EP4090345A1

EP4090345A1 - Composition for the protection of gastric mucosa

Info

Publication number: EP4090345A1
Application number: EP21703983.3A
Authority: EP
Inventors: Umberto DI MAIO
Original assignee: Neilos SRL
Current assignee: Neilos SRL
Priority date: 2020-01-14
Filing date: 2021-01-14
Publication date: 2022-11-23
Also published as: WO2021144727A1; IT202000000502A1

Abstract

A composition containing Hericium erinaceus, at least an extract of Matricaria chamomilla and hyaluronic acid and/or a salt thereof in mixture with suitable carriers is disclosed. The composition is useful in the prevention and in the treatment of ulcers and to protect the gastrointestinal mucosa from aggressive insults.

Description

“Composition for the protection of gastric mucosa”

DESCRIPTION

The present invention relates to a composition containing Hericium erinaceus, at least an extract of Matricaria chamomilla and hyaluronic acid and/or a salt thereof for the prevention and/or the treatment of ulcers and for protecting the gastrointestinal mucosa. Such an invention is based on the synergistic action of the substances contained thereof.

State of the art

Peptic ulcer is a disorder that in recent years affects an increasing percentage of the population. It is possible to identify various types of ulcers that are distinguished by the point where they are located in the digestive tract: duodenal ulcers are located in the duodenum, the first section of the small intestine; gastric ulcers are located within the gastric wall. The stomach wall is made up of four layers (from the innermost to the outermost: mucous, submucosal, muscular and serous) that cooperate to mix food with acids, mucus and pepsin, and release chyme into the duodenum for the absorption process. A gastric ulcer is a necrotic lesion that penetrates the entire thickness of the mucosa characterized by severe epigastric pain. Other symptoms that often go with the appearance of ulcerative tissue in the stomach are a sense of fullness, bloating, early satiety and nausea. Patients with duodenal ulcer generally suffer from epigastric pain in the initial stages of the disease process or during the night and this sense of pain decreases following the intake of food or anti-acid agents. A peptic ulcer is formed due to an imbalance of the mechanisms that regulate tissue homeostasis. In particular, there is an increase in aggressive factors that break the mechanisms that the gastric mucosa uses to protect itself, and a decrease in the defensive factors that normally maintain the integrity of the mucosa. This imbalance favors the establishment of a pathological situation in which the tissue is affected by aggressive insults and fails to activate the necessary defensive mechanisms with a consequent thinning of the mucosa, perforation and, therefore, bleeding. Aggressive factors that contribute to damage to the gastro-duodenal mucosa include, in the first place, dyspepsia. In particular, the increase in acid and pepsin secretions increase the insults on the mucosa and predispose it to a continuous pro-inflammatory state. The presence of Helicobacter pylori contributes to damage the mucosa as it alters the immune and inflammatory mechanisms put in place by the cells in response to an insult. Other factors involved in the onset of peptic ulcers are: the intake of non steroidal anti-inflammatory drugs which by inhibiting cyclooxygenases (both COX1 and COX2) reduce the formation of prostaglandins with a protective effect on the mucosa; psychological stress; oxidative stress and an increase in free radicals that favor the onset of the aforementioned imbalance between aggressive factors and the protective mechanism; inhibition of cell proliferation and infiltration of pro- inflammatory molecules. Pharmacological treatment generally involves a combination of various active ingredients and each therapy is studied and chosen based on the patient's age, general health, medical history and tolerance to the treatment. The goals of the treatment are to reduce pain and improve the healing process. Commonly used drug therapies involve the use of an anti-secretory agent, such as H₂ receptor antagonists and pump inhibitors, of a mucosal protective agent, such as prostaglandin analogues, an antacid or a complex bismuth salt. Therapies with the aforementioned agents present, in the long run, several side effects. The use of cimetidine, an antagonist of H₂ receptors, has, for example, recorded the onset of gynecomastia. Toxicological studies on omeprazole have revealed that the use of the aforementioned pump inhibitor can have, as a side effect, the formation of carcinoids, endocrine tumors of the stomach that are generated by an increase in the proliferation of enterochromaffin cells. As a result, in recent times, attention has grown towards safer alternatives that have fewer side effects and natural products are advancing in the pharmaceutical industry as new potential sources of bioactive molecules. Many natural substances have already proven to be effective in the field of gastro- protection, as well as safe and well tolerated.

It has been surprisingly found that an association of Hericium erinaceus, at least an extract of Matricaria chamomilla and hyaluronic acid and/or a salt thereof is particularly effective in protecting the gastrointestinal mucosa and in reducing ulcers thanks to the synergistic action of its components. Therefore the present invention aims at providing a valid alternative to the currently available therapies to protect the gastrointestinal mucosa from ulcerative phenomena.

The terms used in the present description are as generally understood by the skilled person, unless otherwise indicated.

The term "extract", in the context of this description, means any product attributable to a plant drug including all products derived from mechanical treatments (pulverization, grinding, mixing and/or other methods) or from extraction treatments (solvent extraction, distillation, and/or other specific methods) operated on a drug. Detailed description of the invention

Object of the present invention is a composition containing an association of Hericium erinaceus, at least an extract of Matricaria chamomilla and hyaluronic acid and/or a salt thereof for the protection of gastrointestinal mucosa, particularly useful for the treatment of ulcers.

The components of the association according to the present invention are all known components, widely used in therapy.

Hericium erinaceus

Fungi have always played a central role in traditional oriental therapies and, even today, they are widely used in modern oriental medicine. Fungi represent an important source of interesting bioactive substances which appear to be promising for the development of new compositions with a phytotherapeutic action. A large number of data obtained from medical and scientific studies carried out in various countries of the world have demonstrated the unique and promising properties of substances extracted from mushrooms for the prevention and treatment of multiple pathological states. Hericium erinaceus is a culinary fungus that has a rounded structure and a diameter of about 25 cm. The fresh fungus is white in color and tends to yellow with age. Aging, in addition to causing yellowing, alters the flavor, making it bitter. Hericium does not have a well-defined cap but consists of a short stem from which a series of filaments branch off which have a length of about 4 cm. It generally grows on the trunks, branches and stumps of trees (mainly walnut, oak, elm and beech) in the northern hemisphere. In China it is commonly known as "monkey fungus" and, in the rest of the world, it is known by a number of common names including "lion's mane" or "old man's beard". In addition to its excellent flavor, Hericium erinaceus is also appreciated for its multiple health properties. In the oriental tradition it has always been appreciated and used for its remarkable benefits and, to date, an increasing number of evidence confirm its scientific validity. An important number of studies have highlighted the promising chemotherapeutic effect that the substances extracted from the fruiting body of the fungus have shown in various types of cancer such as stomach, esophageal and skin cancers. Recent studies have shown that this fungus has an important physiological effect on the regulation of lipid and blood sugar levels. Hericium also has promising anti-inflammatory, immunomodulatory, antimicrobial and nerve regeneration activities. Chemical analyzes performed to identify the bioactives present in Hericium erinaceus have revealed, in recent years, that polysaccharides are the most abundant constituents and the main ones responsible for most of the therapeutic actions of the fungus. In addition to polysaccharides, other low molecular weight substances have been identified such as erinacins, ericenones and erinacerins, belonging to the family of terpenoids, proteins, enzymes and amino acids. The polysaccharides extracted from the fruiting body of Hericium in recent years have also attracted attention for the potential beneficial effects exerted in the field of gastro- protection and, in particular, in the healing of peptic ulcers. Scientific evidence shows that treatment with Hericium extract has a positive effect on ulcer-type lesions both in terms of number and size of lesions. The effect is attributed to both a decrease in acid secretions and an increase in the production of protective mucus and an antioxidant effect (due to an increase in antioxidant enzymes SOD and CAT) and a scavenger action on free radicals. An in vitro study has also shown that the bismuth salts of some polysaccharides identified in the fungus have shown an anW- Helicobacter pylori action comparable to that of CBS (colloidal bismuth subcitrate)

Another in vitro study on human GES-1 gastric epithelial cells showed the protective effect exerted by the HEPwl polysaccharides isolated from Hericium against cell damage induced by oxidative stress. The damage was induced by treating cells with concentrations between 150 and 900 mM of H₂0₂. This procedure caused an increase in free radicals and ROS with consequent accumulation of DNA damage and activation of the cell death mechanism by apoptosis. The cells treated with HEPwl (at concentrations of 125, 250, 500, 1000, 2000 and 4000 pg/mL) were protected from damage caused by oxidative stress. Thanks to the activation of antioxidant mechanisms, the treatment reduced DNA damage and protected gastric cells from apoptosis.

An in vivo study in Spague-Dawley rats evaluated the positive effect of Hericium extract on ethanol-induced ulcer or pyloric ligament ulcer. The animals used were divided into groups as follows:

- Group 1 : control, administration of saline solution;

- Group 2: control group with ulcer;

- Groups 3 and 4: pretreatment with 100 mg/kg of fresh or refined extract;

- Groups 5 and 6: pre-treatment with 200 mg/kg of fresh or refined extract; - Groups 7 and 8: pretreatment with 400 mg/kg of fresh or refined extract.

Both models used caused disseminated ulcers with diffuse haemorrhagic phenomena and mucosal edema in the animals undergoing the study. Pretreatment with the extracts used caused a dose-dependent reduction in lesions for both models. The treatment reduced hemorrhagic phenomena and mucosal edema (already with 200 mg/kg). The extracts reduced gastric volume and acidity, demonstrating a regulatory effect on gastric secretion. In the groups treated with the extracts, a decrease in the levels of TNF-a and I L-1 b and an increase in the activity of antioxidant enzymes were recorded.

Hericium extract is useful in the present invention as it represents a valid alternative to conventional drug therapies in preventing and alleviating ulcerative damage to the gastric mucosa without showing any type of side effect.

In the present invention Hericium erinaceus can be used as such or in another suitable form, for example and preferably in the form of an extract.

Matricaria chamomilla

Matricaria chamomilla, commonly known as common chamomile, is an annual herbaceous plant belonging to the Asteraceae family and to the genus Matricaria. The plant has a bushy habit with multiple stems that start from the base and go upwards. Generally it reaches a height between 50 and 80 cm. Chamomile is a particularly aromatic plant, the flowers have a pleasant aromatic smell and contain a characteristic essence consisting of azulene and other substances. The plant is native to Europe and Asia and has also been naturalized in other continents.

Chamomile contains different types of substances that have been isolated and widely used in both medical and cosmetic preparations. Chamomile flower extract contains about 1 - 2% volatile oils. The main components identified in the essential oil are the a-bisabolol terpenoids and its oxide, azulene and chamazulene and acetylene derivatives. The flowers also contain a large number of flavonoids, terpenoids and mucilages a-bisabolol and its oxides A and B, chamazulene and azulene, farnesene, flavonoids including apigenin, luteolin, patuletin and quercetin, coumarins, terpenoids and mucilages are considered the main bioactive ingredients of chamomile. Chamomile is one of the best known and most used medicinal plants in the world. Several studies boast multiple properties including antioxidant, anti-inflammatory, anti-allergic, neuro-protective, antimicrobial and anti-carcinogenic properties. It has traditionally been used for centuries as an anti-inflammatory and antioxidant. As a traditional medicine it is used to treat wounds, ulcers, eczema, skin irritations, burns, sciatica, rheumatic pain, hemorrhoids and various types of infections. In the form of a decoction or aqueous extract, it is used to relieve anxiety states, as a sedative and to calm the nerves. Chamomile infusions are also used for digestive purposes and to treat gastrointestinal disorders including flatulence, indigestion, diarrhea, anorexia, nausea and vomiting, especially in children. Scientific evidence reveals that chamomile extracts have a powerful beneficial action on wounds and ulcerations. Some clinical studies show, in fact, that the use of chamomile reduces the area of the treated wounds, dries the wound and promotes re-epithelialization. Therefore, starting from these results, the attention of many researchers has shifted to the use of chamomile in gastro-protection and the prevention of peptic ulcers. Several studies show that chamomile extracts rich in flavonoids, and in particular in glycosylated derivatives of apigenin, have a protective effect on the gastric mucosa and prevent the formation of ulcerations induced by strong external insults. Interesting data also reveal the potential anti -Helicobacter pylori effect exerted by chamomile extracts, making the use of chamomile in this area even more interesting.

An in vivo pre-clinical study evaluated the protective effect that a chamomile extract by decoction exerts on ulcers induced by ethanol. For this purpose, the animals selected for the experiment are divided into seven groups, each containing ten animals:

- Group 1 : control, administration of physiological solution;

- Group 2: induction of the ulcer by administering 4 g/kg of ethanol;

- Group 3, 4 and 5: pre-treatment with 25, 50 and 100 mg/kg of extract;

- Group 6: famotidine 20 mg/kg;

- Group 7: vitamin C 250 mg/kg.

In all groups except group 1 , the ulcer is induced by administering 4 g/kg of ethanol. Treatment with ethanol causes gastric ulceration, haemorrhagic erosion, edema and leukocyte infiltration in animals undergoing tests. Pre-treatment reduces the structural changes induced by ethanol and reduces gastric lesions in a dose-dependent manner with a percentage of 46.77%, 63.30% and 90.95% respectively. The treatment also reduces the levels of MDA, an index of lipid peroxidation induced by ethanol and inhibits the reduction of SOD, CAT and Glutathione peroxidase levels. The administration of alcohol causes an increase in the levels of hydrogen peroxide, free iron and calcium. This effect is significantly reversed by the chamomile extract. The data obtained, therefore, demonstrate that chamomile exerts a protective effect on the gastric mucosa through an antioxidant mechanism and by activating the mucosal defense mechanisms.

In another in vivo study, sixty albino rats were selected for testing and tested as follows:

- Group 1 : control group treated with deionized water for 28 days;

- Group 2-5: treatment for 27 days with 0, 0.5, 1 and 2 mg/kg of aqueous extract of chamomile.

Ulceration is induced in all animals by administering a single dose of 70% ethanol on day 28. At the end of the administration phase each animal was sacrificed and the stomach was removed and opened. The lesions were measured macroscopically and the performed analyzes revealed that the pretreatment reduces the ulcerative index in a dose-dependent manner. Analyzes carried out on homogenated gastric tissues showed that the aqueous extract of chamomile increased the levels of glutathione (GSH). Histological studies on the tissues examined have reported the harmful effect of ethanol on the mucosa which is reflected in the disorganization of the cell nuclei and the morphology of the glands as well as disorganization of the mucosa with areas of interruption of the muscular layer. The mice pretreated with chamomile showed an improvement in the structural organization of the mucous membranes up to a total disappearance of the signs at the highest doses.

In conclusion, the scientific evidence shows that chamomile is a powerful protective and anti-ulcer agent and constitutes, within the scope of the present invention, a valuable aid in the protection of the mucosa and in the prevention of ulcerative phenomena.

Hyaluronic acid

Hyaluronic acid is a high molecular weight glycosaminoglycan consisting of repeated di-saccharide units. Each disaccharide consists of a molecule of D-glucuronic acid and N-acetyl-glucosamine. Hyaluronic acid is the main component of the extracellular matrix of many tissues. Fibroblasts are the main components responsible for the secretion of hyaluronic acid in the extracellular matrix. This glycosaminoglycan is involved in multiple key processes within tissues and cells. These include cell signaling mechanisms, tissue regeneration and wound repair processes, tissue morphogenesis and differentiation processes, organization of the cellular matrix. Clinically it is used in the treatment of various pathological conditions, in particular it is widely used to promote the healing of wounds, ulcers and canker sores. Thanks to its hydrophilic and hydrodynamic properties, hyaluronic acid is able to retain water and therefore play an important structural role in cells. Its high molecular weight structure and its ability to form macro-aggregates, known as proteoglycans, gives it an efficient anti-hyaluronidase action which is reflected in the protective action of the proteoglycans themselves in the connective tissue. Under normal conditions, proteoglycans help to make the extracellular matrix resistant to compression forces and represent an effective barrier against bacterial infections. When a tissue is damaged, the use of high molecular weight hyaluronic acid helps to rebuild the barrier and the formation of proteoglycans contributes to the reduction of edema formation. To the properties just mentioned, it adds an interesting regulatory action of the anti inflammatory process. Hyaluronic acid acts by exerting a scavenger effect on prostaglandins and metalloproteinases, reducing inflammation mediators, reactive oxygen species and free radicals and reducing leukocyte infiltration phenomena, factors potentially involved in ulceration processes. Several pre-clinical and clinical studies enhance the protective and healing properties of hyaluronic acid, highlighting its benefit in the prevention and treatment of peptic ulcers. The positive action of glycosaminoglycan in the treatment of ulcers is ascribed to a direct barrier effect on the mucosa and to the activation of the mechanisms of repair and healing of the connective tissue under the mucosa. Furthermore, the anti-inflammatory action on the injured mucosa allows to slow down the pro-inflammatory mechanisms triggered in the damaged tissues, reduce the formation of edema, accelerate healing and provide immediate relief of symptoms related to burning.

A pre-clinical study on experimental animals tested the efficacy of two formulations of hyaluronic acid in a model of gastric ulcer induced by ethanol. The selected animals were randomized and divided into four groups each containing six rats. After 48 hours of fasting, the supplementation was organized as follows:

- Control group: administration of distilled water 5 mL/kg;

Positive control group: administration of omeprazole 20 mg/kg;

Group A: administration of high molecular weight hyaluronic acid 240 mg/100 g;

- Group B: administration of a 0.8% hyaluronic acid gel.

One hour after the pre-treatment, absolute ethanol (5mL/kg) was administered in order to induce ulceration. After about an hour, the animals were sacrificed and the stomachs removed and opened along the major curve. The lesion area in terms of mm² was evaluated on the tissues examined. The results obtained from the test showed that the pre-treatment with gel significantly reduces the area of gastric ulcers compared to the control. Pretreatment with high molecular weight hyaluronic acid had the most prominent effect on mucosal protection compared to both control and omeprazole. Histological evaluations have shown that hyaluronic acid reduces damage to the gastric mucosa, edema and leukocyte infiltration.

A clinical study evaluated the protective effect of hyaluronic acid in patients with gastric or duodenal ulcer by injecting a 0.2% solution of hyaluronic acid into the sub mucosal area surrounding the bleeding site. The injected solution was prepared using sodium hyaluronate and saline. The patients included in the study received 20 ml. of solution injected into the bleeding area and in most of the considered cases the bleeding was stopped already at the first administration and a persistent control of the bleeding was recorded in the follow-up period.

Another clinical study evaluated the effect of a hyaluronic acid-based formulation in patients with gastroesophageal reflux in combination with proton pump inhibitors. The 154 patients included in this multicenter, randomized, double-blind study were divided into two groups. The treated group (n = 76) received one stick per day of the test formulation (equivalent to 10 ml.) for 14 days. The placebo group (n = 78) received a placebo for the same time. The study assessed the remission of symptoms (including retrosternal pain, chest burning, acid regurgitation and sour taste in the mouth) and the patients' quality of life. The data collected from this study show that more patients achieved total symptom remission following combined treatment. In addition, the use of mucosal protective agents appears to prolong the period of remission and to delay relapses.

According to a preferred embodiment, the composition object of the present invention comprises the association of Hericium erinaceus, extract of Matricaria chamomilla and hyaluronic acid and/or a salt thereof optionally in mixture with one or more pharmaceutically acceptable carrier.

Hericium erinaceus can be present in an amount from 0.1 mg to 8000 mg, preferably from 5 mg to 5000 mg, still more preferably from 10 mg to 3500 mg.

The extract of Matricaria chamomilla can be present in an amount from 0.1 mg to 5000 mg, preferably from 5 mg to 3500 mg, still more preferably from 10 mg to 2000 mg. Hyaluronic acid and/or a salt thereof can be present in an amount from 0.1 mg to 5000 mg, preferably from 1 mg to 3500 mg, still more preferably from 5 mg to 2000 mg. Hyaluronic acid and/or a salt thereof can be a low-weight, medium-weight or high- weight hyaluronic acid.

Suitable pharmaceutically acceptable carriers are those commonly known to the skilled person for the preparation of pharmaceutical compositions for oral administration, such as powders, granulates, capsules, tablets, solutions, suspensions, etc. By way of non-limiting example, such pharmaceutically acceptable carriers can consist of diluents (for example dibasic calcium phosphate, lactose, microcrystalline cellulose and cellulose derivatives), thickeners (for example gums, hydroxypropylmethylcellulose and other cellulose derivatives), sweeteners (for example sorbitols, mannitol and other polyols, acesulfame K, aspartame, cyclamates, saccharin, sucralose), lubricants (e.g. magnesium stearate, stearic acid, waxes), dispersants, surfactants (e.g. sodium lauryl sulfate and polysorbates), flavoring, adsorbents (e.g. silica gel, talc, starch, bentonite, kaolin), glidants and anti-adhesives (e.g. talc, colloidal silica, corn starch, silicon dioxide), dyes (e.g. iron oxides), opacifiers (e.g. oxide titanium), antioxidants, binders (e.g. gum, starch, gelatin, cellulose derivatives, sucrose, sodium alginate), disaggregating agents (starch, cellulose microcrystalline, alginic acid, crospovidone), plasticizers (e.g. ethylcellulose and other cellulose derivatives, acrylates and methacrylates, glycerol and sorbitol), preservatives (e.g. parabens, sulfur dioxide), rheological modifiers, emulsifiers, humectants, wetting agents, chelators and their mixtures .

The compositions of the present invention can be for example, in the form of a medical device, a food supplement, a nutraceutical, dietetic and nutritional composition, a food product, a beverage, a food, a nutraceutical product, a medicated food, a food for special medical purposes or a pharmaceutical composition.

The compositions of the present invention can also be for example, in the form of a simple, complementary or complete animal feed, a medicated animal feed or an animal feed for particular nutritional purposes.

The compositions according to any of the embodiments here disclosed, can be used both in human and animal.

The composition object of the present invention is preferably in form of a liquid or solid pharmaceutical composition for oral use. According to an embodiment, the composition object of the present invention is in a form selected from powder, mouth-soluble powder, granules, hard capsule, soft-gel capsule, tablet, sachet, solution, suspension, syrup.

The composition of the present invention is particularly effective in protecting the gastrointestinal mucosa, reducing ulcers and reducing the phenomena of inflammation and tissue oxidation thanks to the synergistic action of its components. In fact, the present composition constitutes a valid aid in protecting the gastrointestinal mucosa from aggressive agents that can increase the risk of tissue ulceration and contributes to the repair of damaged tissue. The protective action of the composition of the invention is due to the synergistic action of the components that constitute it. Hericium erinaceus extract reduces areas of ulceration and regulates gastric secretions by decreasing acidity and stimulating the production of protective basic mucus. Furthermore, the fungus acts on inflammatory and oxidative tissue mechanisms favoring the functionality of the mucosa. Chamomile extract enhances the healing action of ulcerative areas and helps to maintain the functionality of the mucosa by supporting antioxidant mechanisms and improving tissue functionality. Both extracts have an interesting anti -Helicobacter pylori action. Hyaluronic acid exerts a barrier effect on the mucous membranes and contributes to the activation of the healing mechanisms of the sub-mucosal connective tissue. With its anti inflammatory action it also helps to reduce the formation of edema and soothe symptoms.

The action of the individual components and of the association object of the present invention is evaluated by in vitro and/or in vivo tests.

To evaluate the protective effect of the composition object of the present invention, in vitro tests on cell cultures are useful. Human gastric epithelial cells (e.g. GES-1 ) are cultured in complete DMEM growth medium and incubated at 37°C and in a humid atmosphere (e.g. 5% C0₂). Following a 24-hour treatment with different concentrations of the substances to be evaluated, an MTT assay is performed to evaluate the cell viability and cytotoxicity of the active ingredients of interest. After a short period of incubation with the reagent, the absorbance is measured (for example at 570 nm). To evaluate the antioxidant effect of the present composition and of its components, the intracellular levels of ROS are evaluated by assay with DCFH-DA (2,7-dichlorofluorescein diacetate) or other assays known to the skilled in the art, the differently treated cells are washed and incubated with DCFH-DA and the fluorescence intensity is measured. The total antioxidant capacity (T-AOC) is evaluated using the ABTS method, or other method known to the skilled person, using a special kit and measuring the optical density of the supernatant, for example at 420 nm. To analyze the levels of SOD, GSH-Px, MDA and LDH, the cells treated with the test substances at different concentrations are subjected to sonification. The supernatant is collected and used to determine the levels of SOD, GSH-Px, MDA and LDH with special kits.

The anti-inflammatory action of the association object of the present invention can be analyzed on an appropriate cellular model by evaluating the levels before and after treatment of cytokines or other markers such as iNOS, COX-2, TNF-a, I L- 1 b , p-NF - kB, p65, p-lkBa.

The potential anti -Helicobacter pylori action of the test substances is assessed by measuring the minimum inhibitory concentration (MIC) using the dilution method. Bacteria grew in base agar medium supplemented with e.g. 10% lysed calf serum. The substances to be tested are dissolved in sterile distilled water and subjected to a series of dilutions. The MIC is determined after incubation at 37°C with the various concentrations. Colloidal bismuth subcitrate, or other suitably selected reagent, can be used as a positive control.

The effectiveness of the composition of the present invention can also be evaluated by means of an in vivo test on experimental animals in line with the directives of the European Community and the Ministry of Health and approved by an Ethics Committee. The tests are performed on mice, for example Sprague-Dawley, pretreated with the test substances. Following pretreatment, a high dose of an ulcerative agent was administered and after a variable incubation period of 1 to 5 hours, the animals are sacrificed and each one's stomach was removed and opened along the major curvature. On the gastric mucosa, the number and size of the ulceration areas are evaluated (expressed in mm²). In a variant of the aforementioned assay, the ulcer can be induced by ligament of the pylorus. Assessment of the areas of ulceration is performed as previously described. In order to evaluate gastric secretions, the volume of gastric juice is collected and used for measuring pH, pepsin activity (using a special kit) and total acidity by titration with sodium hydroxide. The amount of mucus produced can be assessed by scraping the mucosa and the collected mucus is weighed using a precision electronic balance. Ex vivo tests are performed on the collected tissues. Isolated tissue sections are homogenized and the obtained samples are used for the quantization of the levels of MDA, SOD, CAT and GPx. MDA levels are measured by analysis of lipid peroxidation levels. For this purpose, a reactivity test is performed using TBA (thiobarbituric acid) and carrying out an absorbance measurement at 532 nm with a UV-visible spectrophotometer. The levels of SOD are measured by an epinephrine assay and by evaluating, at the end of the test, the absorbance at 480 nm. The activity of the CAT is evaluated by means of a specific kit. The levels of free iron are evaluated on blood samples taken from animals subjected to the assays at the time of sacrifice by colorimetric measurement with ferrozine. A variant of the aforementioned assay involves the use of EDTA as a reaction standard. Plasma calcium levels are measured with a colorimetric method using the reaction with cresolphthalein and measuring the absorbance of the complex formed at 570 nm. The anti-inflammatory action is also evaluated on plasma samples by determining the levels of TNF-a, IL-1 b, gastrin, NO and PGE2.

EXAMPLES Some examples of formulations containing the active ingredients object of the present invention are now provided for illustrative purposes. The daily doses are intended to be administered in suitable oral dosage form and divided into one or more dosage units such as, for example, a capsule, a tablet or a sachet.

Modifications or variations of the embodiments exemplified here, obvious to a skilled person, are included in the appended claims.

Example 1

Example 2

Example 3

Esempio 4

The combinations of active ingredients according to the invention shown in the examples are mixed with one or more suitable carriers to obtain the finished dosage form. The preparation of the desired dosage form is carried out by conventional techniques.

The skilled person will be able to identify both the most suitable carrier(s) and the technique for preparing the finished dosage form.

Claims

1 ) A composition comprising an association of Hericium erinaceus, at least an extract of Matricaria chamomilla and hyaluronic acid and/or a salt thereof.

2) A composition according to claim 1 wherein Hericium erinaceus is present in an amount from 0.1 mg to 8000 mg, preferably from 5 mg to 5000 mg, still more preferably from 10 mg to 3500 mg.

3) A composition according to claim 1 wherein the extract of Matricaria chamomilla is present in an amount from 0.1 mg to 5000 mg, preferably from 5 mg to 3500 mg, still more preferably from 10 mg to 2000 mg.

4) A composition according to claim 1 wherein hyaluronic acid is present in an amount from 0.1 mg to 5000 mg, preferably from 1 mg to 3500 mg, still more preferably from 5 mg to 2000 mg.

5) A composition according to claims 1 to 4 in form of a liquid or solid composition for oral use.

6) A composition according to any of the previous claims in a form selected from powder, mouth-soluble powder, granules, hard capsule, soft-gel capsule, tablet, sachet, solution, suspension, syrup.

7) A composition according to any of the previous claims wherein in the form of a medical device, a food supplement, a nutraceutical, dietetic and nutritional composition, a food product, a beverage, a food, a medicated food, a food for special medical purposes or a pharmaceutical composition or also in the form of a simple, complementary or complete animal feed, a medicated animal feed or an animal feed for particular nutritional purposes.

8) A composition according to any of the previous claims for use in the protection of the gastro-intestinal mucosa in human and animal.

9) A composition according to any of the previous claims for use in the prevention and/or treatment of ulcerative events in human and animal.