EP4694879A1

EP4694879A1 - Composition for oral use comprising quercetin and bromelain

Info

Publication number: EP4694879A1
Application number: EP24723432.1A
Authority: EP
Inventors: Sabrina FIORENTINO; Isabella PISANO; Maria Lisa Clodoveo; Filomena CORBO; Marilena MURAGLIA; Anna BORRACCINO
Original assignee: Sestre SRLS
Current assignee: Sestre SRLS
Priority date: 2023-04-11
Filing date: 2024-04-04
Publication date: 2026-02-18
Also published as: WO2024213970A1; IT202300006855A1

Abstract

Composition for oral use comprising a mixture the components of which are at least quercetin and bromelain for the treatment of chronic inflammatory pathologies, said composition being characterized in 5 that quercetin and bromelain are present together in a proportion respectively equal to 5:3.

Description

COMPOSITION FOR ORAL USE COMPRISING QUERCETIN AND BROMELAIN

DESCRIPTION

Technical field of the invention

The present invention relates to a composition for oral use comprising a combination of nutraceutical molecules such as Quercetin and Bromelain . In particular, the composition of the present invention is used as a prebiotic and anti-inflammatory agent for the treatment of chronic inflammatory pathologies , such as endometriosis .

Background art

As is known, endometriosis is a chronic and recurrent , estrogen-dependent inflammatory disease , characteri zed by the presence of endometrial tissue in ectopic sites . It signi ficantly compromises the women quality of li fe , as it is characteri zed by very painful symptoms represented above all by dysmenorrhea, dyspareunia, dyschetia and chronic disabling pelvic pain, which af flicts especially young women causing serious physical discomfort , relational and emotional di f ficulties . Endometriosis is characteri zed by a multi factorial etiology which sees a close interrelationship between genetic, metabolic, environmental and immune factors. Several studies evaluate the impact of endometriosis on quality of life with specific reference to work productivity. A cross-sectional multicenter study, conducted in 2011 in ten Countries (including Italy) on a total of 1,418 women, shows how endometriosis damages health-related quality of life (HRQoL) and work productivity. Each affected woman loses an average of 10.8 hours of work per week, mainly due to reduced efficiency at work (https : // win .mnlpub limed . com/ public/ 1233A02. pdf .

Data from the Istituto Superiore di Sanita (ISS) report that in Italy 10-15% of women of reproductive age and 30-50% of infertile women are affected by endometriosis. The social impact of the disease is high and is connected to the reduction in quality of life and direct and indirect healthcare costs. In the more advanced clinical stages ("moderate or third degree" and "severe or fourth degree") , endometriosis is included in the list of chronic and disabling pathologies, granting patients the right to benefit in exemption from certain specialized control services . The limited awareness of the pathology is the cause of the serious diagnostic delay, estimated at around seven years .

Currently, therapeutic strategies make use exclusively of hormonal therapies , which are unsatis factory and cause numerous side effects . Antiinflammatories , such as FANs or opioids or progestins , are used for the treatment of these pathologies , to induce forced amenorrhea and limit the symptoms associated with the pathology . However, preliminary studies have highlighted the role of some health foods and nutraceuticals in modulating the inflammatory processes involved in the symptoms and in the progression of endometriosis and its co-morbilities (Nutrients 2021 , 13 ( 6 ) , 1747 ) .

Among the most common symptoms o f endometriosis are : intestinal disorders , dyschetia and constipation .

It is also known that the use of antioxidants in the treatment of endometriosis can inhibit the proli feration of endometrial cells in adenomyosis and reduce their mobility and invasiveness . These inhibitory effects may be related to the under regulation of MMP2, MMP9 proteins

(https : // pubmed . ncbi . nlm. nih . gov/ 33061289/)

Dietary compounds are the main components that regulate gene expression via epigenetic mechanisms such as DNA methylation and histone modification via histone-acetyltransf erases (HAT) and histonedeacetylases (HDAC) . Long-term consumption of bioactive compounds can alter the epigenome and significantly contribute to the development of nutritional programs to prevent and treat chronic metabolic diseases.

As demonstrated by various studies, quercetin is a naturally occurring polyphenolic flavonoid. Known for its antioxidant, anti-inflammatory, antiproliferative and anticancer effects, it has shown great benefits in the treatment of cancer and endometriosis, without obvious side effects. Quercetin is present in apples, onions and citrus fruits. It can activate the enzyme sirtuin-1 NAD- dependent deacetylase (SIRT1) and NAD-dependent deacetylase. It is able to inhibit the expression of tumor necrosis factor ( TNF) -induced interferon gamma ( IFNy) -inducible protein 10 (IP10) and macrophage inflammatory protein 2 (MIP2 ) . Consequently, it blocks posttranslational modi fications ( acetylation and phosphorylation) of H3 histones through the induction of p300/CBP . Consequently, promoters of proinf lammatory genes associated with H3 histones are af fected by inhibition of cofactor recruitment at the chromatin level . Furthermore , through inhibition of HDAC and DNMT1 , quercetin inhibits the cell cycle and consequently induces apoptosis , suppressing tumor growth and angiogenesis (NagisaNosrati , Int . J . Mol . Sci . 2017 , 18 , 2050 ) .

Furthermore , it is also known that the cellular bioavailability of quercetin can be compromised by a close binding to plasma proteins , in particular albumin . The ef fect of the combination of quercetin and bromelain in an attempt to improve the transport of the bioflavonoid across the intestinal membrane is also known . In this blind, open-label study, there was a greater impact on symptom score , percentage of improvement , and percentage of patients with at least 25% improvement , compared to pure quercetin capsules alone . Although the exact mechanism of action of quercetin in improving the symptoms of men with chronic prostatitis is not known, in all four patients taking the quercetin-papain-bromelain combination who were tested, there were strong decreases in the levels of iso-prostane in the prostatic fluid, a putative marker of oxidative stress in the prostate . Quercetin treatment can therefore prevent oxidatively mediated cell damage induced by an infectious , inflammatory or autoimmune mechanism .

A further publication highlights how proteolytic enzymes exert notable ef fects on the barrier function of cell monolayers of the Caco-2 human colorectal adenocarcinoma line , facilitating the transport of normally non-absorbable compounds . This suggests that the reported, but so far unexplained, systemic absorption of proteolytic enzymes after oral administration in vivo may also occur by sel f-enhanced paracellular transport

(https : / / pubmed . ncbi . nlm . nih . gov/ 9755891 / ) .

There is therefore a need to define a pharmaceutical composition that solves or at least mitigates chronic inflammatory pathologies . Summary of the invention

The obj ect of the present invention is a composition for oral use and comprising a mixture , the components of which are at least quercetin and bromelain for the treatment of chronic inflammatory pathologies such as endometriosis or irritable bowel syndrome ( IBS ) . The speci fic composition of the present invention is used as a nutraceutical and antiinflammatory agent .

Advantageously, this composition promotes an antioxidant and anti-inflammatory activity which acts on compromised biochemical pathways through a physiological approach, constant over time which counteracts/reduces chronic inflammation .

Advantageously, this composition acts on symptoms collateral to the pathology, such as intestinal disorders in the case of endometriosis as the use of nutraceuticals is a valid innovative therapeutic tool for the treatment of all conditions characteri zed by neuroinflammation and chronic painful states , in the absence of side ef fects .

Furthermore , the action of nutraceuticals is not immediate but occurs gradually over time , therefore prolonged administration is necessary to obtain the therapeutic effect . Therefore , according to the present invention, a composition for oral use is defined, as speci fied in the attached independent claim .

The dependent claims outline particular and further advantageous aspects of the invention .

Brief description of the drawings

These and other advantages of the invention will now be described in detail , with reference to the attached Figures , which represent an exemplary embodiment of the invention, in which :

- Figure 1 shows the diagram relating to the growth curve of SaccaromycesCerevisiae as a model for studying oxidative stress , according to known technique ;

- Figure 2 shows the diagram relating to the tests carried out on the growth of Saccaromyces Cerevisiae in the presence of the composition in two di f ferent concentrations , which is the obj ect of the present invention,

- Figure 3 shows the diagram relating to the growth curve of Saccaromyces Cerevisiae according to a first concentration of the composition obj ect of the present invention, - Figure 4 shows the diagram relating to the growth curve of Saccaromyces Cerevisiae according to a second concentration of the composition object of the present invention,

- Figure 5 shows the comparison diagram between the curves of Figure 3 and Figure 4, according to the present invention,

- Figure 6 shows the analysis of cell viability in flow cytometry, defined by a gate fixed on the basis of the FCS (forward scatter - frontal dispersion) and the SSC (side scatter - lateral dispersion - SSC_Q2) , according to the present invention,

- Figure 7 shows the identification of the three cell populations, analyzed after staining with 7AAD in flow cytometry via a density plot as a function of FSC according to the present invention,

- Figure 8A-D shows the graph of the cell viability analysis performed by flow cytometry by staining 7AAD. 8A: samples stimulated with LPSvs . unstimulated samples; 8B: samples treated with A, B, A+B + LPS vs. Ctrl+ LPS; 8C: samples treated with A+B + LPS vs.

B+LPS. *p <0.05, **p <0.01,***p <0.001; 8D: dead cells, according to the present invention, - Figure 9A-C shows the structure of H2DCFDA (9A) and its reaction products of hydrolysis of the acetyl groups (9B) and oxidation (9C) which leads to the formation of a fluorescent compound which allows the production of ROS to be estimated, according to the present invention,

- Figure 10 shows the analysis of intracellular ROS production, using a "density plot vs FSC" which analyzes cells selected with the initial gate based on their physical characteristics, according to the present invention,

- Figure 11 A-B shows the analysis of intracellular ROS production, calculated on a reference histogram (11A) which analyzes cells selected with the initial gate through a quantification of the MFI (11B) with the indicated treatments in basal conditions and in the presence of a pro-inflammatory stimulus a: samples stimulated with LPS vs. unstimulated samples: b: samples treated with A, B, A+B + LPS vs. Ctrl + LPS. **p <0.01, ***p <0.001, according to the present invention,

- Figure 12 shows the analysis of intracellular ROS production (DCF+ cells) in relation to the percentage of dead (12A) and live (12B) cells in the indicated treatments in basal conditions and in the presence of a pro-inflammatory stimulus, a: samples stimulated with LPS vs. unstimulated samples; b: samples treated with A, B, A+B + LPS vs. Ctrl + LPS. **p <0.01, ***p <0.001, according to the present invention.

Detailed description

According to the present invention, a composition for oral use is described comprising at least quercetin (2 (3,4 Dihydroxyphenyl) 3, 5, 7trihydroxy4Hl benzopyran-4-one dihydrate) from Alliumcepa L. titrated 30% in Quercetin-100 mcg and bromelain 2500 GDU- 60 mcg. In particular, the ratio between Quercetin and Bromelain is equal to 5:3.

According to a further embodiment of the present invention, the composition further comprises at least one further active compound: for example Inulin, Citrus Aurantium dry extract tit. 6% Synephrine, Artichoke dry extract tit. 5% chlorogenic acid, Broccoli dry extract tit. 0.3%-0.5% Sulforaphane, Melissa officinalis dry extract tit. 4% rosmarinic acid, Quercetin, Mentha piperita dry extract, Fennel dry extract tit. 1% Essential Oil, Selenium and Chromium .

Advantageously, the presence of further active compounds guarantees the balance of the Microbiota, the presence of Inulin for example favors the proli feration of some bacterial strains which contribute to the synthesis of some Enzymes , such as glucuronidase , which regulate the levels of circulating estrogens and therefore contribute to the regulation of the hormonal profile .

Again, according to a further embodiment of the present invention, the composition further comprises at least one inactive excipient : for example calcium phosphate inactive , magnesium stearate inactive and silicon dioxide inactive .

Advantageously, the presence of excipients , for example inactive Mg and/or silicon dioxide , are used as anti-caking agents , or for the purpose of guaranteeing smoothness and stability during the production phase .

According to a further aspect , the composition for oral use is in the form of capsules or soluble powder contained in a special sachet .

In particular, a preferred composition for oral use , for a 700 mg capsule is , for example , shown in

Table 1 where the dosages refer to two capsules .

T a b 1 e 1

The excipients contained in the composition according to the present invention contribute to the above-mentioned advantages . In particular, such excipients for a 700 mg capsule are , for example : inactive calcium phosphate in a weight quantity of

135 mg, inactive magnesium stearate in a weight quantity of 25 mg, inactive silicon dioxide in a weight quantity of 8 . 5 mg .

Advantageously, the recommended dose is equal to two capsules per day . Advantageously, the composition obj ect of the present invention is also used for the preparation of a food supplement , for the treatment of inflammatory disorders of the intestinal tract .

As shown in Table 2 below, the clinical tests were carried out exclusively on women with an average age between 25 and 40 years .

Gender di fferences have implications for human health, risk of assessment and toxicology . There are several effects that influence the results and toxic responses to some drugs or biologically active molecules: in addition to the obvious modifying effects of sexual hormones, there are conditions that affect male and female reproductive organs differently, as well as genetic and hormonal ones.

All aspects of toxicology should consider gender-balanced designs so that a more complete understanding of the differences and similarities can be obtained. Differential gene expression is a new frontier in toxicology. Risk assessment should take into account gender and life cycle differences.

As shown in Figures 1-5, preclinical studies were tested on the growth curve of Saccaromyces Cerevisiae yeasts as a study model of oxidative stress, at tested concentrations of H2O2 : 012 mM.

As shown in Figure 2, two different concentrations of Quercetin and Bromelain were used. Specifically, the first concentration includes Quercetin 15 mg and Bromelain 10 mg (COMBO C) and the second concentration includes Quercetin 5 mg and Bromelain 20 mg (COMBO E) in presence of 2mM H2O2.

Figures 3 and 4 show the effect of the two concentrations of Quercetin and Bromelain (COMBO C and COMBO E) on the growth of Saccaromyces Cerevisiae, in the presence of a 2mM H2O2 oxidizing agent. The concentrations tested are 0.04 (1) and 0.08 (2) g/L. Both concentrations demonstrated an improving effect on the growth of Saccaromyces Cerevisiae colonies.

Finally, Figure 5 shows the comparison diagram between the effect of the two concentrations of Quercetin and Bromelain in the two concentrations used for the tests (COMBO C and COMBO E) . The second concentration (COMBO E) includes Quercetin 5 mg and Bromelain 20 mg and is the one that significantly reduces the production of reactive oxygen species (ROS) in Saccaromyces Cerevisiae yeast cells in presence of a 2mM H2O2 oxidizing agent, at a concentration tested at 0.04 g/L.

Chemical assays on antioxidant activity and in vitro studies were performed on Caco-2 intestinal cells, the latter carried out in basal conditions and in response to a pro-inflammatory stimulus. Different chemical assays were performed: with DPPH (2,2- diphenyl-l-picrylhydrazy) solution and "Fluorescence recovery after photo-bleaching" (FRAP) . The in vitro ef fect of the Quercetin + Bromelain combination was evaluated in terms of cell viability and antioxidant activity, compared to the individual components through :

- the culture and treatment of Caco-2 cells with the substances under investigation;

- analysis of cell viability by cytof luorometry .

Subsequent studies were performed to study the ability of the A+B combination ( respectively Quercetin + Bromelain) to modulate , compared to single substances , the cellular response in basal conditions and following a pro-inflammatory stimulus in an in vitro model of Caco-2 cells , in terms of cell viability and antioxidant potential in the context of intestinal inflammation . It was first necessary to carry out a dose-response experiment , in order to identi fy the dose that had the least impact on cell viability .

Considering the dose indicated in the composition under study, a dilution factor was considered that took into account the concentration ranges usually used in in vitro assays to test later, starting from here , two scalar concentrations . This preliminary analysis allowed to de fine the dose to be used in the experiments reported here , which is the one containing 25 pg/ml A and 15 pg/ml B . Therefore , the Caco-2 cells were treated with the selected dose of substance A, substance B and their combination and after one hour they were stimulated with LBS for 24 hours . At the end of the stimulation with a macrophage stimulator, released from gram-negative bacteria ( LPS ) , the cells were recovered, stained with a cell viability marker ( 7AAD) and analyzed by flow cytometer . The analysis was conducted by carrying out an initial gate based on the physical characteristics of the Caco-2 cells ( si ze and granularity, analyzed respectively by the Forward Scatter, FSC and the Side Scatter, SSC ) , ef fectively excluding cellular debri s . As shown in Figure 6 , there is the identi fication of the population analyzed for the analysis of cell viability in flow cytometry, defined by a gate set based on the FCS and SSC ( Q2 ) .

On the population thus selected, the fluorescence was evaluated for the 7AAD which has an excitation/emission wavelength of 546/ 647 nm, respectively . The fluorescence emitted in the far red allowed to identify, in a density plot as a function of the FSC, three distinct populations: one negative (7AAD-) and one positive for 7AAD within which 2 subpopulations are distinguished, one with an intermediate fluorescence (7AADint. and one with a higher fluorescence (7AAD+) . It is likely that these three populations, identified in terms of percentage, refer, in order, to live cells (Live cells) , to cells that are dying (Dying cells) and dead cells (Dead cells) (Figure 7) .

The results of this analysis showed that the percentage of live cells after stimulation with LPS, unlike what was seen in the untreated sample where it is significantly reduced, is almost unchanged compared to the reference sample not stimulated with LPS; this is true both for single substances and for the A+B combination (Figure 8A and 8B,a) by restricting the analysis to the effect of the three treatments in the presence of a pro-inflammatory stimulus, it was possible to note that the percentage of live cells increased compared to untreated cells, with a more marked increase in the case of treatment with A and with the combination A+B (Figure 8B,b) comparing the ef fect of the combination compared to the single components in the presence of LPS , it was possible to observe a signi ficant increase in the percentage of live cells compared to the treatment with B ( Figure 8B, c ) which is in line with the less marked increase of live cells when treated with B and stimulated with LPS .

In the case of the two subpopulations of 7AAD- positive cells , the dying cells are more represented than the dead cells , as can also be seen from Figure 8A which provides a global view of the distribution of the three populations identi fied following labeling with 7AAD all inside the single sample , allowing to better appreciate their modulation following the stimulation with LPS . Focusing on the cells that are dying, only the sample treated individually with A does not signi ficantly increase the percentage of cells that are dying compared to the relevant sample not stimulated with LPS ( Figure 8C, a ) however, this increasing trend found with A and the signi ficant increase detected after treatment with B and with A+B in presence of a pro-inflammatory stimulus ( Figure 8C, a ) is signi ficantly lower than the increase found in the non-treated sample (Figure 8C,b) . Furthermore, in the case of dying cells, the effect due to treatment with the A+B combination does not differ from the treatment observed with the single substances in the presence of LPS . Regarding dead cells, although generally less represented in all samples as already mentioned, the results obtained demonstrate that these are significantly reduced only by treatment with the A+B combination compared to the basal equivalent (not stimulated with LPS) (Figure 8D,a) . Furthermore, the A+B combination was able to reduce dead cells, like treatment with A alone, compared to cells not stimulated in presence of LPS (Figure 8D, b) . Finally, in line with the modulation demonstrated for the percentage of live cells, the decrease found in dead cells is statistically significant compared to those treated only with B and stimulated with LPS (Figure 8D,c) . From the data presented in Figure 8 it can be seen that treatment with the A+B combination, like the single components, does not significantly modulate the viability of Caco- 2 cells in basal conditions. On the contrary, in the presence of a pro-inflammatory stimulus, it is able to signi ficantly increase the percentage of live cells and reduce that o f dying cells and that of dead cells compared to untreated cells . This being the case , the ef fect on cell viability seems to be more attributable to the A component of the combination, in particular as regards the modulation of live and dead cells . As regards the evaluation of the antioxidant capacity of the A+B combination compared to single molecules , again on Caco-2 cells , the ability of the compounds under examination was studied, in order to reduce the formation of ROS using 2 ' , 7 ' -dichloro- f luorescein- diacetate (H2DCFDA) as a marker, Figure 9A) .

The presence of the two acetyl groups makes the compound lipophilic, allowing it to easily di f fuse inside the cells where it remains trapped due to the action of intracellular esterases which, by removing the acetyl groups , make the compound ( DCFH, Figure 9B ) polar and therefore waterproof . The possible presence of ROS inside the cells induces the oxidation of DCFH into a highly fluorescent product , 2 , 7- dichloro- f luorescein ( DCF, Figure 9C ) .

Fluorescence analysis will provide an estimate of the levels of ROS produced by the cells . This analysis can be done using different detection systems; in our case, the system chosen was the flow cytometer. Also for this analysis, an initial gate was defined based on the FCS and SSC on which the expression of DCF was evaluated in percentage terms through a density plot (DCF+ cells vs FSC, Figure 10) and in terms of median fluorescence (MFI) using a histogram (Figure 11A) .

The MFI analysis demonstrates that, although the compound B in the presence of LPS is the only one, like untreated cells, which is capable of increasing ROS production compared to its reference sample not stimulated with LPS (Figure 11B, a) , this modulation is much less relevant in terms of the median fluorescence analyzed. In fact, comparing the results obtained following stimulation with LPS, cells treated with the A+B combination and its single components (including B) significantly reduce the MFI of DCF compared to untreated cells (Figure 11B, b) .

To confirm that the observed ROS modulation was not a consequence of alterations in cell viability, the percentage of dead cells was first related to that of cells expressing DCF (Figure 12A) . In line with what is shown in Figure 12A, the aforementioned ratio does not appear to be signi ficantly modulated, except in the case of the A+B combination, where the LPS stimulus induces a reduction . Similarly, Figure 12B which analyzes the percentage of live cells compared to DCF+ cells , confirms that the modulation of ROS is not correlated to cell viability; indeed, in the case of non-stimulated cells , compared to a reduction in this ratio detected in the presence of LPS , DCF MFI , it is strongly up-regulated ( Figure 12B ) .

Therefore , also with regard to the ability to reduce oxidative stress in Caco-2 cells , the A+B combination, although demonstrating an antioxidant ef fect in the presence of a pro-inflammatory stimulus , does not present , at least at the tested dosage , a synergistic action as it does not confer an additional benefit compared to the administration of the individual components .

The interpretation of the results obtained from the preliminary study conducted in vitro on the combination of nutraceutical substances A and B present in the food supplement studied through the FRAP and DPPH chemical assays , in agreement with what was demonstrated in the assays on human intestinal cell lines , allows to state that the combination in question shows an interesting antioxidant activity . The results on Caco-2 cells demonstrate , in addition, that treatment with the A+B combination in the presence of a pro-inflammatory stimulus is able to regulate cell viability, by increasing the percentage of live cells and reducing that of dead cells compared to untreated cells ; this ef fect would appear to be attributable to the action of component A.

The reported results allow to demonstrate that the A+B combination does not af fect the activity of the individual components in terms of cell viability and antioxidant potential .

Although at least one exemplary embodiment has been presented in the summary and detailed description, it must be understood that there are a large number of variations within the scope of the invention . Furthermore , it must be understood that the embodiment or embodiments presented are merely examples , which are not intended to limit in any way the scope of protection of the invention or its application or configurations . Rather, the summary and detailed description provide the skil led technician in the sector with a convenient guide to implement at least one exemplary embodiment , it being clear that numerous variations can be made in the function and assembly of the elements described herein, without departing from the scope of protection of the invention as established by the attached claims and their technical-legal equivalents .

Claims

1. Composition for oral use comprising a mixture the components of which are at least quercetin and bromelain for the treatment of chronic inflammatory pathologies, said composition being characterized in that quercetin and bromelain are present together in a proportion respectively equal to 5:3.

2. Composition for oral use according to claim 1 further comprising at least one active compound among the following compounds: Inulin, Citrus Aurantium dry extract tit. 6% Synephrine, Artichoke dry extract tit. 5% chlorogenic acid, Broccoli dry extract tit. 0.3%-0.5% Sulforaphane, Melissa officinalis dry extract tit. 4%, rosmarinic acid, Quercetin, Mentha piperita dry extract, Fennel dry extract tit. 1%, Essential Oil, Selenium and Chromium.

3. The composition for oral use according to claim 1 or 2, further comprising at least one inactive excipient among the following excipients:

- calcium phosphate,

- magnesium stearate,

- silicon dioxide.

4. Composition for oral use according to claim 1 or 2 in the form of a capsule or soluble powder.

5. Composition for oral use according to one of the preceding claims, wherein the mixture of which for a 700 mg capsule comprises the following components :

- Inulin in a weight amount equal to 195 mg;

- Citrus Aurantium dry extract tit. 6% Synephrine in a weight amount equal to 50 mg;

- Artichoke dry extract tit. 5% chlorogenic acid in a weight amount equal to 50 mg;

Broccoli dry extract tit. 0.3%-0.5% sulforaphane in a weight amount equal to 50 mg;

Melissa officinalis dry extract tit. 4% rosmarinic acid in a weight amount equal to 50 mg;

- Quercetin in a weight amount equal to 50 mg;

- Mentha piperita dry extract in a weight amount equal to 30 mg;

- Bromelain 2500 GDU/g in a weight amount equal to 30 mg;

- Fennel dry extract tit. 1% Essential Oil in a weight amount equal to 25 mg;

- Selenium in a weight amount equal to 6.32 mcg; - Chromium in a weight amount equal to 3 . 45 mcg;

- Calcium phosphate in a weight amount equal to 135 mg ;

- Magnesium stearate in a weight amount equal to 25 mg ;

- Silicon dioxide in a weight amount equal to 8 . 5 mg .

6 . Use of a composition as specified in one of claims froml to 5 for the preparation of a food supplement for the treatment of endometriosis .

7 . Use according to claim 6 of the composition according to claim 5 for a dosage of two 700 mg capsules per day .