The present invention relates to a composition of substances preferably obtained from natural sources, which is effective in the protection of the gastrointestinal mucosa and in the prevention and treatment of diseases associated therewith.

Nowadays, gastric disorders are more and more common and debilitating. A significant proportion of the world’s population (3-28%), including mainly women and the elderly, is subject to gastric disorders, and sometimes so severely as to affect the individual’s quality of life. Gastric disorders are intended to mean gastritis, gastric reflux, gastric hyperacidity, delay in the process of gastric emptying, heartburn and dyspepsia. Studies carried out especially over the last decade have highlighted some possible risk factors for the development of typical digestive disorders: among these, the first appears to be Helicobacter pylori , and the second place appears to be occupied by smoking, alcohol abuse, stress, obesity and pregnancy. However, eating habits, the way of eating and what is eaten certainly also have an important effect on the gastric contents and the motility of the digestive tract. Some drugs, especially if taken for prolonged periods of time, can alter the gastric secretion mechanisms, resulting in increased hydrochloric acid concentration in the stomach, and consequently cause gastric hyperacidity. Dyspepsia, generally defined as “bad digestion”, indicates a pathological condition characterised by the predominant presence of persistent or recurrent pain and/or discomfort localized in the epigastrium. This gastric disorder can be classified into: secondary dyspepsia mainly caused by diseases of the upper digestive tract such as esophagitis, gastritis, or by prolonged periods of substance or drug abuse; primary or functional dyspepsia, on the other hand, is not attributable to any particular cause and is a chronic and/or recurrent disorder characterised by pain and discomfort in the upper abdomen. Many subjects suffering from dyspepsia experience increased gastric emptying time, mainly due to altered gastric motility. The onset of this condition results in prolonged permanence of the ingested food, therefore the digestive process is slowed down. More generally, typical symptoms of dyspepsia include: premature satiety during meals, feeling of fullness, abdominal bloating and discomfort, heartburn, and gastric hyperacidity. The term “hyperacidity” refers to a condition characterised by an increase in the acidity of the gastric contents of the stomach compared to the physiological values ranging from 1.5 to 2, and by a set of clinical symptoms such as a sense of fullness, distress and burning of the epigastrium, often associated with regurgitation of acidic material. One of the causes of gastric hyperacidity is abnormal hormonal secretion during and after meals, for example hypergastrinemia. Gastrin is a hormone which, by acting on the CCK2 receptors of the parietal cells of the stomach, stimulates the production of histamine which, in turn, activates acid secretion. Gastric hyperacidity is a condition predisposing to a whole series of alterations of the normal gastroesophageal physiology. Hyperacidity, in fact, increases the risk of peptic ulcers and gastroesophageal reflux. Gastroesophageal reflux syndrome, commonly referred to as GERD, is an ever-increasing clinical problem. This syndrome is defined as a set of uncomfortable symptoms and complications due to the reflux of gastric contents into the oesophagus. Although one of the main etiological factors is gastric hyperacidity, relaxation of the lower gastroesophageal sphincter is a factor frequently encountered in the course of GERD. The classic symptom of gastroesophageal reflux is heartburn, which manifests itself as a burning and heat sensation in the chest that radiates towards the mouth. Gastric hyperacidity and gastroesophageal reflux are both factors predisposing to ulceration of the mucous membranes of the gastroesophageal tract and duodenum. In particular, the increase in acid and pepsin secretions increases the insults to the mucosa and predisposes it to a continuous pro-inflammatory state. Various types of ulcers can be identified, which are distinguished by their location 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, and oesophageal ulcers are located in the oesophageal mucosa. The latter typically result in reflux. An ulcer is a necrotic lesion that penetrates the whole thickness of the mucosa and is characterised by severe epigastric pain. A peptic ulcer occurs due to an imbalance in the mechanisms that regulate tissue homeostasis. In particular, there is an increase in the aggressive factors, which break the mechanisms that the gastric mucosa uses to protect itself, and a decrease in the defensive factors, which normally contribute to maintaining the integrity of the mucosa. The increase in acid and pepsin secretions that increases the insults to the mucosa and predisposes it to a continuous pro-inflammatory state should be mentioned, in the first place, among the aggressive factors that contribute to damaging the mucosa. The presence of Helicobacter pylori contributes to damaging the mucosa as it disrupts the immune and inflammatory mechanisms implemented by 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; oxidative stress and increase in free radicals which favour the occurrence of the above-mentioned imbalance between aggressive factors and protection mechanisms; inhibition of cell proliferation and infiltration of pro-inflammatory molecules. Pharmacological therapies commonly used to reduce hyperacidity, improve digestion and protect the gastric mucosa from any aggressive insults involve the use of an antisecretory agent, such as Eb receptor antagonists and pump inhibitors, a mucosal protective agent, such as prostaglandin analogs, an antacid, or a complex bismuth salt. In the long run, therapies with the aforementioned agents exhibit several side effects. The use of cimetidine, an Eb receptor antagonist, for example, resulted in the occurrence of gynecomastia. Toxicological studies on omeprazole revealed that the use of the above mentioned pump inhibitor may have, as a side effect, the formation of carcinoids, i.e., endocrine tumours of the stomach which 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 potential new sources of bioactive molecules. Many natural substances have already proven effective in gastroprotection, as well as safe and well- tolerated. Therefore, the object of the present invention is to provide a valid alternative to currently available therapies for protecting the gastric mucosa, promoting the digestive process, improving situations of abdominal discomfort and heartburn, as well as preventing the onset of a more serious pathological disorder.

Hyaluronic acid is a high molecular weight glycosaminoglycan consisting of repeating disaccharide units. Each disaccharide consists of one molecule of D-glucuronic acid and N-acetyl-glucosamine. Hyaluronic acid is the main component of the extracellular matrix in many tissues. Fibroblasts are the main components responsible for the secretion of hyaluronic acid in the extracellular matrix. This glycosaminoglycan is involved in many key processes within tissues and cells. These include cell signalling 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 aphthae. Thanks to its hydrophilic and hydrodynamic properties, hyaluronic acid is able to retain water, and therefore to play an important structural role in cells. Its high molecular weight structure and its ability to form macro-aggregates, known as proteoglycans, give it an efficient anti-hyaluronidase action, which results in protection of the same proteoglycans in the connective tissue. Under normal conditions, proteoglycans contribute to making the extracellular matrix resistant to compressive forces and represent an effective barrier against bacterial infections. When a tissue is damaged, the use of high molecular weight hyaluronic acid helps rebuild the barrier, and the formation of proteoglycans contributes to the reduction of oedema formation. In addition to the above-mentioned properties, there is an interesting regulatory action on the anti inflammatory process. Hyaluronic acid acts by exerting a scavenger effect on prostaglandins and metalloproteinases, thereby reducing inflammation mediators, reactive oxygen species and free radicals, and reduces leukocyte infiltration phenomena, which are factors potentially involved in ulceration processes. Several pre-clinical and clinical studies praise the protective and healing properties of hyaluronic acid, highlighting its benefit in the prevention and treatment of peptic ulcers. The positive action of this 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 sub-mucosal connective tissue. Therefore, hyaluronic acid would be effective both in protecting the mucous membrane of the stomach and oesophagus in all cases of hyperacidity and preventing the formation of damage and ulceration, and in promoting healing and reducing the uncomfortable sensations felt in the event that the integrity of the epithelium has already been affected by acid hyper-secretion. Furthermore, the anti-inflammatory action on the injured mucosa allows the pro-inflammatory mechanisms triggered in damaged tissues to be slowed down, oedema formation to be reduced, healing to be accelerated, and burning-related symptoms to be immediately relieved.

A pre-clinical study on experimental animals tested the efficacy of two hyaluronic acid formulations in an ethanol-induced gastric ulcer model. The selected animals were randomized and divided into four groups: control group, positive control group (20mg/kg omeprazole), group A (240mg/100g high molecular weight hyaluronic acid), and group B (0.8% hyaluronic acid gel). The animals were fasted for 48 hours and were administered absolute ethanol (5mL/kg) one hour after pre-treatment with the above-mentioned substances, in order to induce ulceration. After approximately one hour, the animals were sacrificed, and the stomachs collected and opened along the greater curvature. The area of the lesion in terms of mm ² was assessed on the tissues examined. The results obtained from the test showed that pre-treatment with the gel significantly reduces the area of the gastric ulcers compared to the control. Pre-treatment with high molecular weight hyaluronic acid had the most prominent effect on mucosal protection compared to both the control and omeprazole. Histological assessment showed that hyaluronic acid reduces gastric mucosa damage, oedema and leukocyte infiltration.

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

Another clinical study assessed the effect of a hyaluronic acid-based formulation in combination with proton pump inhibitors in patients with gastroesophageal reflux. The 154 patients included in this multicentre, 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 period of time. The study assessed the remission of symptoms (intended as retrosternal pain, heartburn, acid regurgitation and sour taste in the mouth) and the quality of life of the patients. Data from this study show that a greater number of patients achieved total remission of symptoms following combined treatment. In addition, the use of mucosal protective agents appears to prolong the remission period and delay relapse.

Opuntia ficus-indica is a tropical and subtropical plant belonging to the Cactaceae family, to which more than 1500 species of cactus belong. The best known of these is certainly the prickly pear. Opuntia is a plant that can grow in arid and semi-arid climates and is more widely distributed, from a geographical point of view, in countries such as Mexico, Latin America, South Africa and the Mediterranean countries, particularly in Sicily. The plant can reach 3 - 5 m in height, with a thick trunk ending in succulent and oblong stems called cladodes. After 1 or 2 years of life, cladodes produce flowers and fruits that have very different colours, from pale green to deep red. The fruit of the prickly pear has great nutritional value, as it is rich in water and nutrients, and is an important source of livelihood for the populations of the arid areas where it grows. Prickly pear seed infusions, juice or oil are widely used for nutritional, health and cosmetic purposes. It is also known and used as a medicinal remedy for various pathological conditions: in the sub-Saharan area, the flowers and fruits are claimed to have anti-ulcerative and anti-diarrheal properties; the flowers are often used as an oral remedy for haemorrhoids, and the sap of the cladode is used in the treatment of whooping cough. Several scientific reports have investigated the chemical composition of the prickly pear. From the chemical analyses carried out it appeared that Opuntia has a very high content of polyphenols and flavonoids which give it antioxidant and anti-inflammatory properties. In particular, the flowers of the cactus appear to be the main source of polyphenols and flavonoids and for this reason they are promising in the prevention of inflammatory, cardiovascular and neurodegenerative diseases. The analyses also showed that each part of the plant has its own specific compositional profile and stands out for the presence of a given nutrient. For example, the flowers of Opuntia ficus indica are particularly rich in gallic acid, kaempferol and quercetin; the cladode contains nicotiflorin which is a potent neuroprotective agent that has proven effective in reducing ganglionic degeneration following damage induced by hypoxia, oxidative stress or glutamate. Furthermore, the cladode, more than any other part, is rich in polyunsaturated fatty acids. 90% of the total fatty acids contained in the extract are palmitic acid, oleic acid, linoleic acid and linolenic acid. The prickly pear is also rich in minerals, especially potassium and zinc, and vitamins: the skin is very rich in vitamin E and a-tocopherol. Recent studies have shown the high enzymatic content present in the fruit of Opuntia ficus-indica, in particular the presence of proteolytic enzymes is of considerable interest since they promote digestion and reduce the gastric emptying time. In addition, the presence of mucilages (mainly consisting of arabinogalactan and galacturonic acid) in Opuntia ficus-indica has been shown to guarantee the fruit a gastro-protective action thanks to the formation of a layer that covers and protects the gastric mucosa. The mucilages of the above fruit are highly viscous as the negative charges cause a strong molecular repulsion and this results in expansion of these molecules. It is believed that the change in shape of the molecules is responsible for the protection of the gastric mucosa. In conclusion, an Opuntia ficus-indica extract is interesting from the nutritional and health point of view due to its very particular content in micronutrients. Flavonoids appear to be the most interesting of the substances present in the plant. Within the scope of the present invention, the prickly pear is interesting in that it exerts a protective action at the gastroesophageal level and can therefore be useful in case of hyperacidity and reflux due to its protective action and its ability to improve and enhance the defensive factors of the mucosa. Well-established scientific evidence shows that flavonoids stimulate the production of prostaglandins in gastric mucosal cells and, in this respect, exert an anti- ulcerative action, as prostaglandins promote the secretion of bicarbonate and protective mucus, thus counteracting acid hyper-secretion. In addition to the above-mentioned action, the anti-inflammatory and antioxidant action of flavonoids has been widely emphasized, which action, within the scope of the present composition, reduces the inflammatory state to which subjects with gastric hyperacidity are continuously exposed.

A preclinical study assessed the antioxidant effect and the anti-ulcerative action of Opuntia ficus-indica juice. To assess the antioxidant effect, an antiradical activity assay was performed by mixing a standard DPPH solution with different concentrations of the juice. The antioxidant action is measured by measuring the absorbance of the various samples to be tested against the standard DPPH solution at 517 nm. The results obtained showed that the prickly pear juice produced a 50% decrease in the measured absorbance, resulting in a marked antioxidant effect.

The anti-ulcerative activity was assessed in an in vivo study in rats. The animals selected for the test were fed a standard diet and divided into three groups before proceeding with the treatments: control group 1, group 2 in which the mice were treated for 9 days with 3 ml of prickly pear juice, and group 3 in which the mice were treated for 9 days with 100 mg/kg sucralfate. At the end of the treatment, all mice were treated with an ulcerative agent. After 60 minutes, the animals were sacrificed and the stomach of each was collected and opened along the greater curvature in order to run the necessary tests. Macroscopic and microscopic analyses were carried out on the collected tissues. Macroscopic observations showed that treatment with the ulcerative agent causes the formation of disseminated ulcerative lesions, hyperaemia and thickening of the same. Pre-treatment with prickly pear juice exhibited a protective effect on the mucosa, resulting in an epithelium very close to the physiological situation, significantly reduced hyperaemia areas, and low presence of lesions. These data were confirmed by microscopic observations: in the treated group, the tissue shows areas of necrosis alternating with areas with mucus overproduction as a compensatory response to the damage caused. In tissues collected from pre-treated mice, the epithelium has the same characteristics as normal tissue.

A clinical study tested the effect of a formulation containing Opuntia ficus-indica extract on gastroesophageal reflux symptoms. This study included 118 subjects aged between 36 and 64 years old. Based on the pre-established selection criteria, healthy subjects, but who were experiencing disseminated reflux episodes at least 2 to 6 times a week, were selected. The study was double-blinded and randomized, with a control. The enrolled subjects were divided into two groups: placebo group (n=59) and test group (n=59) treated for 8 weeks with 6 g/die of the Mucosave formulation (extract of prickly pear and Olea europea). At the end of the treatment, the Mucosave formulation is well tolerated and effective in controlling GERD-associated symptoms. It also reduces heartburn and acid regurgitation (with a consequent decrease in the abnormal acid exposure of the oesophagus).

Kiwi is an edible berry representing the fruit of plants belonging to the genus Actinidia. In particular, the fruit of Actinidia chinensis (whose best-known variants are Actinidia chinensis var. chinensis and Actinidia chinensis var. deliciosa ) is the largest among those of all plants of the genus Actinidia. Actinidia chinensis is a woody perennial plant belonging to the Actinidiaceae family. It is native to China but today it is also cultivated in New Zealand, United States, Greece, Italy, Chile, France, Japan and Korea, and is mainly distributed in temperate or temperate- warm zones. The kiwi fruit has great economic, nutritional and medicinal significance in terms of production and use. Generally, the fruit is oval in shape, the skin is yellow to green and covered with a yellowish brown down. It can weigh approximately 120 g and can be eaten fresh, although on the market there are many kiwi-based food preparations, juices, yoghurts, wines, dehydrated or candied fruits. The whole Actinidia chinensis plant has always played a leading role in traditional Chinese folk medicine. The fruit, with a sweet and sour taste, is claimed to have beneficial effects on the spleen, stomach and kidneys, and the ability to improve conditions such as dyspepsia, loss of appetite and vomiting. The branches and leaves were used to treat joint pain, bleeding and ulcers. The roots and the bark, with a bitter and astringent taste, were used to improve the micro-circulation and for their anti-inflammatory and draining effect, for example, applicable to rheumatoid arthritis. Today there is a lot of scientific evidence that confirms many of the properties that have always been attributed to this plant and that introduce the possibility of other promising applications. Recent studies carried out on Actinidia have suggested that this plant can be promising for its anti-tumour, antioxidant, anti-inflammatory, antimicrobial, immunoregulatory, hypolipidemic and anti-diabetic action, and for its protective action on the cardiovascular system and hypnotic action. The results obtained from complex chemical analyses showed the complex nutritional profile of the kiwi which is rich in phytocompounds such as polyphenols, triterpene compounds and derivatives thereof, carotenoids, polysaccharides, amino acids, vitamins, essential oils and micronutrients. Among all the compounds identified, the main bioactive molecules are phenolic compounds, triterpenes, vitamin C, vitamin E, fibres and microelements. In addition to the already mentioned compounds to which antioxidant and anti-inflammatory actions are mainly ascribed, which can be applied in different fields, it should be noted that the kiwi is rich in proteases, of which the most abundant is actinidine. Proteases are enzymes that help cut proteins and are involved in digestive processes. The presence of proteases in the kiwi apparently explains their beneficial effect on the gastroesophageal tract, since by promoting the digestive processes they reduce the gastric emptying time. The slowing down of the gastric emptying, in fact, causes an increase in the time of contact of the mucosa with the acidic digestive gastric juices and increases the risk of gastroesophageal reflux. An in vitro study measured the effect of the kiwi on protein hydrolysis by mimicking the acidic and duodenal digestion. In this experiment, 600 mg of a protein sample were subjected, with or without kiwi treatment, to a simulated gastric digestion for 60 minutes at 37°C with various pepsin and hydrochloric acid concentrations using a pH range between 1.3 and 6.2. For the duodenal digestion, the samples were incubated for 120 minutes at pH 6.4. The experiment showed that protein digestion mainly occurs at the gastric level and that the use of kiwi enzymes doubles protein hydrolysis in the gastric environment, measured as a percentage of ammonia nitrogen in the supernatant.

A preclinical study in rats assessed the effect of several variants of kiwi on gastric emptying and protein digestion. The kiwi variants used for the test were Actinidia chinensis var. deliciosa, with a high content of actinidine, and Actinidia chinensis var. chinensis, with a low content of actinidine. The action of these variants was tested on different protein samples from different sources to which two different kiwi extracts were added. 24 rats were selected for assessing gastric emptying and 8 rats were used for each treatment. A total of 14 protein diets were carried out: 2 controls, 6 with A. deliciosa added and 6 with A. chinensis added. For assessing gastric emptying, more than one diet is administered to each group of 8 rats. On the day of the test, rats were administered 2 mL of one of the above-mentioned mixtures, and the transit was recorded by magnetic resonance spectroscopy for 150 minutes. The results obtained show that actinidine accelerates gastric emptying to a greater extent for some protein sources than for others, particularly for beef and zein proteins. To assess the effect on protein digestion, 104 rats were selected and divided into groups and subjected to treatments as indicated above. At the end of the supplementation, the rats were sacrificed, and chyme samples were taken and examined for their protein digestion levels in terms of free and total amino groups. The results obtained confirmed the previous data showing that actinidine increases gastric protein digestion.

A randomized, cross-over, pilot clinical trial was conducted to assess the effect of taking two kiwi varieties on gastric emptying in terms of satiety and overall gastric comfort. Ten healthy subjects between the ages of 21 and 48 were selected for the following trial. The trial was set up as follows: the selected subjects were asked to show up on the day of the test after fasting from food and liquids from the previous evening. The test was carried out by administering to each a high protein content meal and two kiwis of two different varieties (var. chinensis and var. deliciosa). Before and after meals, and at hourly intervals for the following 5 hours, subjects were asked to complete a questionnaire on their sense of satiety, bloating and general well-being. To assess the gastric emptying time, subjects were administered a Smartpill, i.e., a wireless cylindrical capsule that measures the pH and temperature values in real time and transmits the recorded data to a receiver attached to the clothes of each subject. Based on the drastic change in pH, the device allows the recording of the passage from the gastric to the intestinal environment. The same clinical procedure was repeated three times; each test was separated from the previous one by a period of at least 7 days. The results obtained showed that both kiwi variants have an almost overlapping effect as regards the gastric emptying time, but the deliciosa variant containing a higher percentage of actinidine reduces the sense of swelling more than chinensis and decreases the general gastric discomfort.

As indicated above, the composition of the present invention is effective in the protection of the gastrointestinal mucosa and in the prevention and treatment of diseases from gastric disorders in humans or other animals (for example dogs or cats). Examples of gastric disorders suitable to be treated with the composition of the invention are gastritis, gastric reflux, gastric hyperacidity, delay in the gastric emptying process, heartburn, and dyspepsia. The combination of active ingredients that characterises the composition of the present invention allows:

Gastroprotection

Healing of gastric lesions and ulcers

Reduction of tissue inflammation and oxidation phenomena

Reduction of the gastric emptying time

Neutralization of gastric hyperacidity

Resolution of symptoms related to gastroesophageal reflux

The composition of the present invention is therefore a valid aid in protecting the gastric mucosa from aggressive agents, which may 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 make it up. The extract of a plant belonging to the Actinidia genus, besides promoting digestion and reducing gastric emptying time, performs an antioxidant and anti-inflammatory action. The Opuntia ficus- indica extract ensures a protective effect at the gastroesophageal level and has the ability to improve and increase the defensive factors of the mucosa thanks to the presence of a high content of flavonoids. Hyaluronic acid exerts a barrier effect on the mucous membranes and contributes to the activation of the healing mechanisms in the submucosal connective tissue. With its anti-inflammatory action, hyaluronic acid also contributes to reducing oedema formation and relieving symptoms.

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

The antioxidant activity of the formulation comprising the three active ingredients of interest was determined, for example, by following a radical scavenging assessment method in which DPPH (2,2-diphenyl- 1-picrylhydrazyl) is used as a reagent. Ascorbic acid was prepared as a standard sample. Different concentrations of the test formulation were tested, to which a DPPH solution in ethanol was added, and the mixture was incubated at room temperature in the dark. The scavenging activity of the test substances was determined by measuring the absorbance at a wavelength of 517 nm. The percentage of scavenging activity was calculated by using the following formula:

Radical scavenging activity (%) = [l-(A _sampie / A _biank )] x 100.

In vitro tests on cell cultures are useful for assessing the protective effect of the test formulation. For example, GES-1 human gastric epithelial cells cultured in complete DMEM growth medium were used and incubated at 37°C and in a 5% CO2 humid atmosphere. After a 24-hour treatment with different concentrations of the test substances, an MTT assay is carried out to assess the cell viability and the cytotoxicity of the active ingredients of interest. Following a short period of incubation with the reagent, absorbance at 570 nm is measured. To assess the antioxidant effect of the above formulation, intracellular ROS levels are assessed by DCFH-DA (2,7-dichlorofluorescein diacetate) assay, the differently treated cells are washed and incubated with DCFH-DA and the fluorescence intensity is measured. The total antioxidant capacity (T-AOC) is assessed by the ABTS method using a suitable kit and measuring the optical density of the supernatant at 420 nm. To analyse the levels of SOD, GSH-Px, MDA and LDH, cells treated with the test substances at different concentrations are sonicated. The supernatant is collected and used to determine the levels of SOD, GSH-Px, MDA and LDH with appropriate kits.

An in vitro inflammation model was used to assess the anti-inflammatory action of the test formulation. For example, RAW264.7 murine macrophages stimulated with bacterial LPS were used. Cells were cultured in complete DMEM growth medium and incubated at 37°C in a 5% C02 humid atmosphere. 1 hour after treatment with different concentrations of the test formulation, LPS was added for a total time of 24 hours. At the end of this time, the cell viability and the cytotoxicity of the active ingredients of interest were assessed by the MTT assay. NO content in the growth medium was also determined by using Griess reagent. NO concentration was assessed by measuring the absorbance at 540 nm and the calibration curve was obtained by dilution with NaN02. Each treatment was performed in triplicate. In order to determine the anti-inflammatory effect, the cells were treated with different concentrations of the test formulation for 1 hour, after which LPS was added and the cells were incubated again for 1 hour and for 18 hours. At the end of the treatment, the cells were harvested, and protein extraction was carried out. The samples thus obtained were assayed by western blotting so as to determine the levels of inflammatory markers such as iNOS, COX-2, TNF-a, IL-Ib, p-NF-kB p65, p-IkBa.

An in vitro test for the inhibition of the H ⁺ /K ⁺ ATPase enzyme, i.e., the gastric proton pump, was carried out in order to verify the antacid activity of the present formulation comprising Kiwi extract, prickly pear extract and hyaluronic acid. In order to obtain the enzymatic samples to be tested, gastric parietal cells were obtained from sheep stomach, homogenized and centrifuged for 10 minutes. The supernatant (enzyme extract) was used to determine the inhibitory activity of the test substances. The reaction mixture containing the enzyme extract and the formulation containing the three active ingredients of interest was preincubated for 60 minutes at 37°C. The reaction was induced by adding ATP (substrate), MgCE and KC1. After 30 min of incubation at 37°C, the reaction was stopped by adding a mixture containing 4.5% ammonium molybdate and 60% perchloric acid, then it was centrifuged for 10 minutes and the inorganic phosphate produced by the reaction was measured spectrophotometrically at the wavelength of 660 nm following the Fiske- Subbarow method. mQ water, ammonium molybdate and ANSA were added to the supernatant and the mixture was left to stand for 10 minutes at room temperature. The absorbance of the released inorganic phosphate was measured at 660 nm. The enzymatic activity was calculated as micromoles of Pi released per hour at the various doses of the mixture of the two extracts (0-100 pg). The percentage of enzymatic inhibition was calculated by using the following formula:

Percentage of inhibition = [Activity(control) - Activity(test)/Activity(control)] x 100.

The effectiveness of the composition of the present invention can also be assessed by an in vivo test on experimental animals in accordance with the directives of the European Community and the Ministry of Health and approved by an Ethics Committee. The tests are carried out on Sprague-Dawley mice pre-treated with the test substances. Following pre-treatment, a high dose of an ulcerative agent is administered, and after a variable incubation period of 1 to 5 hours, the animals are sacrificed and the stomach of each is removed and opened along the greater curvature. The gastric mucosa is assessed for the number and size of the ulceration areas (expressed in mm ² ). Assessment of the ulceration areas is carried out as described above. The gastric secretions are assessed by collecting the volume of the gastric juice, which is used for measuring the pH, pepsin activity (by means of 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 quantizing the MDA, SOD, CAT, and GPx levels. MDA levels are measured by testing the lipid peroxidation levels. For this purpose, a reactivity test is carried out by using TBA (thiobarbituric acid) and measuring the absorbance at 532 nm with a UV-visible spectrophotometer. SOD levels are measured by epinephrine assay and by assessing the absorbance at 480 nm at the end of the assay. CAT activity is assessed by means of a special kit. Free iron levels are assessed in blood samples taken from the tested animals at the time of the sacrifice by colorimetric measurement using ferrozine. A variant of the above assay involves the use of EDTA as the reaction standard. Plasma calcium levels are measured by a colorimetric method using the reaction with cresolphthalein and measuring the absorbance of the complex formed at 570 nm. Plasma samples are also assessed for their anti-inflammatory action by determining the levels of TNF-a, IE-1b, gastrin, NO and PGE2.

A further in vivo test on animals, which is useful for assessing the gastro-protective activity of the present invention, comprises the following protocol. The selected mice were fasted for 18 hours prior to surgery to induce an esophagitis condition. Subsequently, 2 hours before surgery, the mice were treated with the test substances. The mice were anesthetized, and a 2 cm incision was made in the central part of the abdomen in order to expose the stomach, after which the pylorus was ligated to induce reflux, while maintaining the vagal nerve intact. After 5 hours of surgery, all mice were sacrificed. The oesophagus was immediately removed, washed with saline and photographed to assess mucosal lesions. Western blotting assessed the anti-inflammatory activity of the substances by determining the levels of inflammatory markers such as COX-2, IL-Ib, TNFa, and IkBa and NF-kB. Furthermore, in order to determine the histamine content, a blood sample was taken from the supraorbital plexus using the microcapillary technique and the plasma was separated. The plasma was treated with perchloric acid and centrifuged for 30 minutes at 4°C. HPLC was carried out on the supernatant to assess the histamine content, which was expressed in IU/mg protein.

Further, the effect of the formulation on gastric emptying time was assessed, for example, by following the model described by Smits and Lefebvre (1996). Male mice were fasted for 18 hours and then fed 20 minutes prior to the start of the experiment in order to ensure that the food content in the stomach was as similar as possible. The formulation comprising Actinidia chinensis extract, Opuntia ficus-indica extract and hyaluronic acid and/or a salt thereof was administered orally; after 30 minutes, a marker (a suspension containing 50 mg of phenol red in 100 ml of 1.5% carboxymethylcellulose) was administered to the animals to assess the gastric emptying rate. After 20 minutes, the animals were sacrificed, and the stomach was removed. The stomach was subsequently placed in tubes containing saline, after 20 seconds of stirring 1M NaOH was added in each tube so as to obtain the maximum colorimetric intensity. At this point, a spectrophotometric analysis was carried out at a wavelength of 560 nm. The percentage of gastric emptying was calculated by using the following formula:

Gastric emptying (%) = [1 -(amount of phenol red present in the stomach after 20 min)/(amount of phenol red present in the stomach at time 0)] x 100.

Assessment of the gastric emptying may be performed by Magnetic Resonance Spectroscopy (MRS). This is a non-invasive technique that allows more than one experiment to be performed on the same animal. Mice should have a weight of between 275 and 315 g.

For the study, mice will be given a standard diet with the addition of an indigestible marker (titanium dioxide) and the treatment assigned for that study group. Prior to MRS analysis, the mice will be fasted for 14 hours without food or water, then 1 g of the assigned diet will be given to each group. Rats will be scanned every 25 min/150 min.

Assessment of the reflux and gastric secretion can be carried out after ligating the pylorus, followed by assessment of the parameters in question. Before the start of the experiment, the animals will be fasted for 24 hours; water will be supplied ad libitum.

The animals will be anesthetized and subjected to pyloric ligation. A longitudinal myotonia 1 centimetre thick will be made at the gastroesophageal junction in order to promote gastric reflux. The treatment will be administered immediately after the pyloric ligation surgery. The mice will be fasted for 24 hours after surgery and then sacrificed in a CO2 saturated atmosphere; followed by removal of the oesophagus.

The oesophageal mucosa will be separated from the muscle layer; followed by assessment for the presence of any lesions.

The area (mm ² ) of the oesophageal mucosa exhibiting lesions will be assessed by microscopic analysis.

The animals will be sacrificed after 24 hours from the pyloric ligation surgery; this will be followed by the collection of the contents of the stomach, for assessing the pH and the total amount of gastric juice, and by a gastrectomy, for assessing the presence of lesions in the gastric mucosa.

For the study, the gastric juice will be added with 2 ml of distilled water and centrifuged at 5000 rpm for 15 min at 4°C. After centrifugation, the supernatant will be used for assessing the volume (mL/mouse), pH and acidity (mEq/L). Total acidity will be assessed by titration with 0.01 N NaOH at PH=7 (Phenolphthalein will be used as an indicator). Total acidity will be expressed as mequiv. [H ⁺ ]/ml/4 h.

The stomach will be opened by cutting along the greater curvature and stretched out on a polystyrene support for assessing gastric lesions. The total area of the gastric lesions will be measured, and the degree of severity of the gastric lesions will be expressed by means of a gastric lesion index (mm ² ).

One object of the present invention is a composition as defined in appended claim 1. Further features and advantages of the invention are defined in the dependent claims. All claims form an integral part of the present invention.

In a preferred embodiment, the composition comprises from 0.01 to 50% by weight of hyaluronic acid, preferably from 0.1 to 20% by weight of hyaluronic acid, even more preferably from 0.2 to 10% by weight of hyaluronic acid.

In another preferred embodiment, the composition comprises from 0.01 to 50% by weight of Actinidia chinensis extract, preferably from 0.1 to 20% by weight of Actinidia chinensis extract, even more preferably from 0.2 to 10% by weight of Actinidia chinensis extract.

In a further preferred embodiment, the composition comprises from 0.01 to 50% by weight of Opuntia ficus-indica extract, preferably from 0.1 to 20% by weight of Opuntia ficus- indica extract, even more preferably from 0.15 to 10% by weight of Opuntia ficus-indica extract.

In yet another preferred embodiment, the composition is a pharmaceutical composition, a food supplement, a simple feed, a complementary feed, a complete feed, formulated in an oral dosage form, which is preferably solid, liquid or semi-solid.

As indicated, the pharmaceutical form is preferably a pharmaceutical form for oral administration. Pharmaceutical forms for oral administration include, but are not limited to, a stick gel, a tablet, a capsule, a powder, a granulated orosoluble powder, a granulated orosoluble syrup, a solution, a suspension and a pellet.

The concentrations indicated above refer to the quantity of each of the indicated active ingredients in the pharmaceutical embodiment and, depending on whether the pharmaceutical form is liquid, solid or semi-solid, are expressed as w/w or w/v. For example, in the case of the most preferred pharmaceutical form, i.e., the stick gel for oral administration, concentrations are expressed as w/v.

Suitable pharmaceutically acceptable carriers and/or excipients are those commonly known to those skilled in the art for the preparation of compositions for oral administration such as powders, granulates, capsules, tablets, solutions, suspensions, etc. By way of non limiting example, such pharmaceutically acceptable carriers and/or excipients are selected from the group consisting of diluents (e.g., dibasic calcium phosphate, lactose, microcrystalline cellulose and cellulose derivatives), thickeners (e.g., gums, hydroxypropyl methylcellulose and other cellulose derivatives), sweeteners (e.g., 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 sulphate and polysorbates), flavourings, adsorbents (e.g., silica gel, talc, starch, bentonite, kaolin), glidants and anti-adherents (e.g., talc, colloidal silica, maize starch, silicon dioxide), dyes (e.g., iron oxides), opacifiers (e.g., titanium oxide), antioxidants, binders (e.g., gums, starch, gelatin, cellulose derivatives, sucrose, sodium alginate), disintegrants (starch, microcrystalline cellulose, alginic acid, crospovidone), plasticizers (e.g., ethyl cellulose and other cellulose derivatives, acrylates and methacrylates, glycerol and sorbitol), preservatives (e.g., parabens, sulfur dioxide), viscosifiers, emulsifiers, humectants, wetting agents, chelating agents and/or any combination thereof.

A further aspect of the invention is the above-described composition for use in the therapeutic treatment of gastric disorders in a subject (human or non-human animal).

In a preferred embodiment, said gastric disorders are selected from the group consisting of gastritis, gastric reflux, gastric hyperacidity, delay in the process of gastric emptying, heartburn and dyspepsia. In a preferred embodiment, treatment comprises administering a therapeutically effective amount of hyaluronic acid or a salt thereof comprised between 0.1 and 3000 mg/die, preferably comprised between 1 mg and 2000 mg/die, more preferably comprised between 10 mg and 1000 mg/die.

In another preferred embodiment, treatment comprises administering a therapeutically effective amount of Actinidia chinensis extract comprised between 0.1 and 5000 mg/die, preferably comprised between 5 mg and 3500 mg/die, more preferably comprised between 10 mg and 1000 mg/die.

In a still further preferred embodiment, treatment comprises administering a therapeutically effective amount of Opuntia ficus-indica extract comprised between 0.1 mg and 5000 mg/die, preferably comprised between 5 mg and 3500 mg/die, more preferably comprised between 10 mg and 1000 mg/die.

All of the above embodiments can be combined with each other.

EXAMPLES

Some examples of quantities per single dosage unit of the active components of the compositions object of the present invention are given for illustrative and non-limiting purposes.

EXAMPLE 1: 10 mL stick gel for oral administration EXAMPLE 2: 15 mL stick gel for oral administration

EXAMPLE 3: Oro soluble powder

EXAMPLE 4: Granules for oral use