BR112019001362B1 - USES OF FRAXINUS ANGUSTIFOLIA SAMARA EXTRACT - Google Patents

Abstract

The present invention relates to samara extracts from Fraxinus angustifolia, to processes for providing such extracts and to methods and uses of the obtained extracts. In particular, the present invention relates to the use of such extracts in the reversal of treatment of dysbiosis of the intestinal microbiota related to obesity and/or related to the metabolic syndrome, in the treatment or prevention of hepatic steatosis, non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) and in the modulation and/or adjustment of the intestinal microbiota.

Description

[0001] The present invention relates to extracts of Fraxinus angustifolia (in particular, the samara of the same), to processes for providing such extract and to methods and uses related to such extracts. In particular, the present invention relates to methods for reversing the treatment of obesity-related and/or metabolic syndrome-related gut microbiota dysbiosis, treating or preventing conditions such as hepatic steatosis, non-alcoholic fatty liver disease (NAFLD) and steate -non-alcoholic hepatitis (NASH), and modulate and/or adjust the intestinal microbiota.

[0002] The listing or discussion of an apparent and previously published document in this specification should not necessarily be considered an acknowledgment that the document is part of the prior art or is common general knowledge.

[0003] Non-alcoholic fatty liver disease (NAFLD) is a condition defined by excessive accumulation of fat in the form of triglycerides (steatosis) in the liver (designated as a histological accumulation of more than 5% hepatocytes). It is the most common liver disorder in developed countries; for example, it affects around 30% of adults in the US. If left untreated, the condition can progressively worsen and may ultimately lead to cirrhosis of the liver. NAFLD is particularly prevalent in obese patients, with around 80% of people having the disease.

[0004] A subset of patients with NAFLD exhibit liver cell damage and inflammation, in addition to excessive fat accumulation. This condition, designated as non-alcoholic steatohepatitis (NASH), is histologically virtually indistinguishable from alcoholic steatohepatitis (ASH) (as described by the World Gastroenterology Organization (WGO) in WGO Global Guidelines: Non- alcoholic Fatty Liver Disease and Non-alcoholic Steatohepatitis (2012)). Although the simple steatosis seen in NAFLD is not directly correlated with increased short-term morbidity or mortality, progression from this condition to NASH dramatically increases the risks of cirrhosis, liver failure, and hepatocellular carcinoma (HCC).

[0005] Although morbidity and mortality from hepatic causes are greatly increased in patients with NASH, they are even more strongly correlated with morbidity and mortality from cardiovascular disease. NASH is widely considered the hepatic expression of the conditions commonly referred to as the metabolic syndrome, which include diseases related to type 2 diabetes mellitus, insulin resistance, central (trunk) obesity, hyperlipidemia, low high-density lipoprotein (HDL) cholesterol, hypertriglyceridemia, and hypertension. (see, e.g., Wiernsperger, N., Diabetes Metab Syndr Obes, 6, 379-388 (2013)).

[0006] There is currently a worldwide epidemic of diabetes and obesity. At least 1.46 billion adults were overweight or obese, and since 2008, around 170 million of the world's children were designated overweight or obese. These numbers continue to grow, indicating that NASH will become an increasingly common liver problem in rich and developing countries alike, increasing the global burden of liver disease and affecting public health and healthcare costs globally.

[0007] In 2012, it was estimated that NAFLD and NASH will increase direct and indirect medical costs over five years by approximately 26%. As indicated above, it is also now estimated that about 30% of all adults in developed countries have NAFLD, and about 2 to 6% of such adults are expected to have NASH. In particular, NAFLD is thought to affect up to 70 to 80% of obese individuals (see, e.g., Younossi, Z.M. et al., Clin Gastroenterol Hepatol, 9, 524 to 530 (2011)).

[0008] The exact cause of NASH has not been elucidated, and it is almost certainly not the same in all patients. It is most closely related to insulin resistance, obesity and the metabolic syndrome; however, not all patients with these conditions have NAFLD/NASH, and not all patients with NAFLD/NASH have one of these conditions. However, given that NASH is a potentially fatal condition that leads to cirrhosis, liver failure and CHC, effective treatment is urgently needed.

[0009] There is currently no approved evidence-based drug therapy for NAFLD/NASH. Lifestyle change is critical in any attempt to reverse the course of NAFLD/NASH, and targets for therapy are insulin resistance and oxidative stress. Although several treatment options are being evaluated, the value of most treatments remains uncertain, or the effects are reversed when they are discontinued. Treatment goals for NASH are to reduce histologic features and improve insulin resistance and liver enzyme levels.

[0010] The human intestinal microbiota consists of trillions of microorganisms, most of which have bacterial and viral origin, which are considered non-pathogenic. The microbiota works in conjunction with host defenses and the immune system to protect against pathogen colonization and invasion. It also performs an essential metabolic function, acting as a source of essential nutrients and vitamins and aiding in the extraction of energy and nutrients, such as short-chain fatty acids (SCFA) and amino acids, from food (see, for example, Carding, S. et al, Microb Ecol Health Dis, 26, 26191 (2015)).

[0011] Microbial culture studies detect only a small number of the species of intestinal bacteria. Currently, intestinal microbiota composition and diversity are revealed by culture-independent metagenomic and genetic techniques. Metagenomic analysis and 16S ribosomal RNA gene sequencing showed that, at the phylum level, Firmicutes and Bacteriodetes dominate, with Actinobacteria, Proteobacteria, Fusobacteria, Spirochaetae, Verrucomicrobia, and Lentisphaerae also present (ibid.). Although the dominant phyla are relatively constant between individuals, diversity increases along the taxonomic line, with each individual harboring over a hundred unique species.

[0012] The gut microbiota evolved with humans as a mutualistic partner; however, it was found that changes in the composition of the intestinal microbiota, that is, alteration of the ecological organization of the intestinal microbiota (commonly known as dysbiosis), are related to several clinical conditions, such as obesity, diabetes, atherosclerosis, allergic diseases, gastrointestinal diseases , autoimmune diseases, and cancer (see, e.g., Serino, M., et al., Curr Cardiol Rep, 16(11), 540 (2014)), and also NAFLD (see, e.g., Boursier, J. and Diehl, A.M., PLoS Pathhog, 11(1), e1004559 (2015)). In fact, it is understood that intestinal microbiota dysbiosis could lead to altered intestinal defense, increased bacterial translocation, thus triggering tissue inflammation and hepatic steatosis.

[0013] The possible role of the gut microbiota in the progression of hepatic steatosis includes several potential mechanisms of action: induction of obesity through energy capture from otherwise indigestible dietary polysaccharides; regulation of intestinal permeability and stimulation of low-grade inflammation; modulation of dietary choline metabolism; and stimulation of endogenous ethanol production by enteric bacteria (see Arslan, N., World J Gastroenterol, 20(44), 16452-16463 (2014)).

[0014] In addition, it is understood that an obesogenic microbiota can alternate liver function by stimulating hepatic triglycerides and modulating systemic lipid metabolism, which can indirectly affect the storage of fatty acids in the liver. Restoration of an optimal intestinal microbial system could therefore be a promising strategy to prevent the progression of steatosis and, in particular, halt the progression from NAFLD to NASH.

[0015] The present inventors have now surprisingly found that extracts obtained from samara or seed of Fraxinus angustifolia (hereinafter referred to as FA) (particularly from samara) have a potent activity in reversing dysbiosis of the intestinal microbiota by modulating or adjusting of intestinal microbiota. These effects suggest that such Fraxinus angustifolia extracts may have numerous therapeutic and non-therapeutic (eg cosmetic) uses and uses in the prevention of medical conditions.

FRAXINUS ANGUSTIFOLIA EXTRACTS

[0016] According to the present invention, an extract of Fraxinus angustifolia (FA) (in particular, a seed or samara extract of Fraxinus angustifolia (particularly, samara)) is provided, which may be referred to hereinafter as "extract of the invention”.

[0017] Typically, the extract of the invention may be an extract obtained from FA (in particular, samara or FA seed) using processes as described herein.

[0018] For the sake of clarity, all references in this document to an extract of Fraxinus angustifolia (FA) will refer, in particular, to extracts obtained from extracts of samara or FA seed (more particularly, samara). Furthermore, since the YF Samara will contain YF seed, it will be understood that YF Samara extracts will comprise (or will essentially consist of) YF (Samra) seed extracts.

[0019] The extract of the invention may be an aqueous extract, an alcoholic extract or a hydroalcoholic extract. Preferably, the extract of the invention is a hydroalcoholic extract, such as a hydromethanolic or hydroethanolic extract. For example, the extract of the invention can be a hydroethanolic extract obtained using an extraction solvent comprising from about 1 to about 99% ethanol in water, such as from about 30% to about 75% ethanol in water, or from about 30% to about 50% ethanol in water, such as from about 35% or about 40% ethanol in water.

[0020] The term “aqueous extract”, as used in the present document, refers to the extract obtained from Fraxinus angustifolia (FA) when the extraction of the plant (particularly, sámara) was carried out using water as the only solvent .

[0021] The term “alcohol extract”, as used in the present document, refers to the extract obtained from Fraxinus angustifolia (FA) when the extraction of the plant (particularly, sámara) was carried out using alcohol as the only solvent. For example, 100% methanol and/or 100% ethanol.

[0022] The term “hydroalcoholic extract”, as used in this document, refers to the extract obtained from Fraxinus angustifolia (FA) when the plant has been extracted using a mixture of water and alcohol. For example, from about 1% to about 99% alcohol (eg, ethanol) in water, such an extract would be termed a hydroethanolic extract.

[0023] In certain embodiments, the extract of the invention may comprise (or consist essentially of) the following compounds (secoiridoids): (i) from about 1% to about 16% by weight of nuzenide, such as from about from 1% to about 15% by weight; (ii) from about 1% to about 18% by weight of GL3, such as from about 1% to about 17% by weight; (iii) from about 0.5% to about 1% by weight oleoside methyl ester; (iv) from about 0.03% to about 0.12% by weight excelside B; (v) from about 0.1% to about 1.7% by weight of GL5; and/or (for example e) (vi) from about 0.08% to about 0.8% by weight of salidroside, such as from about 0.08% to about 0.7% by weight.

[0024] Unless otherwise specified herein, the weight percentages listed are based on the total weight of the extract (dry) obtained.

[0025] For clarity, preferences, options, specific features and the like indicated for a particular aspect, feature or parameter of the invention shall, unless the context otherwise indicates, be considered disclosed in combination with any and all other preferences, options, specific and similar features indicated for the same or other aspects, features and parameters of the invention.

[0026] When the term "comprising" or "comprises" is used, it is understood that the extract or composition described must contain the listed ingredient (or listed ingredients), but may optionally contain additional ingredients. When the term "consisting essentially of" or "consisting essentially of" is used, it is understood that the extract or composition described must contain the listed ingredient (or ingredients listed) and may also contain a little (e.g. up to 5 % by weight, or up to 1% or 0.1% by weight) of other ingredients, provided that any additional ingredients do not affect the essential properties of the extract or composition. When the term "consisting of" or "consisting of" is used, it is understood that the extract or composition described must contain only the listed ingredient (or ingredients).

[0027] The term “about”, as used herein, for example, when referring to a measurable value (such as an amount or weight of a specific component in the reaction mixture), refers to variations of ±20 %, ±10%, ±5%, ±1%, ±0.5% or particularly ±0.1% of the specified amount.

[0028] For example, in certain embodiments, the extract of the invention may comprise (or essentially consist of) about 10% by weight of nuzenide and/or (for example, e) about 10% by weight of GL3.

[0029] For the sake of clarification, the structures of the compounds mentioned above are depicted below.

[0030] Additionally, other compounds may also be present in the extract of the invention. In certain embodiments, other compounds that may be present include, but are not limited to, coumarins, such as fraxin, fraxetin, esculin, esculetin, scopoline, 7-methyl eculin, and fraxidine glycoside.

[0031] For example, in certain embodiments, the extract of the invention may further comprise (or consist essentially of/consist of): Fraxin (about 0.095%); fraxetine (about 0.117%); esculin (about 0.017%); esculetin (about 0.017%); scopoline (about 0.038%); 7-methyl eculin (about 0.040%); and/or (for example, e) fraxidine glycoside (about 0.061%).

[0032] In specific embodiments, the total coumarins (estimated based on detected coumarins such as fraxin) was about 0.39%. In addition, other coumarins can be identified (e.g. by LC/MS) at levels too low to be quantified (as their presence was below 5 ppm), but may include cichoriin, scopoletin, kalyncantoside, mandshurin, fraxidine, isofraxidine and fraxinol.

[0033] The person skilled in the art will understand that the extract of the invention can be provided in solid form. By solid form is meant that the compound can be provided as an amorphous solid or as a crystalline or partially crystalline solid.

COMPOSITIONS AND ADMINISTRATION

[0034] According to the present invention, the extract of the invention may be provided in the form of a (suitable) composition, such as a pharmaceutical composition or a food composition (which may be called a functional food composition or a dietary composition).

[0035] In specific embodiments, the extract of the invention can be provided in the form of a pharmaceutical composition (which may also be called a pharmaceutical formulation) or functional food composition comprising the extract of the invention and, optionally, a pharmaceutically acceptable excipient or ingredient acceptable for food use (functional), as appropriate.

[0036] As used herein, references to pharmaceutically acceptable excipients may refer to pharmaceutically acceptable adjuvants, diluents and/or carriers, as known to those skilled in the art.

[0037] Ingredients acceptable for use in food include those known in the art (including those also referred to herein as pharmaceutically acceptable excipients) and which may be natural or unnatural, that is, their structure may or may not occur in nature. In certain cases, they may originate from natural compounds and be subsequently modified (eg maltodextrin).

[0038] In specific embodiments, the extract of the invention may be provided in the form of a pharmaceutical composition or a functional food composition, further comprising an unnatural carrier or a modified natural carrier, such as maltodextrin.

[0039] By "pharmaceutically acceptable" is meant that the additional components of the composition are sterile and pyrogen-free. Such components must be "acceptable" in the sense of being compatible with the extract of the invention and not harmful to the receptors thereof. Thus, "pharmaceutically acceptable" includes any compound (or compounds) used in forming a part of the formulation that is intended to act merely as an excipient, i.e., is not intended to have biological activity in itself. Thus, the pharmaceutically acceptable excipient is generally safe, non-toxic and not biologically or otherwise undesirable.

[0040] The person skilled in the art will understand that the extracts of the invention (for example, in the form of compositions, such as pharmaceutical compositions, as known to those skilled in the art, such as those described herein) can be administered to a patient or subject (e.g., a human or animal patient or subject) by any suitable route, such as orally, rectally, nasally, pulmonaryly, buccally, sublingually, transdermally, intracisternally, intraperitoneally, and parenterally (including subcutaneously, intramuscularly, intrathecally, intravenously and intradermally).

[0041] In particular, the extracts of the invention can be administered orally. In such cases, pharmaceutical compositions according to the present invention can be specifically formulated for administration by the oral route.

[0042] Pharmaceutical compositions for oral administration include solid dosage forms such as hard or soft capsules, tablets, tablets, dragees, pills, lozenges, powders and granules. Where appropriate, they may be prepared with coatings, such as enteric coatings, or they may be formulated so as to provide controlled release of the active ingredient, such as sustained or sustained release, according to methods well known in the art.

[0043] Liquid dosage forms for oral administration include solutions, emulsions, aqueous or oily suspensions, syrups and elixirs.

[0044] The compositions (e.g., pharmaceutical or food compositions) described herein, such as those intended for oral administration, can be prepared according to methods known to those skilled in the art, such as by placing the components of the composition in the addition mixture.

[0045] Such compositions, as described herein, may contain one or more additional components selected from the group consisting of food ingredients, such as sweetening agents, flavoring agents, coloring agents and preserving agents. Tablets may contain the active ingredient (or active ingredients) in admixture with non-toxic pharmaceutically acceptable excipients (or ingredients) which are suitable for the manufacture of tablets. Such excipients (or ingredients) may, for example, be: inert diluents, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, for example corn starch, maltodextrin or alginic acid; binding agents, for example starch, gelatin or acacia; and lubricating agents, for example magnesium stearate, stearic acid or talc. Tablets may be uncoated or may be coated using known techniques to delay disintegration and absorption in the gastrointestinal tract and thus provide a sustained action over a longer period. For example, a time delay material such as glyceryl monostearate or glyceryl distearate may be employed.

[0046] Suitable pharmaceutical carriers include inert solid diluents or fillers, sterile aqueous solutions, and various organic solvents. Examples of solid carriers are lactose, terra alba, sucrose, cyclodextrin, maltodextrin, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid, acacia, modified starch and lower alkyl ethers of cellulose. Examples of liquid carriers are syrup, peanut oil, olive oil, phospholipids, fatty acids, fatty acid amines, polyoxyethylene and water. Furthermore, the carrier or diluent can include any sustained release material known in the art, such as glyceryl monostearate or glyceryl distearate, alone or mixed with a wax.

[0047] Depending on the disorder, and the patient, to be treated, as well as the route of administration, the extracts of the invention can be administered in varying doses (i.e., therapeutically effective doses, as administered to a patient in need thereof) . In this regard, one skilled in the art will understand that the dose administered to a mammal, particularly a human being, in the context of the present invention should be sufficient to affect a therapeutic response in the mammal over a reasonable period of time. The person skilled in the art will recognize that selection of the exact dose and composition and the most appropriate delivery regimen will also be influenced by, inter alia, the pharmacological properties of the formulation, the nature and severity of the condition being treated, and physical condition and mental acuity. of the recipient, as well as by the potency of the specific compound, the age, condition, body weight, sex and response of the patient being treated, and by the stage/severity of the disease.

[0048] Typically, in the use or method of the invention described herein, the extract or the composition comprising the extract is administered in an amount of from about 100 mg/day to about 2,000 mg/day, or from about 500 mg/day to about 1500 mg/day or about 1000 mg/day.

[0049] In any event, the medical professional, or other individual skilled in the art, will be able to routinely determine the actual dosage that will be most suitable for an individual patient. The dosages mentioned above are exemplary of the average case; there can, of course, be individual instances where higher or lower dosage ranges are required, and such dosages are within the scope of this invention.

[0050] When included within a composition (e.g., a pharmaceutical composition) as described herein, the extract is typically present in an amount of from about 1% by weight to about 100% by weight, e.g. from about 10% by weight to about 90% by weight or from about 20% by weight to about 80% or from about 30% by weight to about 70% or from about 40% by weight to about 60% by weight.

PROCESSES FOR OBTAINING EXTRACTS

[0051] The extract of the invention can be isolated from FA seeds or samara (in particular, FA samara) using separation techniques that select the required extract, which can be determined by those skilled in the art.

[0052] Typically, the extract of the invention can be obtained by means of extraction and isolation processes, as generally described herein below, or routine modifications thereof.

[0053] For example, processes for extracting and isolating extracts of the invention may comprise (or essentially consist of) the following steps: (i) extraction of samara from FA (which may be ground) by means of a suitable solvent ; (ii) solvent evaporation; and, if necessary, (iii) purifying the extract (eg, by chromatography).

[0054] Typically, FA samaras are milled into granules with a particle size in the range of about 0.1 mm to about 30 mm, to increase the surface area for solvent contact and increase the effectiveness of extraction.

[0055] Specific solvents that can be used in the extraction process include alcohols (such as methanol) and alcohol/water mixtures (such as methanol and water mixtures). For example, the extraction solvents can be water, a water-alcohol mixture (from about 1% to about 99% alcohol in water. For example, from about 30% to about 75% alcohol in water , or from about 30% to about 50% alcohol in water, such as from about 35% or about 40% alcohol in water) or alcohol. Specific alcohols that may be mentioned include ethanol (EtOH) and methanol (MeOH).

[0056] In specific embodiments, the extraction solvent can be an ethanol-water mixture, such as from about 30% to about 75% ethanol in water, or from about 30% to about 50% ethanol in water. For example about 35% or about 40% ethanol in water.

[0057] In one embodiment, the extraction temperature is in a range of about 20 °C to about 100 °C. In a specific embodiment, the temperature for extraction is in a range of about 50°C to about 70°C.

[0058] Typically, the ratio of plant material to solvent mixture used in the extraction process ranges from about 1:1 to about 1:10 on a gram to milliliter basis, such as from about 1:3 to about from 1:8.

[0059] The incubation period (that is, the period during which the plant material is in contact with the solvent) is typically from about 2 hours to about 24 hours.

[0060] After the plant materials and solvent have been incubated, the solvent is separated from the residual plant material, and the extraction composition is concentrated (that is, the solvent is removed) until the extraction composition has a solid component. Typically, the solid component may comprise (or consist essentially of) from about 1% to about 35% FA secoiridoids. Other components include phenolic compounds, salidroside, coumarins and flavonoids.

[0061] After completion of the extraction process, the secoiridoid (or secoiridoides) can be isolated from the FA extract (i.e., purified) with the use of a chromatographic process, if necessary.

[0062] Typically, the extract of the invention can be obtained using the following process: - the powdered FA extract (that is, obtained by preparing ground samara) is dissolved in an alcohol and the secoiridoid (or secoiridoides) is extracted by alcohol from the powder. The alcohol is then evaporated, and the remaining residue, including secoiridoid (or secoiridoides), is isolated on a reverse-phase C-18 resin-loaded chromatography column; - several fractions containing different compounds are eluted with a series of water and 10% MeOH/90% water, and MeOH system. Fractions are compared by high performance liquid chromatography (HPLC) analysis, and those eluents that have similar HPLC patterns are combined; - the combined fractions are separated on normal phase silica gel column chromatography and eluted with chloroform (CHCl3), CHCl3-methanol mixture starting from 90%, 80% CHCl3 to 100% MeOH to generate several subfractions. The subfractions are compared by HPLC and the fractions containing excelside A and excelside B are combined, respectively. The pooled fractions are further purified by a combination of column chromatography on C-18 resins, MCI GEL CHP-20P and/or Sephadex LH-20 to provide pure excelside A and excelside B.

[0063] The terms "isolated" and "purified", as used herein, refer to the extract or secoiridoide (or secoiridoides) being separated from at least one other component (for example, a polypeptide or cellulose derivative) present with the extract or secoiridoide (or secoiridoides) in its natural source. In one embodiment, the extract or secoiridoid (or secoiridoides) is provided in pure form or in the presence of a solvent, buffer, ion, or other component normally present in a solution thereof.

[0064] Thus, the terms "isolated" and "purified" do not refer to the extract or secoiridoide (or secoiridoides) present in its natural source. Similarly, the term extract refers to components of the natural material that have been obtained through an extraction process, rather than those components present in their natural source (eg as FA seeds).

[0065] In specific embodiments, the extract of the invention, as obtained from such processes, may be: - substantially free of other plant material (for example, free of plant cellulose); - substantially free of plant cells; and/or - substantially free of plant cell matter.

[0066] As used herein, references to a material that is “substantially free” of another material may refer to material consisting of less than 1% by weight (e.g., less than 0.1%, such as less than 0.01% or less than 0.001% by weight) of that other material.

[0067] In alternative embodiments, the method for extracting and isolating an FA extract from an FA seed may be described comprising (or essentially consisting/consisting of) the steps of: (a) grinding an FA seed into particles; (b) treating the particles with a solvent mixture; (c) separating the ground particles from the solvent mixture; and (d) evaporating the solvent mixture.

[0068] In such additional embodiments, the process may also comprise (or essentially consist of) the steps of: (e) dissolving the product of (d) in alcohol; and (f) evaporating the alcohol.

[0069] Typically, in the extraction of FA extract from an FA seed (i.e. steps (a) to (d) as described in this document above): the milled particles have a diameter of about 0.1 mm to 30mm; and/or the temperature is from about 20°C to about 100°C; and/or the ratio of ground particles to solvent mixture is from about 1 g to 1 ml to about 1 g to 8 ml; and/or the ground particles are in contact with the solvent mixture from about 2 hours to about 24 hours; and/or the solvent mixture is water, a water-alcohol mixture or alcohol.

[0070] In specific embodiments, the extract of the invention as described herein may be an extract obtained from (or obtainable by means of) a process as described herein.

THERAPEUTIC AND NON-THERAPEUTIC USES

[0071] As described herein, the extract of the invention may have specific biological effects, which may be useful in the treatment of medical conditions. Thus, according to the present invention, the use of the extract of the invention in medicine (or as a pharmaceutical) is provided.

[0072] Additionally, as described herein, the extract of the invention may be particularly useful in the treatment of diseases, such as those described herein, such as hepatic steatosis (steatosis in the liver).

[0073] The extract of the invention can also assist patients in reducing or limiting weight gain. For example, the extract of the invention can reduce weight gain in a subject who has a high fat diet (as shown in Figure 1).

[0074] The extract of the invention can also reduce glucose intolerance. Typically, the extract of the invention can reduce glucose intolerance in a subject having a high fat diet (as shown in Figure 2).

[0075] The extract of the invention can also reduce the level of fat deposited in the liver (hepatic steatosis). Typically, the extract of the invention can reduce the level of fat deposited in the liver in a subject having a high fat diet (as shown in Figure 3).

[0076] Thus, in one aspect of the invention, there is provided an extract of Fraxinus angustifolia (i.e., an extract of the invention) for use: (a) in reversing dysbiosis of the gut microbiota related to obesity and/or related to metabolic syndrome ; (b) in the treatment or prevention of fatty liver disease, non-alcoholic fatty liver disease (NAFLD) and/or non-alcoholic steatohepatitis (NASH); (c) in the treatment or prevention of leaky gut and/or intestinal hyperpermeability; (d) in the treatment or prevention of cardiometabolic diseases and/or cardiovascular diseases induced by dysbiosis of the intestinal microbiota; (e) in the treatment or prevention of low-grade inflammation; (f) in the treatment or prevention of atherosclerosis; (g) in the treatment or prevention of obesity; and/or (h) in the treatment or prevention of insulin resistance, glucose intolerance, prediabetes and/or diabetes (such as type 2 diabetes).

[0077] In an alternative aspect of the invention, the use of an extract of Fraxinus angustifolia is provided in the manufacture of a medicine for: (a) reversing dysbiosis of the intestinal microbiota related to obesity and/or related to the metabolic syndrome; (b) treat or prevent fatty liver disease, non-alcoholic fatty liver disease (NAFLD) and/or non-alcoholic steatohepatitis (NASH); (c) treating or preventing leaky gut and/or intestinal hyperpermeability; (d) treating or preventing cardiometabolic diseases and/or cardiovascular diseases induced by gut microbiota dysbiosis; (e) treating or preventing low-grade inflammation; (f) treating or preventing atherosclerosis; (g) treating or preventing obesity; and/or (h) treating or preventing insulin resistance, glucose intolerance, pre-diabetes and/or diabetes (such as type 2 diabetes).

[0078] In another alternative aspect of the invention, a method is provided for: (a) reversing dysbiosis of the intestinal microbiota related to obesity and/or related to the metabolic syndrome; (b) treat or prevent fatty liver disease, non-alcoholic fatty liver disease (NAFLD) and/or non-alcoholic steatohepatitis (NASH); (c) treating or preventing leaky gut and/or intestinal hyperpermeability; (d) treating or preventing cardiometabolic diseases and/or cardiovascular diseases induced by gut microbiota dysbiosis; (e) treating or preventing low-grade inflammation; (f) treating or preventing atherosclerosis; (g) treating or preventing obesity; and/or (h) treating or preventing insulin resistance, glucose intolerance, prediabetes and/or diabetes (such as type 2 diabetes), which comprises administering a therapeutically effective amount of an extract of Fraxinus angustifolia to a subject in need of the same.

[0079] In specific embodiments, the disease or disorder to be reversed, treated or prevented is selected from the group (or groups) consisting of: - obesity-related gut microbiota dysbiosis, metabolic syndrome-related gut microbiota dysbiosis; and/or - fatty liver disease, non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH).

[0080] As used herein, the term “treatment” (and similarly “treat”) has its normal meaning in the field of medicine. In particular, the term can refer to achieving a reduction in the severity of one or more clinical symptoms associated with the disease or disorder (e.g., the fungal infection), as can be determined using techniques known to those skilled in the art. (e.g., by a physician) and/or to delay the progression of the disease or disorder (i.e., increase the amount of time it takes for the disease or disorder to progress to a more serious state, e.g., when compared to the time it takes to expect to take in an untreated patient).

[0081] As used herein, the term “prevention” (and similarly “prevent”) includes references to prophylaxis of the disease or disorder (and vice versa). In particular, the term may refer to achieving a reduction in the likelihood that the patient (or healthy subject) will develop the condition (e.g., at least a 10% reduction, such as at least a 20%, 30%, or 40%, for example, at least a 50% reduction].

[0082] For clarification, in the context of the present invention, the terms "treat" and "prevent" include therapeutic or palliative treatment of subjects/patients in need, as well as prophylactic and/or diagnostic treatment of patients who are susceptible relevant disease states.

[0083] As used herein in relation to medical conditions, the term "reduce" may refer to making the observed amount smaller or decreasing in size.

[0084] As used herein in relation to medical conditions, the term "revert" may refer to regressing the relevant trait to a normal state as known to those skilled in the art. For example, reversal of various types of gut microbiota dysbiosis may refer to altering the levels and/or nature of gut microbiota to levels and/or a nature that are the same, or more similar, than those skilled in the art. technique would expect to observe in a healthy subject.

[0085] For clarity, in specific embodiments, the Fraxinus angustifolia extract comprises (or essentially consists of): (i) from about 1% to about 15% by weight of nuzenide; (ii) from about 1% to about 17% by weight of GL3; (iii) from about 0.5% to about 1% by weight oleoside methyl ester; (iv) from about 0.03% to about 0.12% by weight excelside B; (v) from about 0.1% to about 1.7% by weight of GL5; and (vi) from about 0.08% to about 0.7% by weight of salidroside.

[0086] In particular, the FA extract may comprise (or essentially consist of) about 10% by weight of nuzenide and about 10% by weight of GI3.

[0087] Furthermore, for clarity, the Fraxinus angustifolia extract may be in the form of a composition (eg, a pharmaceutical composition or food composition) as described herein.

[0088] As used herein, the terms "subject" and "patient" may be used interchangeably and include mammalian species (particularly humans).

[0089] As used herein, the term "therapeutically effective amount" may refer to an amount of the extract of the invention, or composition comprising the LA extract of the invention, that confers a therapeutic effect on the treated patient (e.g., an amount sufficient to treat or prevent disease). The effect can be objective (that is, measurable by some test or marker) or subjective (that is, the subject provides an indication or perceives an effect).

[0090] As described in this document, changes in the composition of the intestinal microbiota, such as a change in the typical ecological organization of the intestinal microbiota (dysbiosis), are related to conditions such as obesity, insulin resistance, glucose intolerance, pre- diabetes, diabetes, hepatic steatosis, non-alcoholic fatty liver disease, low-grade inflammation, leaky gut, intestinal hyperpermeability, cardiovascular diseases induced by gut microbiota dysbiosis, cardiometabolic diseases and atherosclerosis. The potential for restoring an optimal gut microbial system therefore provides a strategy to prevent the progression of steatosis and the progression from NAFLD to NASH. Furthermore, this effect may also be helpful in promoting general health in patients who are not suffering from a specific disease or medical disorder.

[0091] Thus, in specific embodiments, the use (including the extract for use) or method as described in this document may comprise (or consist of) delay or detention (where the latter term may be called impediment or prevention) the progression of steatosis; particularly slowing or arresting (eg delay) progression from NAFLD to NASH.

[0092] As used herein, references to arresting the progression of a disease state shall refer to treating that disease in such a way that one skilled in the art will not be able to observe any significant worsening of that disease during a period of time after onset of such treatment and/or during which such treatment is provided. Similarly, references to arresting progression from one (first) disease state to another (second) disease state will refer to treating that first disease in such a way that one skilled in the art will not be able to observe the onset of the second state. of illness for a period of time after initiation of such treatment and/or during which such treatment is provided.

[0093] As used herein, references to delaying the progression of a disease state shall refer to treating that disease in such a way that one skilled in the art can observe that, after initiation of such treatment and/or during a period in which treatment is given, the time taken for the disease state to worsen is longer (i.e., for a medically significant period of time, such as for at least one week or, particularly, at least four or eight weeks) than that which would have been expected if such treatment had not been carried out. Similarly, references to delaying progression from one (first) disease state to another (second) disease state will refer to the treatment of that first disease so that one skilled in the art can observe that, after initiation of such treatment and/or during the period in which such treatment is provided, the elapsed time for the first disease state to progress to the second disease state is longer than that which would be expected if such treatment had not been performed.

[0094] Furthermore, in a further aspect of the invention, there is provided the use (e.g., non-therapeutic use) of an extract of Fraxinus angustifolia (i.e., an extract of the invention): (i) in the modulation or adjustment of gut microbiota; (ii) in the reduction of body fat; and/or (iii) in the reduction of blood glucose concentration.

[0095] In an alternative aspect of the invention, there is provided a method (eg, a non-therapeutic method) for: (i) modulating or adjusting the gut microbiota; (ii) reduce body fat; and/or (iii) reducing blood glucose concentration, which comprises administering an effective amount of a Fraxinus angustifolia extract to a subject in need thereof.

[0096] As used in this document, the terms “modulate” (or “modulation”) or “adjust” (or “adjustment”) may refer to the increase (enrichment) and/or decrease of certain taxonomic groups (phylum, class, order, family and gender) present in the intestine. For example, reference to modulation or tuning may refer to an effect that increases and/or decreases certain Taxonomic Operating Units (OTU) present in the gut.

[0097] The term "effective amount" refers to an amount of the extract of the invention, or composition comprising the extract of the invention, that confers an effect on the subject to which the extract or composition was administered (e.g., a sufficient amount to cause the desired effect, such as an increase and/or decrease of certain taxonomic groups (phylum, class, order, family and genus) present in the gut). The effect can be objective (that is, measurable by some test or marker) or subjective (that is, the subject provides an indication or perceives an effect).

[0098] As described in this document, the use or method may result in the increase and/or decrease of certain taxonomic groups (phylum, class, order, family and genus) present in the intestine, such as an increase (enrichment) and/or a decrease in certain taxonomic operational units (OTU) present in the gut.

[0099] In specific embodiments, modulation or adjustment refers to: - increasing the levels of bacterial groups selected from among the genera consisting of Burkholderiales, Sutterellacae, Parasutterella, Betaproteobacteria and Enterorhabdus; and/or - the reduction of the levels of selected bacterial groups among the genera consisting of Prevotellaceae, Flavonifractor, Clostridium IV and Butyricicoccus.

[0100] In more specific embodiments, modulation or adjustment refers to increasing the level of bacterial groups selected from the families consisting of Coriobacteriaceae, Lactobacillaceae, and Rikenellaceae.

[0101] In even more specific modalities, modulation or adjustment refers to: - the increase in the levels of bacterial groups selected from among the genera consisting of Coriobacteriaceae Olsenella, Lactobacillaceae, Lactobacillus and Rikenellaceae Alistipes; and/or - the reduction of Ruminococcaceae Butyricicoccus levels.

[0102] Since the presence of certain bacterial taxonomic groups is linked to high levels of steatosis in the liver, the enrichment of certain bacterial taxonomic groups that result from modulation or adjustment by the extract of the invention, or composition comprising the extract of the invention, can reduce or prevent the development or progression of fatty liver and/or the progression from NAFLD to NASH.

[0103] Thus, in specific embodiments, the modulation or adjustment of the gut microbiota is for use in (ie, results in or has the effect of) preventing or treating non-alcoholic fatty liver disease (NAFLD) and/or delaying ( or arrest) of progression from NAFLD to non-alcoholic steatohepatitis (NASH).

[0104] In a further aspect of the invention, a method is provided for modulating or adjusting the intestinal microbiota in order to prevent or treat NAFLD and/or delay (or halt) the progression of NAFLD to NASH, which comprises administering a effective amount of a Fraxinus angustifolia extract to a subject in need thereof.

[0105] The prevention or treatment of non-alcoholic fatty liver disease (NAFLD) and/or the delay (or arrest) of the progression of NAFLD to non-alcoholic steatohepatitis (NASH) can be achieved by: - increasing the levels of groups bacterial selected from the genera consisting of Burkholderiales, Sutterellacae, Parasutterella, Betaproteobacteria, and Enterorhabdus; and/or - reducing the levels of bacterial groups selected from the genera consisting of Prevotellaceae, Flavonifractor, Clostridium IV and Butyricicoccus; and/or - increasing the level of bacterial groups selected from the families consisting of Coriobacteriaceae, Lactobacillaceae and Rikenellaceae; and/or - increasing the levels of bacterial groups selected from among the genera consisting of Coriobacteriaceae Olsenella, Lactobacillaceae Lactobacillus and Rikenellaceae Alistipes; and/or - reducing the levels of Ruminococcaceae Butyricicoccus

BRIEF DESCRIPTION OF THE FIGURES

[0106] Figure 1 represents: (A) body weight, (B) body weight gain of mice during the 12 weeks of consumption of a high-fat diet (60%) with or without Fraxinus angustifolia extract at 200 mg/kg of body weight and (C) body composition of mice after 12 weeks treatment.

[0107] * indicates that the result is statistically different from the control, p < 0.05.

[0108] Figure 2 represents: (A) Blood glucose before and after 15, 30, 60, 90 and 120 minutes after oral administration of glucose by oral tube and (B) respective values of Area under the curve (AUC) of mice at the end of 12 weeks of consumption of a high-fat diet (60%) with or without Fraxinus angustifolia extract at 200 mg/kg of body weight.

[0109] * indicates that the result is statistically different from the control, p < 0.05.

[0110] Figure 3 represents: (A) liver section prepared from frozen liver and stained with red O oil from control mice and treated with Fraxinus angustifolia (magnification of x20); and (B) percentage of fat per area; Slides stained with oil red O were analyzed with ImageJ analysis software to obtain a quantitative histological measurement of steatosis; a pixel intensity histogram was generated from the image, the area was measured and the results were expressed as percentage of fat per area. Data are represented by plotting in boxes that show median, first quartile, third quartile, minimum and maximum.

[0111] ** indicates that the result is statistically different from the control, p = 0.004 (Mann-Whitney test)

[0112] Figure 4 represents the relative proportion of taxonomic groups at the class level showing individual study samples for each type of sample per group: Mice treated with a high-fat diet (HFD60) at 1 month (_T1) or 3 months (_T3) or mice treated with a high-fat diet and Fraxinus angustifolia extract (HFD60 + F. angust) at 1 month (_T1) or 3 months (_T3).

[0113] Figure 5 represents the relative proportion of taxonomic groups at the class level showing the average for each type of sample per group: Mice treated with a high-fat diet (HFD60) at 1 month (_T1) or 3 months (_T3 ) or mice treated with a high-fat diet and Fraxinus angustifolia extract (HFD60 + F. angust) at 1 month (_T1) or 3 months (_T3).

[0114] Figure 6 represents the relative proportion of taxonomic groups at the family level showing individual study samples for each type of sample per group: Mice treated with a high-fat diet (HFD60) at 1 month (_T1) or 3 months (_T3) or mice treated with a high-fat diet and Fraxinus angustifolia extract (HFD60 + F. angust) at 1 month (_T1) or 3 months (_T3).

[0115] Figure 7 represents the relative proportion of taxonomic groups at the family level showing the average for each type of sample per group: Mice treated with a high-fat diet (HFD60) at 1 month (_T1) or 3 months (_T3 ) or mice treated with a high-fat diet and Fraxinus angustifolia extract (HFD60 + F. angust) at 1 month (_T1) or 3 months (_T3).

[0116] Figures 8 and 9 represent Principal Coordinate Analysis (PCoA) to compare samples from different groups of mice (mice treated with a high-fat diet (HFD60) at 1 month (_T1) or 3 months (_T3) or mice treated with a high-fat diet and Fraxinus angustifolia extract (HFD60 + F. angust) at 1 month (_T1) or 3 months (_T3)) based on Generalized UniFrac distance metrics. Both PCoA plots with alpha values of 0 and 1 are shown.

[0117] Figure 10 represents the 3-month comparison between mice fed a high-fat diet without supplementation and mice fed a high-fat diet supplemented with Fraxinus angustifolia extract. The effect sizes of the linear discriminant analysis were determined using standard values (alpha value of 0.5 for both the Kruskal-Wallis factorial test between classes and the paired Wilcoxon test between subclasses, threshold of 2.0 for the LDA logarithmic score for discriminative characteristics) and the strategy for multiclass analysis defined for “all against all”. (B) The LEfSe cladogram from the LDS effect size data was generated with Bacteria as the tree root with six maximum taxonomic levels of the genus. The highlights (green and red) represent the enrichment of the taxonomic groups indicated in the corresponding group.

[0118] Figure 11 represents the 3-month comparison between mice fed a high-fat diet without supplementation and mice fed a high-fat diet supplemented with Fraxinus angustifolia extract. The LEfSe cladogram from the LDS effect size data was generated with Bacteria as the tree root with six maximum taxonomic levels of the genus. The highlights (green and red) represent the enrichment of the taxonomic groups indicated in the corresponding group.

[0119] Figures 12, 13 and 14 represent regression analyzes of taxonomic groups of the Family Identified by Random Forest and the percentage of liver staining with oil red O as severity of steatosis.

[0120] The present invention will be further described with reference to the following non-limiting examples.

EXAMPLES EXAMPLE 1 - EXTRACTION OF FRAXINUS ANGUSTIFOLIA WITH WATER

[0121] A total of 2.5 kg of the F. angustifolia samara were air-dried and then ground into a coarse powder with a particle size of approximately 1 to 2 mm. The coarse powder was soaked in water in a percolator at 80 to 90 °C for 5 hours and the water extract was drained from the percolator. The extraction process was repeated three times. All water extracts were combined and concentrated on a rotary vacuum evaporator. After the water was evaporated, a total of 550 grams of dry powder extract was obtained. HPLC analysis indicates that this powdered extract contained two major secoiridoids, 11.4% (w/w) nuzenide and 6.2% GB. The composition also contained 0.19% oleoside-1 1-methyl ester, 0.41% excelside B, 0.63% GI5, 0.2% salidroside, along with some minor secoiridoids including ligstroside, ester oleoside dimethyl and excelside A.

EXAMPLE 2- EXTRACTION OF FRAXINUS ANGUSTIFOLIA WITH WATER, WATER-EtOH TO EtOH

[0122] 5 samples were prepared, and each sample contained 5 grams of F. angustifolia samara. Each sample was ground to a powder and subjected to solvent extraction with 200 mL of water, 25% EtOH/75% water, 50% EtOH/50% water, 75% EtOH/25% water and EtOH , respectively. After extracting for 24 hours at room temperature (22 to 24 °C), the solvents were evaporated and the residual solids were analyzed by HPLC. Secoiridoid and salidroside content are listed in Table 1. TABLE 1: MAIN SECOIRIDOID AND SALIDROSIDA CONTENT USING DIFFERENT SOLVENTS (RESULTS EXPRESSED IN PERCENTAGE BY WEIGHT)

EXAMPLE 3 - ISOLATION OF SECOIRIDOIDS FROM FRAXINUS ANGUSTIFOLIA

[0123] 3.5 l of methanol were added and mixed with 500 grams of powdered extract obtained from the procedure shown in Example 1, for 3 hours at room temperature. The methanol solution was separated from the powder by a filtration process. The same process was repeated once and the two methanol extracts were combined and concentrated under reduced pressure to yield a total of 54 grams of dry methanol extract. The methanol extract was redissolved in water and filtered to remove non-water soluble substances. The filtrate was subsequently subjected to reverse phase column chromatographic separation on C-18 resin washed with water and MeOH-water gradient solvent system from 10% MeOH in water to 100% MeOH. A total of 7 fractions were collected. Each fraction eluted from the column was evaporated under vacuum and combined by HPLC analysis. Fractions 2, 3 and 7 were loaded onto a chromatographic column loaded with silica gel resin and eluted with a chloroform-methanol system initiated from CHCl3, 10% MeOH/CHCl3, 20% MeOH/CHCl3, to MeOH at 100%. The fractions collected from the silica gel column were compared by HPLC analysis and each separated eluate was repeatedly subjected to column chromatography on MCI GEL CHP-20P and/or Sephadex LH-20 resins and eluted with a water-methanol system until a single pure compound has been obtained. The compounds excelside A, excelside B, nuzenide, GI3, GI5, ligstroside, oleoside dimethyl ester, oleoside-1,1-methyl ester and salidroside were identified. All chemical structures were elucidated by spectroscopic methods.

EXAMPLE 4 - TEST OF THE EFFECT OF FRAXINUS ANGUSTIFOLIA EXTRACT ON HEPATIC STEatosis IN MICE

[0124] 9-week-old male adult C57BL/6 mice were purchased from Charles River (Charles River Laboratories, L'Arbresle, Rhône, France) and housed at constant room temperature and humidity and maintained on a light/light cycle. 12/12 h dark under SPF conditions. They were fed a high-fat diet (HFD) with 60% energy from fat obtained by SAFE (Scientific Animal Food & Engineering, Augy, France) for 12 weeks, and water was provided ad libitum. Tables 2 and 3 provide the HFD ingredient list and nutritional values, respectively. TABLE 2: LIST OF INGREDIENTS FOR THE PURIFIED DIET 260HF BY SAFE (AUGY, FRANCE) TABLE 3: NUTRITIONAL VALUES OF THE PURIFIED DIET 260HF BY SAFE (AUGY, FRANCE)

[0125] In the treatment group, extract of Fraxinus angustifolia (Vahl) was directly mixed into the diet and thus administered orally at 200 mg/kg/day, which represents a human equivalent dose of 1 g/day according to the FDA (2005) formula: HED equivalent human dose (mg/kg) = animal dose in mg/kg x (animal weight in kg/human weight in kg). The dry extract of Fraxinus angustifolia samara was obtained by extraction with 30% (v/v) ethanol in water as described in this document. The extract may preferably contain approximately 10% (% w/w) nuzenide and GL3 based on the total dry weight of the herbal extract. The effect of Fraxinus angustifolia (Vahl) extract consumption was analyzed by comparing the different parameters in rats consuming both the HFD and the extract (F.angustifolia group) compared to rats consuming only the HFD (control group ).

[0126] Body weight and body weight gain were followed during the 12 weeks and body composition (percent fat mass, lean mass and water) was assessed by NMR at the end of treatment. An oral glucose tolerance test (OGTT) was performed after 12 weeks of treatment by administering 2 g per kg of glucose body weight in fasting mice and monitoring blood glucose during the 2 hours after glucose administration.

[0127] At sacrifice, the liver was carefully removed, weighed, and conditioned for both histological analyses. For detection of liver lipid deposition, liver section was prepared from frozen liver and stained with oil red O as previously reported (Fowler, S.D., Greenspan, P., O.J. Histochem Cytochem, 33, 833-836 ( 1985)). Slides stained with oil red O were analyzed with ImageJ analysis software (National Institute of Mental Health, Bethesda, Maryland, USA) to obtain a quantitative histological measurement of steatosis. Five random images at x20 magnification for each liver biopsy were taken to ensure a representative sample for each specimen. A pixel intensity histogram was generated from the image, the area was measured and the results were expressed as percentage of fat per area.

[0128] As shown in Figure 1, 12-week consumption of a high-fat diet induced a strong body weight gain that was counteracted by simultaneous consumption of Fraxinus angustifolia extract. Particularly, the Fraxinus angustifolia extract was able to reduce fat mass in mice fed an HFD for 12 weeks.

[0129] As shown in Figure 2, consumption of Fraxinus angustifolia extract was also able to significantly reduce glucose intolerance in mice fed an HFD, as shown by significantly reduced blood glucose concentration 30 and 60 minutes after loading (p<0.05) and by the significant reduction in the area under the curve (glycemia versus time) (AUC) (p=0.07).

[0130] As shown in Figure 3, consumption for 12 weeks of a high-fat diet induced a high level of fat deposition in the liver, i.e., steatosis in the control group (35.2%), which is classic with this type of diet as a model of diet-induced obesity, diabetes, and fatty liver (see: Takahashi Y, Soejima Y, Fukusato T, World J Gastroenterol, 18(19), 2300-2308 (2012); and Zhou, Y and Xie, L., Am J Digest Dis, 2(1), 60 to 67 (2015)). The 12-week treatment with Fraxinus angustifolia extract was able to significantly reduce the severity of steatosis (p= 0.004), since only 22.8% of fat was found in the liver of mice treated with the extract, which corresponds to to a 35% reduction in steatosis. According to the histological scoring system of the World Gastroenterology Organization (2012) and the Kleiner and Brunt classification (Kleiner, D. E. and Brunt, E. M., Semin Liver Dis, 32, 3 to 13 (2012)), which classifies the severity of steatosis according to the fat content (Grade 0: <5%, Grade 1: 5 to 33%, Grade 2: 34 to 66%, Grade 3: >67%), treatment with Fraxinus angustifolia extract ensured the reduction of severity of steatosis from grade 2 to grade 1.

EXAMPLE 5 - TEST OF THE EFFECT OF FRAXINUS ANGUSTIFOLIA EXTRACT ON INTESTINAL MICROBIOTA DYSBIOSE IN MICE

[0131] In order to evaluate the modification of the intestinal microbiota induced by consumption of Fraxinus angustifolia extract in mice fed the high-fat diet, a metagenomic study of 16S rDNA was performed in murine fecal samples at baseline (4 weeks) and after the 12 weeks of HFD consumption with or without Fraxinus angustifolia extract (10 pair groups of mice, total number of 40 mice). The bacterial populations contained in the samples were determined using next-generation high-throughput sequencing of variable regions (V3-V4) of the bacterial 16S rDNA gene.

[0132] The metagenomic workflow is used to classify organisms from a metagenomic sample by applying specific regions in the 16S ribosomal RNA gene. This metagenomic workflow is unique to bacteria. The main output is a classification of readings into several taxonomic levels: phylum, class, order, family, and genus. The microbial population present in the samples was determined using next-generation high-throughput sequencing on variable regions of the bacterial 16S rRNA gene. The workflow included the following steps:

(1) library construction and sequencing

[0133] PCR amplification was performed using 16S universal primers targeting the V3-V4 region of the 16S bacterial ribosomal gene. The splice pair length was set to span a 476 base pair amplicon thanks to the 2 x 300 paired ends V3 MiSeq kit. For each sample, a sequencing library was generated by adding sequencing adapters. Detection of sequencing fragments was performed using MiSeq Illumina® technology.

(2) bioinformatics pipeline

[0134] The target metagenomic sequences of the microbiota were analyzed using the following bioinformatics pipeline; Briefly, after demultiplexing the reads on barcoded Illumina pairs, the single read sequences were cleaned and paired for each sample independently into longer fragments. After quality filtering and alignment against a 16S reference database, grouping into operational taxonomic units (OTU) with an identity threshold of 97% and taxonomic assignment were performed in order to determine community profiles.

[0135] Based on these results, graphical representations were made of the relative proportion of taxonomic groups (phylum, class, order, family and genus) present in 1) individual study samples and 2) the average for each type of sample/group.

[0136] As shown in Figures 4, 5, 6 and 7, the gut microbiota profiles were similar at the beginning of consumption of the high-fat diet (T1), although some minor differences could be observed at class levels and, more intensively , at familial levels in mice that consumed Fraxinus angustifolia extract compared to mice that did not consume the extract concomitantly with HFD.

[0137] Principal Coordinate Analysis (PCoA) was performed to compare samples based on Generalized UniFrac distance metrics (Lozupone C, Lladser ME, Knights D, Stombaugh J, Knight R (2011) UniFrac: an effective distance metric for microbial community comparison.ISME J. 5(2): 169 to 172) in order to illustrate the differences in groups of mice.

[0138] As shown in Figures 8 and 9, although the composition of the intestinal microbiota of mice was similar at the beginning of consumption of a high-fat diet (T1) as demonstrated by the stackable profiles of the distribution of individuals, it can be observed that after 3 months of treatment with Fraxinus angustifolia extract, treated individuals could be differentiated from untreated individuals according to their intestinal microbiota composition.

[0139] The Linear Discriminant Analysis (LDA) Effect Size (LEfSe) method (Segata, N. et al., Genome Biol, 12(6), R60 (2011)) was then used to analyze the comparisons high dimensionality class of metagenomic data. LefSe is an algorithm for high-dimensional biomarker discovery and explanation that can identify taxonomic groups that characterize the differences between two or more biological conditions. It emphasizes both statistical significance and biological relevance, allowing researchers to identify differentially abundant traits that are also consistent with biologically meaningful categories (subclasses). First, LEfSe robustly identifies features that are statistically different between biological classes. It then performs additional tests to assess whether these differences are consistent with expected biological behavior. The effect sizes of the linear discriminant analysis were determined using standard values (alpha value of 0.5 for both the Kruskal-Wallis factorial test between classes and the paired Wilcoxon test between subclasses, threshold of 2.0 for the LDA logarithmic score for discriminative characteristics) and the strategy for multiclass analysis defined for “all against all”.

[0140] As shown in Figures 10 and 11, the LefSe analysis revealed that, at the levels of genus or OTU, there was enrichment of different taxonomic groups (Burkholderiales, Sutterellacae, Parasutterella, Betaproteobacteria, Enterorhabdus and other OTUs) in mice treated with the Fraxinus angustifolia extract compared to untreated mice fed an HFD. On the other hand, Prevotellaceae, Flavonifractor, Clostridium_IV, Butyricicoccus and other taxonomic groups were less represented in mice treated with the Fraxinus angustifolia extract compared to untreated mice fed a HFD. These results highlight the modification of the intestinal microbiota induced by supplementation with Fraxinus angustifolia extract.

[0141] The correlation between microbiome analysis and the severity of steatosis was analyzed using Random Forest Analysis methodology (Touw, WG et al., Brief Bioinform, 14(3), 315 to 326 (2013)). As shown in Figures 12, 13 and 14, the relative abundance of various taxonomic groups at the family or genus level, respectively, is correlated with the severity of steatosis in mice fed a high-fat diet, clearly showing that Fraxinus extract angustifolia was able to modify the intestinal microbiota and, particularly, the relative abundance of some families or genera (Coriobacteriaceae, Lactobacillaceae, Rikenellaceae), and that these modifications could reduce the development of steatosis. The results of the correlation analysis between the abundance of taxonomic groups (by family and genus) and the severity of steatosis are shown in Tables 4 and 5 below. TABLE 4: STATISTICAL ANALYSIS FOR THE CORRELATION BETWEEN THE ABUNDANCE OF TAXONOMIC GROUPS BY FAMILY AND THE SEVERITY OF STEatosis. TABLE 5: STATISTICAL ANALYSIS FOR THE CORRELATION BETWEEN THE ABUNDANCE OF TAXONOMIC GROUPS BY GENRE AND THE SEVERITY OF STEATS.

Claims (11)

1. Use of a Fraxinus angustifolia samara extract characterized by the fact that it is in the manufacture of a pharmaceutical composition for: (a) reversing dysbiosis of the intestinal microbiota related to obesity and/or related to metabolic syndrome; (b) treat or prevent fatty liver disease, non-alcoholic fatty liver disease (NAFLD) and/or non-alcoholic steatohepatitis (NASH); (c) treating or preventing leaky gut and/or intestinal hyperpermeability; (d) treating or preventing cardiometabolic diseases and/or cardiovascular diseases induced by gut microbiota dysbiosis; and/or (e) modulate or adjust gut microbiota. 2. Use, according to claim 1, characterized by the fact that the intestinal microbiota is modulated or adjusted through the increase of selected bacterial groups among the genera consisting of Burkholderiales, Sutterellacae, Parasutterella, Betaproteobacteria and Enterorhabdus. 3. Use, according to claim 1, characterized by the fact that the intestinal microbiota is modulated or adjusted by reducing the levels of bacterial groups selected from the genera consisting of Prevotellaceae, Flavonifractor, Clostridium IV and Butyricicoccus. 4. Use, according to claim 1, characterized by the fact that the intestinal microbiota is modulated or adjusted through the increase of selected bacterial groups among the families comprising Coriobacteriaceae, Lactobacillaceae and Rikenellaceae. 5. Use according to any one of claims 1 to 4, characterized in that the extract comprises: (i) from 1% to 15% by weight of nuzenide; (ii) from 1% to 17% by weight of GL3; (iii) from 0.5% to 1% by weight oleoside methyl ester; (iv) from 0.03% to 0.12% by weight of excelside B; (v) from 0.1% to 1.7% by weight of GL5; and (vi) from 0.08% to 0.7% by weight of salidroside. 6. Use according to any one of claims 1 to 5, characterized in that the extract comprises 10% by weight of nuzenide and 10% by weight of GL3. 7. Use according to any one of claims 1 to 6, characterized in that the extract is a hydroethanolic extract. 8. Use according to claim 7, characterized in that the hydroethanolic extract is obtained using a solvent containing 25% to 75% ethanol. 9. Use according to any one of claims 1 to 8, characterized in that the extract is used in the manufacture of a composition to be administered in the form of: (a) a pharmaceutical composition comprising an extract of samara Fraxinus angustifolia as recited in any one or more of claims 1 to 8 and, optionally, a pharmaceutically acceptable excipient; or (b) a food composition comprising a Fraxinus angustifolia samara extract as recited in any one or more of claims 1 to 8 and, optionally, an ingredient acceptable for use in food. 10. Use according to claim 9, characterized in that the composition is for oral administration. 11. Use according to any one of claims 1 to 10, characterized in that the composition is to be administered to a human subject.