Extract of Ulva algae for the treatment of inflammation and cognitive and / or emotional disorders induced by metabolic syndrome

Abstract

The present invention relates to an extract of algae of the genus Ulva and its use in human and animal nutrition for the treatment of inflammation, preferably inflammation associated with metabolic syndrome, as well as for the treatment of cognitive and / or emotional disorders induced by metabolic syndrome. The present invention also relates to food supplements and food compositions comprising an extract of algae of the genus Ulva.

Description

Title of the invention: Extract of Ulva algae for the treatment of inflammation and cognitive and / or emotional disorders induced by metabolic syndrome technical field

[0001] The present invention relates to an extract of algae of the genus Ulva and its use in human and animal nutrition for the treatment of inflammation, preferably inflammation associated with metabolic syndrome, as well as for the treatment of cognitive and / or emotional disorders induced by metabolic syndrome. The present invention also relates to food supplements and food compositions comprising an extract of algae of the genus Ulva. Prior art

[0002] Genetic predisposition, sedentary lifestyle, dietary habits, environmental factors, and altered metabolism are the main and converging factors in the development of obesity, a well-known complex and multifactorial pathology that has reached pandemic proportions. The incidence of obesity has increased very rapidly over the last 30 years, suggesting that behavioral and environmental factors, particularly greater availability and consumption of high-fat foods (the "High Fat Diet"), have strongly contributed to this condition. Obesity induced by a high-fat diet is linked to several pathological conditions such as diabetes, cardiovascular disease, hypertension, liver disease, and certain forms of cancer (colon, gallbladder, breast, etc.).

[0003] The common feature of all obesity-induced metabolic diseases is inflammation, in particular low-grade chronic inflammation which is closely related to the pathophysiology of adipose tissue.

[0004] Indeed, obesity induced by a high-fat diet is characterized by low-grade inflammation, as well as insulin resistance, oxidative stress, and mitochondrial dysfunction.

[0005] There is therefore a need to develop molecules targeting inflammation in order to treat inflammation and also to treat cognitive and / or emotional disorders induced by metabolic syndrome.

[0006] Numerous compositions have been developed to prevent or treat these pathologies. However, the existing products or methods for meeting these needs are limited by: - either the complexity, and therefore the cost, of formulating these compositions including ingredients of widely varying levels of purity, - or the lack of factual demonstration of the anti-inflammatory or neuro-protective activity of the extracts.

[0007] In this context, algae are of increasing interest to this market, which is demanding new products of natural origin. Indeed, while they have been valued for decades, mainly as a food source, for the texturizing properties of the polysaccharides they produce or, more recently, for their dermo-cosmetic properties, marine macroalgae, in particular, present a wide diversity of molecules that are still little studied.

[0008] Over the period 1999-2015, 99 purified compounds from marine macroalgae exhibiting neuroprotective activity were identified. Of these compounds, 57 came from brown macroalgae, 28 from red macroalgae, and only 14 from green macroalgae.

[0009] From the perspective of green marine macroalgae, there is a substantial body of literature highlighting the properties of compounds isolated from green algae or complex green algae extracts in the nutraceutical or pharmaceutical fields. However, no data relating to the anti-inflammatory activities of these same compounds or extracts have been found.

[0010] It therefore appears that marine macroalgae, and in particular green algae, can still be considered to date as an under-exploited reservoir of anti-inflammatory compounds. Summary of the invention

[0011] The inventors have demonstrated that an extract of algae of the genus Ulva exhibits anti-inflammatory properties.

[0012] Thus, the present invention relates to an extract of algae of the genus Ulva, characterized in that: - the protein content is between 5 and 60% by mass, preferably between 15 and 50% by mass, preferably between 25 and 35% by mass, relative to the total dry mass of the extract, - the total phenolic compound content is between 0.5 and 40% by mass, preferably between 1 and 20% by mass, preferably between 1 and 10% by mass, relative to the total dry mass of the extract, - the total sugar content is between 0.5 and 80% by mass, preferably between 1 and 50% by mass, preferably between 5 and 15% by mass, relative to the total dry mass of the extract.

[0013] In one embodiment, the Ulva seaweed extract according to the invention has a protein content of between 28 and 32% by mass relative to the dry mass total extract.

[0014] In one embodiment, the Ulva seaweed extract according to the invention has a total phenolic compound content of between 2 and 6% by mass relative to the total dry mass of the extract.

[0015] In one embodiment, the Ulva seaweed extract according to the invention has a total sugar content of between 5 and 15% by mass relative to the total dry mass of the extract.

[0016] In one embodiment, the Ulva seaweed extract according to the invention is an Ulva seaweed extract selected from Ulva lactuca, Ulva conglobata, Ulva ohnoi, Ulva reticulata, Ulva prolifera, Ulva flexuosa and Ulva intestinalis.

[0017] The in vitro anti-inflammatory effect of the Ulva algae extract according to the invention was evaluated on a Raw 264.7 murine macrophage cell line provided by ATCC Cell (“American Type Culture Collection”): code ATCC-TIB-71. These are immortalized mouse macrophages used as reference biological study models in metabolic diseases, to measure anti / pro-inflammatory activity.

[0018] Advantageously, the inventors have demonstrated that after 6 hours of pre-incubation with the extract according to the invention, LPS-induced nitric oxide (NO) production is drastically reduced compared to inflammatory control conditions involving LPS alone. These results indicate that the extract according to the invention is highly anti-inflammatory, as it induces a reduction in NO secretion close to that induced by dexamethasone at very low concentrations. Indeed, nitric oxide (NO) is a key mediator of the inflammatory process, regulating the activity, growth, and death of many types of immune and inflammatory cells. A reduction in NO secretion reflects anti-inflammatory activity.

[0019] Furthermore, the modulation of protein signaling pathways involved in inflammation by the extract according to the invention was evaluated. Advantageously, the inventors demonstrated that the extract according to the invention induces a drastic reduction in the expression of NLRP3 and iNOS proteins, involved in the inflammatory process in cultured Raw 264.7 macrophages.

[0020] To complement these tests, the modulation by the extract according to the invention of the secretion of pro-inflammatory cytokines in the extracellular environment was evaluated. Advantageously, the inventors demonstrated that the algae extract according to the invention induces, after 6 h of pre-incubation, a drastic decrease in the secretion of LPS-induced TNF-α and 1TL-6, reaching a level similar to that obtained in the presence of dexamethasone, a reference anti-inflammatory compound, even at the very low concentration of 0.1 pg / ml.

[0021] These results demonstrate the anti-inflammatory effect of the extract according to the invention vis- regarding NO production, the expression of key proteins involved in signaling cascades related to the inflammatory process (NLRP-3 and iNOS), and the secretion of pro-inflammatory cytokines.

[0022] Thus, the present invention also relates to an extract of algae of the genus Ulva according to the invention for its use in the treatment of inflammation.

[0023] In one embodiment, the inflammation is inflammation induced by a metabolic syndrome.

[0024] The inventors also evaluated the effect of the extract according to the invention on metabolic and behavioral alterations by evaluating metabolic parameters (weight, fat mass and lean mass) and behavioral parameters (anxious-type behavior, memory abilities) in a metabolic syndrome model.

[0025] The animals' anxious behavior was assessed using the elevated cross maze test. The inventors demonstrated that supplementation with the extract according to the invention restores the animals' exploration time in open arms (F(2,20) = 7.376, p<0.01). Supplementation with the extract according to the invention thus protects against the development of anxiety-like disorders induced by the HFD diet.

[0026] Mice were also tested in the Morris water maze to evaluate the chronic effects of the treatment on memory. Interestingly, supplementation with the extract according to the invention restores the cognitive abilities of animals on a high-fat diet. Indeed, they discriminate the target quadrant in a statistically significant manner compared to chance (p<0.05).

[0027] The inventors have thus demonstrated that the extract according to the invention makes it possible to protect against a degradation of cognitive abilities and an emotional alteration induced by a metabolic syndrome.

[0028] Thus, the present invention also relates to an extract of algae of the genus Ulva according to the invention for its use in the treatment of cognitive and / or emotional disorders induced by metabolic syndrome.

[0029] According to one embodiment, cognitive and emotional disorders are chosen from among hippocampal-dependent long-term memory disorders and anxiety-type disorders.

[0030] The present invention also relates to a food supplement or food composition for humans or pets comprising an extract of algae of the genus Ulva according to the invention.

[0031] Indeed, the seaweed extract according to the invention can be used in human nutrition but also in animal nutrition. Brief description of the drawings

[0032] Other features, details and advantages will become apparent from reading the detailed description below, and from analyzing the accompanying drawings, in which: Fig. 1

[0033] [Fig. 1] shows the effect of fraction P on the viability of cultured murine Raw 264.7 macrophages after 24 h of incubation (A) or after 6 h of pre-incubation followed by 18 h of incubation in the presence of LPS (1 pg / ml) (B). The results express the relative mean percentage of viable macrophages compared to the control conditions (100%) ± ETM. Significant differences obtained between the samples and the control conditions (A) or between the samples and the conditions with LPS alone (B) are indicated by (n = 4): ns (not significant: p>0.05) (One-Way ANOVA). Fig. 2

[0034] [Fig. 2] shows the effect of fraction P on nitric oxide (NO) secretion by cultured Raw 264.7 murine macrophages after 24 h incubation (A) or after 6 h pre-incubation followed by 18 h incubation in the presence of LPS (1 pg / ml) (B). The results express the concentration of NO secreted into the culture medium ± ETM. Significant differences obtained between the samples and the control conditions (A) or between the samples and the conditions with LPS alone (B) are indicated by (n = 4): ns (not significant: p > 0.05), *** (p < 0.001), and **** (p < 0.0001) (One-way ANOVA). Fig. 3

[0035] [Fig. 3] shows the effect of fraction P on the respective expression of NLRP3 (A) and iNOS (B) proteins by cultured Raw 264.7 murine macrophages after 6 h of pre-incubation followed by 18 h of incubation in the presence of LPS. The results express the level of expression of the target proteins ± ETM, relative to the expression level of [3-actin], as a housekeeping protein. Significant differences obtained between the samples and the conditions with LPS alone are indicated by (n = 6): ns (not significant: p > 0.05), * (p < 0.05), ** (p < 0.01), and *** (p < 0.001) (One-Way ANOVA). Fig. 4

[0036] [Fig. 4] shows the effect of fraction P on the secretion of TNF-α and IL-6 by cultured Raw 264.7 murine macrophages after 24 h of incubation (A, C) or after 6 h of pre-incubation followed by 18 h of incubation in the presence of LPS (B, D). The results express the concentration of TNF-α and IL-6 secreted into the culture medium ± ETM. Significant differences obtained between the samples and the control conditions (A, C) or between the samples and the conditions with LPS alone (B, D) are indicated by (n = 4): ns (not significant: p > 0.05), * (p < 0.05), ** (p < 0.01) and *** (p < 0.001) (One-Way ANOVA). Fig. 5

[0037] [Fig.5] shows the effect of P fraction supplementation on the meta parameters Weights of obese and diabetic animals fed a high-fat diet (HFD): body weight (A), lean mass (B), and fat mass (C) of C57BL / 6J mice fed an HFD or standard A04 diet for 16 weeks and supplemented for the last 4 weeks with fraction P solution at 1 mg / ml in drinking water or with water (controls). Significant differences are indicated by (n = 12 / group): ns (not significant: p > 0.05), ** (p < 0.01), *** (p < 0.001), and **** (p < 0.0001) (One-way ANOVA + Tuckey post-hoc test). Fig. 6

[0038] [Fig.6] shows the effect of fraction P supplementation on exploration duration Open arms (OA) of the elevated cross maze, in obese and diabetic animals fed a high-fat diet (HFD): C57BL / 6J mice fed an HFD or standard A04 diet for 16 weeks and supplemented for the last 4 weeks with fraction P solution at 1 mg / ml in drinking water or with water (controls). Significant differences are indicated by (n = 12 / group): * (p<0.05) and ** (p<0.01) (One-way ANOVA + Tuckey post-hoc test). Fig. 7

[0039] [Fig.7] shows the schematic representation of the behavioral test protocol of the Morris's water maze. Fig. 8

[0040] [Fig.8] shows the effect of P fraction supplementation on animal memory Obese and diabetic mice fed a high-fat diet (HFD) were used. Memory was assessed by the time spent in the quadrant opposite the platform (Opp(E)), the quadrants adjacent to the platform (Adj(N or S)), or in the target quadrant where the platform was located (Target(W)). These mice were fed an HFD or standard A04 diet for 16 weeks and supplemented during the last 4 weeks with fraction P at 1 mg / ml in their drinking water or with water (controls). Significant differences between the obtained values ​​and chance (indicative value of 25%) are indicated by (n = 12 / group): * (p<0.05) and *** (p<0.001) (One-way ANOVA + Tuckey post-hoc test). Detailed Description Extract of algae of the genus Ulva

[0041] The present invention relates to an extract of algae of the genus Ulva characterized in that - the protein content is between 5 and 60% by mass, preferably between 15 and 50% by mass, preferably between 25 and 35% by mass, relative to the total dry mass of the extract, - the total phenolic compound content is between 0.5 and 40% by mass, preferably between 1 and 20% by mass, preferably between 1 and 10% by mass, relative to the total dry mass of the extract, - the total sugar content is between 0.5 and 80% by mass, preferably between 1 and 50% by mass, preferably between 5 and 15% by mass, relative to the total dry mass of the extract. Protein content

[0042] According to one embodiment, the seaweed extract of the genus Ulva is characterized by a protein content of between 5 and 60% by mass relative to the total dry mass of the extract, preferably between 25 and 35% by mass relative to the total dry mass of the extract and preferably, the protein content is between 28 and 32% by mass, even more preferably about 30% by mass relative to the total dry mass of the extract.

[0043] The protein content may be measured by any method known to those skilled in the art. By way of illustration, the methods of Lowry (Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov; 193(1):265-75), Smith (Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, et al. Measurement of protein using bicinchoninic acid. Anal Biochem. 1985 Oct; 150(1):76-85), and Kjeldahl (Kirk PL. Kjeldahl Method for Total Nitrogen. Anal Chem. 1950 Feb 15;22(2):354-8) may be cited. These protocols may be used independently and alternatively to measure the protein content. Total phenolic compound content

[0044] According to one embodiment, the extract of algae of the genus Ulva is characterized by a total phenolic compound content of between 0.5 and 40% by mass relative to the total dry mass of the extract, preferably between 1 and 10% by mass relative to the total dry mass of the extract and preferably, the total phenolic compound content is between 2 and 6% by mass, preferably still about 4% by mass relative to the total dry mass of the extract.

[0045] The term "total phenolic compounds" refers to all secondary metabolites characterized by the presence of an aromatic ring bearing free hydroxyl groups or groups involved with a carbohydrate, present in all parts of higher plants, the most represented being anthocyanins, flavonoids and tannins.

[0046] The total phenolic compound content can be measured by any method known to those skilled in the art. By way of illustration, the method of Singleton and Rossi (Singleton VL, Rossi JA. Colorimetry of total phenolics with phosphomolybdic-phosphotungstic acid reagents. Am J Enol Vitic. 1965 Jan 1; 16(3): 144) may be cited, with some modifications. Total sugar content

[0047] According to one embodiment, the seaweed extract of the genus Ulva is characterized by a total sugar content of between 0.5 and 80% by mass relative to the total dry mass of the extract, preferably between 5 and 15% by mass relative to the total dry mass of the extract and preferably, the total sugar content is about 10% by mass relative to the total dry mass of the extract.

[0048] The term “total sugars” means all carbohydrates: mono-, oligo-, polysaccharides and their derivatives.

[0049] The total sugar content can be measured by any method known to those skilled in the art. By way of illustration, the phenol / sulfuric acid mixture method introduced by Dubois (Dubois M, Gilles K, Hamilton JK, Rebers PA, Smith F. A Colorimetry Method for the Determination of Sugars. Nature. 1951 Jul; 168(4265): 167-167) may be cited.

[0050] According to a preferred embodiment, the extract of algae of the genus Ulva is characterized in that: - The protein content is approximately 30% by mass relative to the total dry mass of the extract, - The total phenolic compound content is approximately 4% by mass relative to the total dry mass of the extract, - The total sugar content is approximately 10% by mass relative to the total dry mass of the extract.

[0051] In the present invention, the term "approximately," when referring to a number or a numerical range, means that the number or numerical range in question is an approximation within the limits of experimental variability (or within the limits of statistical experimental error), and therefore that the number or numerical range may vary between 1% and 15% of the number or numerical range indicated. For example, the use of "approximately X" encompasses + / -1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, and 15% of the value X. Algae of the genus Ulva

[0052] Algae of the genus Ulva are the sea lettuces, multicellular green algae distributed in the oceans with about 400 species.

[0053] According to one embodiment, the alga of the genus Ulva is chosen from all the species of the genus Ulva, preferably from the group consisting of the species Ulva lactuca, Ulva rigida, Ulva fasciata, Ulva pertusa, Ulva califomica, Ulva gigantea, Ulva pseudocurvata, Ulva conglobata, Ulva armoricana, Ulva fasciculata, Ulva ohnoi, Ulva pseudolinza, Ulva reticulata, Ulva rotundata, Ulva elegans, Ulva prolifera, Ulva scandinavica, Ulva umbilicata, Ulva flexuosa, Ulva clathrata, Ulva compressa and Ulva intestinalis.

[0054] According to a preferred embodiment, the alga of the genus Ulva is selected from Ulva lactuca, Ulva conglobata, Ulva ohnoi, Ulva reticulata, Ulva prolifera, Ulva flexuosa and Ulva intestinalis.

[0055] According to one embodiment, the alga of the genus Ulva is Ulva lactuca.

[0056] According to one embodiment, the extract of algae of the genus Ulva according to the invention is capable of being obtained by the process described below. Inflammation

[0057] The inventors have advantageously demonstrated the anti-inflammatory effect of the extract according to the invention with respect to the production of NO, the expression of key proteins involved in signaling cascades related to the inflammatory process (NLRP-3 and iNOS), and the secretion of pro-inflammatory cytokines.

[0058] Thus, the present invention relates to an extract of algae of the genus Ulva according to the invention for its use in the treatment of inflammation.

[0059] According to one embodiment, the present invention relates to an extract of algae of the genus Ulva according to the invention for its use in the treatment of inflammation induced by metabolic syndrome.

[0060] The term "metabolic syndrome" usually refers to disorders of energy metabolism (fasting hyperglycemia, insulin resistance, hypertension, hypertriglyceridemia, visceral fat accumulation, low plasma HDL cholesterol levels) associated with obesity or being overweight. Metabolic syndrome is diagnosed when at least three of the above-listed abnormalities are present.

[0061] According to one embodiment, the present invention relates to an extract of algae of the genus Ulva according to the invention for its use in improving at least one symptom or disorder associated with metabolic syndrome.

[0062] According to one embodiment, the present invention relates to an extract of algae of the genus Ulva according to the invention for its use in the treatment of inflammation induced by obesity or overweight.

[0063] Obesity and overweight can be determined, in particular, by calculating the body mass index (BMI). The BMI is obtained by calculating the ratio between weight and height squared. For example, a person measuring 1.62 m and weighing 55 kg has a BMI of 20 (i.e., 55 / 1.622). Generally speaking, a person is considered to have a normal weight when their BMI is between 18 and 25 kg / m², to be overweight when their BMI is between 25 and 30 kg / m², and to be obese if their BMI exceeds 30 kg / m².

[0064] The present invention also relates to a method for the treatment of inflammation, including the administration of an algae extract according to the invention to a mammal in need of it.

[0065] The present invention also relates to the use of an algae extract according to the invention for obtaining a drug for the treatment of inflammation.

[0066] The present invention also relates to a pharmaceutical or veterinary composition comprising an algae extract according to the invention for its use in the treatment of inflammation.

[0067] Treatment of cognitive and / or emotional disorders induced by metabolic syndrome.

[0068] The inventors have indeed demonstrated that the extract according to the invention makes it possible to protect against a degradation of cognitive abilities and / or an emotional alteration induced by a metabolic syndrome.

[0069] Indeed, it has been demonstrated that the extract according to the invention protects against the development of anxiety-type disorders induced by the HFD diet and that it allows the cognitive abilities of animals fed with an HFD diet to be restored.

[0070] Thus, the present invention also relates to an algae extract according to the invention for the treatment of cognitive and / or emotional disorders induced by metabolic syndrome.

[0071] Among the cognitive and emotional disorders, we will mention hippocampal-dependent long-term memory disorders and anxiety-type disorders.

[0072] According to one embodiment, the present invention relates to an algae extract according to the invention for the treatment of hippocampal-dependent long-term memory disorders and / or anxiety-type disorders.

[0073] The present invention also relates to a method of treating cognitive and / or emotional disorders induced by metabolic syndrome comprising administering an algae extract according to the invention to a mammal in need of it.

[0074] The present invention also relates to the use of an algae extract according to the invention for obtaining a drug intended for the treatment of cognitive and / or emotional disorders induced by metabolic syndrome.

[0075] The present invention also relates to a pharmaceutical or veterinary composition comprising an algae extract according to the invention for its use in the treatment of cognitive and / or emotional disorders induced by metabolic syndrome. Treatment

[0076] The terms "treatment" or "treatment method" are not absolute terms and, when applied to inflammation or to cognitive and / or emotional disorders induced by metabolic syndrome, refer to a procedure or plan A treatment is designed, even with a low probability of success, to induce an overall beneficial effect such as delaying the onset of the disease or reducing the severity of one or more symptoms. Typically, in the case of inflammation, treatment can be understood as reducing the inflammatory response. Typically, in the case of inflammation, treatment can be understood as reducing cognitive and / or emotional disturbances. Typically, in the case of inflammation, treatment can be understood as improving memory capacity and / or reducing anxiety.

[0077] The expressions "induced by metabolic syndrome" and "associated with metabolic syndrome" can be used interchangeably.

[0078] According to one embodiment, the invention aims at the use of an algae extract according to the invention for the improvement of at least one symptom associated with a metabolic syndrome, in which said improvement includes the reduction of inflammation, the reduction of cognitive and / or emotional disorders, the improvement of memory capacities and / or the reduction of anxiety.

[0079] Whether for the treatment of inflammation or the treatment of cognitive and / or emotional disorders induced by metabolic syndrome, the mammal is chosen from among humans and companion animals.

[0080] The seaweed extract according to the invention can indeed be used in human nutrition but also in animal nutrition.

[0081] According to one embodiment, the mammal is a human.

[0082] According to another embodiment, the mammal is a pet such as a dog or a cat.

[0083] According to one embodiment, the seaweed extract according to the invention may be administered in the form of a pharmaceutical or veterinary composition comprising, in addition to the seaweed extract, a pharmaceutically acceptable excipient.

[0084] The term “pharmaceutically acceptable” means a substance that is not biologically or otherwise undesirable, i.e., that can be incorporated into a pharmaceutical composition administered to a patient or animal without causing undesirable biological effects or without interacting adversely with any of the other components of the composition in which it is contained, for example by inhibiting or diminishing the anti-inflammatory properties of the seaweed extract.

[0085] Typically, the pharmaceutically acceptable excipient may be selected from a diluent, a disintegrant, a binder, a sliding agent, a lubricant, a wetting agent, a buffering agent, a suspending agent, an adjuvant, an emulsifier, an absorbent, a preservative, a surfactant, a sweetener, an antioxidant, or a mixture thereof. These excipients are described, for example, in "The Science and Practice of Pharmacy 1995, edited by E.W. Martin, Mack Publishing Company, 19th edition, Easton, PA.

[0086] The quantity of the algae extract according to the invention in the compositions can vary so as to administer an effective amount of the algae extract to obtain the desired therapeutic response for a particular mammal.

[0087] By "effective amount" or "therapeutically effective amount" of a compound is meant a non-toxic but sufficient amount of the compound to provide the desired effect.

[0088] Typically, the administered quantity, or dose, depends on the activity of the seaweed extract according to the invention, the route of administration, the severity of the pathology, as well as the health status and medical history of the mammal being treated, and various factors such as body weight, diet, and any possible combination with other therapeutic agents. However, it is within the competence of a person skilled in the art to determine the appropriate dosage and to initiate treatment at a dose lower than that required to obtain the desired therapeutic effect, then gradually increase the dose until the desired effect is achieved.

[0089] The seaweed extract may be administered orally. Typically, pharmaceutical or veterinary compositions may be presented as a food supplement for humans or pets or as food compositions for humans or pets.

[0090] Method for obtaining the extract of algae of the genus Ulva according to the invention

[0091] Advantageously, the seaweed extract according to the invention can be obtained according to the steps of the process described in patent FR 2 998 894 of the company SEPROSYS, which describes a seaweed processing process comprising: - a grinding stage, - diffusion of the algae to be treated into the water, - filtering the pulp recovered after the diffusion stage in order to obtain pressed juice on the one hand and pressed pulp on the other, - ultrafiltration of the pressed juice in order to obtain a retentate on the one hand and a permeate on the other.

[0092] The inventors advantageously discovered that the permeate obtained by implementing such a process on algae of the genus Ulva exhibited anti-inflammatory properties.

[0093] Thus, the present invention also relates to a process for obtaining an extract of algae of the genus Ulva comprising the steps: - seaweed grinding - algae extraction - filtration to obtain an algae extract with anti-inflammatory properties.

[0094] The term "algae extract exhibiting anti-inflammatory properties" means an algae extract that enables: - decrease NO production, or - reduce the expression of NLRP3 and iNOS proteins, or - reduce the secretion of TNF-α and IL-6.

[0095] According to one embodiment, the seaweed grinding step makes it possible to obtain seaweed particles with a diameter of less than 10 mm, preferably less than 5 mm, preferably less than 2 mm.

[0096] Typically, the algae can be ground in demineralized water, at a temperature between 25 and 100°C, preferably between 40 and 90°C, preferably between 55 and 75°C, and preferably, at a temperature of about 65°C.

[0097] Any utensil known to a person skilled in the art can be used to grind the seaweed, typically a knife grinder.

[0098] According to one embodiment, the process may include a step prior to grinding, of washing the algae, typically in raw water for 10 min at room temperature, followed by spinning.

[0099] According to one embodiment, the algae extraction or diffusion step is carried out at a temperature between 25 and 100°C, preferably between 40 and 90°C and preferably at a temperature of about 80°C.

[0100] According to one embodiment, the duration of the algae extraction or diffusion step is between 15 min and 5 h, preferably between 1 h and 4 h, preferably between 1 h and 3 h. According to a preferred embodiment, the duration of the extraction step is approximately 2 h.

[0101] According to one embodiment, the extraction step is carried out in a reduced volume of water.

[0102] According to one embodiment, the extraction is carried out under constant stirring. By way of illustration, the extraction is carried out under constant stirring using a paddle shaker, at a rotation speed of 10 rpm.

[0103] The extraction step makes it possible to obtain an aqueous extract and algae pulps.

[0104] According to one embodiment, the aqueous extract obtained following the extraction is filtered.

[0105] According to one embodiment, the filtration steps may be carried out according to the The process described in patent FR 2 998 894 of the company SEPROSYS. The process comprises the following steps: - Filtration of the pulp recovered after the diffusion stage, in order to obtain pressed juice on the one hand and pressed pulp on the other, - ultrafiltration of the pressed juice in order to obtain a retentate on the one hand and a permeate on the other.

[0106] Alternatively, the following filtration steps may be carried out after the step extraction.

[0107] Typically, the aqueous extract obtained after the extraction step may undergo a first filtration step and be filtered using a vibrating sieve having a cut-off threshold of 50 µm. This filtration step of the aqueous extract makes it possible to remove algae residues.

[0108] According to one embodiment, the pulps obtained after the extraction step are pressed.

[0109] This step advantageously allows for obtaining pressed juice on the one hand and pressed pulp on the other, and for extracting as much juice as possible. In one embodiment, the pressed juice is added to the aqueous extract.

[0110] According to one embodiment, the process comprises, following the first filtration step, three additional filtration steps.

[0111] According to one embodiment, the process according to the invention comprises a first microfiltration step.

[0112] By way of illustration, a plate filter type microfiltration unit equipped with two filters of 1.2 pm and then 0.8 pm (3M, 40*40, pore size 15 HN and 50 HN) may be used.

[0113] The microfiltration step makes it possible to obtain, from the pressed juice, a retentate on the one hand and a permeate on the other.

[0114] The permeate obtained following the microfiltration step can be filtered via an ultrafiltration unit.

[0115] Thus, according to one embodiment, the process according to the invention includes an ultrafiltration step.

[0116] According to one embodiment, the process according to the invention includes an ultrafiltration step of the permeate obtained following microfiltration so as to obtain a retentate on the one hand and a permeate on the other hand.

[0117] According to one embodiment, the ultrafiltration step is carried out between 60 and 100°C, preferably at about 80°C, at a pressure between 2 and 10 bars, preferably at about 5 bars and at a circulation flow rate between 300 l / h and 600 l / h, preferably at 450 l / h.

[0118] According to one embodiment, the ultrafiltration step is carried out until a retentate of approximately 4 Bx is obtained.

[0119] 4 Bx or Brix corresponds to approximately 4% of dry matter as measured by the refractive index fraction and expressed in degrees brix sucrose scale.

[0120] The retentate can be withdrawn from the unit and the permeate can be recovered and then reverse osmosis a reverse osmosis unit.

[0121] By way of illustration, ultrafiltration can be carried out on an ultrafiltration unit equipped with a Kerasep KBW 15 kDa type membrane (Novasep Process).

[0122] According to one embodiment, the process according to the invention includes a reverse osmosis step.

[0123] According to one embodiment, the process according to the invention includes a reverse osmosis step of the ultrafiltration permeate.

[0124] According to one embodiment, the reverse osmosis step is carried out between 20 and 50°C, preferably between 25 and 40°C and preferably, the reverse osmosis step is carried out at about 30°C, at a pressure between 10 and 50 bars, preferably between 20 and 30 bars and at a circulation flow rate between 80 l / h and 150 l / h, preferably at 120 l / h.

[0125] By way of illustration, the reverse osmosis step can be carried out on a reverse osmosis unit equipped with a SW 30 HR type membrane (Dow).

[0126] According to one embodiment, the reverse osmosis step is continued until a retentate of approximately 13 Bx corresponding to the osmosis ultrafiltration permeate is obtained.

[0127] 13 Bx or Brix corresponds to approximately 13% dry matter as measured by the index of reduction and expressed in degrees Brix sucrose scale.

[0128] According to one embodiment, the obtained osmosed ultrafiltration permeate will undergo a drying step, for example by freeze-drying.

[0129] The fraction obtained of the type of osmosis ultrafiltration permeate will hereinafter be referred to as the extract of algae of the genus Ulva according to the invention or also permeate or also “fraction P”.

[0130] Thus, according to one embodiment, the process for obtaining an extract of algae of the genus Ulva comprises the following steps: a) Washing the algae to be treated b) Grinding of the washed algae c) Water extraction of algae washed in a reduced volume of water d) Filtration of the pulp recovered after step c) then pressing of the pulp to obtain a pressing juice on the one hand and pressing pulp on the other e) Microfiltration of the pressed juice obtained in step d) so as to obtain a retentate on the one hand and a permeate on the other f) Ultrafiltration of the microfiltration permeate obtained in step e) so as to obtain a retentate on the one hand and a permeate on the other g) Reverse osmosis of the ultrafiltration permeate obtained in step f) h) Lyophilization of the osmosis ultrafiltration permeate obtained in step g).

[0131] According to one embodiment, the seaweed extract obtained by the process and having anti-inflammatory properties has the following characteristics: a protein content of about 30%, an average total phenolic compound content of about 4% and about 10% total sugars. Dietary supplement

[0132] According to one embodiment, the present invention also relates to a food supplement comprising an extract of algae of the genus Ulva according to the invention.

[0133] The extract of algae of the genus Ulva is as defined above.

[0134] The term “food supplement” means foodstuffs the purpose of which is to supplement a normal diet and which constitute a concentrated source of nutrients or other substances having a nutritional or physiological effect.

[0135] According to one embodiment, the food supplement comprises, in addition to the seaweed extract according to the invention, at least one ingredient selected from nutrients such as vitamins and minerals, plants and / or plant extracts, substances with a nutritional or physiological purpose, food adjuvants or additives such as colorings, flavorings, preservatives.

[0136] The food supplement may also include any pharmaceutically acceptable excipient as previously defined.

[0137] The food supplement according to the invention can be in any form compatible with oral absorption in one or more daily doses. It can be in particular in the form of capsules, tablets, lozenges or loose powder, the latter preferably being packaged in individual sachets.

[0138] It is particularly interesting to use the food supplement comprising an extract of the genus Ulva according to the invention in a food supplement, intended for the overweight or obese patient, but also for the overweight or obese pet.

[0139] According to one embodiment, the food supplement for humans is presented in the form of unit doses, for example in the form of capsules, caplets, tablets, lozenges or loose powder packaged in unit sachets, each unit dose comprising between 0.05 g and 2 g of seaweed extract according to the invention, preferably between 0.1 and 1 g, and even more preferably between 0.2 and 0.8 g.

[0140] According to another embodiment, the pet food supplement is presented in the form of unit doses, for example in the form of loose powder packaged in unit sachets, each unit dose comprising between 0.01 g and 2 g of seaweed extract according to the invention, preferably between 0.025 and 1.5 g, and even more preferably between 0.03 and 1 g.

[0141] Surprisingly, the extract according to the invention exhibits significant activity at very low concentrations.

[0142] Without wishing to be bound by any theory, it is the opinion of the inventors that Concentration-dependent induction and inhibition mechanisms intervene or cancel each other out below a threshold concentration of the extract according to the invention, given that this extract is a complex mixture of different molecules which can logically combine synergistic and / or antagonistic effects. Dietary composition for humans

[0143] The seaweed extract according to the invention can be incorporated into any food composition intended for human consumption. Therefore, the seaweed extract according to the invention can be incorporated into beverages, meal replacements, biscuits, cereals, desserts, dairy preparations, etc.

[0144] It will be particularly interesting to use the food composition comprising an extract of the genus Ulva according to the invention in a food composition intended for the overweight or obese patient. Pet food composition

[0145] The present invention also relates to a pet food composition comprising an extract of algae of the genus Ulva according to the invention.

[0146] These food compositions are not intended to be limited to a specific list of ingredients, as these ingredients will depend on factors such as, for example, the desired nutritional balance for the specific type of pet, and the availability of the ingredients. In addition to the seaweed extract according to the invention, the pet food composition may generally include vitamins, minerals, and other additives such as flavorings, preservatives, emulsifiers, and humectants. The nutritional balance, including the relative proportions of the seaweed extract according to the invention and the other ingredients, is determined according to dietary standards known in veterinary practice. For example, the nutritional balance of a cat food composition is determined based on the known dietary requirements of cats.

[0147] It will be particularly interesting to use the food composition comprising an extract of the genus Ulva according to the invention in a food composition intended for overweight or obese pets. Examples

[0148] Example 1: Production and characterization of an extract of an alga of the genus Ulva

[0149] A. Extract production

[0150] This method corresponds to an adaptation of the extraction and fractionation process described in patent FR2998894B1 of the company Seprosys, allowing the purification of protein and polysaccharide fractions from green marine macroalgae.

[0151] The process comprises the following sequence of steps: a) Washing the algae to be treated b) Grinding the washed algae c) Diffusion of the washed algae in a reduced volume of water d) Filtration of the pulp recovered after step c) then pressing of the pulp to obtain a pressing juice on the one hand and pressing pulp on the other e) Microfiltration of the pressed juice obtained in step d) so as to obtain a retentate on the one hand and a permeate on the other f) Ultrafiltration of the microfiltration permeate obtained in step e) so as to obtain a retentate on the one hand and a permeate on the other g) Reverse osmosis of the ultrafiltration permeate obtained in step f) h) Lyophilization of the osmosis ultrafiltration permeate obtained in step g).

[0152] 1 kg of dried seaweed is soaked in 30 L of raw water for 10 min at temperature ambient temperature. The algae are then wrung out and pressed using a fabric cone to remove as much water as possible. The washed algae are then ground in 5 liters of demineralized water at 65°C using a knife mill until particles less than 2 mm in diameter are obtained. The 5 liters of ground material are transferred to a thermostatically controlled tank at 80°C containing 10 liters of demineralized water at room temperature. Extraction is carried out under constant agitation using a paddle shaker at a rotation speed of 10 rpm for 2 hours. The aqueous extract is then filtered using a vibrating screen with a 50 µm cutoff (stainless steel mesh, NEGOFILTRE) to remove any remaining algae. The seaweed pulp is then removed from the tank and pressed using a press (60 hl / h) to extract as much juice as possible.The juice from the press is then combined with the initial filtered aqueous extract, and approximately 15 liters of total extracted juice are recovered and filtered through a plate filter microfiltration unit equipped with two filters: 1.2 µm and 0.8 µm (3M, 40 x 40 mm, pore sizes 15 HN and 50 HN). Approximately 15 liters of permeate are recovered and then filtered again through an ultrafiltration unit equipped with a Kerasep KBW 15 kDa membrane (Novasep Process). Filtration is carried out at 80°C, 5 bar, and a flow rate of 450 liters / hour, corresponding to a flow velocity of 5 m / s. Filtration continues until a retentate of approximately 4 Bx is obtained, which is then withdrawn from the unit. The permeate is then collected and treated with reverse osmosis in a unit equipped with a Dow SW 30 HR membrane, allowing for concentration of the fraction. Reverse osmosis is carried out at 30°C, between 20 and 30 bar, and at a flow rate of 120 liters per hour, corresponding to a flow velocity of 1.3 m / s.Reverse osmosis is continued until a retentate of approximately 13 Bx is obtained, which is then withdrawn from the unit and subsequently lyophilized. This fraction obtained, of the osmosis ultrafiltration permeate type, will hereafter be referred to as the permeate-type Ulva algae extract or "fraction P".

[0153] B. Characterization of the extract

[0154] a) Solubility

[0155] Several different solvents are used to evaluate the solubility of the permeate-type extract of Ulva algae (fraction P): water, acetonitrile (ACN), methanol, ethanol, acetone, hexane, a water / ACN mixture (1:1, v / v), and a methanol / acetone mixture (1:1, v / v). Different mass quantities of the solid extract are vortexed with the different solvents, and then the absorbance of the resulting solution or suspension is measured at 600 nm using a FLUOstar Omega microplate spectrophotometer (BMG LABTECH). After subtracting the blank obtained from each pure solvent, the solution is considered clear and the extract soluble if the absorbance obtained is less than 0.02.

[0156] Fraction P is soluble only in water and the water / ACN mixture (1:1, v / v) (Table 1), its solubility being maximum in water (1 g / 1), and minimum in the water / ACN mixture (1:1, v / v).

[0157] [Tables 1] Solvent Solubility of fraction P (mg / ml) Water 1 Water / ACN (1:1, v / v) 0.33

[0158] Table 1: Solubility of fraction P in water and water / acetonitrile mixture (1:1, v / v).

[0159] b) Biochemical characterization

[0160] Two solvents, water and a water / ACN mixture (1:1, v / v), are used to prepare two solutions of fraction P at 20 mg / ml. These are then centrifuged at 15,000 rpm for 2 min and filtered through 0.22 µm to obtain a final solution containing only the soluble compounds. Due to the influence of the color of these two solutions on the absorbance measurement, they are then diluted 1 / 18 with the same solvent before analysis.

[0161] All analyses are carried out in triplicate.

[0162] Protein content

[0163] Protein content is assessed using three biochemical protocols, based respectively on the methods of Lowry (Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265-75), Smith (Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, et al. Measurement of protein using bicinchoninic acid. Anal Biochem. 1985 Oct; 150(1):76-85) and Kjeldahl (Kirk PL. Kjeldahl Method for Total Nitrogen. Anal Chem. 1950 Feb 15;22(2):354-8).

[0164] - Lowry's method:

[0165] This involves the use of two reagents: Lowry's reagent, which consists of a mixture of 100 µl of 2% (w / v) potassium and sodium double tartrate solution, 100 µl of 1% (w / v) copper sulfate solution and 10 ml of 2% (w / v) sodium carbonate solution in 0.1 M NaOH; and Folin's reagent, corresponding to a 1 / 20 dilution of the Folin-Ciocalteu phenol reagent in ultrapure water.

[0166] In a 96-well microplate (NUNC 96 A / S, Denmark), 25 µL of the solution to be assayed are added to 75 µL of Lowry's reagent before incubation for 20 min at room temperature and in the dark. Then, 125 µL of Folin's reagent are added to the reaction mixture. A second incubation of 30 min at room temperature and in the dark is performed before measuring the absorbance at 750 nm using a FLUOstar Omega microplate spectrophotometer (BMG LABTECH). A bovine serum albumin (BSA) standard curve of 50 to 600 pg / ml is prepared simultaneously under the same conditions.

[0167] - Smith method, with bicinchoninic acid (BCA):

[0168] In a 96-well microplate (NUNC 96 A / S, Denmark), 200 µl of BCA reagent (bicinchoninic acid / copper sulfate 4% (w / v) (50:1, v / v)) and 10 µl of the solution to be assayed are added. After incubation at 37°C in an oven for 30 min, the absorbance at 562 nm is measured using a FLUOstar Omega microplate spectrophotometer (BMG LABTECH). A standard curve of BCA ranging from 50 to 600 mg / L is prepared simultaneously under the same conditions.

[0169] - Kjeldahl method:

[0170] This method involves two successive steps: a first step of mineralization and a second step of distillation. For mineralization, 1 g of sample is added to a flask, along with 2 catalyst tablets. A flask containing only the tablets is used as a blank, and another flask containing a known quantity of urea and the 2 catalyst tablets is used as a control. 10 mL of 96% sulfuric acid are added to the flask. The solution is then heated for 2 h at approximately 400°C. After mineralization, distillation is carried out for 6 min after the addition of 50 mL of 30% (w / v) NaOH and 50 mL of ultrapure water. The liquid is then collected in a boric acid solution (4% (w / v), pH 4.65) and titrated with 1 M HCl.

[0171] Total sugar content

[0172] The determination of total sugars is carried out by the phenol / sulfuric acid mixture method introduced by Dubois (Dubois M, Gilles K, Hamilton JK, Rebers PA, Smith F. A Colorimetry Method for the Determination of Sugars. Nature. 1951 Jul; 168(4265): 167-167). In glass vials, 40 µl of the solution to be titrated are added to 40 µl of 5% (w / v) phenol and 200 µl of 96% sulfuric acid. The mixture... The actionable sample is incubated for 30 min at 90°C in the dark. The samples are then cooled on ice. Absorbance is then measured at 485 nm in a 96-well microplate using a FLUOstar Omega microplate spectrophotometer (BMG LABTECH). A standard curve of rhamnose, a representative monomer, ranging from 0.025 to 0.6 mg / ml, is prepared simultaneously under the same conditions.

[0173] Total phenolic compound content

[0174] The determination of total phenolic compounds is carried out using the method of Singleton and Rossi (Singleton VL, Rossi JA. Colorimetry of total phenolics with phos-phomolybdic-phosphotungstic acid reagents. Am J Enol Vitic. 1965 Jan 1; 16(3): 144), with some modifications.

[0175] In centrifuge tubes, 10 µL of the sample to be assayed and 790 µL of ultrapure water are introduced. After mixing, 20 µL of 20% (w / v) Na₂CO₃ and then 50 µL of 2 N Folin-Ciocalteu reagent are added. The tubes are incubated at room temperature in the dark for 2 h. 200 µL of the reaction medium are then transferred to a 96-well microplate, and the absorbance at 730 nm is measured using a FLUOstar Omega microplate spectrophotometer (BMG LABTECH). A standard curve of gallic acid, a reference phenolic compound, ranging from 10 to 150 pg / ml, is prepared simultaneously under the same conditions.

[0176] Biochemical characterization of fraction P shows that this fraction has a significant protein content, ranging from 28.6% to 31.6% depending on the assay method used and the nature of the solvent used for its solubilization. These very similar results allow us to average this total protein content at approximately 30%. Similarly, the total phenolic compounds are quantified at very similar values, regardless of the solvent used, giving an average content of approximately 4%. The results obtained in terms of total sugar content, however, are very different, the content measured in the water / ACN mixture (1:1, v / v) being much lower (3.3%) than that measured in water (10.0%). This is consistent, given the lower solubility of the resuspended oligo- and / or poly-saccharides in a mixture of water and ACN, which is a less polar solvent, whereas they were initially extracted in pure water.In conclusion, the extract contains 10% total sugars.

[0177] [Tables2] Sample Content (% ws) Protein Total Sugars (n=3) Phenolic Compounds (n=3) Lowry Method (n=3) BCA Method (n=3) Kjehldal Method (n=5) Fraction P Water 28.6 ± 7.4% 29.8 ± 8.9% 29.6 ± 6.2% 10.0 ± 8.2% 4.6 ± 5.1% Water / ACN (1:1, v / v) 29.3 ± 4.2% 31.6 ± 9.4% not determined 3.3 ± 3.7% 4.1 ± 3.3%

[0178] Example 2: Production and characterization of an extract of an alga of the genus Ulva

[0179] In order to evaluate the in vitro anti-inflammatory effect of the permeate-type Ulva algae extract (fraction P), the latter was tested on a Raw 264.7 murine macrophage cell line provided by ATCC Cell (“American Type Culture Collection”): code ATCC-TIB-71. These are immortalized mouse macrophages used as reference biological study models in metabolic diseases, to measure anti / pro-inflammatory activity (Jones E, Adcock IM, Ahmed BY, Punchard NA. Modulation of LPS stimulated NF-kappaB mediated Nitric Oxide production by PKCe and JAK2 in RAW macrophages. Journal of Inflammation. 2007 Nov 24;4(1):23).

[0180] For all these evaluations, a one-way ANOVA statistical test was performed using Origin 6 software (OriginLab Corporation).

[0181] A. Effect of an extract of Ulva algae (permeate) on the cell viability of cultured Raw 264.7 macrophages:

[0182] The cells are cultured in Dulbecco's Modified Eagle Medium (DMEM) (PAN Biotech, Dutscher) supplemented with 10% (v / v) fetal bovine serum (PAN Biotech, Dutscher) and 1% (v / v) of an antibiotic solution (10,000 U / ml penicillin, 10 mg / ml streptomycin) (PAN Biotech, Dutscher). This medium, hereinafter referred to as the "complete medium," is sterilized by filtration through 0.22 µm. The cells are cultured in a temperature-controlled incubator at 37°C, with 5% CO2, under a humid atmosphere, in 75 cm² ventilated flasks (BD Biosciences), and maintained by mechanical scraping with a scraper (Sarstedt). The culture medium is renewed every 2 or 3 days. All manipulations are carried out under a class 2 microbiological safety cabinet. For the experiments, the cells are used between the 11th and 16th passages.

[0183] The effect of permeate-type Ulva algae extract (fraction P) on viability Cellular activity is determined via the enzymatic conversion of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (Mosmann T. Rapid colorimetry assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods. 1983 Dec 16;65(1-2):55-63). This assay relies on the reduction of the tetrazolium salt by mitochondrial succinate dehydrogenase to Formazan crystals. Cells are seeded in sterile 96-well microplates (BD Biosciences) at a concentration of 5 x 10⁴ cells / ml in a volume of 100 µl of complete medium. After 24 h of incubation, the complete medium is replaced with 100 µl of complete medium containing fraction P at different concentrations (between 0.01 and 500 pg / ml), or with 1 pM dexamethasone (an anti-inflammatory drug compound, used as a positive control). A negative control containing only complete medium is also used.After 24 h of exposure, 25 µl of a 5 mg / ml MTT solution in PBS are added to each well, which are then incubated for 4 h in the culture incubator. The medium is then removed, and 200 µl of DMSO (dimethyl sulfoxide) are added to each well to solubilize the Formazan crystals that have formed. After 10 min of incubation at room temperature, the absorbance at 550 nm is measured using a Fluostar Omega microplate reader (BMG LABTECH).

[0184] Cell viability is determined according to the following formula:

[0185] [Math.l] J ■ / ■. ' lï ; • / zy \ Absorbentf i Cell viability % =------rr—r-------------— x 100 / Absorbancetimo^^^

[0186] Figure IA shows the impact of fraction P on the viability of Raw 264.7 macrophages, compared to control conditions, after 24 h of exposure. Figure IB shows the same experiment in the presence of Salmonella lipopolysaccharide (LPS) at 1 pg / ml (LPS 0111, reference L2630, Merck), after 6 h of pre-incubation with fraction P at different concentrations. In both cases, the effect of a reference anti-inflammatory drug used as a positive control, dexamethasone (1 pM), is also compared. The results obtained demonstrate that neither fraction P, at any concentration, nor dexamethasone at 1 pM has a significant effect on the cell viability of Raw 264.7 macrophages, whether cultured in the presence of LPS or not.

[0187] B. Effect of an extract of Ulva algae (permeate) on nitric oxide (NO) secretion by cultured Raw 264.7 macrophages under basal or pro-inflammatory conditions

[0188] Nitric oxide (NO) is a central mediator of the inflammatory process, regulating the activity, growth, and death of many types of immune and inflammatory cells (Grisham MB, Jourd'Heuil D, Wink DA. Nitric oxide. I. Physiological chemistry of nitric oxide and its metabolites: implications in inflammation. Am J Physiol. 1999 Feb;276(2):G315-321, Lee M, Rey K, Besler K, Wang C, Choy J. Im-munobiology of Nitric Oxide and Regulation of Inducible Nitric Oxide Synthase. Results Problem Cell Differ. 2017;62:181-207). NO can be measured from RAW 264.7 macrophage culture supernatants according to the protocol described by Li and Shah (Li S, Shah NP. Characterization, Anti-Inflammatory and Antiproliferative Activities of Natural and Sulfonated Exo-Polysaccharides from Strep-tococcus thermophilus ASCC 1275. Journal of Food Science. 2016;81(5):M1167-76), which involves the reaction of Griess reagent with NO, resulting in the formation of a stable chromophore absorbing at 540 nm.

[0189] The cells and culture conditions are the same as those described in Example 2, Part A. The cells are seeded in sterile 96-well microplates (BD Biosciences) at a density of 1.5 x 10⁶ cells / ml in complete DMEM medium. After 24 h of incubation, the initial medium is replaced with 100 µl of complete medium containing fraction P at various concentrations (ranging from 0.01 to 100 pg / ml), or with 1 pM dexamethasone (the reference anti-inflammatory compound used as a positive control), before a further 6 h of incubation. A negative control containing only complete medium is also prepared.To evaluate the anti-inflammatory effects of the permeate-type Ulva algae extract (fraction P), the same experiment is carried out in parallel, this time with 6 h of pretreatment in the presence of permeate at various concentrations, or of dexamethasone at 1 pM, followed by incubation in the presence of salmonella lipopolysaccharide (LPS) at 1 pg / ml (LPS OUI, reference L2630, Merck) for 18 h.

[0190] The "Griess Reagent Kit for Nitrite Determination" (G-7921, Invitrogen™, Thermo Fisher Scientific) is used to determine the concentration of NO in cell culture supernatants, according to the protocol provided by the supplier. 150 µL of cell supernatant is taken for assay using an equivalent volume of Griess reagent. After incubation at room temperature for 30 min, the absorbance is measured at 540 nm using a Fluostar Omega microplate reader (BMG LABTECH).

[0191] Figure 2 shows the effect of fraction P on NO secretion in the culture medium under basal (A) or inflammatory (B) conditions. An increase in NO secretion compared to control conditions is observed in the presence of LPS (positive control), as well as for the two highest concentrations of fraction P (10 and 100 pg / ml) (Figure 2A).

[0192] On the contrary, fraction P induces a very significant decrease in NO production in the presence of LPS (Figure 2B), with the concentration of NO in the culture supernatant being halved at a concentration of 1 pg / ml and divided by approximately 1.5 at Concentrations of 0.01 and 0.1 g / ml, compared to inflammatory control conditions involving LPS alone. These results indicate that the P fraction is very strongly anti-inflammatory, as it induces a reduction in NO secretion close to that induced by dexamethasone at very low concentrations.

[0193] C. Effect of an extract of Ulva algae (permeate) on the expression of key proteins involved in LPS-induced inflammation signaling pathways by cultured Raw 264.7 macrophages

[0194] The evaluation of the modulation by permeate-type Ulva algae extract (fraction P) of protein signaling pathways involved in inflammation complements and clarifies that of nitric oxide secretion by cultured Raw 264.7 macrophages. This evaluation is performed by Western blot analysis of protein biomarkers involved in the inflammatory process: NLRP-3 and iNOS. NLRP-3, also known as the inflammasome, belongs to a class of cytoplasmic protein complexes (NLRPs) that modulate the activation of inflammatory mediators. NLRP-3 plays a pivotal role among the NLRP class and is activated after stimulation by microbial and non-microbial factors such as bacterial toxins, microparticles, or LPS. It promotes the expression, maturation and secretion of a multitude of pro-inflammatory cytokines (Wang Z, Zhang S, Xiao Y, Zhang W, Wu S, Qin T, et al. NLRP3 Inflammasome and Inflammatory Diseases. Oxid Med Cell Longev.2020 Feb 17;2020:4063562). Inducible nitric oxide synthase (iNOS) catalyzes the synthesis of nitric oxide (NO), which is a key mediator of inflammation. LPS, interferon gamma (IFN-γ), TNF-α, and IL-1 can induce iNOS expression. (Lee M, Rey K, Besler K, Wang C, Choy J. Immunobiology of Nitric Oxide and Regulation of Inducible Nitric Oxide Synthase. Results Probl Cell Differ. 2017;62:181-207).

[0195] The Western blot method is a semi-quantitative analytical method for the expression of proteins present in cells. The cells and culture conditions are the same as those described in Example 2, Part A. Raw 264.7 cells are seeded in sterile 6-well microplates (BD Biosciences) at 1 x 10⁶ cells / ml in 1 ml of complete DMEM medium. After 24 h of incubation, the initial medium is replaced with 1 ml of complete medium containing fraction P at various concentrations or dexamethasone at 1 pM (the reference anti-inflammatory compound used as a positive control), before another 6 h of incubation. A negative control containing only complete medium is also prepared. LPS at 1 pg / ml is then added to all wells except the one corresponding to the control conditions, and incubation is continued for 18 h.The cell supernatant is discarded, and then a cell lysate is prepared in lysis buffer (RIPA, Thermo Fisher Scientific) supplemented with a phosphatase inhibitor (Cell Signaling). The cell lysate is... The sample was centrifuged for 5 min at 10,000 g at 4°C. The protein concentration in the supernatant was then determined by applying the Smith method with bicinchonic acid (BCA), according to the protocol given in Example 1. An equivalent amount of protein for each sample (25 µg) was mixed with loading buffer (Bio-Rad) and 3-mercaptoethanol (Merck) to achieve a final volume of 50 µL. The samples were then heated to 95°C for 5 min, and 10 µL were then loaded, after returning to room temperature, into gels containing different concentrations of polyacrylamide depending on the molecular weight of the protein being studied. Size markers were also added to the gel (Seeblue® plus 2, Thermo Fisher Scientific). The gel was run at 150 V for 45 min in a migration buffer.The proteins are then transferred from the gel to a nitrocellulose membrane (Bio-Rad) at 100 V for 7 min in a transfer buffer using the semi-liquid transfer method (Bio-Rad). The membrane is then blocked for 1.5 hours at room temperature using a blocking solution containing 5% bovine serum albumin (BSA) in TBS-Tween 20 (Merck). The membrane is incubated overnight at 4°C in the presence of the primary antibody, and then in the presence of the secondary antibody coupled to horseradish peroxidase for 1.5 hours at room temperature (15).

[0196] [Tables3] Antibody name Dilution Supplier NLRP3 (D4D8T) Rabbit mAb 1 / 500 Cell Signaling iNOS Rabbit Polyclonal antibody 1 / 1000 Proteintech [3-Actin (13E5) Rabbit mAb 1 / 1000 Cell Signaling HRP secondary IgG antibody 1 / 10000 Thermo Fisher Scientific

[0197] Table 3: Primary and secondary antibodies used for Western Blot analysis of signaling pathways involved in LPS-induced inflammation.

[0198] The detection solution (SuperSignal™ West Femto Maximum Sensitivity Substrate, Thermo Fisher Scientific) is applied to the membrane for 1 min, then the acquisition is performed by a Chemidoc system (Bio-Rad). The analysis of the band densities is performed by the ImageLab software (Bio-Rad) and the results are expressed relative to the amount of the [3-actin] protein present in the samples, chosen as the housekeeping protein.

[0199] This Western blot analysis demonstrates that fraction P induces a drastic reduction in the expression of NLRP3 and iNOS proteins, involved in the inflammatory process in cultured Raw 264.7 macrophages ([Fig. 3]). This effect is dose-dependent for NLRP3, with fraction P inhibiting its expression from the first concentration of 1 pg / ml.

[0200] Conversely, iNOS expression is reduced only at low concentrations (0.1 and 1 pg / ml), with fraction P exhibiting no significant activity at 10 pg / ml. This inverted dose-response effect may originate from two main causes. Either the involvement of concentration-dependent induction and inhibition mechanisms, which either activate or cancel each other out below a threshold concentration, fraction P being, in fact, a very complex mixture of different molecules that can logically exhibit synergistic and / or antagonistic effects. Or the possible involvement of a hormesis phenomenon, a toxicology term describing a two-phase biological response to a compound, characterized by stimulation at low doses associated with a beneficial effect, and by inhibition at high doses associated with a toxic effect.The second hypothesis is very unlikely, given that the P fraction is not cytotoxic at the concentrations studied, as demonstrated by the commonly used MTT cell viability test, presented in example 2 (part A), both in the presence and absence of LPS.

[0201] Taken together, these results fully corroborate the anti-inflammatory effect demonstrated by the P fraction with respect to NO production by cultured Raw 264.7 macrophages, presented in Example 1 (Part B).

[0202] D. Effect of an extract of Ulva algae (permeate) on the secretion of pro-inflammatory cytokines by cultured Raw 264.7 macrophages, in the presence of LPS:

[0203] The evaluation of the modulation of pro-inflammatory cytokine secretion into the extracellular environment by the P fraction also complements and clarifies that of nitric oxide secretion by cultured Raw 264.7 macrophages, as well as the protein signaling pathways involved in inflammation. This is performed by ELISA for the quantitative analysis of two pro-inflammatory cytokines secreted by cells and present in the extracellular environment: TNF-α and IL-6.

[0204] The cells and culture conditions are the same as those described in Example 2, Part A. Raw 264.7 cells are seeded in sterile 6-well microplates (BD Biosciences) at 1 x 10⁶ cells / ml in 1 ml of complete DMEM medium. After 24 h of incubation, the initial medium is replaced with 1 ml of complete medium containing fraction P at various concentrations (ranging from 0.01 to 100 pg / ml), or with 1 pM dexamethasone (the reference anti-inflammatory compound used as a positive control) before a further 6 h of incubation. A negative control containing only complete medium is also prepared. LPS at 1 pg / ml is then added to all wells except the one corresponding to the control conditions, and incubation is continued for 18 h. The culture supernatant is then removed and frozen at -80°C. The concentrations of TNF-α and IL-6 in this supernatant are then determined according to the method indicated by the kit supplier (Peprotech), involving two final absorbance measurements, one for evaluation, at 405 nm, and the other for correction, at 650 nm, carried out using a Fluostar Omega microplate reader (BMG LABTECH).

[0205] The secretion of both pro-inflammatory cytokines is considerably increased in the presence of LPS, compared to control conditions ([Fig. 4]). The P fraction also exhibits this pro-inflammatory effect at high concentrations: from 1 pg / ml for TNF-α secretion and 10 pg / ml for IL-6 secretion. Below these threshold concentrations, no significant difference is observed compared to control conditions.

[0206] After 6 h of pre-incubation with fraction P and in the presence of LPS, the secretion of TNF-a and 1TL-6 is drastically reduced, reaching a level similar to that obtained in the presence of dexamethasone, the reference anti-inflammatory compound, including at the very low concentration of 0.1 pg / ml.

[0207] Taken together, these results demonstrate the anti-inflammatory effect of the P fraction with respect to NO production, the expression of key proteins involved in signaling cascades related to the inflammatory process (NLRP-3 and iNOS), and the production of pro-inflammatory cytokines.

[0208] Example 3: Demonstration of the in vivo efficacy of an extract of algae of the genus Ulva (permeate) on a model of behavioral alterations induced by a hypercaloric diet

[0209] Adult male C57B1 / 6J mice, supplied by Janvier and housed in the NutriNeuro laboratory's animal facility, were fed either a nutritionally balanced diet (diet A04) or a high-calorie diet enriched in fat and sugar (diet HFD) for 12 weeks to induce weight gain associated with metabolic syndrome and behavioral alterations, according to a protocol classically described in the literature and used within the NutriNeuro laboratory (Zemdegs J, Quesseveur G, Jarriault D, Pénicaud L, Fioramonti X, Guiard BP. High-fat diet-induced metabolic disorders impairs 5-HT function and anxiety-like behavior in mice. Br J Pharmacol. 2016 Jul;173(13):2095-110, Zemdegs J, Martin H, Pintana H, Bullich S, Manta S, Marked MA, et al. Metformin Promotes Anxiolytic and Antidepressant-Like Responses in Insulin-Resistant Mice by Decreasing Circulating Branched-Chain Amino Acids. J Neurosci. 2019 Jul 24;39(30):5935-48)..

[0210] To evaluate the effect of permeate-type Ulva algae extract (fraction P) on metabolic and behavioral alterations, animals were given a 1 mg / ml fraction P supplement via their drinking water for 4 weeks following a 12-week HFD diet. At the end of this supplementation, metabolic (weight, fat mass, and lean mass) and behavioral parameters (anxious behavior, memory capacity) are assessed.

[0211] For all these evaluations, a one-way ANOVA statistical test associated with a post-hoc Tuckey test was performed using GraphPad Prism 7 software (GraphPadSoftware).

[0212] A. Impact of supplementation with Ulva genus algae extract (permeate) on metabolic parameters

[0213] Following supplementation, the animals were weighed to assess the impact of supplementation on weight gain. As expected, mice fed an HFD diet had a significantly greater weight than mice fed the control diet (AO4) (F(2,22) = 12.34, p<0.001) ([Fig. 5]). Furthermore, supplementation with fraction P induced a statistically significant weight loss in supplemented HFD mice compared to unsupplemented mice (p<0.01). The weight of HFD mice supplemented with fraction P was not statistically different from that of control mice.

[0214] Analysis using a 3T spin-echo MRI device allowed for the quantification of lean and fat mass in mice. As expected, mice fed a high-fat diet (HFD) had significantly less lean mass than control mice (F(2,22) = 35.11, p<0.001), with no effect of supplementation. However, unsupplemented HFD mice had significantly more fat mass than control mice (F(2,22) = 36.59, p<0.001). But supplementation with fraction P significantly reduced fat mass in mice fed an HFD diet (F(2,22) = 36.59, p<0.01).

[0215] Supplementation with fraction P thus protects against the fat mass gain induced by the HFD diet.

[0216] B. Impact of supplementation with Ulva genus algae extract (permeate) on the behavioral parameters

[0217] a) Assessment of anxiety-type disorders

[0218] Anxious-type behavior in animals was assessed in the elevated cross maze test. Two opposing arms are open (without walls), and the other two have 10 cm high walls (closed). In the center is a platform open to all four arms. Mice are placed on the central platform at the start of the test and are free to explore the maze for 5 minutes. The time spent in the different zones was measured using the SMART video-tracking system (Bioseb). This device allows for the determination of the animals' anxiety level, as they spontaneously avoid the naturally anxiety-inducing open arms and prefer the closed arms. Using this device, it has been shown that pharmacological treatments with anxiolytics are effective in counteracting the natural aversion induced by the open arms.

[0219] As expected, animals fed a high-fat diet (HFD) and not supplemented showed a significant decrease in time spent in the open arms of the elevated cross maze (F(2, 20) = 7.376, p < 0.05) and thus exhibited anxiety-like behaviors ([Fig. 5]). Supplementation with fraction P restored the animals' exploration time in the open arms (F(2, 20) = 7.376, p < 0.01). Therefore, fraction P supplementation protected against the development of anxiety-like behaviors induced by the HFD diet.

[0220] Evaluation of memory impairments

[0221] In order to evaluate the chronic effects of the treatment on memory, mice are tested in the Morris water maze.

[0222] The water maze test developed by Morris (Morris R. Developments of a water-maze procedure for studying spatial learning in the rat. J Neurosci Methods. 1984 May;1 1(1):47-60.) allows for the measurement of learning and spatial memory abilities in animals. In this experimental protocol, this test specifically involves hippocampal-dependent reference spatial memory. The animal is placed in a pool and must learn to locate a refuge platform using visual cues distributed on the walls of the room.

[0223] To perform this test, we have a circular pool (150 cm in diameter), filled with water (35 cm deep, temperature: 22±2°C), made opaque by the addition of white paint. The platform (10 cm in diameter) is submerged 5 cm below the surface of the water and is located 30 cm from the edge of the pool. The chosen protocol takes place in 4 steps ([Fig.7]): 1-1'Habituation: allows the mice to become familiar with swimming and climbing onto a platform. This step takes place in a circular basin (65 cm in diameter) filled with clean water. 2- Cued learning: allows for the assessment of motor or visual deficits and enhances familiarization. The animals must find the submerged platform indicated by a clue. 3-1'Reference spatial learning: the animals must find a submerged and unseen platform using distal visual cues. 4- The recall test: allows us to assess spatial memory. Mice are placed in the pool without a platform and must search for 1 minute for the place where the platform was initially positioned.

[0224] Mouse movements are recorded by a video-tracking system (SMART), which allows for each trial to determine the latency to reach the platform as well as the total distance traveled in the pool. The swimming speed of the mice is deduced from these values. The time spent in the different quadrants, as well as the distance traveled and the number of entries in the target quadrant, are calculated.

[0225] In our protocol, control animals fed a balanced diet showed no cognitive impairment and significantly discriminated the target quadrant from random exploration (p<0.001). As expected, animals on a high-fat diet (HFD) were unable to recognize the target quadrant and therefore exhibited impairments in their long-term spatial memory ([Fig. 8]). Interestingly, supplementation with fraction P restored the animals' cognitive abilities, as they significantly differentiated the target quadrant from random exploration (p<0.05). There was no difference in learning between the groups.

Claims

Demands

1. Extract of algae of the genus Ulva characterized in that: - the protein content is between 5 and 60% by mass, preferably between 15 and 50% by mass, preferably between 25 and 35% by mass, relative to the total dry mass of the extract, - the total phenolic compound content is between 0.5 and 40% by mass, preferably between 1 and 20% by mass, preferably between 1 and 10% by mass, relative to the total dry mass of the extract, - the total sugar content is between 0.5 and 80% by mass, preferably between 1 and 50% by mass, preferably between 5 and 15% by mass, relative to the total dry mass of the extract for its use in the treatment of inflammation induced by metabolic syndrome.

2. Extract of algae of the genus Ulva characterized in that: - the protein content is between 5 and 60% by mass, preferably between 15 and 50% by mass, preferably between 25 and 35% by mass, relative to the total dry mass of the extract, - the total phenolic compound content is between 0.5 and 40% by mass, preferably between 1 and 20% by mass, preferably between 1 and 10% by mass, relative to the total dry mass of the extract, - the total sugar content is between 0.5 and 80% by mass, preferably between 1 and 50% by mass, preferably between 5 and 15% by mass, relative to the total dry mass of the extract for its use in the treatment of cognitive and / or emotional disorders induced by metabolic syndrome.

3. Algae extract according to claim 1 or 2, characterized in that the protein content is between 28 and 32% by mass relative to the total dry mass of the extract.

4. Algae extract according to claim 1 or 2, characterized in that the content of total phenolic compounds is between 2 and 6% by mass relative to the total dry mass of the extract.

5. Algae extract according to claim 1 or 2, characterized in that the total sugar content is between 5 and 15% by mass relative to the total dry mass of the extract.

6. Extract of algae of the genus Ulva according to any one of the claims previous, characterized in that the algae of the genus Ulva is chosen from the group consisting of the species Ulva lactuca, Ulva rigida, Ulva fasciata, Ulva pertusa, Ulva califomica, Ulva gigantea, Ulva pseu-docurvata, Ulva conglobata, Ulva armoricana, Ulva fasciculata, Ulva ohnoi, Ulva pseudolinza, Ulva reticulata, Ulva rotundata, Ulva elegans, Ulva prolifera, Ulva scandinavica, Ulva umbilicata, Ulva flexuosa, Ulva clathrata, Ulva compressa and Ulva intestinalis.

7. Algae extract of the genus Ulva according to any one of claims 1 to 6, characterized in that the algae of the genus Ulva is selected from Ulva lactuca, Ulva conglobata, Ulva ohnoi, Ulva reticulata, Ulva prolifera, Ulva flexuosa, and Ulva intestinalis.

8. Extract of algae of the genus Ulva for its use according to claim 2 characterized in that the cognitive and emotional disorders are selected from among hippocampal-dependent long-term memory disorders and anxiety-type disorders.

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