WO2021214364A1 - Therapeutic use of a maqui extract in crohn's disease - Google Patents

Abstract

The present invention relates to a polyphenol extract obtained from the fruit of Aristotelia chilensis, and to the use of pharmaceutical compositions comprising the extract, in the prevention and treatment of Crohn's disease.

Description

DESCRIPTION

Therapeutic use of a maqui extract in Crohn's disease The present invention refers to the use of a polyphenolic extract obtained from the fruit of Aristotelia chilensis, as well as the use of pharmaceutical compositions comprising said extract, in the prevention and treatment of Crohn's disease. Therefore, the invention belongs to the field of medicine or pharmacy, more specifically to the technical field of the use of therapeutic plant extracts for the treatment or prevention of Crohn's disease.

BACKGROUND OF THE INVENTION

The intestinal epithelium is part of one of the largest surfaces that separates our internal environment from the exterior, participates in the digestion and absorption of nutrients and is a key component in the intestinal barrier, preventing the passage to the internal environment of a wide spectrum of microorganisms and Potentially carcinogenic substances present in the lumen of the intestine. A dysregulation of epithelial homeostasis (for example, an imbalance between cell production, differentiation and elimination) compromises the integrity of the intestinal barrier and if it is not restored it will cause intestinal pathologies such as inflammation and cancer (Viggiano d., Et a !. , 2015, Eur. Rev. Med Pharmacol Sci 19, 1077-1085).

Inflammatory bowel disease (Eli) is a chronic inflammatory disorder of the intestinal tract suffered by millions of patients around the world and is made up of two main types of diseases: ulcerative colitis (UC) and Crohn's disease (CD). Although the pathological-clinical phenotypes are similar, it is possible to distinguish both entities by their difference in the location of inflammation in the gastrointestinal tract, immunological and histological patterns. Whereas Crohn's disease is characterized by discontinuous transmural inflammation that can affect any stretch of the digestive tract, ulcerative colitis is distinguished by diffuse mucosal inflammation, extending proximally from the rectum with varying degrees of extension (SatsangiJ ., et al., 2006, Gut. 55 (6): 749-53). The exact origin of Crohn's disease is unknown, but immunological, microbiological, environmental and genetic factors have been recognized that increase the risk of suffering, among them, the deregulation of the microbiota is becoming increasingly important. These factors trigger an exaggerated immune response that, after the release of a large amount of pro-inflammatory cytokines (interferon-γ, tumor necrosis factor (TNF) and interleukins, cause intestinal inflammation (José León A., et al, 2006, Med Clin (barc), 127 (4): 145-52)). This inflammation generates an increase in reactive oxygen species (ROS), causing oxidative stress (EO) and damaging tissues. In Crohn's disease, oxidative stress and inflammation play an important role in the pathogenesis and perpetuation of mucosal damage. Oxidative stress is caused by an imbalance between ROS production and the antioxidant capacity of cells. An excessive increase in ROS (including oxygen ions, free radicals, and peroxides) can lead to damage to cell membranes, proteins, and DNA. Antioxidants are substances that are released at the site of inflammation and provide protection against ROS. This alteration of the redox state could be an etiological factor or trigger of Eli (Achitei D., et al, 2013, Dig Dis Sci, 58 (5): 1244-9).

So far, CD is not curable, therefore, what is intended is to keep it in a state of complete remission permanently, this being the only way to modify the evolution of the disease. Currently, treatments for CD are directed at the immunological and inflammatory mechanisms involved in the development of the disease. Some common drugs or treatments in the state of the art are corticosteroids, immunomodulators, and in some cases surgery in advanced disease phase.

In the document HendrikM. et al., 1995 (Gastroenterology, Volume 109, Issue 1, 129-135) describes the use of a monoclonal anti-TNF antibody for the treatment of Crohn's disease, in particular a human / mouse chimeric monoclonal anti-TNF antibody cA2 .

In Daniel H., et al., 1999, N Engl J Med; 340: 1398-1405, infliximab, another monoclonal antibody to TNFα factor, is described for the treatment of fistulas produced in patients with Crohn's disease.

Also corticosteroids, such as prednisone, are used to treat Crohn's disease, however, they can be substantially toxic and some are not effective in treating Crohn's disease. There are also other pharmacological alternatives such as anti-inflammatories. For example, in Brian G., et al., 1995 N Engl J Med; 332: 292-297 describes the use of methotrexate, an anti-inflammatory, to improve symptoms in patients with Crohn's disease.

All these treatments or drugs are expensive, produce adverse or secondary effects such as allergies, infections or aggravation of gastrointestinal complications, and in a high percentage of patients they are not effective or lose response, making it difficult for doctors to choose the effective therapy for each patient. suffering from Crohn's disease.

Recently, fruit extracts have been proposed to combat inflammatory bowel diseases for their anti-inflammatory activity. For example, in Tanideh N., et al., 2014, World Applied Sciences Journal 31 (3): 281-288, the effect of a strawberry extract in the treatment of ulcerative colitis in rats is described. In the document Pera M., et al., 2016, The Journal of Nutritlonal Biochemistry Volume 28, Pages 103-113 describes the preventive and therapeutic effect of an antioxidant and anti-inflammatory extract of blueberries in the treatment of ulcerative colitis. However, these and other fruit extracts with an anti-inflammatory effect have not been applied for the treatment of Crohn's disease.

Therefore, it is necessary to develop drugs or alternative treatments to those already existing in the state of the art, anti-inflammatory, non-toxic and effective, that improve or eliminate symptoms in patients with Crohn's disease.

DESCRIPTION OF THE INVENTION

The inventors have observed that the polyphenolic extract of A. chilensis or maqui, has a high antioxidant and anti-inflammatory power and reverses the symptoms and tissue changes associated with Crohn's disease, drastically reducing the parameters of inflammation and tissue damage, in addition to restoring the size and weight of the intestine, without having adverse effects.

Obtaining said polyphenolic extract of maqui was carried out by means of an extraction process with acidic methanol followed by several filtration steps and a subsequent lyophilization for its conservation. Subsequently, in vitro tests were carried out using cell cultures of the colon cancer line HT-29 and the mouse macrophage cell line RAW264.7, observing the antioxidant and anti-inflammatory effect of the polyphenolic extract of maqui in said cell lines, which It is described throughout point 2 of the results section relative to "In vitro tests and in figures 5-8 of this document.

Furthermore, the Inventors performed in vivo tests with mice. These mice were treated with TNBS to induce Crohn's disease and they were administered the polyphenolic extract of maqui by the orogastric route, both before and after Induction of Crohn's disease with TNBS, determining the preventive and therapeutic nature of said extract, respectively. . The Inventors observed that the polyphenolic extract of A. chilensis or Ach attenuated weight loss, regulated the expression of several proteins related to inflammation and the oxidative state, and suppressed inflammation of the large intestine, both at the macroscopic and microscopic levels of the tissue. colonic, as well as the consequences of said inflammation such as: shortening of the length and reduction of the weight of the colon, ulcers, hyperemia or soft stools and mucus in the large intestine, which is described along point 3 of the relative results section to "In vivo tests" and in Figures 9-25 herein, thus describing, for the first time, the use of a polyphenolic extract of A. chilensis for the treatment and prevention of Crohn's disease.

Therefore, in a first aspect, the present invention refers to the use of a polyphenolic extract of Aristotelia chilensis for its use in the treatment or prevention of Crohn's disease in a subject, hereinafter "the use of the Invention ”.

Aristotelia chilensis or "maqui" is a plant of the Elaeocarpaceae family, native to southern Chile and adjacent areas in southern Argentina, although it is already cultivated in other countries such as Spain. Its fruit is a small edible fleshy berry, green when immature and purple-black when ripe. It is from this berry that the Inventors have extracted the polyphenolic extract described herein.

The terms "polyphenolic extract of A. chilensis" or "polyphenolic extract of maqui" or "Ach", used interchangeably throughout this document, refer to an extract, compound, substance, sample, concentrate or product that comprises polyphenols and that is isolated, obtained, derived or extracted from the A. chilensis plant through physical and / or chemical extraction methods or procedures. Hereinafter, the "polyphenolic extract of A. chilensis" will be referred to as the "polyphenolic extract of the invention".

It is routine practice for a person skilled in the art to obtain the polyphenolic extract of maqui from extraction methods of plant extracts. Examples of extraction methods or treatments include, but are not limited to, aqueous solvent extraction, alcoholic or hydroalcoholic solvent extraction, nonalcoholic solvent extraction, cold compression, direct distillation, steam injection, steam entrainment, vaporization, indirect distillation , clevenger, supercritical fluids, membranes, filtration, electrical pulses, ultrasound, percolation or leaching, maceration, hot water infusion, decoction, digestion and cold and hot fat enfleurage. In a particular embodiment of the use of the invention, the polyphenolic extract of maqui is obtained by the method described in Genskcwsky, E., et al., Determination of polyphenolic profile, antioxidant activity and antibacterial properties of maqui (Aristotelia chilensis (Molina) Stuntz ) to Chilean blackberry. J Sci Food Agrie, 2016, 96 (12): 4235-42.

It is also routine practice for a person skilled in the art to apply stabilization methods of the obtained vegetable extracts to maintain their organoleptic and functional qualities in their subsequent storage, and any of them can be applied in the context of the present invention. Examples of extract stabilization methods include, but are not limited to, nanoencapsulation, microencapsulation, gelling systems, dehydration, Spray Drying, and lyophilization.

In another particular embodiment of the use of the invention, the polyphenolic extract of maqui is stabilized by lyophilization.

Polyphenols are chemical substances that are characterized by comprising at least one phenol group per molecule. In the state of the art, polyphenols can be classified into hydrolyzable tannins, lignins and flavonoids. Hydrolyzable tannins are polymers, in particular polyesters, made up of phenolic acids and simple sugars, capable of being hydrolyzed by acids, alkalis and enzymes. Examples of hydrolyzable tannins include, but are not limited to, gallic (gallic acid) and ellagic (ellagic acid).

Lignin is a class of complex, amorphous and cross-linked phenolic polymers that results from the union of several phenylpropyl acids and alcohols (cycummarilic, coniferilic and synapilic), insoluble in acids and soluble in alkali, which is characterized by being the main component of the wood from plants.

Flavonoids are water-soluble polyphenolic compounds that are synthesized in plants from one molecule of phenylalanine and 3 of malonyl-CoA, whose product, the base structure, is cyclized thanks to an isomerase enzyme. The base structure, a C6-C3-C6 skeleton, can later undergo many modifications and additions of functional groups, so flavonoids are a very diverse family of compounds that are classified into the following main classes: chalcones, flavones, flavonols, flavandiols, anthocyanins, condensed tannins and aurons. Examples of flavonoids include, but are not limited to, tricetin, apigenin, luteolin, quercetin, myricetin, kaempferol, fisetin, leucocyanidin, leucopelargonidin, leukodelphinidin, pelargonidin, delphinidin, cyanidin, pethunidine, peonidinidin, malvidin, rosinidolinidin, amalvidin, rosinidolin, avidolin, rosinidolin, narpensin , liquiritigenin, eriodictyol, dihydroquercetin, dihydromyricetin and dihydrokampferol, among others.

The concentration of polyphenols in the polyphenolic extract of A. chilensis can vary over a wide range. However, in another particular embodiment of the use of the invention, the A. chilensis polyphenolic extract comprises at least 30 mg of polyphenols for each total gram of extract. In another more particular embodiment of the use of the invention, the A. chilensis polyphenolic extract comprises from 30 to 50 mg of polyphenols for each total gram of extract, or from 35 to 45 mg of polyphenols per total gram of extract. In another still more particular embodiment of the use of the invention, the A. chilensis polyphenolic extract comprises 39 mg of polyphenols for each total gram of extract. It is routine practice for one skilled in the art to estimate the concentration of total polyphenols in a plant extract, particularly maqui. An example of a method for estimating or calculating polyphenols in a plant extract is the Folin-Ciocalteu method.

The inventors observed that said polyphenolic extract of A. chilensis or Ach, described in previous paragraphs of this document, it prevents and reverses the symptoms, inflammation and tissue changes associated with Crohn's disease, so that said extract can be used for the treatment and prevention of Crohn's disease.

The term "treatment", as understood in the present invention, refers to combating the effects caused by a disease, in particular Crohn's disease, in a subject (preferably a mammal, and more preferably a human) including:

(i) inhibiting the disease or pathological condition, that is, arresting its development;

(ii) alleviating the disease or pathological condition, that is, causing regression of the disease or pathological condition or its symptoms;

(iii) stabilize the disease or pathological condition.

The term "prevention" as understood in the present invention consists in preventing the occurrence of a disease, in particular Crohn's disease, that is, preventing the disease or pathological condition from occurring in a subject (preferably mammalian, and more preferably a human), in particular when said subject is predisposed to suffering from the disease. In the present invention, the term "Crohn's disease" refers to an inflammatory bowel disease, of autoimmune origin, that affects any part of the intestine, although it usually has a more localized involvement at the ileocolic level, and that is characterized by producing a chronic inflammation, leading to abdominal pain, loose stools or diarrhea, hyperemia and / or ulcers in the subject.

Maqui polyphenolic extract can be used in the treatment and prevention of Crohn's disease in any subject. As used in the present invention, the term "subject" or "individual" refers to any animal that has a digestive tract, in particular, intestine. Examples of animals include, but are not limited to, fish, birds, amphibians, reptiles, and mammals. Examples of mammals include, but are not limited to, horses, pigs, rabbits, sheep, goats, cows, dogs, cats, guinea pigs, rats, mice, non-human primates, and humans. The term does not indicate a particular age or gender.

In another particular embodiment, the subject of the invention is a mammal, preferably a primate, more preferably, a human being of any race, sex or age. The polyphenolic extract of maqui can be administered to the subject in a therapeutically effective dose, which can vary over a wide range. In the present invention, the terms "therapeutically effective dose" or "therapeutically effective amount", used interchangeably herein, refer to that amount or dose of A. chilensis or Ach polyphenolic extract that when administered to a subject, preferably to a mammal, and more preferably to a human, is sufficient to produce the prevention and / or treatment of Crohn's disease The therapeutically effective amount will vary, for example, according to the metabolic stability and duration of action of Ach; age, body weight, general health, sex and diet of the subject; the mode and time of administration; the rate of excretion, the combination with other drugs; the severity of the disease or the particular pathological condition of It is routine practice for a person skilled in the art, for example a medical specialist, to determine the therapeutically effective dose for each subject, e.g. referable in a human, according to his own knowledge and the conditions or physiological variables of the subject.

In another particular embodiment of the use of the invention, the polyphenolic extract of A. chilensis is administered to the subject in a therapeutically effective dose, preferably daily, of between 10 to 60 mg per Kg of the subject.

In another more particular embodiment of the use of the invention, the therapeutically effective daily dose of the polyphenolic extract of A. chilensis is between 20 to 50 mg per Kg of the subject.

In another even more particular embodiment of the use of the invention, the therapeutically effective daily dose of the polyphenolic extract of A. chilensis per Kg of the subject is selected from the list consisting of: 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg and 50 mg. In another even more particular embodiment of the use of the invention, the therapeutically effective daily dose of the polyphenolic extract of A. chilensis is 50 mg per Kg of the subject.

As understood by a person skilled in the art, the polyphenolic extract of A. chilensis can be comprised in a composition, particularly in a pharmaceutical or nutritional composition, for use in the treatment or prevention of Crohn's disease, as described in paragraphs above of this document. Therefore, another aspect of the invention is a composition comprising a polyphenolic extract of A. chilensis, hereinafter the composition of the invention, for use in the treatment or prevention of Crohn's disease in a subject.

The terms "Aristotelia chilensis polyphenolic extract", "Crohn's disease treatment" and "Crohn's disease prevention" have been previously described herein and apply equally to this aspect of the invention, as well as to all your particular realizations (alone or in combination).

In a particular embodiment, the composition of the invention is a pharmaceutical composition.

The term "pharmaceutical composition" refers to a set, mixture, combination of components or substances that comprises the polyphenolic extract of A. chilensis in any concentration, and that improves the health status of the subject, in particular it implies a preventive or therapeutic effect. of Crohn's disease. The pharmaceutical composition can be for human or veterinary use. The term "pharmaceutical composition for human use" refers to a substance or combination of substances with properties for the treatment or prevention of Crohn's disease in humans or that can be used in humans or administered to humans in order to restore , correct or modify physiological functions by exerting a pharmacological, immunological or metabolic action, or to establish a medical diagnosis. The term "pharmaceutical composition for veterinary use" refers to a substance or combination of substances with curative or preventive properties with respect to animal diseases or that can be administered to the animal in order to restore, correct or modify its physiological functions by exerting an action pharmacological, immunological or metabolic, or to establish a veterinary diagnosis.

In another particular embodiment, the pharmaceutical composition also comprises at least one pharmacologically acceptable carrier and / or excipient.

The term "vehicle" or "carrier" refers to a substance, preferably an inert substance, that facilitates the incorporation of other compounds, that allows a better dosage and administration or improves the consistency and form of the pharmaceutical composition for use in the treatment or prevention of Crohn's disease. Therefore, the vehicle is a substance that is used in the medicine to dilute any of the components of the pharmaceutical composition to a certain volume or weight; or that even without diluting said components it is capable of allowing a better dosage and administration or giving consistency and shape to the medicine. When the presentation form is liquid, the pharmaceutically acceptable carrier is the diluent.

The term "excipient" refers to a substance that helps the absorption of any of the components of the pharmaceutical composition, stabilizes said components, modifies their organoleptic properties or determines the physicochemical properties of the pharmaceutical composition and its bioavailability. Thus, excipients could have the function of keeping the components together, such as starches, sugars or celluloses, a sweetening function, a coloring function, a protective function of the drug, such as for example to isolate it from air and / or humidity, a function filling of a tablet, capsule, pill or any other form of presentation such as, for example, dibasic calcium phosphate, disintegrating function to facilitate the dissolution of the components and their absorption in the intestine, without excluding other types of excipients not mentioned in this paragraph. Furthermore, as understood by the person skilled in the art, the excipient and the vehicle must be pharmacologically acceptable. The term "pharmaceutically acceptable" refers to the fact that the vehicle or excipient must allow the activity of the compounds of the pharmaceutical composition, in particular the polyphenolic extract of A. chilensis, that is, it must be compatible with said components, so that do not cause harm to the organisms to which it is administered.

The pharmaceutical composition or medicament can be presented in any clinically permitted form of administration and in a therapeutically effective amount. For example, it may be in a form adapted for oral, sublingual, nasal, intracatecal, bronchial, lymphatic, rectal, transdermal, intravenous, intraperitoneal, orogastric, intracolonic, inhaled, or parenteral administration.

In another particular embodiment, the pharmaceutical composition for use in the treatment or prevention of Crohn's disease is administered orally, orogastric or intracolonic. The pharmaceutical composition of the invention for use in the treatment or prevention of Crohn's disease can be formulated in solid, semisolid, liquid or gaseous forms, such as tablet, capsule, pill, powder, granule, ointment, solution, suppository, injection, inhalant, gel, bead, or spray. The form adapted for oral administration is selected from the list which includes, but is not limited to, drops, syrup, herbal tea, elixir, suspension, extemporaneous suspension, drinkable vial, tablet, capsule, granule, seal, pill, tablet, lozenge, troche. or lyophilized. Additionally, the polyphenolic extract of A. chilensis may be comprised in a nutridonal composition for use in the treatment or prevention of Crohn's disease. Therefore, in a particular embodiment, the composition of the invention is a nutridonal composition. The term "nutritional composition" or "nutridonal composition" of the present invention refers to a food composition or food that, regardless of providing nutrients to the subject who takes it, beneficially affects one or more functions of the organism, in such a way that it provides a better state of health and well-being. As a consequence, said nutritional composition can be intended for the prevention and / or treatment of Crohn's disease.

In another particular embodiment, the nutridonal composition is selected from a supplement, a nutraceutical, a probiotic or a symbiotic.

The term "supplement", synonymous with any of the terms "dietary supplement", "nutritional supplement", "food supplement" or "food supplement" is a component or components intended to supplement the diet, and can be a food. Some examples of dietary supplements are, but are not limited to, vitamins, minerals, botanicals, amino acids, and food components such as enzymes and glandular extracts. They are not presented as substitutes for a conventional food or as a sole component of a meal or of the diet, but as a supplement to the diet.

The term "nutraceutical" as used in the present invention refers to substances isolated from a food and used in a dosage form that have a beneficial effect on health. Said nutraceutical can be a supplement. The term "probiotic" as used in the present invention refers to a composition comprising microorganisms that, when supplied in adequate amounts, exert beneficial effects on the health of the host or subject, preferably subjects suffering from Crohn's disease and who suffer from intestinal damage.

The term "symbiotic" as used in the present invention refers to those foods or compositions that contain a mixture of prebiotics and probiotics. As a general rule, they contain a prebiotic component that favors growth and / or metabolic activity and ultimately the effect of the probiotic with which it is combined, as for example and without limitation, it may be the association of fructooligosaccharides or galactooligosaccharides to an intestinal bacteria such as For example, a strain of the species B. uniformis, which when supplied in adequate amounts exert beneficial effects on the health of the subject, preferably subjects suffering from Crohn's disease and suffering from intestinal damage.

In another particular embodiment, the nutritional composition is a food that is selected from the list consisting of: a dairy product, a vegetable product, a meat product, a snack, chocolate, a drink and a baby food. The dairy product is selected from the list consisting of, but not limited to, a product derived from fermented milk (for example, but not limited to yogurt or cheese) or non-fermented (for example, but not limited to, ice cream, butter, margarine, whey). The vegetable product is, for example, but not limited to, a cereal in any form of presentation, fermented or non-fermented. The beverage can be, but is not limited to, any fruit juice or non-fermented milk.

Throughout the description and claims the word "comprise" and its variants are not intended to exclude other technical characteristics, additives, components or steps. For those skilled in the art, other objects, advantages and characteristics of the invention will emerge in part from the description and in part from the practice of the invention. The following examples and figures are provided by way of illustration, and are not intended to be limiting of the present invention. BRIEF DESCRIPTION OF THE FIGURES Figure 1. Schematization of the process to obtain the polyphenolic extract of maqui.

Figure 2. Shows microscopy evaluation of slide rolled whole large intestine tissue in a single section.

Figure 3. Shows the AG calibration line.

Figure 4. Shows the trolox calibration line. Figure 5. Shows the cell viability with the Ach treatment in HT-29 exposed to different times and concentrations of Ach. The following statistical significance are considered: * Statistical significance (<0.01) with respect to (a); ** Statistical significance (<0.01) with respect to (b); + Statistical significance (<0.01) between 24 and 48 hours.

Figure 6. Shows the viability of RAW264.7 macrophages treated with LPS, with different concentrations of Ach and 5-ASA. The viability of the cells without LPS and without treatment was 100%. Statistical significance was considered a p-value <0.05.

Figure 7. Shows the percentage (%) of oxidation as an indicator of oxidative stress in the HT-29 cell line exposed to H ₂ O ₂ as an oxidant compound (100% oxidation) and to NAC antioxidant treatments and different concentrations of Ach. Statistical significance was considered a p-value <0.05 with respect to (a).

Figure 8. Shows the percentage (%) of oxidation as an indicator of oxidative stress in RAW264.7 macrophages treated with LPS, with different concentrations of Ach and 5-ASA. Macrophages exposed to LPS was considered 100% oxidation%. It was considered: * Statistical significance with p-value <0.05 and ** statistical significance with p-value <0.01 with respect to (a).

Figure 9. Shows colonic tissue images after TNBS induction of acute Crohn's disease, a) Transmural inflammation (H&E, 20x) (Span spanning span); Mixed infiltration by neutrophils and lymphocytes; Necrosis, b) Extensive glandular destruction (H&E, 10x); c) Loss of mucous secretion (H&E, 20 x): d) Ulcer, vascular congestion in lamina propria (edema) (H&E, 20 x).

Figure 10. Assessment of% weight loss, stool consistency and rectal bleeding on an assessment scale between 0 and 3 to obtain the DAI in the experimental groups. The following statistical significance were considered: ** Statistical significance (<0.001) with respect to the G1 control; + Statistical significance (0.001) with respect to G2.

Figure 11. Body weight recording in the experimental groups from 1 week prior to induction (day -7) until the last day prior to sacrifice (day 4).

Figure 12. Representative photographs of the colon of the different groups; shortening of the colon is considered a marker of tissue inflammation.

Figure 13. Comparison of the means and the maximum and minimum values of the length of the large intestine by experimental groups. Figure 14. Comparison of the means and the maximum and minimum values of the weight of the large intestine by experimental groups.

Figure 15. Macroscopic score according to colonic morphology. ** Statistical significance (<0.001) with respect to Group G1; + Statistical significance (<0.001) with respect to group G2.

Figure 16. Microscopic score according to histological damage (0-12). ** Statistical significance (<0.001) with respect to Group G1; + Statistical significance (<0.001) with respect to Group G2.

Figure 17. Histopathological study of histological preparations of the colon of the control group (G1). H&E stain. (a) H&E 4 x; (b) H&E 10x; (c) H&E 40 x. Panoramic micrograph (a) of the entire colon of a mouse treated with 50% EtOH (Control Groups, G1) where an intact colonic tissue is observed in all its layers. In micrograph (b), a single focus of glandular destruction associated with a slight loss of goblet cells is observed throughout the length of the colon in figure (c).

Figure 18. Histopathological study of histological preparations of the colon of the diseased group (G2). H&E stain. (a) H&E 4 x; (b) H&E 20 x; (c) H&E 40 x. In a panoramic micrograph (a) of the entire colon, a loss of the glandular architecture distributed in foci is observed, ranging from the distal end to becoming more severe in the proximal end, together with the presence of necrosis foci. Micrograph (b) shows severe destruction of the crypts with inflammatory infiltrate and edema in the mucosa and submucosa, evidenced by the open spaces between the cells. Micrograph (c) shows a mixed inflammatory infiltrate (neutrophils and lymphocytes) invading the muscle wall.

Figure 19. Histopathological study of histological preparations of the colon of the sick group treated curatively with Ach (G3). H&E stain. (a) H&E 4 x; (b) H&E 10x; (c) H&E 40 x. Micrograph (a) illustrates a conserved structure throughout the colon with slight inflammation in the mucosa and an inflammatory focus near the cecum where glandular loss is observed. The micrograph (b) shows an area of acute inflammation that is restricted to the mucosa, without the presence of inflammatory cells in the muscle layer or in the serosa, evidencing the absence of transmural inflammation. There is a slight loss of glandular architecture in the inflammatory focus, with preserved glandular structure around the infiltrate and in the tissue proximal to the focus. The micrograph (c) shows a focus of acute inflammation with infiltration of neutrophils in the lamina propria of the mucosa, without destruction of the glands of the large intestine.

Figure 20. Histopathological study of histological preparations of the colon of the sick group treated with Ach preventively (G4). H&E stain. (a) H&E 4 x; (b) H&E 10x; (c) H&E 40 x Micrograph (a) shows a single focus of necrosis, a fairly conserved glandular structure throughout the entire colonic tissue and an insignificant inflammatory infiltrate with a predominance of lymphocytes, without showing a specific focus. Micrograph (b) shows re-epithelialized tissue and hyperchromatic nuclei associated with regenerative changes. A fibrinoleukocyte layer with epithelial renewal tissue is shown in the micrograph (c).

Figure 21. Histopathological study of the integrity and functionality of the colonic mucosa. EtOH50% group (G1): Micrograph (a) indicates PAS + staining for glycoproteins, including mucin, manifesting functional colonic mucosa. Higher magnification (a.1) shows strong staining and crypts arranged in the usual position. Group TNBS + EtOH50% (G2): The cross-section of the micrograph (b) shows a weakly positive PAS staining in the most distal area of the large intestine and a predominance of PAS- in the middle and proximal area (b.1). TNBS + TTO / Ach group (G3): PAS + secreting cells in almost the entire tissue area (c). Some areas (c.1) show weakly positive PAS staining. Group TNBS + PREV / Ach (G4): Recovery of the integrity and functionality when visualizing the mucus-secreting cells compared to the diseased group G2 (d). The micrograph (d.1) shows PAS + staining in the proximal mucosa of the colonic tissue. Figure 22. Expression of Nrf-2 (a) and HO-1 (b) proteins by Western blot analysis Densitometric analysis expressed in percentage (%) in control group G1, sick group G2 and sick groups treated with Ach (curative G3 and preventive G4). The standard deviation of the mean (SEM) is expressed with vertical bars. + Statistical significance (<0.05) compared to G1 control.

Figure 23. Expression of INOS and COX-2 proteins by Western blot analysis Densitometric analysis expressed in percentage (%) of the control group G1, sick group G2 and sick groups treated with Ach (curative G3 and preventive G4). The standard deviation of the mean (SEM) is expressed with vertical bars. ‘Statistical significance (<0.05) with respect to G2; \ Statistical significance (<0.05) with respect to control G1.

Figure 24. a) NLRP3 protein expression by Western blot and normalized densitometric analysis with β-actin, expressed as a percentage (%). The standard deviation of the mean (SEM) is expressed with vertical bars, b) Expression of ASC protein by Western blot and normalized densitometric analysis with β-actin, expressed in percentage (%). The standard deviation of the mean (SEM) is expressed with vertical bars, c) Caspase-1 protein expression by Western blot and normalized densitometric analysis with β-actin, expressed as a percentage (%). The standard deviation of the mean (SEM) is expressed with vertical bars. * Statistical significance (<0.05) with respect to group G2; + Statistical significance (<0.05) compared to G1 control.

Figure 25. IL-1β concentration in colonic tissue by ELISA analysis. The standard deviation of the mean (SEM) is expressed with vertical bars. * Statistical significance (<0.01) with respect to group G2; + Statistical significance (<0.01) compared to G1 control.

EXAMPLES

In the following, the invention will be illustrated by tests carried out by the inventors, which demonstrate the effectiveness of the invention. MATERIALS AND METHODS

1. OBTAINING THE POLYPHENOLIC EXTRACT OF ARISTOTEUA CHILENSIS (MAQUI)

1.1. Extraction from maqui

The inventors obtained the maqui extract from a product packaged and marketed in Chile called “ISLA NATURA DE CHILE®.” The fruit contains seeds and pulp, is wild-harvested in Patagonia and is freeze-dried for sale.

In order to obtain an extract that includes total polyphenols (anthodanins and non-anthocyanins), the inventors carried out the extraction using acid methanol (MeOH / H +) optimizing the method published by Genskowsky E, et al., J Sci Food Agrie. 2016; 96 (12): 4235-4242. The entire procedure (Figure 1) was carried out in the dark and at room temperature and through the following steps:

(a) Extraction: the inventors weighed 50 grams of lyophilized maqui powder and added 250 mL of MeOH / H + (0.1%) at pH 1, mixed it with a spatula in order to obtain a homogeneous solution. Subsequently, the mixture was sonicated with an ultrasound device (Hielscher Ultrasound Technology UP400S) at maximum power for 2 minutes. Then, they centrifuged the samples at 4000 rpm for 10 minutes and allowed it to predpitate for 5 minutes. Finally, the inventors recovered the supernatant in an Erlenmeyer flask and repeated the entire process 5 times.

(b) Evaporation: Once all the supernatants were recovered, the inventors put them in a rotary evaporator (Buchi B-490 Heating Bath) for between 2 to 3 hours at 35 ° C in order to evaporate the organic solvent and concentrate the abstract. They resuspended the extract in distilled water (H ₂ Od), centrifuged it at 4000 rpm for 4 minutes and finally recovered the supernatant.

(c) Filtration: The inventors carried out 3 filtration processes, by means of the vacuum process in 1 L kitasate. For the first filtration, they used celite (Celite® Hyflo Supercel Merck, code: 1907/2006), and subsequently made 2 filtrations with filter paper of porosity # 1 (100-150 MM) and then # 3 (40-100 MM), respectively. (d) Lyophilization: the inventors lyophilized the final product (polyphenolic extract of A. chilensis or lyophilized Ach), obtained after the extraction, evaporation and filtration steps described above, for 24 hours in Telstar model Cryodos at -80 ° C. The lyophilization process is a method that avoids enzymatic and chemical changes, protein denaturation, loss of aromas and easily oxidizable components, without causing loss of activity.

1.2. Estimation of the total polyphenol content (TCC) by Folin-Clocalteu.

The inventors measured the concentration of total polyphenols in the extract of A. chilensis (Ach) by spectrophotometry, as described by Matic P., et al., JAOAC int. 2017; 100 (6): 1795-1803 based on a colorimetric oxide-reduction reaction. The oxidizing agent used was the Folin-Ciocalteu reagent, which contains phosphowolphramic acid (2WO ₄ ² ), which is reduced originating W ₂ O ₅ (tungsten blue), through the addition of the Folin-Ciocalteu reagent in a alkaline medium. The absorption spectrum of this compound is evaluated at 740 nm.

The inventors used in the assay the reagents Folin-Ciocalteu (Sigma Aldrich, code: F9252), Na ₂ CO ₃ (Sigma Aldrich, code: 497-19-8) and gallic acid [AG] (Sigma Aldrich, code. : 149-91-7).

For the preparation of the calibration line, the inventors used a standard solution of GA (0.1 mg / mL) from which volumes of 0.5, 1, 2, 3, 4 and 5 mL were taken and volume was completed. up to 25 mL of H ₂ Od.

For the analysis, the inventors prepared a solution with 12.8 mg of lyophilized A. chilensis polyphenolic extract (lyophilized Ach) and dissolved it in 10 mL of H ₂ Od. The resulting concentration of the lyophilized Ach solution was 1.28 mg / mL. They took a 20 mL aliquot of this solution in a 50 mL volumetric flask and made up to the mark with water. Subsequently, they took a 0.5 mL aliquot of this second previous dilution in a 25 mL volumetric flask and added 1.3 mL of Folin-Ciocalteu reagent and 2.5 mL of _{saturated Na 2} CO ₃ , and homogenized this resulting dilution . The Inventors waited 10 minutes for the appearance of a blue color of the solution. They made a visual comparison of the intensity of the blue color of the sample of polyphenolic extract of A. chilensis (Ach) with the solutions of the GA calibration line, described above, and carried out the absorbance measurement at 740nm. The results were expressed in mg of AG / g lyophilized Ach (maqui extract).

1.3. Estimation of antioxidant power using the FRAP method.

The inventors carried out the estimation of the antioxidant power by the FRAP method, as described by Benzie IF, et al., 1996, Anal Biochem, 239 (1): p. 70-6, based on the use of antioxidants as reducers and inactivation of oxidants (one species reduced at the expense of oxidation of another) in a redox-related colorimetric reaction, where the ferric complex [Fe ³ *] - 2.4, 4-tripyridyl-striazine (Fe ³⁺ - TPTZ) colorless, it is reduced to an intense blue colored complex, the ferrous ion (Fe ²⁺ - TPTZ). The test was carried out using TPTZ reagents (Sigma Aldrich, Code: 3682-35-7), FeCl ₃ -6H ₂ O solution (Sigma Aldrich, Code: 10025-77-1) and Trolox for calibration line (Sigma Aldrich, Code: 53188-07-1).

The inventors made the mixture of the following reagents, following the order of appearance: 1) 40 mM hydrochloric acid (HCl), 535 μL of HCl (37%) was diluted in 100 mL of H ₂ Od, 2) 0 acetate buffer, 3 mM at pH 3.6, for 250 mL, 0.0061 g of sodium acetate (NaAc) was dissolved in 200 mL of dH ₂ O and pH was adjusted using 40 mM HCl and made up to 250 mL, 3) FeCl solution ₃ -6H ₂ O 20mM, 0.1352 g of solution was dissolved in 25 mL of H ₂ Od and 4) 10mM TPTZ solution, 0.0312 g of reagent was weighed and dissolved in a 10 mL flask with 40mM HCl. The inventors kept the final working solution FRAP throughout the process in a bath at 37 ° C. The ratio of acetate buffer: TPTZ: FeCl3-6H ₂ 0 was in a ratio of 10: 1: 1 and HCl at 40nM was used to adjust pH.

For the preparation of the Trolox calibration line, the inventors prepared the stock solution by dissolving 0.01 g of Trolox in 5 mL of MeOH + 5 mL of H ₂ Od. From this solution, they prepared different concentrations for different points on the line (Table 1).

For analysis, the inventors prepared a solution weighing 12.8 mg of lyophilized Ach and dissolving it in 10 mL deH ₂ Od. The concentration of the solution was 1.28 mg / mL. From the 1.28 mg / mL stock solution, they made two solutions at different extract concentrations to obtain sample 1 (M1) = 0.128 mg / mL and sample 2 (M2) = 0.512 mg / mL, to determine its antioxidant activity. For the blank, the inventors used only 2 mL of the FRAP reagent and 1 mL of H ₂ Od. For preparation of the samples, we added 2 mL of FRAP, 0.9 ml deH ₂ Od and 0.1 mL to each sample in duplicate M1 and M2. For the calibration curve they used the Trolox standard. They inverted the different solutions to mix, keeping them in the dark until absorbance was measured at 593nm. The results are expressed in μmol of Trolox Equivalents (TE) / g.

2. IN VITRO TESTS

2.1. Cell culture

The human colon cancer epithelial cell line HT-29 was obtained from the Department of Pharmacy of the University of Seville and the inventors maintained it in McCoy's 5a medium (Biowest, code: L0210) supplemented with fetal bovine serum. ([SFB], Biowest, Code: S1810) at 10% and penicillin-streptomycin ([P / STj Biowest, code: L0014) at 1%. The cells were incubated at 37 ° C and 5% CO ₂ . All tests were carried out with 80% confluence. Mouse macrophage cell line RAW 264.7 was obtained from the Andalusian Center for Molecular Biology and Regenerative Medicine (CABIMER) in Seville. The inventors maintained the cells in high glucose DMEM medium (Biowest, code: L0102) with 10% FBS and 1% P / ST. Subsequently, the inventors incubated the cells at 37 ° C and 5% CO ₂ . All the tests were carried out with a confluence of 80%, while the maintenance of the cell line was carried out by standard techniques.

2.2. Cell Viability Assay To determine cell viability, the inventors used Alamar Blue Cell Viability Reagent (Thermo Fisher Invitrogen ™, code: DAL1025) as an indicator of metabolic activity. Specifically, they used resazurin, a blue, cell-permeable and weakly fluorescent dye in the assay. Upon entering the cell, resazurin is reduced to resorufin, producing a colorimetric change (red-pink) and a fluorescent signal. All tests were carried out in triplicate. The procedure was carried out as directed by the provider.

The Inventors seeded each cell line, HT-29 and RAW 264.7, in a 24-well plate (P24) at a density of 1.2 x 10 ⁵ cells / mL. Once 80% confluence was reached, the inventors treated the cell lines with different concentrations of Ach, N-acetylcysteine (NAC) or 5-aminosalicylic acid (5-ASA) as shown in Table 2.

Table 2: Treatments performed on HT-29 and Raw264.7 cell lines. Ach: polyphenolic extract of maqui; LPS: lipopolysaccharide; NAC: N-acetylcysteine; 5-ASA: 5-aminosalicylic acid.

Subsequently, the inventors removed the medium, washed the cultures 2 times with PBS, and added Alamar Blue and culture medium in a 1:10 ratio (McCoy's 5a medium without phenol red for HT-29; DMEM medium without phenol red for RAW 264.7 ). The medium did not carry phenol red or supplementation, as serum or additional proteins have been shown to interfere with the emission of fluorescence or absorbance.

The inventors incubated the cell cultures for 1 to 4 hours at 37 ° C, 5% CO ₂ and protected from light. Detection sensitivity increases with longer incubation time. The inventors took 100 µL of medium from each well and placed on a 96-well plate (P96) for reading, measuring the absorbance at 570 nm in a plate reader (Multiskan FC, Thermo Fisher Scientific, code: N07710). Given the direct relationship between absorbance and cell viability, the inventors performed the calculation with respect to the control cells according to the following equation:

The absorbance of the sample is that obtained with the different treatments administered and the control absorbance is the untreated cells.

The concentrations and measurement times for each cell line were:

• HT-29: Ach concentrations were [100 µg / mL]; [200 µg / mL]; [300 µg / mL], the treatment time with Ach was 24 and 48 hours post-induction of oxidative therapy. The positive control was known antioxidant N-acetyldsteine (NAC) at 5 mM (Sigma Aldrich, code: A9165) and the oxidizing substance H ₂ O ₂ 0.05% (500 μm) at 30% v / v (PanReac AppliChem, code: 131077).

• RAW 264.7: Ach concentrations were [75 µg / mL]; [100 µg / mL]; [200 µg / mL]; [300 µg / mL] and the treatment time with Ach 12 hours. The positive control was 5-ASA (AGROS Organics TM, code: 89-57-6) at a concentration of 0.1, 0.5 and 1 mM. Synergy was assessed with 100 µg / mL of Ach with 0.1 mM of 5-ASA. The inflammation-inducing substance was 1 µg / mL of LPS (Sigma Aldrich, code: L2630).

23. Determination of ROS

For the evaluation of the oxidative stress state (EO), the inventors used as indicator 2 ', 7'-dichlorofluorescein diacetate (DCFH-DA) according to the method of Wang, H. et al., 1999, Radie Biol Med , 27 (5-6): p.612-6. DCFH-DA is taken up by cells, and through intracellular esterases, it is metabolized to 2 ', 7'-dichlorofluorescein (DCFH), which in the presence of ROS (ONOO ^~ ), is oxidized to dichlorofluroescein (DCF), a fluorescent compound that is used as a quantitative measure of cellular EO.

For analysis, the inventors seeded each cell line in a 96-well plate (P96) at a density of 11.8x1 or ⁴ cells / mL. Once a confluence of the

80%, the inventors treated the cell lines with different concentrations of Ach, NAC or 5-ASA as shown in Table 2 shown above, and incubated for 1 hour at 37 ° C and 5% CO ₂ . The inventors prepared a stock solution of DCFH-DA at 0.01 M / L in DMSO and stored it at -20 ° C.

The inventors removed the supernatant and washed 2 times with PBS. Subsequently, they added 25 pM of DCFH-DA solution in medium without phenol red and without supplementation for 30 minutes, incubating it at 37 ° C and 5% CO ₂ .

They carried out the fluorescence recording with a wavelength excitation of 485 nm with the Infinity Control device, using the Tecan-i-control program. Since DCFH-DA indirectly measures the amount of ROS generated, the inventors performed the calculation through the following equation:

Where 'RFU treatment' is the mean fluorescence detected by each treatment condition described in Table 2; 'RFU without cells' is the mean fluorescence detected in wells containing DCFH-DA, but without cells; and 'RFU positive control', is the mean fluorescence detected in the wells with LPS (in RAW 264.7 cells) or H ₂ O ₂ (in HT-29 cells) and culture medium (McCoy's 5a medium without phenol red for HT-29 ; DMEM medium without phenol red for RAW 264.7). The concentrations and measurement times for each cell line were:

• HT-29: Ach concentrations were [100 µg / mL]; [200 µg / mL]; [300 µg / mL] and the treatment time with Ach was 1 hour. The known antioxidant N-acetylcysteine (NAC) at 5 mM (Sigma Aldrich, code: A9165) was used as a positive control and 0.05% (500 μm) at 30% v / v (PanReac _{) as oxidizing substance H 2} O ₂ AppliChem, code: 131077).

• RAW 264.7: Ach concentrations were [75 µg / mL]; [100 µg / mL]; [200 µg / mL]; [300 µg / mL] and the treatment time with Ach was 12 hours. As a positive control, 5-ASA (AGROS OrganicsTM, code: 89-57-6) were used at a concentration of 0.1, 0.5 and 1 mM. Synergy was assessed with 100 µg / mL of Ach with 0.1 mM of 5-ASA. The inflammation-inducing substance was 1 µg / mL of LPS (Sigma Aldrich, code: L2630).

3. IN VIVO TESTS

3.1. Animal model

The inventors used adult Balb / c mice 12 to 14 weeks old and weighing between 19 and 39 grams from the Animal Facility Service of the Faculty of Medicine of the University of Seville. The animals were kept in pathogen-free conditions and procedures approved by the corresponding Ethics Committee of the Junta de Andalucía were used. The inventors provided the mice with food and water ad libitum and were kept on a 12 hour dark / light cycle, 25 ° C temperature and 55% humidity. Mice were anesthetized by ventilatory anesthesia with 4- 5% ofluorane and an oxygen flow at 0.5-1.0 L / minute for 5 minutes during the induction of Crohn's disease (CD), and under intraperitoneal (ip) anesthesia during sacrifice using 100 mg / Kg of Ketamine and 5mg / Kg of Diazepan, with a duration of between 30 and 45 minutes.

The mice were randomized into 4 groups of 6 mice each:

Group 1 [G1]: Induction control (50% EtOH only)

Group 2 [G2]: Crohn's patient [CD] with induction with TNBS + EtOH 50%, without treatment.

Group 3 [G3]: Crohn's disease [CD] with induction with TNBS + polyphenolic extract of A. chilensis, 1 daily dose started one day after induction with TNBS until sacrifice carried out 5 days after starting treatment with TNBS. CD therapeutic group treatment. Group 4 [G4]: Crohn's patient [CD] with induction with TNBS + polyphenolic extract of A. chilensis, 1 daily dose during the 7 days prior to induction with TNBS and until sacrifice carried out 5 days after starting the induction with TNBS. CD preventive treatment group. For disease induction, the inventors prepared TNBS (Sigma-Aldrich, code: 92822) at a concentration of 100 mg / Kg with 50% EtOH and inoculated intracolonic (ic) inserted at about 3 to 4 cm using a 2 mm thick polyurethane probe, according to the protocol described in section 3.1.1. The day before the intervention, the inventors withdrew the feed for 12 hours in order to keep the colon free of feces. The animals were kept 5 days after a single i.c. of TNBS until slaughter. The inventors administered polyphenolic extract of A. chilensis at a dose of 50 mg / Kg by the orogastric route.

3.1.1. Preparation of TNBS.

Materials:

TNBS (Sigma-Aldrich, code: 92822): keep cold.

100% ethanol

Anesthesia: Ketamine (Ketolar) + Valium (diazepam) / Isofluorane Insulin syringe: Size 1 mL (27-30 -gauge needle)

Polyurethane probe or catheter: 2mm thick. Procedure:

1. For rectal instillation of TNBS (100 mg / Kg) in mice, prepare 2 mg of TNBS / 100 µL of 50% ethanol. Mice with only 50% ethanol will be used as control groups.

TNBS preparation: Density: 1.14 g / mL at 20 ° C

1 mL - 1.14 gx mL - 0.02 gx = 0.0175 ml or 17.5 μL of the product (1M TNBS) in 50 μL H ₂ O + 50 μL EtOH = 100 μL total volume (250 μL for rats).

2. Connect a 1 mL needleless syringe to a polyurethane tube or catheter (2 mm thick).

3. The mouse must be fasting 18 hours prior to the experiment.

4. Gently introduce the catheter (moistened with serum) rectally by turning it on itself, deepening approximately 3-4 cm for mice.

5. Slowly inject 100 µL of TNBS (250 µL for rats). After injecting the TNBS, place the animal upside down for 3-4 minutes to avoid loss of TNBS.

6. Post-administration hydration of TNBS. Option 1: Feed the animals for the first week with commercial or laboratory-prepared oral rehydration salts (ORS): 8% sucrose + 0.2% saline (200 mg NaCI + 8 g sucrose in 100 mL H ₂ O) to prevent dehydration. Option 2: Subcutaneous administration of Ringer Lactate 1OmL / kg (20μL in a mouse with an average weight of 20g) postoperatively and 24 hours later.

7. Track daily weight and signs of dehydration.

3.2. Assessment of the severity of the disease

The Inventors evaluated the clinical activity of colitis during experimentation to determine the disease activity index (DAI). The presence of diarrhea, rectal bleeding and percentage of weight loss were assessed on a scale of 0 to 3 (Table

3).

Table 3: clinical activity index

3.3. Macroscopic study

After intraperitoneal anesthesia of the animals, the inventors extracted the large intestine, weighed and measured it (width and length) and made the photographic record. Subsequently, they opened the large intestine by longitudinal incision. Table 4 shows the criteria to evaluate the damage through the macroscopic scoring system modified from that described by Appleyard CB, et al., 1995 Am J Physiol, 269 (1 Pt 1): p. G119-25.

Table 4: Gross intestinal damage score

3.4. Histopathological study After removal of the colon, the inventors washed it with PBS to remove blood and stool debris. Subsequently, they cut the large intestine lengthwise in two; one half was frozen in liquid nitrogen for later analysis and the other half was wound from the distal to the proximal end (Figure 2). After the roll was made, the inventors fixed it in 4% formaldehyde and embedded it in paraffin. They then made serial 4 μm sections (evaluating the 1st, 5th and 10th sections of each sample) and stained them with hematoxylin and eosin (H&E) and periodic acid-schiff's reagent (PAS) using standard techniques, for subsequent histopathological study. The inventors examined the preparations to assign damage scoring at the microscopic level according to a modified system based on that proposed by Appleyard CB, et al., 1995 Am J Physiol, 269 (1 Pt 1): p. G119-25 (Table 5).

Table 5: Intestinal microscopic damage score

3.5. Obtaining cytoplasmic proteins and Western Blot analysis

For processing the samples, the inventors randomly selected 4 colon tissues per group (n = 4) frozen in liquid nitrogen, weighed about 100 mg of tissue, and homogenized them in a sonicator with ice-cold lysis buffer in the ratio 1: 3 (50 mM Tris-HCl, pH 7.5, 8 mM MgCl2, 5 mM ethylene glycol bis (ethylene glycol-bis (2-aminoethyl ether) -N, N, N'N'-tetra-acetic acid [EGTA] ), 0.5 mM EDTA, 0.01 mg / mL leupeptin, 0.01 mg / mL pepstatin, 0.01 mg / mL aprotinin, 1 mM phenylmethylsulfonyl fluoride (PMSF) and 250 mM NaCl). The inventors centrifuged the homogenates at 12,000g, 15 minutes, 4 ° C and collected the supernatants and stored them at -80 ° C.

To determine the protein concentration of the colon homogenates, the inventors used the colorimetric method of Bradford MM, et al., 1976, Anal Biochem, 72: p. 248-54. The aliquots containing supernatants, the inventors conducted a 1:20 dilution (2μΙ sample: 38μΙ_ deH ₂ Od) to calculate protein / mL milligrams. Subsequently, the inventors carried out a Western blot analysis.They separated the samples from the supernatants with equal amount of protein (30 μg) by 10% and 15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (PAGE / SDS) depending on the protein of interest. Then, they electrophoretically transferred the proteins to a nitrocellulose membrane and stained them with Ponceau red to record the efficiency of protein loading. They then blocked the membranes in blocking solution (5% bovine serum albumin [BSA] dissolved in 0.5% Nonidet ™ P40 [NP-40] and PBS pH 7.4) at room temperature for 1 ½ hour. .

Subsequently, the inventors incubated with specific primary antibodies in blocking solution overnight: polyclonal rabbit anti-iNOS antibody (1: 1000; Stressgen-Enzo Life Sciences, Farmingdale, NY, USA, code: ADI-KAS-NO001-D ), polyclonal rabbit anti-COX2 antibody (1: 300; Cayman Chemical, Ann Arbor, MI, USA, code: 160116), polyclonal rabbit anti-Nrf-2 antibody (1: 1000; Santa Cruz, USA code : sc-722), rabbit anti-HO-1 polyclonal antibody (1: 1000; Stressgen-Enzo Ufe Sciences, Farmingdale, NY, USA, code: ADI-SPA-896), rabbit anti-NLRP3 monoclonal antibody (1 : 1000; Cell Signaling, Danvers, MA, USA; code: D4D8T), rabbit monoclonal antibody ASC (1: 1000; Cell Signaling, Danvers, MA, USA; code: D2WBU) and polyclonal rabbit anti-Caspase antibody 1 (1: 1000; abcam, Cambridge, UK, code: ab1872).

After 3 washes, the membranes were incubated with secondary anti-rabbit horseradish peroxidase (HRP) antibody (1: 1600; Cell Signaling, Danvers, MA, USA; code: 7074P2) or anti-mouse (1: 1600; Dako ·, Atlanta, code: P0447) in blocking solution for 1½ hour at room temperature. To demonstrate equal protein loading, the membranes were incubated with β-actin using an anti β-actin antibody (1: 6000; Sigma Aldrich ·, St. Louis, MO, USA, code: A5316).

The inventors performed immunodetection with a chemiluminescence light detection kit (SuperSignal West Femto, for all proteins and Pico, for β-atin, Pierce, IL, USA). They then monitored immunological signals using an automated molecular imaging system for chemiluminescent proteins. (Amersham 600, Ge Healthcare Ufe Sciences, Buckinghamshire, UK). The inventors analyzed the signals and quantified them with an imaging system (Biophotonics ImageJ Analysis Software, National Institute of Mental Health, Bethesda, MD, USA) and expressed them as a percentage with respect to the control group.

3.6. Determination of IL-1β

The inventors measured IL-1β concentrations in colonic tissue using the quantitative enzyme immunoassay (ELISA) kit according to the manufacturer's instructions (Murine IL-1β, PeproTech, code: 900-K47). They selected a random subset of colon samples (n = 6) for the trial. The inventors weighed and homogenized cold colon samples with sonicator in 1:20 protease inhibitor buffer for ELISA (1X PSS, 0.5 mM EDTA, 0.01 mg / mL leupeptin, 0.01 mg / mL pepstatin, 0.01 mg / mL aprotinin, 1 mM phenylmethylsulfonyl fluoride [PMSF]) · Then, the homogenates were centrifuged at 12000 g for 10 minutes at 4 ° C and the supernatants were collected and stored at -80 ° C. Cytokine levels were expressed as µg / mg of tissue.

3.7. Statistic analysis

All values in figures and tables are expressed as arithmetic mean ± standard error of the mean (SEM). For calculations of standard deviation in data expressed in percentages, the inventors used the% relative standard deviation (% DER). To compare means, first, they checked the normality of the variables using the Shapiro Wilk test. To evaluate the assumption of equality of variance, they used Levene's test. When they verified that the variable had a normal distribution and homogeneity of variance, they applied the studenf t test for the comparison of 2 means. If not, they applied the Man Whitney U test.

For the comparison of 3 means or more, the inventors first used Shapiro Wilk's test to check assumption of normality and Bartlett's test to check for equality of variance. If the assumptions were met, they used the ANOVA test and when the assumptions of normality and variance were not met, they used the Kruskal-Wallis test. When ANOVA or Kruskal-Wallis yielded a level of significance <0.05, they performed a Bonferroni post-hoc test. All data were considered statistically significant with a p-value <0.05 and p-value <0.01. Experiments involving in vitro assays and Western blot analysis were performed in triplicate. ELISA analyzes were performed in duplicate. Histopathological analysis was performed blind or masked in order to avoid observer bias. All data were processed in STATA 12.0 software.

RESULTS

1. OBTAINING THE POLYPHENOLIC EXTRACT OF ARISTOTEUA CHILENSIS (MAQUI)

1.1. Total polyphenol content of the extract of A. chilensis (Ach)

Table 6 shows the absorbance values of different concentrations of gallic acid (GA) between 0.5 ppm and 8 ppm. Figure 3 shows the calibration line, representing absorbance versus AG concentration (ppm). The absorbance of the blank was 0.03.

Table 6: Absorbency at different concentrations of Trolox. AG: Gallic Acid; Abs:

Absorbency

The results of the Folin Ciocalteu method reveal that in each gram of the lyophilized extract of the maqui fruit there is an average of 39.02 mg of polyphenols expressed as Gallic acid (GA) (Table 7).

Table 7: Content of total polyphenols in lyophilized extract of maqui. M1: Sample 1; M2: Sample 2; BM: Blank sample; CTP: Total content of polyphenols

1.2. Antioxidant capacity of A. chilensis extract (Ach)

Table 8 shows the absorbance values of different concentrations of Trolox between 79.9μΜ and 399.5μΜ. Figure 4 shows the calibration line with the data obtained from the FRAP test representing the absorbance versus the T rolox concentration (μΜ).

Table 8: Absorbency at different concentrations of Trolox. Abs: Absorbance

The results regarding the antioxidant capacity (CA) of the maqui extract to reduce Fe ³⁺ to Fe ²⁺ are shown in Table 9. Solution # 1 shows a CA of 20843.75 μmol Trolox equivalents (TE / g) , while solution # 2 presents a CA of 20296.87 μmol TE / g, showing similar values between them. On average, the data indicate that the maqui extract presents a CA of 20,571.31 μmol TE / g.

Table 9: Antioxidant capacity of the polyphenolic extract of maqui

2. IN VITRO TESTS

2.1. Effect of A. chilensis (Ach) extract on cell viability

HT-29 colon epithelial cells were exposed to 0.05% H ₂ O ₂ as an oxidizing agent in this EO model. To test the protective effect of maqui, the inventors incubated the cells with increasing concentrations of Ach for 24 and 48 hours and compared when incubated with NAC, a widely known and clinically used antioxidant. After exposure to these compounds, they evaluated cell viability using the Alamar Blue ^{® test} . The results showed a significant reduction (p-value <0.01) of viability in those cells exposed to H ₂ O ₂ . When the HT-29 were incubated with the polyphenolic extract of maqui (Ach), the cell death caused by EO was reduced, increasing the cell viability in a statistically significant way (p-value <0.01) at 24 hours when it was treated. with Ach at a concentration of 300 μg / mL. At 48 hours, from the lowest to the highest concentrations of Ach (100, 200 and 300 μg / mL), a significant increase in viability was observed compared to cells without treatment exposed to H ₂ O ₂ (Figure 5) .

In order to evaluate the cytotoxic profile of Ach, the inventors evaluated the viability of the RAW264.7 cell line when they were incubated for 12 hours with different concentrations of Ach polyphenolic extract (75, 100, 200 and 300 μg / mL) and LPS (1 μg / mL) as a macrophage activator and inflammatory agent. The viability of cells incubated with LPS was 89.37% (SEM 10.60%), while with Ach it varied from 98.2% (SEM 8.73%) to 106.82% (SEM 11.59%). ) and with 5-ASA between 79.89% (SEM 10.83%) and 90.75% (SEM 10.24%). The synergy between 1 mM of 5-ASA and 100 μg / mL of Ach also did not show a reduction in cell viability (90.7%, SEM 10.24%). The viability test by Alamar Blue ^® revealed that the polyphenolic extract of maqui did not exert any significant effect on cytotoxicity in macrophage cells. RAW264.7 (Figure 6).

2.2. Effect of A. chllensis (Ach) extract on oxidative stress in cell culture

Because DCFH-DA has high cellular permeability and is oxidized by ONOO- to DCF, the inventors evaluated the percentage of oxidation of this compound in the HT-29 and RAW264.7 cell lines. In HT-29 they observed a significant increase in intracellular oxidation with the addition of H ₂ O ₂ (p-value <0.05). The percentage of oxidation shows a dose-dependent inhibition of the polyphenolic extract (reduction of 63.96%, SEM 2.24%; 57.62%, SEM 9.85%; and 43.75% SEM 13.19%) at concentrations of Ach of 100, 200 and 300 μg / mL respectively, reaching a significant reduction at a concentration of 300 μg / mL (p-value <0.05). In parallel, when cells exposed to H ₂ O ₂ were treated with NAC, the inventors observed a decrease in oxidation, although not significant, of 74.61% (SEM 14.87%) compared to cells incubated only with H ₂ O ₂ (Figure 7).

On the other hand, the control macrophages showed a significantly lower oxidation (p-value <0.01) compared to the macrophages activated with LPS, demonstrating the relationship between inflammation and EO. When the cell line is treated with different concentrations of Ach extract, it is possible to observe a protective effect against EO mediated by ONOO- (DCF), while macrophages treated with 5-ASA at concentrations of 0.1, 0.5 and 1 mM did not show significant effects on oxidation, although with a downward trend. The inventors observed that at 0.1 mM 5-ASA, with the addition of the lowest dose of Ach (100 μg / mL), the% oxidation was the lowest of all treatments (20.83%, SEM 6 , 72%), approaching the oxidation values that the cells presented without stimulating with LPS (13.69%, SEM 6.34%) (Figure 8). 3. IN VIVO TESTS

3.1. Evidence for the induction of acute Crohn's disease by TNBS

Mice treated with TNBS exhibited intense inflammation of the colonic tissue wall. The large intestine presented an atypical morphology with obvious distortion and loss of glandular architecture. There was a marked impact on the mucosa and submucosa related to infiltration of inflammatory cells, especially neutrophils and lymphocytes, associated with acute inflammation and thickening due to edema of the lamina propria. The inventors detected some areas with ulceration and necrosis. Additionally, the damage extended towards the muscular and serous layer, being a transmural inflammation (Figure 9 AD). The inventors did not observe thickening of the muscle layer or granulomas, since these parameters are associated with a chronic phase of the disease, verifying that the experimental model in this study is of the acute type.

3.2. Effect on clinical activity and body weight of Ach in TNBS-induced Crohn's disease

During the active phase of the disease, the inventors observed an obvious alteration in the health status of the animals. All diseased mice presented hypomotility, prostration, and piloerection the day after induction with TNBS. However, the sick groups treated with Ach (G3 and G4) recovered daily activity, with evident physical and health improvement.

All the experimental groups had a daily follow-up, controlling body weight and evaluating the stool for consistency, presence of mucus and bleeding. These indicators were processed using the “DAI” clinical indicator, one of the most important parameters, together with body weight, to determine the clinical severity of the disease. Mice inoculated with TNBS (G2) without treatment showed a significant increase (<0.001) in DAI compared to the control group with EtOH50% (G1). Consistent with the findings evidenced in the state of health, the animals treated both post-induction of the disease and preventively (G3 and G4, respectively) present a DAI similar to the control group, with a highly significant decrease compared to sick mice. with CD (<0.001) (Figure 10).

In particular, the induction of acute Crohn-type inflammation caused by the administration of TNBS was associated with a significant loss of body weight, whereas treatment with Ach attenuated weight loss. The diseased mice in group G2 (TNBS + EtOH50%) showed a decrease in weight from the day of induction until sacrifice. The G3 group (TNBS + TTO / Ach) presented a slight loss on the day after induction that stabilized until the day of sacrifice. For its part, Group G4 (TNBS + PREV / Ach) managed to regain weight equal to the days prior to induction (Figure 11).

3.3. Preventive and curative effect of Ach on morphological and macroscopic parameters of colonic tissue in the animal model of Crohn's disease induced by TNBS

The inventors treated the mice with 50 mg / Kg of Ach by the orogastric route 4 days after induction of EC by TNBS (group G3) and 7 days prior to induction and continuously until the end of the experiment (group G4). . Four days after induction, the mice were sacrificed and the large intestine removed for gross evaluation. The colon of diseased mice without treatment (G2) revealed a shortening compared to the mice of the control group (G1). The colon of Ach treated mice (G3 and G4) showed marked suppression of tissue inflammation, evidenced by the length of the colon (Table 10 and Figure 12).

Table 10: Average (± SEM) of the colon length of the experimental groups

The differences between the sick group and the control group are statistically significant (p-value <0.001). Significant differences were also found when the G2 group with TNBS + EtOH50% was compared with the Ach treated groups (Figure 13). Additionally, the inventors assessed the weight of the large intestine, since it is another marker of tissue inflammation. The mean colon weight was significantly higher in the G2 group (p-value <0.01). The mice treated preventively and curatively with Ach showed a marked reduction in colonic weight (p-value <0.001), reaching values equal to or even lower than that of the mice in the control group G1 (Figure 14). When the inventors carried out the macroscopic evaluation, it revealed the presence of mucus and soft and liquid stools inside the large intestine of those mice that were administered TNBS + EtOH50% by the ic route. EC water. In addition, during the extraction of colonic tissue at sacrifice, it was possible to demonstrate adhesion of the colon to adjacent tissues. The presence of hyperemia, congestion and ulcerations in mice treated with TNBS was observed at the time of opening the large intestine longitudinally. These morphological characteristics were almost totally restored in the Ach treated groups (G3 and G4).

When the inventors applied the macroscopic score, the observed colonic damage was evident in the sick group G2 (p-value <0.001) with respect to the i.c. of EtOH50% in the control group G1, which yielded a minimum score of the macroscopic score. Interestingly, the groups treated with Ach (G3 and G4) presented a strong statistical significance in the reduction of the score with respect to the sick group. The values of the G3 and G4 groups were similar to the control group G1 (Figure 15).

3.4. Preventive and curative effect of Ach on microscopic evaluation and histopathological study of colonic tissue in the animal model of Crohn's disease induced by TNBS

Microscopic evaluation showed a significant increase in histological damage in mice treated with TNB + 50% EtOH. The score of this group was 3 times higher than the group that only intervened with 50% EtOH. When the inventors compared with the groups that received treatment, they observed that the G2 group had twice the score than the group treated with Ach both preventively and curatively (Figure 16).

On the other hand, the histological evaluation showed a transmural inflammation distributed in foci characterized by infiltration of inflammatory cells, ulceration in the mucosa and submucosa and loss of goblet cells in the entire colon of diseased mice (G2), while in the colon samples of control mice (G1), typical characteristics of a normal structure were observed. The administration of Ach by the orogastric route to diseased mice showed a significant recovery from pathological alterations, restoring the normal histology of the colonic tissue (Figure 17 AC: Intact colonic tissue in all its layers (mucosa, submucosa, muscular and serosa) in the control group (G1), Figure 18 AC: Severe loss of glandular architecture distributed in foci, mixed inflammatory infiltrate (neutrophils and lymphocytes) and edema (G2), Figure 19 AC: Histological structure preserved throughout the colon with mild inflammation in the lamina propria (G3), Figure 20 AC: Single focus of necrosis with preserved architecture throughout the colonic tissue without transmural inflammation (G4).

3.5. Preventive and curative effect of Ach on the integrity of the colonic mucosa in animals with Crohn's disease induced by TNBS The histological characteristics of the integrity of the colonic mucosa and its muco-secretory capacity were examined with PAS staining, which allowed The inventors distinguish mucus-secreting goblet cells. In group G1 it was possible to observe a positive PAS staining (PAS +) for glycoproteins and goblet cells were clearly distinguished. The sick group (G2) presented a negative PAS stain (PAS) to a large extent, mainly in the middle and proximal parts, showing a histologically significant decrease in the integrity and functionality of the colonic mucosa. The groups treated with Ach extract (curative: G3 and preventive: G4), showed a relevant recovery of the integrity of the mucosa of the large intestine. The changes were evidenced by the adequate glandular profile and an improvement in PAS staining, achieving PAS + staining in practically all the tissue (Figure 21 A-D).

3.6. Expression of Nrf-2 and HO-1 proteins related to oxidative stress in Crohn's disease and its regulation with Ach

Nrf-2 is a transcription factor responsible for the cellular redox balance and regulator of the expression of HO-1, an important protein with antioxidant and anti-inflammatory effects. The inventors performed a Western blot analysis to identify the expression levels of Nrf-2 and HO-1 in colonic tissue of the model of CD elicited by TNBS and in the groups treated with Ach. Nrf-2 showed upregulation in the sick group with respect to the control group G1, without presenting significant differences when the densitometric and statistical analysis was performed. The groups treated with Ach, both preventive and curative, showed a slight upward trend compared to the control group (G1) and slightly lower than the sick group (G2) (Figure 22A). Consistent with these observations, the expression of the HO-1 protein increased in the G2 group, with statistical significance when compared to the G1 group. Following The same trend as Nrf-2, HO-1 showed a slight increase when the mice were administered Ach, although this difference was not statistically significant (Figure 22 B).

3.7. Expression of INOS and COX-2 proteins involved in inflammatory processes in Crohn's disease and their levels post-treatment with Ach

To investigate the inflammatory processes related to tissue damage observed in the histopathological analysis of the IG of the experimental CD model, the inventors performed an analysis of the expression of INOS and COX-2 proteins in colonic tissue. Western blot analysis showed that INOS, a protein related to the initiation and perpetuation of intestinal inflammation, was significantly increased (p-value <0.05) in animals with CD by TNBS. However, levels of this protein were significantly attenuated when treated with Ach after induction of the disease (p-value <0.05) and a notable downward trend when Ach extract was administered prior to induction. (Figure 23 A). As for COX-2, widely recognized for its participation in the acute inflammatory activity in inflammatory and epithelial cells, its expression increased in group G2, while in group G3 it presented a significant decrease (p-value <0.05) with respect to the sick group (G1). Additionally, the group of mice treated preventively with Ach shows a significant reduction, reaching levels similar to the control group G1 (Figure 23 B)

3.8. Expression of proteins of the multiprotein inflammasome complex NLRP3 / ASC / Caspase 1 in Crohn's disease and its levels after treatment with Ach

To evaluate the impact of the treatment on the molecular mechanisms involved that underlie the inflammatory process, the inventors performed a protein expression analysis by Western blot analysis of the inflammasome N LRP3 / ASC / caspase 1 complex, whose expression and activity has recently been described as a pathway of great relevance in the homeostasis and immunity of the gastrointestinal system and a critical pathway in intestinal inflammation. The results of the analysis showed that the expression of the NLRP3 protein is significantly increased (p-value <0.05) in the sick group G2, while with Ach it was able to strongly decrease (p-value <0.05) the expression of the NLRP3 protein (Figure 24 A). Consequently, the ASC protein, which plays a primary role in the formation of the inflammasome, showed an increase in Crohn's diseased mice, while down-regulation was observed in the Ach treated groups, although without presenting statistically significant differences (Figure 24B). When caspase 1 was observed and quantified, the last protein of the inflammatory cascade of the NLRP3 flammasome and responsible for the maturation of IL-1β and IL-18, the results showed an increase in its expression in the group with untreated CD ( G2), while a significant reduction was observed in mice with Ach (G3) -based curative treatment. The groups administered Ach 7 days prior to induction (G4) showed an expression with a strong downward trend when compared to the sick group G2 (Figure 24C).

3.9. Effect of Ach on the levels of the inflammatory cytokine IL-1β in the animal model of Crohn's disease induced by TNBS

In order to explore the mechanisms by which Ach decreases transmural inflammation in the colon of mice with CD by TNBS, the inventors evaluated the production of inflammatory cytokines. Due to the strong expression of the flamasome in intestinal tissue of animals with CD, the production of IL-1β was specifically evaluated in colonic tissue, whose maturation and biological activity require the assembly of the NLRP3 flamasome and activation of caspase 1. The researchers determined that in the CS model by TNBS there is a significant increase (p-value <0.01) in IL-1β production, consistent with the intense activation of the NLRP3 nflamasome found in the large intestine by Western blot. They verified that Ach was able to attenuate with a high power of statistical significance (p-value <0.01) the levels of IL-1β in colonic tissue (Figure 25), proving the anti-inflammatory effect of Ach through a significant reduction in the production of the inflammatory cytokine IL-1β.

Claims

1. Polyphenolic extract of Aristotelia chilensis for use in the treatment or prevention of Crohn's disease in a subject.

2. The extract for use according to claim 1 wherein the subject is a mammal, preferably a human.

3. The extract for use according to claim 1 or 2 comprising at least 30 mg of polyphenols for each total gram of extract.

4. The extract for use according to claim 3 comprising 39 mg of polyphenols.

5. The extract for use according to any one of claims 1 to 4 wherein the therapeutically effective daily dose of said extract is between 1 to 60 mg per Kg of the subject.

6. The extract for use according to claim 5 wherein the therapeutically effective daily dose is 50 mg per Kg of the subject.

7. The extract for use according to any one of claims 1 to 6, wherein the polyphenolic extract of Aristotelia chilensis is comprised within a composition.

8. The extract for use according to claim 7 wherein the composition is a pharmaceutical composition or a nutritional composition.

The extract for use according to claim 8, wherein the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.

10. The extract for use according to any one of claims 7 to 9 wherein the composition is administered orally, orogastric or intracolonic.

The extract for use according to claim 8, wherein the nutritional composition is selected from a supplement, a nutraceutical, a probiotic or a symbiotic.