis aimed to understand whether intake of the probiotic treatment, compared to placebo, was able to increase the relative abundance of the target strains (L. rhamnosus in this specific case) and, above all, whether the increase observed at the end of administration (T2) was maintained at follow-up (T3). For this purpose, measurements were collected for each subject at three different time points: TO (baseline, before treatment), T2 and T3. This analysis demonstrated that the treated sample showed a greater and, above all, more durable increase in relative abundance of the target compared to placebo. Indeed, the significant Group x Time interaction indicates that the trajectories are different: in the Treated group the increase continues up to follow-up, whereas in the Placebo group it tends to attenuate.

[0689] This pattern is consistent with colonization / persistence in the treated group.

[0690] CONCLUSIONS

[0691] In conclusion, between the treated group and the placebo group at the beginning of the study there are no statistically significant differences with regard to the IBS-SSS score, the physical and mental component of quality of life, the Bristol scale score and the number of bowel movements per week, indicating correct randomization of the subjects. Following treatmentwith the food supplement, the scores vary in a statistically significant manner over time, indicating an improvement in the condition. This trend indicates the clinically significant efficacy of the supplement whereby subjects, after two months of treatment and in some cases also after discontinuation of treatment, on average present an improved condition compared to baseline. With regard to the typical symptoms of IBS-C, the sensation of bloating, abdominal distension and sensation of heaviness decrease in the treated group compared to placebo, whereas abdominal pain shows a decrease but not statistically significant, unlike flatulence which remains unchanged compared to the placebo group, as well as the VAS score and the blood parameters serotonin, gastrin and cortisol. Furthermore, the group treated with the supplement resorted to rescue treatments significantly less frequently compared to the group treated with placebo. Use of the kit according to the invention therefore shows a profile consistent with a clinical improvement of IBS symptoms in IBS-C, measured with standard endpoints and with statistical models suitable for repeated measures, with favorable signals particularly on overall severity dimensions and, more generally, on a structured set of clinical variables.

[0692] Bibliography

[0693] [1] Sidhu, D., Vasundhara, M., & Dey, P. (2023). The intestinal-level metabolic benefits of green tea catechins: Mechanistic insights from preclinical and clinical studies. Phytomedicine, 155207;

[0694] [2] Presti, I., D’orazio, G., Labra, M., La Ferla, B., Mezzasalma, V, Bizzaro, G., ... & Di Gennaro, P. (2015). Evaluation of the probiotic properties of new Lactobacillus and Bifidobacterium strains and their in vitro effect. Applied Microbiology and Biotechnology, 99, 5613-5626;

[0695] [3] Dey, P. (2025). Phytochemical- Microbiota Interaction at the Interface of Human Health and Disease . In Body Recomposition (pp. 150-168). CRC Press;

[0696] [4] Radical scavenging activity of tea catechins and their related compounds - PubMed;

[0697] [5] A pilot study to evaluate the safety and efficacy of an oral dose of (-)-epigallocatechin-3-gallate-rich polyphenon E in patients with mild to moderate ulcerative colitis - PubMed;[6] Therapeutic Effects of Green Tea Polyphenol (-)-Epigallocatechin-3 -Gallate (EGCG) in Relation to Molecular Pathways Controlling Inflammation, Oxidative Stress, and Apoptosis - PMC); An Update on the Health Benefits of Green Tea,

[0698] [7] A Review on the Biological Activity of Camellia Species - PubMed.

Claims

Claims1. A kit of products for the treatment or prevention of intestinal dysfunctions comprising:- a first product comprising at least 40% catechins, wherein at least 60% of said catechins is formed by (-)-epigallocatechin-3 -gallate (EGCG);- a second product comprising a mixture of probiotics having a bacterial load of at least 5 billion CFU / g, preferably said intestinal dysfunctions comprise gut-brain interaction disorders, in particular: functional dyspepsia (FD); irritable bowel syndrome (IBS); functional abdominal discomfort; defecation disorders; gastrointestinal motility disorders and / or combinations thereof.

2. The kit according to claim 1, wherein said first product comprises at least 50%, preferably between 50% and 60%, of said catechins, and said catechins comprise at least 70% of said EGCG.

3. The kit according to claim 1 or 2, comprising at least 50% of dry extract of green tea leaves, preferably between 50% and 80%, more preferably 60%, the dry extract of green tea leaves, preferably of the species Camellia Sinensis L. Kuntze, in turn comprising said catechins.

4. The kit according to one or more of the preceding claims, wherein said second product comprises said mixture of probiotics having a bacterial load of at least 6 billion CFU / g, preferably between 6 billion CFU / g and 15 billion CFU / g, more preferably 12 billion CFU / g.

5. The kit according to one or more of the preceding claims, wherein:- said first product comprises between 100 mg and 600 mg of catechins, wherein said catechins comprise (-)-epigallocatechin-3 -gallate (EGCG) in an amount between 80 mg and 550 mg;- said second product comprises between 100 mg and 600 mg of a mixture of probiotics to promote and maintain intestinal functions, preferably said first product comprises hydroxypropyl methylcellulose, gellan gum, corn starch, cellulose, silicon dioxide and magnesium salts of fatty acids and iron oxides and / or hydroxides and / orpatent blue V, and said second product comprises hydroxypropyl methylcellulose, pectin, corn starch, silicon dioxide and magnesium salts of fatty acids.

6. The kit according to one or more of the preceding claims, wherein said catechins are between 200 mg and 350 mg, preferably 270 mg, and wherein, of said catechins, EGCG is between 190 mg and 300 mg, preferably 195 mg.

7. The kit according to one or more of the preceding claims, comprising dry extract of green tea leaves between 200 mg and 800 mg, more preferably 300 mg, the dry extract of green tea leaves, preferably of the species Camellia Sinensis L. Kuntze, in turn comprising said catechins.

8. The kit according to one or more of the preceding claims, wherein said catechins are selected from the group consisting of:• (-)-epigallocatechin-3 -gallate (EGCG),• (-)-epigallocatechin (EGC),• (-)-epicatechin-3 -gallate (ECG),• (+)-gallocatechin-3-gallate (GCG),• (-)-epicatechin (EC),• (+)-gallocatechin (GC),• (+)-catechin (C);and / or combinations thereof.

9. The kit according to one or more of the preceding claims, wherein said mixture of probiotics comprises Lactobacillus and / or Bifidobacterium, each genus being between 2 billion CFU and 4 billion CFU, preferably 2 billion CFU.

10. The kit according to one or more of the preceding claims, wherein said probiotics are selected from the group consisting of Bifidobacterium animalis subspecies lactis BL050, Lactobacillus rhamnosus LRH020, Lactobacillus plantarum PBS067 and / or combinations thereof, wherein, preferably, each probiotic type is between 2 billion and 4 billion, preferably 2 billion.

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